Breast Cancer

Lasers Med Sci. 2016 Dec;31(9):1775-1782. Epub 2016 Aug 12.

The effects of low-level laser irradiation on breast tumor in mice and the expression of Let-7a, miR-155, miR-21, miR125, and miR376b.

Khori V1, Alizadeh AM2, Gheisary Z3, Farsinejad S3, Najafi F4, Khalighfard S3, Ghafari F3, Hadji M3, Khodayari H3.

Author information

  • 1Ischemic Disorders Research Center, Golestan University of Medical Sciences, Gorgan, Iran.
  • 2Cancer Research Center, Tehran University of Medical Sciences, Tehran, Iran, Zip Code: 1419733141. aalizadeh@sina.tums.ac.ir.
  • 3Cancer Research Center, Tehran University of Medical Sciences, Tehran, Iran, Zip Code: 1419733141.
  • 4Medical Engineering, Faculty of Biomedical Engineering, Amir Kabir University, Tehran, Iran.

Abstract

Low-level laser therapy (LLLT) is a form of photon therapy which can be a non-invasive therapeutic procedure in cancer therapy using low-intensity light in the range of 450-800 nm. One of the main functional features of laser therapy is the photobiostimulation effects of low-level lasers on various biological systems including altering DNA synthesis and modifying gene expression, and stopping cellular proliferation. This study investigated the effects of LLLT on mice mammary tumor and the expression of Let-7a, miR155, miR21, miR125, and miR376b in the plasma and tumor samples. Sixteen mice were equally divided into four groups including control, and blue, green, and red lasers at wavelengths of 405, 532, and 632 nm, respectively. Weber Medical Applied Laser irradiation was carried out with a low power of 1-3 mW and a series of 10 treatments at three times a week after tumor establishment. Tumor volume was weekly measured by a digital vernier caliper, and qRT-PCR assays were performed to accomplish the study. Depending on the number of groups and the p value of the Kolmogorov-Smirnov test of normality, a t test, a one-way ANOVA, or a non-parametric test was used for data analyses, and p?<?0.05 was considered to be statistically significant. The average tumor volume was significantly less in the treated blue group than the control group on at days 21, 28, and 35 after cancerous cell injection. Our data also showed an increase of Let-7a and miR125a expression and a decrease of miR155, miR21, and miR376b expression after LLLT with the blue laser both the plasma and tumor samples compared to other groups. It seems that the non-invasive nature of laser bio-stimulation can make LLLT an attractive alternative therapeutic tool.

Lasers Med Sci. 2016 Aug 19. [Epub ahead of print]

The use of low-level light therapy in supportive care for patients with breast cancer: review of the literature.

Robijns J1,2, Censabella S3, Bulens P4,3, Maes A4,3, Mebis J5,4,3.

Author information

  • 1Faculty of Medicine & Life Sciences, Hasselt University, Martelarenlaan 42, 3500, Hasselt, Belgium. jolien.robijns@uhasselt.be.
  • 2Limburg Oncology Center, Stadsomvaart 11, 3500 Hasselt, Belgium. jolien.robijns@uhasselt.be.
  • 3Division of Medical Oncology, Jessa Hospital, Campus Virga Jesse, Stadsomvaart 11, 3500 Hasselt, Belgium.
  • 4Limburg Oncology Center, Stadsomvaart 11, 3500 Hasselt, Belgium.
  • 5Faculty of Medicine & Life Sciences, Hasselt University, Martelarenlaan 42, 3500, Hasselt, Belgium.

Abstract

Breast cancer is the most common cancer in women worldwide, with an incidence of 1.7 million in 2012. Breast cancer and its treatments can bring along serious side effects such as fatigue, skin toxicity, lymphedema, pain, nausea, etc. These can substantially affect the patients’ quality of life. Therefore, supportive care for breast cancer patients is an essential mainstay in the treatment. Low-level light therapy (LLLT) also named photobiomodulation therapy (PBMT) has proven its efficiency in general medicine for already more than 40 years. It is a noninvasive treatment option used to stimulate wound healing and reduce inflammation, edema, and pain. LLLT is used in different medical settings ranging from dermatology, physiotherapy, and neurology to dentistry. Since the last twenty years, LLLT is becoming a new treatment modality in supportive care for breast cancer. For this review, all existing literature concerning the use of LLLT for breast cancer was used to provide evidence in the following domains: oral mucositis (OM), radiodermatitis (RD), lymphedema, chemotherapy-induced peripheral neuropathy (CIPN), and osteonecrosis of the jaw (ONJ). The findings of this review suggest that LLLT is a promising option for the management of breast cancer treatment-related side effects. However, it still remains important to define appropriate treatment and irradiation parameters for each condition in order to ensure the effectiveness of LLLT.

Antioxid Redox Signal. 2015 Sep 28. [Epub ahead of print]

Phototherapy-induced antitumor immunity: long-term tumor supression effects via photoinactivation of respiratory chain oxidase-triggered superoxide anion burst.

Lu C1,2, Zhou F3, Wu S4,5,6, Liu L7, Xing D8.
Author information
1Guangzhou, China.
2MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University , No. 55 Zhongshan Avenue West, Tianhe District,Guangzhou , guangzhou, China , 510631 ; lucx@scnu.edu.cn.
3MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University , No. 55 Zhongshan Avenue West, Tianhe District,Guangzhou , guangzhou, China , 510631 ; zhouff@scnu.edu.cn.
4South China Normal UniversityGuang Zhou, China , 510631.
5China.
6MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University , No. 55 Zhongshan Avenue West, Tianhe District,Guangzhou , guangzhou, China , 510631 ; wushn@scnu.edu.cn.
7MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University , No. 55 Zhongshan Avenue West, Tianhe District,Guangzhou , guangzhou, China , 510631 ; liulei@scnu.edu.cn.
8South China Normal University , No. 55 Zhongshan west road, Tianhe district , guangzhou, China , 510631 ; xingda@scnu.edu.cn.

Abstract
AIMS:
Our previous studies have demonstrated that as a mitochondria-targeting cancer phototherapy, high-fluence low-power laser irradiation (HF-LPLI) results in oxidative damage that induces tumor cell apoptosis. In this study, we focused on the immunological effects of HF-LPLI phototherapy and explored its antitumor immune regulatory mechanism.
RESULTS:
We found not only that HF-LPLI treatment induced tumor cell apoptosis but also that HF-LPLI-treated apoptotic tumor cells activated macrophages. Due to mitochondrial superoxide anion burst after HF-LPLI treatment, tumor cells displayed a high level of phosphatidylserine oxidation, which mediated the recognition and uptake by macrophages with the subsequent secretion of cytokines and generation of cytotoxic T lymphocytes. In addition, in vivo results showed that HF-LPLI treatment caused leukocyte infiltration into the tumor and efficaciously inhibited tumor growth in an EMT6 tumor model. These phenomena were absent in the respiration-deficient EMT6 tumor model, implying that the HF-LPLI-elicited immunological effects were dependent on the mitochondrial superoxide anion burst.
INNOVATION:
Here, for the first time, we show that HF-LPLI mediates tumor-killing effects via targeting photoinactivation respiratory chain oxidase to trigger a superoxide anion burst, leading to a high level of oxidatively modified moieties, which contributes to the phenotypic changes in macrophages and mediates the antitumor immune response.
CONCLUSION:
Our results suggest that HF-LPLI may be an effective cancer treatment modality that both eradicates the treated primary tumors and induces an antitumor immune response via photoinactivation of respiratory chain oxidase to trigger superoxide anion burst.
Discov Med. 2015 Apr;19(105):293-301.

Advances in strategies and methodologies in cancer immunotherapy.

Lam SS1, Zhou F2, Hode T1, Nordquist RE1, Alleruzzo L1, Raker J1, Chen WR3.

Author information

  • 1Immunophotonics Inc., 4320 Forest Park Ave. #303, St. Louis, MO 63108, USA.
  • 2Biophotonics Research Laboratory, Center for Interdisciplinary Biomedical Education and Research, University of Central Oklahoma, Edmond, OK 73034, USA.
  • 3Biophotonics Research Laboratory, Center for Interdisciplinary Biomedical Education and Research, University of Central Oklahoma, Edmond, OK 73034, USA and Immunophotonics Inc., 4320 Forest Park Ave. #303, St. Louis, MO 63108, USA.

Abstract

Since the invention of Coley’s toxin by William Coley in early 1900s, the path for cancer immunotherapy has been a convoluted one. Although still not considered standard of care, with the FDA approval of trastuzumab, Provenge and ipilimumab, the medical and scientific community has started to embrace the possibility that immunotherapy could be a new hope for cancer patients with otherwise untreatable metastatic diseases. This review aims to summarize the development of some major strategies in cancer immunotherapy, from the earliest peptide vaccine and transfer of tumor specific antibodies/T cells to the more recent dendritic cell (DC) vaccines, whole cell tumor vaccines, and checkpoint blockade therapy. Discussion of some major milestones and obstacles in the shaping of the field and the future perspectives is included. Photoimmunotherapy is also reviewed as an example of emerging new therapies combining phototherapy and immunotherapy.

 J Biomed Opt.  2012 Oct;17(10):101516. doi: 10.1117/1.JBO.17.10.101516.

Low-level laser therapy on MCF-7 cells: a micro-Fourier transform infrared spectroscopy study.

Magrini TD, dos Santos NV, Milazzotto MP, Cerchiaro G, da Silva Martinho H.

Source

Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Rua Santa Adélia 166, Bangu, Santo André, SP 09210-170, Brazil.

Abstract

Low-level laser therapy (LLLT) is an emerging therapeutic approach for several clinical conditions. The clinical effects induced by LLLT presumably scale from photobiostimulation/photobioinhibition at the cellular level to the molecular level. The detailed mechanism underlying this effect remains unknown. This study quantifies some relevant aspects of LLLT related to molecular and cellular variations. Malignant breast cells (MCF-7) were exposed to spatially filtered light from a He-Ne laser (633 nm) with fluences of 5, 28.8, and 1000 mJ/cm². The cell viability was evaluated by optical microscopy using the Trypan Blue viability test. The micro-Fourier transform infrared technique was employed to obtain the vibrational spectra of each experimental group (control and irradiated) and identify the relevant biochemical alterations that occurred due to the process. It was observed that the red light influenced the RNA, phosphate, and serine/threonine/tyrosine bands. We found that light can influence cell metabolism depending on the laser fluence. For 5 mJ/cm², MCF-7 cells suffer bioinhibition with decreased metabolic rates. In contrast, for the 1 J/cm² laser fluence, cells present biostimulation accompanied by a metabolic rate elevation. Surprisingly, at the intermediate fluence, 28.8 mJ/cm², the metabolic rate is increased despite the absence of proliferative results. The data were interpreted within the retrograde signaling pathway mechanism activated with light irradiation.

Photomed Laser Surg.  2012 Sep;30(9):551-8. doi: 10.1089/pho.2011.3186. Epub 2012 Aug 1.

A preliminary study of the safety of red light phototherapy of tissues harboring cancer.

Myakishev-Rempel M, Stadler I, Brondon P, Axe DR, Friedman M, Nardia FB, Lanzafame R.

Source

Department of Dermatology, University of Rochester, Rochester, New York, USA. max.rempel@gmail.com

Abstract

OBJECTIVE:

Red light phototherapy is known to stimulate cell proliferation in wound healing. This study investigated whether low-level light therapy (LLLT) would promote tumor growth when pre-existing malignancy is present.

BACKGROUND DATA:

LLLT has been increasingly used for numerous conditions, but its use in cancer patients, including the treatment of lymphedema or various unrelated comorbidities, has been withheld by practitioners because of the fear that LLLT might result in initiation or promotion of metastatic lesions or new primary tumors. There has been little scientific study of oncologic outcomes after use of LLLT in cancer patients.

METHODS:

A standard SKH mouse nonmelanoma UV-induced skin cancer model was used after visible squamous cell carcinomas were present, to study the effects of LLLT on tumor growth. The red light group (n=8) received automated full body 670 nm LLLT delivered twice a day at 5 J/cm(2) using an LED source. The control group (n=8) was handled similarly, but did not receive LLLT. Measurements on 330 tumors were conducted for 37 consecutive days, while the animals received daily LLLT.

RESULTS:

Daily tumor measurements demonstrated no measurable effect of LLLT on tumor growth.

CONCLUSIONS:

This experiment suggests that LLLT at these parameters may be safe even when malignant lesions are present. Further studies on the effects of photoirradiation on neoplasms are warranted.

J Biomed Opt. 2012 Oct 25;17(10):101516-1. doi: 10.1117/1.JBO.17.10.101516.

Low-level laser therapy on MCF-7 cells: a micro-Fourier transform infrared spectroscopy study.

Magrini TD, Dos Santos NV, Milazzotto MP, Cerchiaro G, da Silva Martinho H.

Abstract

ABSTRACT. Low-level laser therapy (LLLT) is an emerging therapeutic approach for several clinical conditions. The clinical effects induced by LLLT presumably scale from photobiostimulation/photobioinhibition at the cellular level to the molecular level. The detailed mechanism underlying this effect remains unknown. This study quantifies some relevant aspects of LLLT related to molecular and cellular variations. Malignant breast cells (MCF-7) were exposed to spatially filtered light from a He-Ne laser (633 nm) with fluences of 5, 28.8, and 1000 mJ/cm2. The cell viability was evaluated by optical microscopy using the Trypan Blue viability test. The micro-Fourier transform infrared technique was employed to obtain the vibrational spectra of each experimental group (control and irradiated) and identify the relevant biochemical alterations that occurred due to the process. It was observed that the red light influenced the RNA, phosphate, and serine/threonine/tyrosine bands. We found that light can influence cell metabolism depending on the laser fluence. For 5 mJ/cm2, MCF-7 cells suffer bioinhibition with decreased metabolic rates. In contrast, for the 1 J/cm2 laser fluence, cells present biostimulation accompanied by a metabolic rate elevation. Surprisingly, at the intermediate fluence, 28.8 mJ/cm2, the metabolic rate is increased despite the absence of proliferative results. The data were interpreted within the retrograde signaling pathway mechanism activated with light irradiation.

Photomed Laser Surg.  2012 Aug 1. [Epub ahead of print]

A Preliminary Study of the Safety of Red Light Phototherapy of Tissues Harboring Cancer.

Myakishev-Rempel M, Stadler I, Brondon P, Axe DR, Friedman M, Nardia FB, Lanzafame R.

Source

1 Department of Dermatology, University of Rochester , Rochester, New York.

Abstract

Abstract Objective: Red light phototherapy is known to stimulate cell proliferation in wound healing. This study investigated whether low-level light therapy (LLLT) would promote tumor growth when pre-existing malignancy is present.

Background data: LLLT has been increasingly used for numerous conditions, but its use in cancer patients, including the treatment of lymphedema or various unrelated comorbidities, has been withheld by practitioners because of the fear that LLLT might result in initiation or promotion of metastatic lesions or new primary tumors. There has been little scientific study of oncologic outcomes after use of LLLT in cancer patients.

Methods: A standard SKH mouse nonmelanoma UV-induced skin cancer model was used after visible squamous cell carcinomas were present, to study the effects of LLLT on tumor growth. The red light group (n=8) received automated full body 670 nm LLLT delivered twice a day at 5 J/cm(2) using an LED source. The control group (n=8) was handled similarly, but did not receive LLLT.

Measurements on 330 tumors were conducted for 37 consecutive days, while the animals received daily LLLT. Results: Daily tumor measurements demonstrated no measurable effect of LLLT on tumor growth.

Conclusions: This experiment suggests that LLLT at these parameters may be safe even when malignant lesions are present. Further studies on the effects of photoirradiation on neoplasms are warranted.

Vopr Kurortol Fizioter Lech Fiz Kult.  2012 Jul-Aug;(4):23-32.

The efficacy of polychromatic visible and infrared radiation used for the postoperative immunological rehabilitation of patients with breast cancer.

[Article in Russian]
[No authors listed]

Abstract

The immunological rehabilitation of the patients with oncological problems after the completion of standard anti-tumour therapy remains a topical problem in modern medicine. The up-to-date phototherapeutic methods find the increasingly wider application for the treatment of such patients including the use of monochromatic visible (VIS) and near infrared (nIR) radiation emitted from lasers and photodiodes. The objective of the present study was to substantiate the expediency of postoperative immune rehabilitation of the patients with breast cancer (BC) by means of irradiation of the body surface with polychromatic visible (pVIS) in combination with polychromatic infrared (pIR) light similar to the natural solar radiation without its minor UV component. The study included 19 patients with stage I–II BC at the mean age of 54.0 +/- 4.28 years having the infiltrative-ductal form of the tumour who had undergone mastectomy. These patients were randomly allocated to two groups, one given the standard course of postoperative rehabilitation (control), the other (study group) additionally treated with pVIS + pIR radiation applied to the lumbar-sacral region from days 1 to 7 after surgery. A Bioptron-2 phototherapeutic device, Switzerland, was used for the purpose (480-3400 nm, 40 mW/cm2, 12 J/cm2, with the light spot diameter of 15 cm). The modern standard immunological methods were employed. It was found that mastectomy induced changes of many characteristics of cellular and humoral immunity; many of them in different patients were oppositely directed. These changes were apparent within the first 7 days postoperatively. The course of phototherapy (PT) was shown to prevent the postoperative decrease in the counts of monocytes and natural killer (NK) cells, the total amount of CD3+ -T-lymphocytes (LPC), CD4+ -T-helpers, activated T-lymphocytes (CD3+ HLA-DR+ cells) and IgA levels as well as intracellular digestion rate of neutrophil-phagocyted bacteria. Moreover PT promoted faster normalization of postoperative leukocytosis and activation of cytotoxic CD8+ -T-LPC, reduced the elevated concentration of immune complexes in blood. Among the six tested cytokines, viz. IL-1beta, TNF-alpha, IL-6, IL-10, IFN-alpha, and IFN-gamma, only the latter two underwent significant elevation of their blood concentrations (IL-6 within 1 day) and IFN-gamma (within 7 days after mastectomy). The course of PT resulted in the decrease of their levels to the initial values. The follow-up of the treated patients during 4 years revealed neither recurrence of the disease nor the appearance of metastases.

Photomed Laser Surg. 2010 Feb;28(1):115-23.

The effect of laser irradiation on proliferation of human breast carcinoma, melanoma, and immortalized mammary epithelial cells.

Powell K, Low P, McDonnell PA, Laakso EL, Ralph SJ.

School of Medical Science, Griffith University, Gold Coast, Queensland, Australia.

Abstract

OBJECTIVE: This study compared the effects of different doses (J/cm(2)) of laser phototherapy at wavelengths of either 780, 830, or 904 nm on human breast carcinoma, melanoma, and immortalized human mammary epithelial cell lines in vitro. In addition, we examined whether laser irradiation would malignantly transform the murine fibroblast NIH3T3 cell line.

BACKGROUND: Laser phototherapy is used in the clinical treatment of breast cancer-related lymphoedema, despite limited safety information. This study contributes to systematically developing guidelines for the safe use of laser in breast cancer-related lymphoedema. METHODS: Human breast adenocarcinoma (MCF-7), human breast ductal carcinoma with melanomic genotypic traits (MDA-MB-435S), and immortalized human mammary epithelial (SVCT and Bre80hTERT) cell lines were irradiated with a single exposure of laser. MCF-7 cells were further irradiated with two and three exposures of each laser wavelength. Cell proliferation was assessed 24 h after irradiation.

RESULTS: Although certain doses of laser increased MCF-7 cell proliferation, multiple exposures had either no effect or showed negative dose response relationships. No sign of malignant transformation of cells by laser phototherapy was detected under the conditions applied here.

CONCLUSION: Before a definitive conclusion can be made regarding the safety of laser for breast cancer-related lymphoedema, further in vivo research is required.

Vopr Kurortol Fizioter Lech Fiz Kult. 2009 Nov-Dec;(6):49-52.

Application of low-power visible and near infrared radiation in clinical oncology.

[Article in Russian]

Zimin AA, Zhevago NA, Buniakova AI, Samoilova KA.

Although low-power visible (VIS) and near infrared (nIR) radiation emitted from lasers, photodiodes, and other sources does not cause neoplastic transformation of the tissue, these phototherapeutic techniques are looked at with a great deal of caution for fear of their stimulatory effect on tumour growth. This apprehension arises in the first place from the reports on the possibility that the proliferative activity of tumour cells may increase after their in vitro exposure to light. Much less is known that these phototherapeutic modalities have been successfully used for the prevention and management of complications developing after surgery, chemo- and radiotherapy. The objective of the present review is to summarize the results of applications of low-power visible and near infrared radiation for the treatment of patients with oncological diseases during the last 20-25 years. It should be emphasized that 2-4 year-long follow-up observations have not revealed any increase in the frequency of tumour recurrence and metastasis.

Photomed Laser Surg. 2009 Oct;27(5):763-9.

Managing postmastectomy lymphedema with low-level laser therapy.

Lau RW, Cheing GL.

Department of Rehabilitation Sciences, The Hong Kong Polytechnic University, Hong Kong SAR, China.

OBJECTIVE: We aimed to investigate the effects of low-level laser therapy (LLLT) in managing postmastectomy lymphedema. BACKGROUND DATA: Postmastectomy lymphedema (PML) is a common complication of breast cancer treatment that causes various symptoms, functional impairment, or even psychosocial morbidity. A prospective, single-blinded, controlled clinical trial was conducted to examine the effectiveness of LLLT on managing PML.

METHODS: Twenty-one women suffering from unilateral PML were randomly allocated to receive either 12 sessions of LLLT in 4 wk (the laser group) or no laser irradiation (the control group). Volumetry and tonometry were used to monitor arm volume and tissue resistance; the Disabilities of Arm, Shoulder, and Hand (DASH) questionnaire was used for measuring subjective symptoms. Outcome measures were assessed before and after the treatment period and at the 4 wk follow-up.

RESULTS: Reduction in arm volume and increase in tissue softening was found in the laser group only. At the follow-up session, significant between-group differences (all p < 0.05) were found in arm volume and tissue resistance at the anterior torso and forearm region. The laser group had a 16% reduction in the arm volume at the end of the treatment period, that dropped to 28% in the follow-up. Moreover, the laser group demonstrated a cumulative increase from 15% to 33% in the tonometry readings over the forearm and anterior torso. The DASH score of the laser group showed progressive improvement over time.

CONCLUSION: LLLT was effective in the management of PML, and the effects were maintained to the 4 wk follow-up.

Clin Rehabil. 2009 Feb;23(2):117-24

Efficacy of pneumatic compression and low-level laser therapy in the treatment of postmastectomy lymphoedema: a randomized controlled trial.

Kozanoglu E, Basaran S, Paydas S, Sarpel T.

Department of Physical Medicine and Rehabilitation, Faculty of Medicine, Cukurova University, Adana, Turkey.

Objective: To compare the long-term efficacy of pneumatic compression and low-level laser therapies in the management of postmastectomy lymphoedema.

Design: Randomized controlled trial.Setting: Department of Physical Medicine and Rehabilitation of Cukurova University, Turkey.

Subjects: Forty-seven patients with postmastectomy lymphoedema were enrolled in the study.Interventions: Patients were randomly allocated to pneumatic compression (group I, n=24) and low-level laser (group II, n=23) groups. Group I received 2 hours of compression therapy and group II received 20 minutes of laser therapy for four weeks. All patients were advised to perform daily limb exercises.Main measures: Demographic features, difference between sum of the circumferences of affected and unaffected limbs (triangle upC), pain with visual analogue scale and grip strength were recorded.

Results: Mean age of the patients was 48.3 (10.4) years. triangle upC decreased significantly at one, three and six months within both groups, and the decrease was still significant at month 12 only in group II (P = 0.004). Improvement of group II was greater than that of group I post treatment (P = 0.04) and at month 12 after 12 months (P = 0.02). Pain was significantly reduced in group I only at posttreatment evaluation, whereas in group II it was significant post treatment and at follow-up visits. No significant difference was detected in pain scores between the two groups. Grip strength was improved in both groups, but the differences between groups were not significant.

Conclusions: Patients in both groups improved after the interventions. Group II had better long-term results than group I. Low-level laser might be a useful modality in the treatment of postmastectomy lymphoedema.

Photomed Laser Surg. 2008 Aug;26(4):393-400.

Low-level laser therapy in the prevention and treatment of chemotherapy-induced oral mucositis in young patients.

Abramoff MM, Lopes NN, Lopes LA, Dib LL, Guilherme A, Caran EM, Barreto AD, Lee ML, Petrilli AS.

Private practice, São Paulo, Brazil.

Abstract Objective: A pilot clinical study was conducted to evaluate the efficacy and feasibility of low-level laser therapy (LLLT) in the prevention and treatment of chemotherapy (CT)-induced oral mucositis (OM) in young patients. Background Data: Besides compromising the patient’s nutrition and well-being, oral mucositis represents a portal of entry into the body for microorganisms present in the mouth, which may lead to sepsis if there is hematological involvement. Oncologic treatment tolerance decreases and systemic complications may arise that interfere with the success of cancer treatment. LLLT appears to be an interesting alternative to other approaches to treating OM, due to its trophic, anti-inflammatory, and analgesic properties. Materials and Methods: Patients undergoing chemotherapy (22 cycles) without mucositis were randomized into a group receiving prophylactic laser-irradiation (group 1), and a group receiving placebo light treatment (group 2). Patients who had already presented with mucositis were placed in a group receiving irradiation for therapeutic purposes (group 3, with 10 cycles of CT). Serum granulocyte levels were taken and compared to the progression of mucositis. Results: In group 1, most patients (73%) presented with mucositis of grade 0 (p = 0.03 when compared with the placebo group), and 18% presented with grade 1. In group 2, 27% had no OM and did not require therapy. In group 3, the patients had marked pain relief (as assessed by a visual analogue scale), and a decrease in the severity of OM, even when they had severe granulocytopenia. Conclusion: The ease of use of LLLT, high patient acceptance, and the positive results achieved, make this therapy feasible for the prevention and treatment of OM in young patients.

Ann Oncol. 2007 Apr;18(4):639-46. Epub 2006 Oct 3

A systematic review of common conservative therapies for arm lymphoedema secondary to breast cancer treatment.

Moseley AL, Carati CJ, Piller NB.

School of Nursing & Midwifery, University of South Australia, Adelaide, Australia. amanda.moseley@yahoo.com.au

Secondary arm lymphoedema is a chronic and distressing condition which affects a significant number of women who undergo breast cancer treatment. A number of health professional and patient instigated conservative therapies have been developed to help with this condition, but their comparative benefits are not clearly known. This systematic review undertook a broad investigation of commonly instigated conservative therapies for secondary arm lymphoedema including; complex physical therapy, manual lymphatic drainage, pneumatic pumps, oral pharmaceuticals, low level laser therapy, compression bandaging and garments, limb exercises and limb elevation. It was found that the more intensive and health professional based therapies, such as complex physical therapy, manual lymphatic drainage, pneumatic pump and laser therapy generally yielded the greater volume reductions, whilst self instigated therapies such as compression garment wear, exercises and limb elevation yielded smaller reductions. All conservative therapies produced improvements in subjective arm symptoms and quality of life issues, where these were measured. Despite the identified benefits, there is still the need for large scale, high level clinical trials in this area.

Lasers Med Sci. 2006 Jul;21(2):90-4. Epub 2006 May 4.

Low-level laser therapy in management of postmastectomy lymphedema.

Kaviani A, Fateh M, Yousefi Nooraie R, Alinagi-zadeh MR, Ataie-Fashtami L.

Tehran University of Medical Sciences and Iranian Center for Medical Laser Research, Tehran, Iran. akaviani@sina.tims.ac.ir

The aim of this paper was to study the effects of low-level laser therapy (LLLT) in the treatment of postmastectomy lymphedema. Eleven women with unilateral postmastectomy lymphedema were enrolled in a double-blind controlled trial. Patients were randomly assigned to laser and sham groups and received laser or placebo irradiation (Ga-As laser device with a wavelength of 890 nm and fluence of 1.5 J/cm2) over the arm and axillary areas. Changes in patients’ limb circumference, pain score, range of motion, heaviness of the affected limb, and desire to continue the treatment were measured before the treatment and at follow-up sessions (weeks 3, 9, 12, 18, and 22) and were compared to pretreatment values. Results showed that of the 11 enrolled patients, eight completed the treatment sessions. Reduction in limb circumference was detected in both groups, although it was more pronounced in the laser group up to the end of 22nd week. Desire to continue treatment at each session and baseline score in the laser group was greater than in the sham group in all sessions. Pain reduction in the laser group was more than in the sham group except for the weeks 3 and 9. No substantial differences were seen in other two parameters between the two treatment groups. In conclusion, despite our encouraging results, further studies of the effects of LLLT in management of postmastectomy lymphedema should be undertaken to determine the optimal physiological and physical parameters to obtain the most effective clinical response.

J Photochem Photobiol B. 2000 Dec;59(1-3):1-8.

Magnetic resonance imaging (MRI) controlled outcome of side effects caused by ionizing radiation, treated with 780 nm-diode laser –preliminary results.

Schaffer M, Bonel H, Sroka R, Schaffer PM, Busch M, Sittek H, Reiser M, Duhmke E.
Department of Radiation Therapy, University of Munich, Germany.

sroka@life.med.uni-muenchen.de

BACKGROUND and OBJECTIVE: Ionizing radiation therapy by way of various beams such as electron, photon and neutron is an established method in tumor treatment. The side effects caused by this treatment such as ulcer, painful mastitis and delay of wound healing are well known, too. Biomodulation by low level laser therapy (LLLT) has become popular as a therapeutic modality for the acceleration of wound healing and the treatment of inflammation. Evidence for this kind of application, however, is not fully understood yet. This study intends to demonstrate the response of biomodulative laser treatment on the side effects caused by ionizing radiation by means of magnetic resonance imaging (MRI). STUDY

DESIGN/PATIENTS and METHODS: Six female patients suffering from painful mastitis after breast ionizing irradiation and one man suffering from radiogenic ulcer were treated with lambda=780 nm diode laser irradiation at a fluence rate of 5 J/cm2. LLLT was performed for a period of 4-6 weeks (mean sessions: 25 per patient, range 19-35). The tissue response was determined by means of MRI after laser treatment in comparison to MRI prior to the beginning of the LLLT.

RESULTS: All patients showed complete clinical remission. The time-dependent contrast enhancement curve obtained by the evaluation of MR images demonstrated a significant decrease of enhancement features typical for inflammation in the affected area.

CONCLUSION: Biomodulation by LLLT seems to be a promising treatment modality for side effects induced by ionizing radiation.

Central Nervous System Disorders

Curr Alzheimer Res. 2015;12(9):860-9.

Cognitive Improvement by Photic Stimulation in a Mouse Model of Alzheimer’s Disease.

Zhang Y, Wang F, Luo X, Wang L, Sun P, Wang M, Jiang Y, Zou J, Uchiumi O, Yamamoto R, Sugai T, Yamamoto K, Kato N1.

Author information

  • 1Department of Physiology, Kanazawa Medical University, Ishikawa 920-0293, Japan. kato@kanazawa-med.ac.jp.

Abstract

We previously reported that activity of the large conductance calcium-activated potassium (big-K, BK) channel is suppressed by intracellular A? in cortical pyramidal cells, and that this suppression was reversed by expression of the scaffold protein Homer1a in 3xTg Alzheimer’s disease model mice. Homer1a is known to be expressed by physiological photic stimulation (PS) as well. The possibility thus arises that PS also reverses A?-induced suppression of BK channels, and thereby improves cognition in 3xTg mice. This possibility was tested here. Chronic application of 6-hour-long PS (frequency, 2 Hz; duty cycle, about 1/10; luminance, 300 lx) daily for 4 weeks improved contextual and tone-dependent fear memory in 3xTg mice and, to a lesser extent, Morris water maze performance as well. Hippocampal long-term potentiation was also enhanced after PS. BK channel activity in cingulate cortex pyramidal cells and lateral amygdalar principal cells, suppressed in 3xTg mice, were facilitated. In parallel, neuronal excitability, elevated in 3xTg mice, was recovered to the control level. Gene expression of BK channel, as well as that of the scaffold protein Homer1a, was found decreased in 3xTg mice and reversed by PS. It is known that Homer1a is an activity-dependently inducible immediate early gene product. Consistently, our previous findings showed that Homer1a induced by electrical stimulation facilitated BK channels. By using Homer1a knockouts, we showed that the present PS-induced BK channel facilitation is mediated by Homer1a expression. We thus propose that PS might be potentially useful as a non-invasive therapeutic measure against Alzheimer’s disease.

Exp Brain Res. 2016 Jul 5. [Epub ahead of print]

Near-infrared light treatment reduces astrogliosis in MPTP-treated monkeys.

El Massri N1, Moro C2, Torres N2, Darlot F2, Agay D2, Chabrol C2, Johnstone DM3, Stone J3, Benabid AL2, Mitrofanis J4.

Author information

  • 1Department of Anatomy F13, University of Sydney, Sydney, 2006, Australia.
  • 2University Grenoble Alpes, CEA, LETI, CLINATEC, MINATEC Campus, 38000, Grenoble, France.
  • 3Department of Physiology F13, University of Sydney, Sydney, 2006, Australia.
  • 4Department of Anatomy F13, University of Sydney, Sydney, 2006, Australia. john.mitrofanis@sydney.edu.au.

Abstract

We have reported previously that intracranial application of near-infrared light (NIr) reduces clinical signs and offers neuroprotection in a subacute MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) monkey model of Parkinson’s disease. In this study, we explored whether NIr reduces the gliosis in this animal model. Sections of midbrain (containing the substantia nigra pars compacta; SNc) and striatum were processed for glial fibrillary acidic protein (to label astrocytes; GFAP) and ionised calcium-binding adaptor molecule 1 (to label microglia; IBA1) immunohistochemistry. Cell counts were undertaken using stereology, and cell body sizes were measured using ImageJ. Our results showed that NIr treatment reduced dramatically (~75 %) MPTP-induced astrogliosis in both the SNc and striatum. Among microglia, however, NIr had a more limited impact in both nuclei; although there was a reduction in overall cell size, there were no changes in the number of microglia in the MPTP-treated monkeys after NIr treatment. In summary, we showed that NIr treatment influenced the glial response, particularly that of the astrocytes, in our monkey MPTP model of Parkinson’s disease. Our findings raise the possibility of glial cells as a future therapeutic target using NIr.

BMC Neurosci. 2016 May 18;17(1):21. doi: 10.1186/s12868-016-0259-6.

Comparative assessment of phototherapy protocols for reduction of oxidative stress in partially transected spinal cord slices undergoing secondary degeneration.

Ashworth BE1,2, Stephens E1,2, Bartlett CA1, Serghiou S3, Giacci MK1, Williams A3, Hart NS1,4, Fitzgerald M5.

Author information

  • 1Experimental and Regenerative Neurosciences, School of Animal Biology, The University of Western Australia, Crawley, WA, Australia.
  • 2Department of Biology and Biochemistry, The University of Bath, Bath, UK.
  • 3Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
  • 4Department of Biological Sciences, Macquarie University, Sydney, NSW, 2109, Australia.
  • 5Experimental and Regenerative Neurosciences, School of Animal Biology, The University of Western Australia, Crawley, WA, Australia. lindy.fitzgerald@uwa.edu.au.

Abstract

BACKGROUND:

Red/near-infrared light therapy (R/NIR-LT) has been developed as a treatment for a range of conditions, including injury to the central nervous system (CNS). However, clinical trials have reported variable or sub-optimal outcomes, possibly because there are few optimized treatment protocols for the different target tissues. Moreover, the low absolute, and wavelength dependent, transmission of light by tissues overlying the target site make accurate dosing problematic.

RESULTS:

In order to optimize light therapy treatment parameters, we adapted a mouse spinal cord organotypic culture model to the rat, and characterized myelination and oxidative stress following a partial transection injury. The ex vivo model allows a more accurate assessment of the relative effect of different illumination wavelengths (adjusted for equal quantal intensity) on the target tissue. Using this model, we assessed oxidative stress following treatment with four different wavelengths of light: 450 nm (blue); 510 nm (green); 660 nm (red) or 860 nm (infrared) at three different intensities: 1.93 × 10(16) (low); 3.85 × 10(16) (intermediate) and 7.70 × 10(16) (high) photons/cm(2)/s. We demonstrate that the most effective of the tested wavelengths to reduce immunoreactivity of the oxidative stress indicator 3-nitrotyrosine (3NT) was 660 nm. 860 nm also provided beneficial effects at all tested intensities, significantly reducing oxidative stress levels relative to control (p ? 0.05).

CONCLUSIONS:

Our results indicate that R/NIR-LT is an effective antioxidant therapy, and indicate that effective wavelengths and ranges of intensities of treatment can be adapted for a variety of CNS injuries and conditions, depending upon the transmission properties of the tissue to be treated.

 

Acta Cirurgica Brasileira

On-line version ISSN 1678-2674

Acta Cir. Bras. vol.30 no.9 São Paulo Sep. 2015

http://dx.doi.org/10.1590/S0102-865020150090000005

ORIGINAL ARTICLES

The influence of low-level laser irradiation on spinal cord injuries following ischemia- reperfusion in rats1

Amir Sotoudeh I   , Amirali Jahanshahi II   , Saeed Zareiy III   , Mohammad Darvishi IV   , Nasim Roodbari V   , Ali Bazzazan VI  

IAssistant Professor, Faculty of Veterinary Science, Kahnooj Branch, Islamic Azad University (IAU), Kerman, Iran. Design, analysis and interpretation of data; manuscript writing

IIResearcher, Elite Club, Kahnooj Branch, IAU, Kerman, Iran. Design and acquisition of data

IIIResident, Aerospace and Subaquatic Medicine School, AJA University of Medical Sciences, Tehran, Iran Branch, and Islamic Azad University, Tehran, Iran. Technical procedures, acquisition and interpretation of data

IVAssociate Professor, Department of Infection Medicine, AJA University of Medical Sciences, Tehran, Iran. Analysis and interpretation of data, statistical analysis

VAssistant Professor, Faculty of Experimental Science, Kahnooj Branch, Islamic Azad University, Kerman, Iran. Analysis of data, manuscript writing

VIGraduate student, Faculty of Veterinary Science, Garmsar Branch, IAU, Semnan, Iran. Acquisition and interpretation of data.

ABSTRACT

PURPOSE:

To investigate if low level laser therapy (LLLT) can decrease spinal cord injuries after temporary induced spinal cord ischemia-reperfusion in rats because of its anti-inflammatory effects.

METHODS:

Forty eight rats were randomized into two study groups of 24 rats each. In group I, ischemic-reperfusion (I-R) injury was induced without any treatment. Group II, was irradiated four times about 20 minutes for the following three days. The lesion site directly was irradiated transcutaneously to the spinal direction with 810 nm diode laser with output power of 150 mW. Functional recovery, immunohistochemical and histopathological changes were assessed.

RESULTS:

The average functional recovery scores of group II were significantly higher than that the score of group I (2.86 ± 0.68, vs 1.38 ± 0.09; p<0.05). Histopathologic evaluations in group II were showed a mild changes in compare with group I, that suggested this group survived from I-R consequences. Moreover, as seen from TUNEL results, LLLT also protected neurons from I-R-induced apoptosis in rats.

CONCLUSION:

Low level laser therapy was be able to minimize the damage to the rat spinal cord of reperfusion-induced injury.

INTRODUCTION

Neurologic injuries due to I-R of the spinal cord has an incidence of between 2.9% and 23%1. Pathogenic mechanisms of neuronal cell death after spinal cord I-R injury include energy failure, excitotoxicity, and oxidative stress2 , 3.There are some applications which can reduce spinal cord I-R injuries such as hypothermia, vascular shunting, left heart bypass, drainage of cerebrospinal fluid, monitoring of somatosensory evoked potentials, single clamp technique and reimplantation of major intercostal arteries4  6. Also, there are experimental studies like ischemic preconditioning and adjunctive medications for reducing the incidence of this complication7. Despite several surgical modifications and pharmacologic approaches, postoperative spinal cord dysfunction has not been totally eliminated8.

Low level laser therapy (LLLT) has photochemical reactions with cell membranes, cellular organelles and enzymes. LLLT can induce a complex chain of physiological reactions by increasing mitochondrial respiration, activating transcription factors, reducing key inflammatory mediators, inhibiting apoptosis, stimulating angiogenesis, and increasing neurogenesis to enhance wound healing, tissue regeneration and reduce acute inflammation9 , 10. LLLT has been clinically applied to treatment of rheumatoid arthritis, periodontal disease, pain management and healing of wounds and burns11  13. Many studies approved that LLLT has the potential to be an effective noninvasive therapy for spinal cord injury14 , 15.

The aim of this study is to evaluate if LLLT can protect rats spinal cord from I-R injury, so we hypothesized that LLLT would attenuate immunohistochemical and histopathological changes and improve functional recovery after the ischemia/ reperfusion-induced spinal cord injury in rats.

METHODS

Animal care and experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publications No. 8023). Forty eight male Wistar rats weighing 400-450g were used in this study. Anesthesia was induced by intramuscular injection of ketamine hydrochloride 60 mg/kg and xylazine 10 mg/kg. A longitudinal incision was made through the skin on the abdominal region and the abdominal aorta was exposed through midline laparotomy. Heparin (250 UI/kg) was administered intravenously before aortic clamping. Spinal cord ischemia was induced by crossclamping for 60 min, using Bulldog forceps (Figure 1).

Vascular clamps were placed under the left renal vein and above the bifurcation in the aorta. Then the forceps were removed and the chest closed routinely. Animals were placed in their cages after recovery. Rats were randomly assigned to two groups.

FIGURE 1 Surgical site: the ventral aorta was exposed and clamped by Bulldog forceps for 60 minutes. 

In control group (group I), I-R injury was induced but not irradiated with the laser beam. The irradiation protocol was applied as Byrnes described previously16. Briefly in treatment group (group II), 15 minutes after I-R induction on the spinal cord, the lesion site as a rectangular, about 3 cm2(3 cm length×1 cm width) was irradiated transcutaneously to the spinal direction with 810 nm diode laser (Thor International, UK;) with output power of 150 mW. The dosage applied to the surface of the skin was 1,589 J/cm2 per day (0.53 W/cm2, 450 J). Irradiation was repeated daily for the following 3 consecutive days. In each day, irradiation was applied 4 times about 20 minutes with contact mode.

Neurologic scoring system

The Neurologic deficits of animals were evaluated on postoperative 72 hour by a single trained blinded observer by using the following scoring:

Grade 0: paraplegia with no lower extremity motor function;

Grade 1: poor lower extremity motor function;

Grade 2: good movement of the hind limbs, but unable to stand;

Grade 3: able to stand but unable to walk normally; Grade 4: complete recovery17.

Spinal cord histopathologic examination

All animals were anesthetized with lethal dose of pentobarbital (25 mg/kg). Spinal cords were dissected totally and fixed in 10% formalin and embedded in paraffin with routine procedures. Sections from fourth to sixth lumbar segment were obtained. The spinal cord tissues were embedded in paraffin and serial transverse sections (5 µm) cut from paraffin blocks and stained with hemotoxylin and eosin for histopathologic examination. Histopathologic evaluations were performed with means of light microscopy by a neuropathologist who was blinded to experimental conditions.

TUNEL staining

TUNEL staining was performed by an in situ cell death detection kit (Roche, Germany). Hematoxylin was used to counterstain the sections. Quantitative analysis was performed blindly by counting the number of TUNEL positive neurons in the ventral horns in five microscopic fields as described previously18.

Statistical analysis

All data are expressed as mean ± standard deviation. Statistical analysis of the neurologic scores were analyzed by using KruskalWallis one-way analysis of variance (ANOVA). The investigators were blinded to the treatments. Values for statistical analyses were considered significant at p<0.05. All analyses were performed by using the SPSS software package (SPSS, Inc, Chicago, Ill).

RESULTS

Neurological evaluations presented in Table 1. In the group I, the neurological scores was lower. Although in the group II, the Tarlov scale increased and showed a significant difference after 72h of reperfusion (p<0.05).

TABLE 1 – Neurologic status 72 hours after reperfusion as evaluated by the modified Tarlov neurologic recovery scale. 

score Group I (Control) N=24 Group II (Treatment) N=24
0 8 1
1 7 3
2 3 5
3 4 4
4 2 11
Mean ± SD 1.38 ± 0.09 2.86 ± 0.68*

*Mean neurologic scores showed a significant difference between control and treatment groups (p<0.05) both at 72h after reperfusion.

Histopathologic evaluations in group I, presented that had severe ischemic injury with inclusive necrosis of gray matter, which enclosed typically necrotic nuroglia cells with eosinophilic cytoplasm, and loss of cytoplasmic structures. In addition the numbers of normal nuroglia cells were apparently reduced in this group and neuronal structural alterations were observed, which included oligodendrocytes pyknosis, light staining tigroid body, nucleus’s atrophy of nuroglia cell and nucleolus disappearance of oligodendrocytes. Furthermore, hemorrhagic macules were scattered into tissue structures and vacuolar changes were observed in the cytoplasm (Figure 2). The histopathologic changes in group II were milder than that observed in the group I, and the gray matter architecture was generally preserved, with most nuroglia cells appearing to have survived the ischemic consequences (Figure 3).

FIGURE 2 The neurons of spinal cord anterior horn of group I were assessed by H&E staining and viewed at the magnification of 200 times which presented group necrotic changes with prominent vacuolization, intensely eosinophilic cytoplasm, Nissl granule loss, and pyknosis (arrows) as well as by the presence of infiltrating neutrophils and mononuclear phagocytes severe percellular edema and glial cell proliferation. 

FIGURE 3 The neurons of spinal cord anterior horn of group II were assessed by H&E staining and viewed at the magnification of 200 times which showed relative preservation of tissue architecture along with almost complete protection of the neurons, vascular structures, and glial cells along with only mild per cellular edema. The arrows indicate ischemia neuron cells showing mildly eosinophilic cytoplasm, Nissl body loss, and pyknosis. 

Average TUNEL-positive cell counts are shown in Table 2. These data show that the group II exhibited significantly fewer TUNEL-positive cells compared with the group I.

TABLE 2 – Quantitative analysis of the number of TUNEL-positive cells in the ventral horn of spinal cord of all groups, 72h after reperfusion. 

Group I (Control) II (Treatment)
Number of TUNEL-posetive motor neurons Mean SD (n=24) 73.04 0.3 Mean SD (n=24) 36.50** 0.6

*Mean Quantitative analysis showed a significant difference between control and treatment groups (p<0.05) both at 72h after reperfusion.

It is understandable that the number of TUNEL-positive neurons decreased significantly after laser therapy, suggesting that LLLT may protect spinal cords from I-R apoptosis. Spinal cord sections were stained with TUNEL and observed at the light microscopic level (400 times magnification). In the spinal cord ventral horn of the group I, amount of vacuoles appeared and numerous TUNEL-positive neurons were observed (Figure 4). By contrary, very few positively stained neurons were observed in group II (Figure 5).

FIGURE 4 TUNEL staining and quantification of apoptotic motor neurons after reperfusion (×400). Many TUNEL-positive neurons with intense nucleus staining were visible in group I. The arrows indicate TUNEL-positive motor neurons. 

FIGURE 5 TUNEL staining and quantification of apoptotic motor neurons after reperfusion (×400). Only a small number of positively stained neurons were observed in the group II. The arrows TUNELpositive motor neurons. 

DISCUSSION

Our results showed that LLLT will be able to reduce the damages of spinal cord after I-R in rats. This result was verified by both neurological and histological and observations. Additionally, Functional recovery of LLLT group was significantly improved when compared with control group.

Spinal cord I-R injury is a persistent clinical problem in surgical repair of thoracic and thoracoabdominal aneurism surgeries19 , 20. The major cause of spinal cord injury, during and after aortic surgery to the occurrence of one or more of the three following events: (I) the duration and degree of ischaemia; (II) failure to re-establish blood flow to the spinal cord after repair; (III) a biochemically mediated reperfusion injury21. Reperfusion is the restoration of blood flow to the organ after a period of ischaemia. Reperfusion of ischaemic neuronal tissues leads to release production of oxygen derived free radicals, produced as a result of incomplete oxygenation during the period of ischaemia22. Inflammatory response with production of cytokines by microglia and activated neutrophils also contributes to generation of these radicals23 , 24. Several different surgical strategies and laboratory studies have been developed in attempt to decrease the risk of this devastating complication25  27. However, neurological injury in thoracoabdomial surgery remains one of the greatest unsolved mysteries28  30.

The therapeutic effects of LLLT have been reported, being associated with production of anti-apoptotic, pro-proliferative, antioxidant, and angiogenic factors31  33. LLLT also known as photobiomodulation, is an emerging therapeutic approach in which cells or tissues are exposed to low-levels of red and near-IR light. Its experimental applications have broadened to include serious diseases such as heart attack, stroke, and spinal cord injury. Oron et al, suggested that a transcranial application of LLLT after traumatic brain injury provides a significant long-term functional neurological benefit and decreases brain tissue loss34. In another research applied LLLT in acute Spinal cord injury caused by of trauma which promotes axonal regeneration and functional recovery35.

LLLT may have beneficial effects in the acute treatment of I-R by reducing inflammatory mediators, inhibiting apoptosis, stimulating angiogenesis, and increasing neurogenesis9. Transcranial LLLT applied after ischemic stroke in rats caused a significant improvement of neurological score compared to sham animals36.

We hypothesized that LLLT would effectively protect spinal cord by its antioxidant and anti-inflammatory. To our knowledge, the present study probably is the first study to evaluating the neuroprotective effects of LLLT in attenuating I-R induced neurologic injury to the rat spinal cord. It is known that functional recovery after I-R is highly correlated with the volume of remaining normal nerve fibers in spinal tissue37. Adno et al.11, demonstrated transcutaneous application of 810-nm nonpolarized laser significantly promoted axonal regrowth, our results are in agreement with that and show association of improved neurologic status.

Byrnes et al.16, found that 810 nm light, at a dosage of 1.589 J/cm2, significantly improves axonal regrowth, functional improvement and statistically significant suppression of immune cell invasion and pro-inflammatory cytokine and chemokine gene expression. Similarly we documented that LLLT had efficient protection on neural cells from apoptosis or necrosis. Also decreased inflammatory cell accumulation in the spinal cords of animals that received LLLT as compared with the control group also supports LLLT proposed anti-inflammatory property and may contribute to neuroprotection.

CONCLUSION

Low level laser therapy protects the spinal cord from ischemia-reperfusion injury spinal cord ischemia and provide better locomotor function in rats which may be related to antiinflammatory properties of that.

REFERENCES

1.  Cambria RP, Davison JK, Zannetti S, L’Italien G, Brewster DC, Gertler JP, Moncure AC, LaMuraglia GM, Abbott WM. Clinical experience with epidural cooling for spinal cord protection during thoracic and thoracoabdominal aneurysm repair. J VascSurg. 1997;25:234-41. PMID: 9052558. [ Links ]

2.  Choi DW. Glutamate neurotoxicity and diseases of the nervous system. Neuron. 1998;1:623-34. PMID: 2908446. [ Links ]

3.  Dawson TM, Dawson VL, Synder SH. A novel messenger in brain: the free radical, nitric oxide. Ann Neurol. 1992;32:297-311. PMID: 1384420. [ Links ]

4.  Akgun S, Tekeli A, Kurtkaya O, Civelek A, Isbir SC, Ak K, Arsan S, Sav A. Neuroprotective effects of FK-506, L-carnitine and azathioprine on spinal cord ischemia-reperfusion injury. Eur J Cardiothorac Surg. 2004;25:105-10. PMID: 14690740. [ Links ]

5.  Okita Y, Takamoto S, Ando M, Morota T, Yamaki F, Matsukawa R, Kawashima Y. Repair of aneurysms of the entire descending thoracic aorta or thoracoabdominal aorta using a deep hypothermia. Eur J Cardiothorac Surg. 1997;12:120-6. PMID: 9262092. [ Links ]

6.  Cambria RP, Giglia JS. Prevention of spinal cord ischemic complications after thoracoabdominal aortic surgery. Eur J Vasc Endovasc Surg. 1998;15:96-109. PMID: 9551047. [ Links ]

7.  Isbir CS, Ak K, Kurtkaya O, Zeybek U, Akgun S, Scheitauer BW, Sav A, Cobanoglu A. Ischemic preconditioning and nicotinamide in spinalcord protection in an experimental model of transient aortic occlusion. Eur J Cardiothorac Surg. 2003;23:1028-33. PMID: 12829083. [ Links ]

8.  Kiziltepe U, Turan NND, Han U, Ulus AT, Akar F. Resveratrol, a red wine polyphenol, protects spinal cord from ischemia-reperfusion injury. J Vasc Surg. 2004;40(1):138-45. PMID: 15218474. [ Links ]

9.  Hashmi JT, Huang YY, Osmani BZ, Sharma SK, Naeser MA, Hamblin MR. Role of low-level laser therapy in neurorehabilitation. PM R. 2010 Dec;2(12 Suppl 2):S292-305. doi: 10.1016/j. pmrj.2010.10.013. [ Links ]

10.  Hamblin M, Huang YY, Wu Q, Xuan W, Ando T, Xu T, Sharma S, Kharkwal G. Low-level light therapy aids traumatic brain injury. Biomed Opt Med Imaging. 2011;10:1-3. doi: 10.1117/2.1201102.003573. [ Links ]

11.  Ando T, Sato S, Kobayashi H, Nawashiro H, Ashida H, Hamblin MR, Obara M. Low-level laser therapy for spinal cord injury in rats: effects of polarization. J Biomed Opt. 2013;18(9):1-6. PMID: 24030687. [ Links ]

12.  Ekim A, Armagan O, Tascioglu F, Oner C, Colak M. Effect of low level laser therapy in rheumatoid arthritis patients with carpal tunnel syndrome. Swiss Med Wkly. 2007;137(23-24):347-52. PMID: 17629805. [ Links ]

13.  Simunovic Z, Ivanovich AD, Depolo A. Wound healing of animal and human body sport and traffic accident injuries using low-level laser therapy treatment: a randomized clinical study of seventy-four patients with control group. J Clin Laser Med Surg. 2000;18(2):6773. PMID: 11800105. [ Links ]

14.  Rochkind S, Shahar A, Amon M, Nevo Z. Transplantation of embryonal spinal cord nerve cells cultured on biodegradable microcarriers followed by low power laser irradiation for the treatment of traumatic paraplegia in rats. Neurol Res. 2002;24(4):355-60. PMID: 12069281. [ Links ]

15.  Rochkind S. Photoengineering of neural tissue repair processes in peripheral nerves and the spinal cord: research development with clinical applications. Photomed Laser Surg. 2006;24(2):151-7. PMID: 16706693. [ Links ]

16.  Byrnes KR, Waynant RW, Ilev IK, Wu X, Barna L, Smith K, Heckert R, Gerst H, Anders JJ. Light promotes regeneration and functional recovery and alters the immune response after spinal cord injury. Lasers Surg Med. 2005;36:171-85. PMID: 15704098. [ Links ]

17.  Tarlov IM, Klinger H. Spinal cord compression studies. II. Time limits for recovery after acute compression in dogs. AMA Arch Neurol Psychiatry. 1954 Mar;71(3):271-90. PMID: 13123590. [ Links ]

18.  Shan LQ, Ma S, Qiu XC, Zhou Y, Zhang Y, Zheng LH, Ren PC, Wang YC, Fan QY, Ma BA. Hydroxysafflor Yellow A protects spinal cords from ischemia/reperfusion injury in rabbits. Neuroscience. 2010;11:98. doi: 10.1186/1471-2202-11-98. [ Links ]

19.  Liang CL, Lu K, Liliang PC, Chen TB, Chan SHH, Chen HJ. Ischemic preconditioning ameliorates spinal cord ischemiareperfusion injury by triggering autoregulation. J Vasc Surg. 2012 Apr;55(4):1116-23. doi: 10.1016/j.jvs.2011.09.096. [ Links ]

20.  Ilhan A, Koltuksuz U, Ozen S, Uz E, Ciralik H, Akyol O. The effects of caffeic acid phenethyl ester (CAPE) on spinal cordischemia/ reperfusion injury in rabbits. Eur J Cardiothorac Surg. 1999;16:45863. PMID: 10571095. [ Links ]

21.  Svensson LG. New and future approaches for spinal cord protection. Semin Thorac Cardiovasc Surg. 1997;9(3):206-21. PMID: 9263340. [ Links ]

22.  Wan IYP, Angelini GD, Bryan AJ, Ryder I, Underwood MJ. Prevention of spinal cord ischaemia during descending thoracic and thoracoabdominal aortic surgery. Eur J Cardiothorac Surg. 2001;19:203-13. PMID: 11167113. [ Links ]

23.  Ilhan A, Koltuksuz U, Ozen S, Uz E, Ciralik H, Akyol O. The effects of caffeic acid phenethyl ester (CAPE) on spinal cord ischemia/ reperfusion injury in rabbits. Eur J Cardiothorac Surg. 1999;16:45863. PMID: 10571095. [ Links ]

24.  Garcia JH, Liu KF, Yoshida Y, Lian J, Chen S, Del Zoppo G. Influx of leukocytes and platelets in an evolving brain infarct. Am J Pathol. 1994;144:188-99. PMID: 8291608. [ Links ]

25.  Etz CD, Luehr M, Kari FA, Bodian CA, Smego D, Plestis KA, Griepp RB. Paraplegia after extensive thoracic and thoracoabdominal aortic aneurysm repair: does critical spinal cord ischemia occur postoperatively? J Thorac Cardiovasc Surg. 2008;135:324-30. doi: 10.1016/j.jtcvs.2007.11.002. [ Links ]

26.  Bisdas T, Redwan A, Wilhelmi M, Haverich A, Hagl C, Teebken O, Pichlmaier M. Less-invasive perfusion techniques may improve outcome in thoracoabdominal aortic surgery. J Thorac Cardiovasc Surg. 2010;(21)140:1319-24. doi: 10.1016/j.jtcvs.2010.01.012. [ Links ]

27.  Matsuda H, Ogino H, Fukuda T, Iritani O, Sato S, Iba Y, Tanaka H, Sasaki H, Minatoya K, Kobayashi J, Yagihara T. Multi disciplinary approach to prevent spinal cord ischemia after thoracic endovascular aneurysm repair for distal descending aorta. Ann Thorac Surg. 2010;90:561-5. doi: 10.1016/j.athoracsur.2010.04.067. [ Links ]

28.  Cunningham JJN, Laschinger JC, Merkin HA, Nathan IM, Colvin S, Ransohoff J, Spencer FC. Measurement of spinal cord ischemia during operations upon the thoracic aorta. Ann Surg. 1982;144:574. doi: 10.1097/00000658-198209000-00007. [ Links ]

29.  Laschinger JC, Cunningham JJN, Nathan IM, Knopp EA, Cooper MM, Spencer FC. Experimental and clinical assessment of the adequacy of partial bypass in maintenance of spinal cord blood flow. Ann Thorac Surg. 1983;36:417-26. PMID: 6625737. [ Links ]

30.  Cunningham JNJ. Spinal cord ischemia. Introduction. Semin Thorac Cardiovasc Surg. 1998;10:3-5. PMID: 9469770. [ Links ]

31.  Huang YY, Chen ACH, Carroll JD, Hamblin MR. Biphasic dose response in low level light therapy. Dose Response. 2009;7(4):35883. doi: 10.2203/dose-response.09-027.Hamblin. [ Links ]

32.  Huang YY, Sharma SK, Carroll JD, Hamblin MR. Biphasic dose response in low level light therapy, an update. Dose Response. 2011;9(4):602-18. doi: 10.2203/dose-response.11-009.Hamblin. [ Links ]

33.  Xuan W, Vatansever F, Huang L, Wu Q, Xuan Y, Dai T, Ando T, Xu T, Huang YY, Hamblin MR. Transcranial low-level laser therapy improves neurological performance in traumatic brain injury in mice: effect of treatment repetition regimen. PLoS One. 2013;8(1):e53454. doi: 10.1371/journal.pone.0053454. [ Links ]

34.  Oron A, Oron U, Streeter J, de Taboada L, Alexandrovich A, Trembovler V, Shohami E. Low-level laser therapy applied transcranially to mice following traumatic brain injury significantly reduces long-term neurological deficits. J Neurotrauma. 2007;24(4):651-6. PMID: 17439348. [ Links ]

35.  Wu X, Dmitriev AE, Cardoso MJ, Viers-Costello AG, Borke RC, Streeter J, Anders JJ. 810nm Wavelength light: an effective therapy for transected or contused rat spinal cord. Lasers Surg Med. 2009 Jan;41(1):36-41. doi: 10.1002/lsm.20729. [ Links ]

36.  Detaboada L, Ilic S, Leichliter-Martha S, Oron U, Oron A, Streeter J. Transcranial application of low-energy laser irradiation improves neurological deficits in rats following acute stroke. Lasers Surg Med. 2006 Jan;38(1):70-3. PMID: 16444697. [ Links ]

37.  You SW, Chen BY, Liu HL, Lang B, Xia JL, Jiao XY, Ju G. Spontaneous recovery of locomotion induced by remaining fibers after spinal cord transection in adult rats. Restor Neurol Neurosci. 2003;21(1-2):39-45. PMID: 12808201. [ Links ]

Financial source: Islamic Azad University

1Research performed at Department of Surgery, Faculty of Veterinary, Islamic Azad University (IAU), Kahnooj Branch.

Received: May 06, 2015; Revised: July 07, 2015; Accepted: August 04, 2015

Correspondence:Amir Sotoudeh Islamic Azad University Kahnooj Branch Kahnooj, Iran Phone: 00989121768066 Fax: 00983495230203 dramirsotoudeh@kahnoojiau.ac.ir

Conflict of interest: none

 This is an open-access article distributed under the terms of the Creative Commons Attribution License

Neurophotonics. 2016 Jul;3(3):031404. doi: 10.1117/1.NPh.3.3.031404. Epub 2016 Mar 4

Review of transcranial photobiomodulation for major depressive disorder: targeting brain metabolism, inflammation, oxidative stress, and neurogenesis.

Cassano P1, Petrie SR2, Hamblin MR3, Henderson TA4, Iosifescu DV5.

Author information

  • 1Massachusetts General Hospital, Depression Clinical and Research Program, One Bowdoin Square, 6th Floor, Boston, Massachusetts 02114, United States; Harvard Medical School, Department of Psychiatry, 401 Park Drive, Boston, Massachusetts 02215, United States.
  • 2Massachusetts General Hospital, Depression Clinical and Research Program, One Bowdoin Square, 6th Floor, Boston, Massachusetts 02114, United States.
  • 3Massachusetts General Hospital, Wellman Center for Photomedicine, 50 Blossom Street, Boston, Massachusetts 02114, United States; Harvard Medical School, Department of Dermatology, 55 Fruit Street, Boston, Massachusetts 02114, United States; Harvard-MIT Division of Health Sciences and Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
  • 4Synaptic Space, 3979 East Arapahoe Road, Littleton, Colorado 80122, United States; Neuro-Laser Foundation, Suite 420, 215 South Wadsworth, Lakewood, Colorado 80226, United States.
  • 5Mount Sinai Medical School, Mood and Anxiety Disorders Program, 1428 Madison Avenue, New York, New York 10029, United States; Mount Sinai Medical School, Department of Psychiatry and Neuroscience, 1 Gustave L. Levy Place, New York, New York 10029, United States.

Abstract

We examined the use of near-infrared and red radiation (photobiomodulation, PBM) for treating major depressive disorder (MDD). While still experimental, preliminary data on the use of PBM for brain disorders are promising. PBM is low-cost with potential for wide dissemination; further research on PBM is sorely needed. We found clinical and preclinical studies via PubMed search (2015), using the following keywords: “near-infrared radiation,” “NIR,” “low-level light therapy,” “low-level laser therapy,” or “LLLT” plus “depression.” We chose clinically focused studies and excluded studies involving near-infrared spectroscopy. In addition, we used PubMed to find articles that examine the link between PBM and relevant biological processes including metabolism, inflammation, oxidative stress, and neurogenesis. Studies suggest the processes aforementioned are potentially effective targets for PBM to treat depression. There is also clinical preliminary evidence suggesting the efficacy of PBM in treating MDD, and comorbid anxiety disorders, suicidal ideation, and traumatic brain injury. Based on the data collected to date, PBM appears to be a promising treatment for depression that is safe and well-tolerated. However, large randomized controlled trials are still needed to establish the safety and effectiveness of this new treatment for MDD.

J Exp Neurosci. 2016 Feb 1;10:1-19. doi: 10.4137/JEN.S33444. eCollection 2016.

Neuroprotective Effects Against POCD by Photobiomodulation: Evidence from Assembly/Disassembly of the Cytoskeleton.

Liebert AD1, Chow RT2, Bicknell BT3, Varigos E4.
Author information
1University of Sydney, Sydney, NSW, Australia.
2Brain and Mind Institute, University of Sydney, Sydney, NSW, Australia.
3Australian Catholic University, Sydney, NSW, Australia.
4Olympic Park Clinic, Melbourne, VIC, Australia.
Abstract
Postoperative cognitive dysfunction (POCD) is a decline in memory following anaesthesia and surgery in elderly patients. While often reversible, it consumes medical resources, compromises patient well-being, and possibly accelerates progression into Alzheimer’s disease. Anesthetics have been implicated in POCD, as has neuroinflammation, as indicated by cytokine inflammatory markers. Photobiomodulation (PBM) is an effective treatment for a number of conditions, including inflammation. PBM also has a direct effect on microtubule disassembly in neurons with the formation of small, reversible varicosities, which cause neural blockade and alleviation of pain symptoms. This mimics endogenously formed varicosities that are neuroprotective against damage, toxins, and the formation of larger, destructive varicosities and focal swellings. It is proposed that PBM may be effective as a preconditioning treatment against POCD; similar to the PBM treatment, protective and abscopal effects that have been demonstrated in experimental models of macular degeneration, neurological, and cardiac conditions.
J Neurosurg. 2015 Nov 27:1-13. [Epub ahead of print]

Intracranial application of near-infrared light in a hemi-parkinsonian rat model: the impact on behavior and cell survival.

 Reinhart F1, Massri NE2, Chabrol C1, Cretallaz C1, Johnstone DM3, Torres N1, Darlot F1, Costecalde T1, Stone J3, Mitrofanis J2, Benabid AL1, Moro C1.
 Author information
1CEA, Leti, and Clinatec Departments, University Grenoble Alpes, Minatec Campus, Grenoble, France; and
2Departments of 2 Anatomy and.
3Physiology, University of Sydney, New South Wales, Australia.
Abstract
OBJECT The authors of this study used a newly developed intracranial optical fiber device to deliver near-infrared light (NIr) to the midbrain of 6-hydroxydopamine (6-OHDA)-lesioned rats, a model of Parkinson’s disease. The authors explored whether NIr had any impact on apomorphine-induced turning behavior and whether it was neuroprotective.
METHODS Two NIr powers (333 nW and 0.16 mW), modes of delivery (pulse and continuous), and total doses (634 mJ and 304 J) were tested, together with the feasibility of a midbrain implant site, one considered for later use in primates. Following a striatal 6-OHDA injection, the NIr optical fiber device was implanted surgically into the midline midbrain area of Wistar rats. Animals were tested for apomorphine-induced rotations, and then, 23 days later, their brains were aldehyde fixed for routine immunohistochemical analysis.
RESULTS The results showed that there was no evidence of tissue toxicity by NIr in the midbrain. After 6-OHDA lesion, regardless of mode of delivery or total dose, NIr reduced apomorphine-induced rotations at the stronger, but not at the weaker, power. The authors found that neuroprotection, as assessed by tyrosine hydroxylase expression in midbrain dopaminergic cells, could account for some, but not all, of the observed behavioral improvements; the groups that were associated with fewer rotations did not all necessarily have a greater number of surviving cells. There may have been other “symptomatic” elements contributing to behavioral improvements in these rats.
CONCLUSIONS In summary, when delivered at the appropriate power, delivery mode, and dosage, NIr treatment provided both improved behavior and neuroprotection in 6-OHDA-lesioned rats.
J Cereb Blood Flow Metab. 2014 Aug;34(8):1391-401. doi: 10.1038/jcbfm.2014.95. Epub 2014 May 21.

Low-level laser therapy effectively prevents secondary brain injury induced by immediate early responsive gene X-1 deficiency.

Zhang Q1, Zhou C1, Hamblin MR2, Wu MX2.

Author information

  • 11] Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, USA [2] Department of Dermatology, Harvard Medical School, Boston, Massachusetts, USA.
  • 21] Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, USA [2] Department of Dermatology, Harvard Medical School, Boston, Massachusetts, USA [3] Affiliated faculty member of the Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts, USA.

Abstract

A mild insult to the brain can sometimes trigger secondary brain injury, causing severe postconcussion syndrome, but the underlying mechanism is ill understood. We show here that secondary brain injury occurs consistently in mice lacking immediate early responsive gene X-1 (IEX-1), after a gentle impact to the head, which closely simulates mild traumatic brain injury in humans. The pathologic lesion was characterized by extensive cell death, widespread leukocyte infiltrates, and severe tissue loss. On the contrary, a similar insult did not induce any secondary injury in wild-type mice. Strikingly, noninvasive exposure of the injured head to a low-level laser at 4 hours after injury almost completely prevented the secondary brain injury in IEX-1 knockout mice. The low-level laser therapy (LLLT) suppressed proinflammatory cytokine expression like interleukin (IL)-1? and IL-6 but upregulated TNF-?. Moreover, although lack of IEX-1 compromised ATP synthesis, LLLT elevated its production in injured brain. The protective effect of LLLT may be ascribed to enhanced ATP production and selective modulation of proinflammatory mediators. This new closed head injury model provides an excellent tool to investigate the pathogenesis of secondary brain injury as well as the mechanism underlying the beneficial effect of LLLT.

  J Biomed Opt. 2013;18(12):128005. doi: 10.1117/1.JBO.18.12.128005.

Near-infrared stimulation on globus pallidus and subthalamus.

Yoo M1, Koo H2, Kim M2, Kim HI3, Kim S4.

 1Gwangju Institute of Science and Technology (GIST), Department of Medical System Engineering, Gwangju, Republic of Korea.

  • 2Wonkwang University School of Medicine, Department of Physiology, Iksan, Republic of Korea.
  • 3Gwangju Institute of Science and Technology (GIST), Department of Medical System Engineering, Gwangju, Republic of KoreacGwangju Institute of Science and Technology (GIST), School of Mechatronics, Gwangju, Republic of KoreadPresbyterian Medical Center, Department of Neurosurgery, Jeonju, Republic of Korea.
  • 4Gwangju Institute of Science and Technology (GIST), Department of Medical System Engineering, Gwangju, Republic of KoreacGwangju Institute of Science and Technology (GIST), School of Mechatronics, Gwangju, Republic of Korea.

Abstract

Near-infrared stimulation (NIS) is an emerging technique used to evoke action potentials in nervous systems. Its efficacy of evoking action potentials has been demonstrated in different nerve tissues. However, few studies have been performed using NIS to stimulate the deep brain structures, such as globus pallidus (GP) and subthalamic nucleus (STN). Male Sprague-Dawley rats were randomly divided into GP stimulation group (n=11) and STN stimulation group (n=6). After introducing optrodes stereotaxically into the GP or STN, we stimulated neural tissue for 2 min with continuous near-infrared light of 808 nm while varying the radiant exposure from 40 to 10 mW. The effects were investigated with extracellular recordings and the temperature rises at the stimulation site were also measured. NIS was found to elicit excitatory responses in eight out of 11 cases (73%) and inhibitory responses in three cases in the GP stimulation group, whereas it predominantly evoked inhibitory responses in seven out of eight cases (87.5%) and an excitatory response in one case in STN stimulation group. Only radiation above 20 mW, accompanying temperature increases of more than 2°C, elicited a statistically significant neural response (p<0.05). The responsiveness to NIS was linearly dependent on the power of radiation exposure.

J Neurotrauma. 2014 Jun 1;31(11):1008-17. doi: 10.1089/neu.2013.3244. Epub 2014 May 8.

Significant improvements in cognitive performance post-transcranial, red/near-infrared light-emitting diode treatments in chronic, mild traumatic brain injury: open-protocol study.

Naeser MA1, Zafonte R, Krengel MH, Martin PI, Frazier J, Hamblin MR, Knight JA, Meehan WP 3rd, Baker EH.

Author information

  • 11 VA Boston Healthcare System , Boston, Massachusetts.

Abstract

This pilot, open-protocol study examined whether scalp application of red and near-infrared (NIR) light-emitting diodes (LED) could improve cognition in patients with chronic, mild traumatic brain injury (mTBI). Application of red/NIR light improves mitochondrial function (especially in hypoxic/compromised cells) promoting increased adenosine triphosphate (ATP) important for cellular metabolism. Nitric oxide is released locally, increasing regional cerebral blood flow. LED therapy is noninvasive, painless, and non-thermal (cleared by the United States Food and Drug Administration [FDA], an insignificant risk device). Eleven chronic, mTBI participants (26-62 years of age, 6 males) with nonpenetrating brain injury and persistent cognitive dysfunction were treated for 18 outpatient sessions (Monday, Wednesday, Friday, for 6 weeks), starting at 10 months to 8 years post- mTBI (motor vehicle accident [MVA] or sports-related; and one participant, improvised explosive device [IED] blast injury). Four had a history of multiple concussions. Each LED cluster head (5.35 cm diameter, 500 mW, 22.2 mW/cm(2)) was applied for 10 min to each of 11 scalp placements (13 J/cm(2)). LEDs were placed on the midline from front-to-back hairline; and bilaterally on frontal, parietal, and temporal areas. Neuropsychological testing was performed pre-LED, and at 1 week, and 1 and 2 months after the 18th treatment. A significant linear trend was observed for the effect of LED treatment over time for the Stroop test for Executive Function, Trial 3 inhibition (p=0.004); Stroop, Trial 4 inhibition switching (p=0.003); California Verbal Learning Test (CVLT)-II, Total Trials 1-5 (p=0.003); and CVLT-II, Long Delay Free Recall (p=0.006). Participants reported improved sleep, and fewer post-traumatic stress disorder (PTSD) symptoms, if present. Participants and family reported better ability to perform social, interpersonal, and occupational functions. These open-protocol data suggest that placebo-controlled studies are warranted.

Lasers Med Sci. 2014 May 24. [Epub ahead of print]

 “Low-intensity laser therapy effect on the recovery of traumatic spinal cord injury”

Paula AA1, Nicolau RA, Lima MD, Salgado MA, Cogo JC.
  • 1Instituto de Pesquisa e Desenvolvimento (IP&D), Universidade do Vale do Paraíba (Univap), São José dos Campos, São Paulo, Brazil.

Abstract

Scientific advances have been made to optimize the healing process in spinal cord injury. Studies have been developed to obtain effective treatments in controlling the secondary injury that occurs after spinal cord injury, which substantially changes the prognosis. Low-intensity laser therapy (LILT) has been applied in neuroscience due to its anti-inflammatory effects on biological tissue in the repairing process. Few studies have been made associating LILT to the spinal cord injury. The objective of this study was to investigate the effect of the LILT (GaAlAs laser-780 nm) on the locomotor functional recovery, histomorphometric, and histopathological changes of the spinal cord after moderate traumatic injury in rats (spinal cord injury at T9 and T10). Thirty-one adult Wistar rats were used, which were divided into seven groups: control without surgery (n?=?3), control surgery (n?=?3), laser 6 h after surgery (n?=?5), laser 48 h after surgery (n?=?5), medullar lesion (n?=?5) without phototherapy, medullar lesion?+?laser 6 h after surgery (n?=?5), and medullar lesion?+?laser 48 h after surgery (n?=?5). The assessment of the motor function was performed using Basso, Beattie, and Bresnahan (BBB) scale and adapted Sciatic Functional Index (aSFI). The assessment of urinary dysfunction was clinically performed. After 21 days postoperative, the animals were euthanized for histological and histomorphometric analysis of the spinal cord. The results showed faster motor evolution in rats with spinal contusion treated with LILT, maintenance of the effectiveness of the urinary system, and preservation of nerve tissue in the lesion area, with a notorious inflammation control and increased number of nerve cells and connections. In conclusion, positive effects on spinal cord recovery after moderate traumatic spinal cord injury were shown after LILT.

J Cereb Blood Flow Metab.  2014 May 21. doi: 10.1038/jcbfm.2014.95. [Epub ahead of print]

Low-level laser therapy effectively prevents secondary brain injury induced by immediate early responsive gene X-1 deficiency.

Author information

  • 11] Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, USA [2] Department of Dermatology, Harvard Medical School, Boston, Massachusetts, USA.
  • 21] Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, USA [2] Department of Dermatology, Harvard Medical School, Boston, Massachusetts, USA [3] Affiliated faculty member of the Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts, USA.

Abstract

A mild insult to the brain can sometimes trigger secondary brain injury, causing severe postconcussion syndrome, but the underlying mechanism is ill understood. We show here that secondary brain injury occurs consistently in mice lacking immediate early responsive gene X-1 (IEX-1), after a gentle impact to the head, which closely simulates mild traumatic brain injury in humans. The pathologic lesion was characterized by extensive cell death, widespread leukocyte infiltrates, and severe tissue loss. On the contrary, a similar insult did not induce any secondary injury in wild-type mice. Strikingly, noninvasive exposure of the injured head to a low-level laser at 4 hours after injury almost completely prevented the secondary brain injury in IEX-1 knockout mice. The low-level laser therapy (LLLT) suppressed proinflammatory cytokine expression like interleukin (IL)-1? and IL-6 but upregulated TNF-?. Moreover, although lack of IEX-1 compromised ATP synthesis, LLLT elevated its production in injured brain. The protective effect of LLLT may be ascribed to enhanced ATP production and selective modulation of proinflammatory mediators. This new closed head injury model provides an excellent tool to investigate the pathogenesis of secondary brain injury as well as the mechanism underlying the beneficial effect of LLLT.Journal of Cerebral Blood Flow & Metabolism advance online publication, 21 May 2014; doi:10.1038/jcbfm.2014.95.

Alzheimers Res Ther. 2014; 6(1): 2.
Published online Jan 3, 2014. doi:  10.1186/alzrt232

Photobiomodulation with near infrared light mitigates Alzheimer’s disease- related pathology in cerebral cortex – evidence from two transgenic mouse models.

Sivaraman Purushothuman,1,2 Daniel M Johnstone,corresponding author1,2 Charith Nandasena,1,2 John Mitrofanis,1,3 and Jonathan Stone1,2
Author information ? Article notes ? Copyright and License information ?

Introduction

Alzheimer’s disease (AD) is a chronic, debilitating neurodegenerative disease with limited therapeutic options; at present there are no treatments that prevent the physical deterioration of the brain and the consequent cognitive deficits. Histopathologically, AD is characterised by neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein and amyloid-beta (A?) plaques [1,2]. The extent of these histopathological features is considered to vary with and to determine clinical disease severity [2]. While the initiating pathogenic events underlying AD are still debated, there is strong evidence to suggest that oxidative stress and mitochondrial dysfunction have important roles in the neurodegenerative cascade [35]. Therefore, it has been proposed that targeting mitochondrial dysfunction could prove valuable for AD therapeutics [6].

One safe, simple yet effective approach to the repair of damaged mitochondria is photobiomodulation with near-infrared light (NIr). This treatment, which involves the irradiation of tissue with low intensity light in the red to near-infrared wavelength range (600 to 1000 nm), was originally pioneered for the healing of superficial wounds [7] but has been recently shown to have efficacy in protecting the central nervous system. While the mechanism of action remains to be elucidated, there is evidence that NIr preserves and restores cellular function by reversing dysfunctional mitochondrial cytochrome c oxidase (COX) activity, thereby mitigating the production of reactive oxygen species and restoring ATP production to normal levels [8,9].

To date, NIr treatment has yielded neuroprotective outcomes in animal models of retinal damage [9,10], traumatic brain injury [11,12], Parkinson’s disease [1315] and AD [16,17]. Furthermore, NIr therapy has yielded beneficial outcomes in clinical trials of human patients with mild to moderate stroke [18] and depression [19]. This treatment represents a promising alternative to drug therapy because it is safe, easy to apply and has no known side-effects at levels even higher than optimal doses [20].

The aim of this study was to assess the efficacy of NIr in mitigating the brain pathology and associated cellular damage that characterise AD. We utilised two mouse models, each manifesting distinct AD-related pathologies: the K3 tau transgenic model, which develops NFTs [21,22]; and the APP/PS1 transgenic model, which develops amyloid plaques [23]. Here, we present histochemical evidence that NIr treatment over a period of 1 month reduces the severity of AD-related pathology and oxidative stress and restores mitochondrial function in brain regions susceptible to neurodegeneration in AD, specifically the neocortex and hippocampus. The findings extend our previous NIr work in models of acute neurodegeneration [13,14] to demonstrate that NIr is also effective in protecting the brain against chronic insults due to AD-related genetic aberrations, a pathogenic mechanism that is likely to more closely model the human neurodegenerative condition.

Methods

Mouse models

The K3 transgenic mouse model, originally generated as a model of frontotemporal dementia [21,22], harbours a human tau gene with the pathogenic K369I mutation; expression is driven by the neuron-specific mThy1.2 promoter. This model manifests high levels of hyperphosphorylated tau and NFTs by 2 to 3 months of age and cognitive deficits by about 4 months of age [21,22]. We commenced our experiments on K3 mice and matched C57BL/6 wildtype (WT) controls at 5 months of age, when significant neuropathology is already present.

The APPswe/PSEN1dE9 (APP/PS1) transgenic mouse model, obtained from the Jackson Laboratory (Stock number 004462; Bar Harbor, ME, USA), harbours two human transgenes: the amyloid beta precursor protein gene (APP) containing the Swedish mutation; and the presenilin-1 gene (PS1) containing a deletion of exon 9 [23]. The APP/PS1 mice exhibit increased A? and amyloid plaques by 4 months of age [24] and cognitive deficits by 6 months of age [25]. We commenced our experiments on APP/PS1 mice and matched C57BL/6 × C3H WT controls at 7 months of age, when numerous amyloid plaques and associated cognitive deficits are present.

Genotyping of mice was achieved by extracting DNA from tail tips through a modified version of the Hot Shot preparation method [26] and amplifying the transgene sequence by polymerase chain reaction. As reported previously, K3 mice were identified using the primers 5-GGGTGTCTCCAATGCCTGCTTCTTCAG-3 (forward) and 5-AAGTCACCCAGCAGGGAGGTGCTCAG-3 (reverse) [21,22] and APP/PS1 mice were genotyped using primers 5-AGGACTGACCACTCGACCAG-3 (forward) and 5-CGGGGGTCTAGTTCTGCAT-3 (reverse) [23].

Experimental design

For each series of experiments on K3 mice (aged 5 months) or APP/PS1 mice (aged 7 months) there were three experimental groups: untreated WT mice, untreated transgenic mice and NIr-treated transgenic mice (n = 5 mice per experimental group for the K3 series, 15 mice in total; n = 6 mice per experimental group for the APP/PS1 series, 18 mice in total). Our design did not include a WT control group exposed to NIr because NIr has no detectable impact on the survival and function of cells in normal healthy brain [1315]. Given the consistency of the previous results, use of animals for this extra control group did not seem justified [27].

Mice in the NIr-treated groups were exposed to one 90-second cycle of NIr (670 nm) from a light-emitting device (LED) (WARP 10; Quantum Devices, Barneveld, WI, USA) for 5 days per week over 4 consecutive weeks. Light energy emitted from the LED during each 90-second treatment equates to 4 Joule/cm2; a total of 80 Joule/cm2 was delivered to the skull over the 4 weeks. Our measurements of NIr penetration across the fur and skull of a C57BL/6 mouse indicate that ~2.5% of transmitted light reaches the cortex.

For each treatment, the mouse was restrained by hand and the LED was held 1 to 2 cm above the head. The LED light generated no heat and reliable delivery of the radiation was achieved [1315]. For the sham-treated WT, K3 and APP/PS1 groups, animals were restrained in the same way and the device was held over the head, but the light was not switched on. This treatment regime is similar to that used in previous studies where beneficial changes to neuropathology and behavioural signs were reported [1315].

Experimental animals were housed two or more to a cage and kept in a 12-hour light (<5 lux)/dark cycle at 22°C; food pellets and water were available ad libitum. All protocols were approved by the Animal Ethics Committee of the University of Sydney.

Histology and immunohistochemistry

At the end of the experimental period, mice were anaesthetised by intraperitoneal injection of sodium pentobarbital (60 mg/kg) and perfused transcardially with 4% buffered paraformaldehyde. Brains were post fixed for 3 hours, washed with phosphate-buffered saline and cryoprotected in 30% sucrose/phosphate-buffered saline. Tissue was embedded in OCT compound (ProSciTech, Thuringowa, QLD, Australia) and coronal sections of the neocortex and the hippocampus (between bregma ?1.8 and ?2.1) were cut at 20 ?m thickness on a Leica cryostat (Nussloch, Germany).

Immunohistochemistry

For most antibodies, antigen retrieval was achieved using sodium citrate buffer with 0.1% Triton. Sections were blocked in 10% normal goat serum and then incubated overnight at 4°C with a mouse monoclonal antibody – paired helical filaments-tau AT8, 1:500 (Innogenetics, Ghent, Belgium); 4-hydroxynonenal (4-HNE), 1:200 (JaICA, Fukuroi, Shizuoka, Japan); 8-hydroxy-2?-deoxyguanosine (8-OHDG), 1:200 (JaICA); COX, 1:200 (MitoSciences, Eugene, OR, USA) – and/or a rabbit polyclonal antibody (200 kDa neurofilament, 1:500; Sigma, St. Louis, MO, USA). Sections were then incubated for 3 hours at room temperature in Alexa Fluor-488 (green) and/or Alexa Fluor-594 (red) tagged secondary antibodies specific to host species of the primary antibodies (1:1,000; Molecular Probes, Carlsbad, CA, USA). Sections were then counterstained for nuclear DNA with bisbenzimide (Sigma).

Two different but complementary antibodies were used to label A? peptide: 6E10, which recognises residues 1 to 16; and 4G8, which recognises residues 17 to 24. We have previously used these two antibodies in combination to validate A? labelling, demonstrating identical labelling patterns in the rat neocortex and hippocampus [28]. For double labelling using 6E10 antibodies (1:500; Covance, Princeton, NJ, USA) and anti-glial fibrillary acidic protein antibodies (1:1,000; DAKO, Glostrup, Denmark), antigen retrieval was achieved by incubation in 90% formic acid for 10 minutes, and primary antibody incubation was carried out overnight at room temperature. For labelling using the 4G8 (1:500; Covance) antibody, slides were treated with 3% H2O2 in 50% methanol, incubated in 90% formic acid and then washed several times in dH2O before the blocking step, as described previously [28]. After blocking, sections were incubated overnight at room temperature with 4G8 antibody. Sections were then incubated in biotinylated goat anti-mouse IgG for 1 hour followed by ExtrAvidin peroxidase for 2.5 hours. The sections were then washed and developed with 3,3?-Diaminobenzidine.

Negative control sections were processed in the same fashion as described above except that primary antibodies were omitted. These control sections were immunonegative. Fluorescent images were taken using a Zeiss Apotome 2, Carl Zeiss, Oberkochen, Germany. Brightfield images were taken using a Nikon Eclipse E800, Nikon Instruments, Melville, NY, USA.

Histology

NFTs were assessed using the Bielschowsky silver staining method, as described previously [21,22]. Briefly, sections were placed in prewarmed 10% silver nitrate solution for 15 minutes, washed and then placed in ammonium silver nitrate solution at 40°C for a further 30 minutes. Sections were subsequently developed for 1 minute and then transferred to 1% ammonium hydroxide solution for 1 minute to stop the reaction. Sections were then washed in dH2O, placed in 5% sodium thiosulphate solution for 5 minutes, washed, cleared and mounted in dibutyl phathalate xylene.

As described previously [28], A? plaques were studied by staining with Congo red, a histological dye that binds preferentially to compacted amyloid with a ?-sheet secondary structure [29]. Briefly, sections were treated with 2.9 M sodium chloride in 0.01 M NaOH for 20 minutes and were subsequently stained in filtered alkaline 0.2% Congo red solution for 1 hour.

Morphological analysis

Staining intensity and area measurements

To quantify the average intensity and area of antibody labelling within the neocortex and hippocampal regions, an integrated morphology analysis was undertaken using MetaMorph software. For each section, the level of nonspecific staining (using an adjacent region of unstained midbrain) was adjusted to a set level to ensure a standard background across different groups. Next, outlines of retrosplenial cortex area 29 and hippocampal CA1 region were traced and the average intensity and area of immunostaining were calculated by the program. Measurements were conducted on ?4 representative sections per animal and ?3 animals per experimental group. Statistical analyses were performed in Prism 5.0 (Graphpad, La Jolla, CA, USA) using one-way analysis of variance with Tukey’s multiple comparison post test. All values are given as mean ± standard error of mean.

Amyloid-beta plaque measurements

Digital brightfield images of 4G8 staining in the neocortical and hippocampal regions (between bregma ?1.8 and ?2.1) were taken at 4× magnification and analysed with Metamorph, Molecular Devices LLC, Sunnyvale, CA, USA. The software was programmed to measure the number of plaques and the average size of plaques after thresholding for colour. The percentage of area covered by plaques (plaque burden) was calculated by multiplying the number of plaques by the average size of plaques, divided by the area of interest, as described previously [30]. The average number of Congo red-positive plaques in the APP/PS1 brain regions was estimated using the optical fractionator method (StereoInvestigator; MBF Science, Williston, VT, USA), as outlined previously [14]. Briefly, systematic random sampling of sites was undertaken using an unbiased counting frame (100 ?m × 100 ?m). All plaques that came into focus within the frame were counted. Measurements were conducted on ?4 representative sections per animal and ?3 animals per experimental group. Plaque numbers and size were analysed using a two-tailed unpaired t test (when variances were equal) or Welch’s t test (when variances were unequal). All values are given as mean ± standard error of mean. For all analyses, investigators were blinded to the experimental groups.

Results

Evidence of NIr-induced neuroprotection is presented from the neocortex (retrosplenial area) and the hippocampus (CA1 and subiculum), two cortical regions affected in the early stages of human AD [2].

Near-infrared light mitigates the tau pathology of K3 cortex

Hyperphosphorylation of the neuronal microtubule stabilising protein tau and the resulting NFTs are much studied features of dementia pathology [2,31]. The K3 mouse model manifests hyperphosphorylated tau and NFTs by 2 to 3 months of age and cognitive deficits by about 4 months of age [21,22]. We observe strong labelling for hyperphosphorylated tau in the neocortex and the hippocampus at 6 months of age; expression appears to plateau after this age, with similar labelling observed in 12-month-old mice (Figure 1A,B,C,D,E,F).

Figure 1

Figure 1

Time course of the natural development of cortical pathology in K3 and APP/PS1 mice. (A), (B), (C), (D), (E), (F) Micrographs of hyperphosphorylated tau labelling (red), using the AT8 antibody, in the neocortex (A to C) and hippocampus (D toF) of untreated 

In the retrosplenial area of the neocortex there was a significant overall difference in AT8 immunolabelling for tau between the experimental groups, both when considering average intensity of labelling (P < 0.01 by analysis of variance; Figure 2A) and labelled area (P < 0.01; Figure 2B). Tukeypost hoc testing revealed significant differences between the untreated K3 group and the other two groups; labelling was much stronger and more widespread in K3 mice than WT controls (17-fold higher intensity, P < 0.01), and this labelling was reduced by over 70% in NIr-treated mice (P < 0.05). Interestingly, there was no significant difference between the WT and K3-NIr groups, suggesting that NIr treatment had reduced hyperphosphorylated tau to control levels in K3 mice. A similar trend was observed when considering the NFT pathology (Figure 2C,D,E). In contrast to WT brain, which showed no NFT-like lesions (Figure 2C), the K3 brain contained many ovoid shaped NFT-like lesions (that is, spheroids; Figure 2D). Such structures were less frequent in the K3-NIr brain (Figure 2E).

Figure 2

Figure 2

Effect of near-infrared light treatment on hyperphosphorylated tau and neurofibrillary tangles in the neocortex of K3 mice. (A), (B) Quantification of tau AT8 immunolabelling, based on average labelling intensity (A) and labelled area (B). All error bars 

Similar effects were observed in the hippocampus (Figure 3). There was a significant overall difference between the experimental groups in AT8 immunolabelling of the CA1 pyramidal cells (P < 0.01). As for the neocortex, K3 mice showed far greater labelling than WT mice (17-fold higher intensity, P < 0.01) and this was reduced over 65% by NIr treatment (P < 0.01). Again, there were no significant differences between the WT and K3-NIr groups (P > 0.05). Bielschowsky silver staining of the subiculum (Figure 3C,D,E) revealed axonal swellings and spheroids in the hippocampal region of K3 mice (Figure 3D), which were less pronounced in mice from the K3-NIr group (Figure 3E). No pathology was observed in the hippocampus of WT mice (Figure 3C).

Figure 3

Figure 3

Effect of near-infrared light treatment on hyperphosphorylated tau and neurofibrillary tangles in the hippocampus of K3 mice. (A), (B) Quantification of tau AT8 immunolabelling, based on average labelling intensity (A) and labelled area (B). All error 

One should note that the large white matter pathways associated with the hippocampus were labelled intensely by silver staining in all three groups (Figure 3C,D,E). This labelling has been described previously and is not associated with any neuropathology [32].

Near-infrared light reduces oxidative stress in K3 cortex

Oxidative stress and damage are common features of neurodegenerative diseases such as AD, and may be a precursor to neuronal death [35]. We assessed two common markers of oxidative stress: 4-HNE, a toxic end-product of lipid peroxidation that may bind to proteins that then trigger mitochondrial dysfunction and cellular apoptosis in AD [33]; and 8-OHDG, a marker for nuclear and mitochondrial DNA oxidation, which is elevated in AD brains [34].

Overall, 4-HNE immunoreactivity in the neocortex was significantly different between the experimental groups (Figure 4), by both average labelling intensity (P < 0.01) and labelled area (P < 0.001). As with AT8 labelling above, the K3 group showed a much higher average 4-HNE labelling intensity and area than the WT group (fivefold and 20-fold, respectively) and this labelling was significantly reduced (by 50% and 80%, respectively) in the K3-NIr group. Again, these measures showed no significant differences between the WT and K3-NIr groups (P > 0.05).

Figure 4

Figure 4

Effect of near-infrared light treatment on oxidative stress markers in the neocortex of K3 mice. (A), (B), (F), (G)Quantification of immunolabelling of two oxidative stress markers, 4-hydroxynonenal (4-HNE; A, B) and 8-hydroxy-2?-deoxyguanosine 

Similar patterns were observed for 8-OHDG immunoreactivity. Overall, there was a significant difference between the groups for 8-OHDG immunolabelling, by both average intensity (P < 0.0001) and labelled area (P < 0.0001). Again the K3 group showed significantly higher 8-OHDG labelling intensity and area than the WT group (sixfold and 17-fold, respectively), and the 8-OHDG labelling intensity and area were significantly reduced in the K3-NIr group relative to untreated K3 (65% and 85% reduction, respectively). The intensity and area of 8-OHDG labelling did not differ significantly between the WT and the K3-NIr groups (P > 0.05), suggesting that NIr treatment reduces markers of oxidative stress to control levels. The representative photomicrographs of 8-OHDG immunoreactivity in the retrosplenial area (Figure 4H,I,J) reflect the quantitative data, with many 8-OHDG+ structures in the K3 group (Figure 4I) but not in the WT and K3-NIr groups (Figure 4H,J)

Near-infrared light mitigates mitochondrial dysfunction in K3 cortex

We assessed expression patterns of the mitochondrial enzyme COX in the neocortex and the hippocampus as a marker of mitochondrial function. Overall, there were statistically significant differences in the patterns of COX immunoreactivity between the different experimental groups, both in the neocortex and the hippocampus (both P < 0.0001; Figure 5). Relative to WT mice, the COX labelling intensity and area were reduced in K3 mice in both the neocortex and the hippocampus (>70% and >75% reductions, respectively). The K3-NIr mice showed a significant recovery of COX immunoreactivity relative to untreated K3 mice in both the neocortex (>1.7-fold increase, P < 0.05) and the hippocampus (>3.4-fold increase, P < 0.001). However, recovery was not complete, with K3-NIr mice having significantly lower COX immunoreactivity than WT mice in the neocortex (~50%, P < 0.001) and significantly lower COX labelling intensity (~20%, P < 0.05) in the hippocampus. These two groups did not differ significantly in COX labelling area in the hippocampus (P > 0.05).

Figure 5

Figure 5

Effect of near-infrared light treatment on cytochrome coxidase labelling in the neocortex and hippocampus of K3 mice. (A), (B), (F), (G) Quantification of immunolabelling of the mitochondrial marker cytochrome c oxidase (COX) in the neocortex retrosplenial 

Near-infrared mitigates amyloid pathology in APP/PS1 cortex

Along with NFTs, A? plaques are considered a primary pathological hallmark of AD and A? load is often used as a marker of AD severity [1,35]. We assessed the distribution of A? plaques and more immature forms of the A? peptide in the neocortex and hippocampus of APP/PS1 mice aged 7 months; this age is after the first signs of intracellular A? within cells (at 3 months; Figure 1G) and extracellular A? plaques (at 4.5 and 12 months; Figure 1H and ?and1I,1I, respectively).

Three quantitative measures of plaque pathology were used: percentage plaque burden, average plaque size and number of plaques. Immunohistochemical labelling with the anti-A? antibody 4G8 revealed a significant reduction in percentage plaque burden (Figure 6A,D), average plaque size (Figure 6B,E) and number of plaques (Figure 6C,F) in both the neocortex and the hippocampus of NIr-treated APP/PS1 mice relative to untreated APP/PS1 controls. Percentage plaque burden was reduced by over 40% in the neocortex (Figure 6A; P < 0.001) and over 70% in the hippocampus (Figure 6D; P < 0.01), average plaque size was reduced 25% in the neocortex (Figure 6B) and 30% in the hippocampus (Figure 6E), and the number of plaques was reduced by over 20% in the neocortex (Figure 6C) and by over 55% in the hippocampus (Figure 6F; all P < 0.05).

Figure 6

Figure 6

Effect of near-infrared light on amyloid-beta and plaque pathology in APP/PS1 mice. (A), (B), (C), (D), (E), (F)Quantification of amyloid-beta (A?) 4G8 immunolabelling of amyloid plaques in the neocortex (A, B, C) and hippocampus (D, E, F), based 

The photomicrographs of the 4G8 immunoreactivity in Figure 6 reflect the quantitative data described earlier. The WT brain is free of plaques (Figure 6H,K); many 4G8+ plaques (arrows) are present in the neocortex (Figure 6I) and the hippocampus (Figure 6L) of untreated APP/PS1 mice, and fewer plaques are present in NIr-treated APP/PS1 mice (Figure 6J,M). Comparable immunolabelling was achieved using the 6E10 anti-A? antibody (data not shown).

A similar but less pronounced trend was observed when staining with Congo red (Figure 7), which stains only mature plaques. Mean counts of plaques in the neocortex (Figure 7A) and the hippocampus (Figure 7B) of NIr-treated APP/PS1 brains were lower than mean counts in untreated APP/PS1 brains (reductions >30%). However, the differences did not reach statistical significance; given the findings described above with the 4G8 and 6E10 anti-A? antibodies, this suggests that NIr may have greatest effect on recently formed A? deposits. The micrographs in Figure 7 show that mature plaques were absent from the WT brain (Figure 7C,D) but were present in the neocortex (Figure 7E) and hippocampus (Figure 7F) of untreated APP/PS1 brains. There appeared to be fewer plaques in the NIr-treated APP/PS1 brains (Figure 7G,H).

Figure 7

Figure 7

Effect of near-infrared light on Congo red-positive plaque numbers in APP/PS1 mice. (A), (B) Quantification of Congo red-positive plaque counts in the neocortex (A) and hippocampus(B). All error bars indicate standard error of the mean. (C), (D), (E), 

Discussion

Using two mouse models with distinct AD-related pathologies (tau pathology in K3, amyloid pathology in APP/PS1), we report evidence that NIr treatment can mitigate the pathology characteristic of AD as well as reduce oxidative stress and restore mitochondrial function in brain regions affected early in the disease. Further, the extent of mitigation – to levels less than at the start of treatment – suggests that NIr can reverse some elements of AD-related pathology.

The present results add to our previous findings of NIr-induced neuroprotection in models of toxin-induced acute neurodegeneration (that is, MPTP-induced parkinsonism). When incorporated into the growing body of evidence that NIr can also protect against CNS damage in models of stroke, traumatic brain injury and retinal degeneration [912,36], the findings provide a basis for trialling NIr treatment as a strategy for protection against neurodegeneration from a range of causes. Present evidence is based on the use of multiple methods, immunohistochemical and histological, to demonstrate pathological features (for example, 4G8 antibody labelling and Congo red staining for amyloid plaques, AT8 antibody labelling and Bielschowsky silver staining for NFTs).

Relationship to previous studies

The present study focused on pathological features considered characteristic of AD, as well as on signs of cellular damage (for example, oxidative stress, mitochondrial dysfunction) that have been demonstrated in AD and in animal models [24]. Our observations in the K3 strain add to previous studies by providing the first evidence in this strain of extensive oxidative damage and mitochondrial dysfunction [27].

Our findings are consistent with previous reports of the effects of red to infrared light on AD pathology in animal models. De Taboada and colleagues assessed the capacity of 808 nm laser-sourced infrared radiation, delivered three times per week over 6 months, to reduce pathology in an APP transgenic model of A? amyloidosis [17]. Treatment led to a reduction in plaque number, amyloid load and inflammatory markers, an increase in ATP levels and mitochondrial function, and mitigation of behavioural deficits. De Taboada and colleagues commenced treatment at 3 months of age, before the expected onset of amyloid pathology and cognitive effects. Similarly, Grillo and colleagues reported that 1,072 nm infrared light, applied 4 days per week for 5 months, reduces AD-related pathology in another APP/PS1 transgenic mouse model (TASTPM) [16]. These investigators also initiated light treatment before the onset of pathology, at 2 months of age. Both studies thus provide evidence that infrared radiation can slow the progression of cerebral degeneration in these models. The present results confirm these observations, in two distinct transgenic strains; they also confirm that the wound-healing and neuroprotective effects of red-infrared length do not vary qualitatively with wavelength, over a wide range.

Evidence of reversal of pathology

Previous reports have described the natural history of the K3 [21,22] and APP/PS1 transgenic models [24,37]. Based on these previous reports and our own baseline data (Figure 1), significant brain pathology and functional deficits are present in both models at the ages when we commenced treatment. Our results therefore suggest that significant reversal of pathology has been induced by the NIr treatment. This has implications for clinical practice, where most patients are not diagnosed until pathogenic mechanisms have already been initiated and resultant neurologic symptoms manifest [15,27].

This evidence that AD-related neuropathology can be transient – appear then disappear – is not novel. Garcia-Alloza and colleagues described evidence of the transient deposition of A?, including the formation of plaque-like structures, in a transgenic model of A? deposition [24]. Reversal of such pathology, by interventions such as NIr treatment, may therefore be possible. However our results suggest that reversal may also be limited to recently formed, immature plaques, as we observed a significant NIr-induced reduction in immunolabelling with the 4G8 and 6E10 antibodies but no significant difference in Congo red staining. Because the 4G8 and 6E10 antibodies recognise various forms of A?, while Congo red stains only mature, compacted plaques, a reasonable deduction is that NIr treatment reduces only the transient, recently formed A? deposits, with no substantial effect on mature plaques. As there is still no consensus as to the pathogenic roles of different forms of A?, it is unclear how this might impact on the therapeutic potential of NIr in a clinical setting.

Mechanisms

The mechanisms underlying the neuroprotective actions of red to infrared light are not completely understood. There is considerable evidence that NIr photobiomodulation enhances mitochondrial function and ATP synthesis by activating photoacceptors such as COX and increasing electron transfer in the respiratory chain, while also reducing harmful reactive oxygen species [3840]. NIr photobiomodulation could also upregulate protective factors such as nerve growth factor and vascular endothelial growth factor [41,42] and mesenchymal stem cells [43] that could target specific areas of degeneration.

The ability of NIr to reduce the expression of hyperphosphorylated tau, which in turn reduces oxidative stress [44], may be key to its neuroprotective effect. Oxidative stress and free radicals increase the severity of cerebrovascular lesions [45,46], mitochondrial dysfunction [4,47], oligomerisation of A? [5,48] and tauopathies and cell death [48,49] in AD. Considering the brain’s high consumption of oxygen and consequent susceptibility to oxidative stress, mitigating such stressors would probably have a pronounced protective effect [50].

 Conclusions

Overall, our results in two transgenic mouse models with existing AD-related pathology suggest that low-energy NIr treatment can reduce characteristic pathology, oxidative stress and mitochondrial dysfunction in susceptible regions of the brain. These results, when taken together with those in other models of neurodegeneration, strengthen the notion that NIr is a viable neuroprotective treatment for a range of neurodegenerative conditions. We believe this growing body of work provides the impetus to begin trialling NIr treatment as a broad-based therapy for AD and other neurodegenerations.

 Abbreviations

A: Amyloid-beta; AD: Alzheimer’s disease; APP: Amyloid beta precursor protein gene; COX: Cytochrome c oxidase; 4-HNE: 4-hydroxynonenal; LED: Light-emitting diode; NFT: Neurofibrillary tangle; NIr: Near-infrared light; 8-OHDG: 8-hydroxy-2?-deoxyguanosine; PS1: Presenilin 1; WT: Wildtype.

Competing interests

The authors declare that they have no competing interests.

 Authors’ contributions

SP undertook the bulk of the experimental work and analysis and wrote the manuscript. DMJ and JM were involved with the analysis of the data and the writing of the manuscript. CN was involved with genotyping and treating the animals. JS was involved in conceiving and designing the study and the writing of the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors thank Tenix Corporation, Sir Zelman Cowen Universities Fund and Bluesand Foundation for funding. They are grateful to Prof. Lars Ittner for providing the breeding litter for K369I mice, and to Dr Louise Cole and the Bosch Advanced Microscopy facility for the help with MetaMorph. Sharon Spana was splendid for her technical help. DMJ is supported by a National Health and Medical Research Council of Australia (NHMRC) Early Career Fellowship.:

References

  • Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science. 2002;6:353–356. doi: 10.1126/science.1072994. [PubMed][Cross Ref]
  • Braak H, Braak E. Staging of Alzheimer’s disease-related neurofibrillary changes. Neurobiol Aging. 1995;6:271–278. doi: 10.1016/0197-4580(95)00021-6. [PubMed] [Cross Ref]
  • Nunomura A, Perry G, Aliev G, Hirai K, Takeda A, Balraj EK, Jones PK, Ghanbari H, Wataya T, Shimohama S, Chiba S, Atwood CS, Petersen RB, Smith MA. Oxidative damage is the earliest event in Alzheimer disease. J Neuropathol Exp Neurol. 2001;6:759–767. [PubMed]
  • Yao J, Irwin RW, Zhao L, Nilsen J, Hamilton RT, Brinton RD. Mitochondrial bioenergetic deficit precedes Alzheimer’s pathology in female mouse model of Alzheimer’s disease. Proc Natl Acad Sci USA. 2009;6:14670–14675. doi: 10.1073/pnas.0903563106. [PMC free article][PubMed] [Cross Ref]
  • Stone J. What initiates the formation of senile plaques? The origin of Alzheimer-like dementias in capillary haemorrhages. Med Hypotheses. 2008;6:347–359. doi: 10.1016/j.mehy.2008.04.007. [PubMed] [Cross Ref]
  • Calabrese V, Guagliano E, Sapienza M, Panebianco M, Calafato S, Puleo E, Pennisi G, Mancuso C, Butterfield DA, Stella AG. Redox regulation of cellular stress response in aging and neurodegenerative disorders: role of vitagenes. Neurochem Res. 2007;6:757–773. doi: 10.1007/s11064-006-9203-y. [PubMed] [Cross Ref]
  • Whelan HT, Smits RL Jr, Buchman EV, Whelan NT, Turner SG, Margolis DA, Cevenini V, Stinson H, Ignatius R, Martin T, Martin T, Cwiklinski J, Philippi AF, Graf WR, Hodgson B, Gould L, Kane M, Chen G, Caviness J. Effect of NASA light-emitting diode irradiation on wound healing. J Clin Laser Med Surg. 2001;6:305–314. doi: 10.1089/104454701753342758.[PubMed] [Cross Ref]
  • Desmet KD, Paz DA, Corry JJ, Eells JT, Wong-Riley MT, Henry MM, Buchmann EV, Connelly MP, Dovi JV, Liang HL, Henshel DS, Yeager RL, Millsap DS, Lim J, Gould LJ, Das R, Jett M, Hodgson BD, Margolis D, Whelan HT. Clinical and experimental applications of NIR-LED photobiomodulation. Photomed Laser Surg. 2006;6:121–128. doi: 10.1089/pho.2006.24.121.[PubMed] [Cross Ref]
  • Eells JT, Wong-Riley MT, VerHoeve J, Henry M, Buchman EV, Kane MP, Gould LJ, Das R, Jett M, Hodgson BD, Margolis D, Whelan HT. Mitochondrial signal transduction in accelerated wound and retinal healing by near-infrared light therapy. Mitochondrion. 2004;6:559–567. doi: 10.1016/j.mito.2004.07.033. [PubMed] [Cross Ref]
  • Natoli R, Zhu Y, Valter K, Bisti S, Eells J, Stone J. Gene and noncoding RNA regulation underlying photoreceptor protection: microarray study of dietary antioxidant saffron and photobiomodulation in rat retina. Mol Vis. 2010;6:1801–1822. [PMC free article] [PubMed]
  • Xuan W, Vatansever F, Huang L, Wu Q, Xuan Y, Dai T, Ando T, Xu T, Huang YY, Hamblin MR. Transcranial low-level laser therapy improves neurological performance in traumatic brain injury in mice: effect of treatment repetition regimen. PLoS One. 2013;6:e53454. doi: 10.1371/journal.pone.0053454. [PMC free article] [PubMed] [Cross Ref]
  • Oron A, Oron U, Streeter J, de Taboada L, Alexandrovich A, Trembovler V, Shohami E. Low-level laser therapy applied transcranially to mice following traumatic brain injury significantly reduces long-term neurological deficits. J Neurotrauma. 2007;6:651–656. doi: 10.1089/neu.2006.0198. [PubMed] [Cross Ref]
  • Moro C, Torres N, El Massri N, Ratel D, Johnstone DM, Stone J, Mitrofanis J, Benabid AL. Photobiomodulation preserves behaviour and midbrain dopaminergic cells from MPTP toxicity: evidence from two mouse strains. BMC Neurosci. 2013;6:40. doi: 10.1186/1471-2202-14-40.[PMC free article] [PubMed] [Cross Ref]
  • Shaw VE, Spana S, Ashkan K, Benabid AL, Stone J, Baker GE, Mitrofanis J. Neuroprotection of midbrain dopaminergic cells in MPTP-treated mice after near-infrared light treatment. J Comp Neurol. 2010;6:25–40. doi: 10.1002/cne.22207. [PubMed] [Cross Ref]
  • Peoples C, Spana S, Ashkan K, Benabid AL, Stone J, Baker GE, Mitrofanis J. Photobiomodulation enhances nigral dopaminergic cell survival in a chronic MPTP mouse model of Parkinson’s disease. Parkinsonism Relat Disord. 2012;6:469–476. doi: 10.1016/j.parkreldis.2012.01.005. [PubMed] [Cross Ref]
  • Grillo SL, Duggett NA, Ennaceur A, Chazot PL. Non-invasive infra-red therapy (1072 nm) reduces beta-amyloid protein levels in the brain of an Alzheimer’s disease mouse model, TASTPM. J Photochem Photobiol B. 2013;6:13–22. [PubMed]
  • De Taboada L, Yu J, El-Amouri S, Gattoni-Celli S, Richieri S, McCarthy T, Streeter J, Kindy MS. Transcranial laser therapy attenuates amyloid-beta peptide neuropathology in amyloid-beta protein precursor transgenic mice. J Alzheimers Dis. 2011;6:521–535. [PubMed]
  • Lampl Y, Zivin JA, Fisher M, Lew R, Welin L, Dahlof B, Borenstein P, Andersson B, Perez J, Caparo C, Ilic S, Oron U. Infrared laser therapy for ischemic stroke: a new treatment strategy: results of the NeuroThera Effectiveness and Safety Trial-1 (NEST-1) Stroke. 2007;6:1843–1849. doi: 10.1161/STROKEAHA.106.478230. [PubMed] [Cross Ref]
  • Schiffer F, Johnston AL, Ravichandran C, Polcari A, Teicher MH, Webb RH, Hamblin MR. Psychological benefits 2 and 4 weeks after a single treatment with near infrared light to the forehead: a pilot study of 10 patients with major depression and anxiety. Behav Brain Funct.2009;6:46. doi: 10.1186/1744-9081-5-46. [PMC free article] [PubMed] [Cross Ref]
  • Tuby H, Hertzberg E, Maltz L, Oron U. Long-term safety of low-level laser therapy at different power densities and single or multiple applications to the bone marrow in mice. Photomed Laser Surg. 2013;6:269–273. doi: 10.1089/pho.2012.3395. [PubMed] [Cross Ref]
  • Ittner LM, Fath T, Ke YD, Bi M, van Eersel J, Li KM, Gunning P, Gotz J. Parkinsonism and impaired axonal transport in a mouse model of frontotemporal dementia. Proc Natl Acad Sci USA. 2008;6:15997–16002. doi: 10.1073/pnas.0808084105. [PMC free article] [PubMed][Cross Ref]
  • van Eersel J, Ke YD, Liu X, Delerue F, Kril JJ, Gotz J, Ittner LM. Sodium selenate mitigates tau pathology, neurodegeneration, and functional deficits in Alzheimer’s disease models. Proc Natl Acad Sci USA. 2010;6:13888–13893. doi: 10.1073/pnas.1009038107. [PMC free article][PubMed] [Cross Ref]
  • Jankowsky JL, Fadale DJ, Anderson J, Xu GM, Gonzales V, Jenkins NA, Copeland NG, Lee MK, Younkin LH, Wagner SL, Younkin SG, Borchelt DR. Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase. Hum Mol Genet. 2004;6:159–170. [PubMed]
  • Garcia-Alloza M, Robbins EM, Zhang-Nunes SX, Purcell SM, Betensky RA, Raju S, Prada C, Greenberg SM, Bacskai BJ, Frosch MP. Characterization of amyloid deposition in the APPswe/PS1dE9 mouse model of Alzheimer disease. Neurobiol Dis. 2006;6:516–524. doi: 10.1016/j.nbd.2006.08.017. [PubMed] [Cross Ref]
  • Cao D, Lu H, Lewis TL, Li L. Intake of sucrose-sweetened water induces insulin resistance and exacerbates memory deficits and amyloidosis in a transgenic mouse model of Alzheimer disease.J Biol Chem. 2007;6:36275–36282. doi: 10.1074/jbc.M703561200. [PubMed] [Cross Ref]
  • Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, Warman ML. Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT) Biotechniques.2000;6:52–54. [PubMed]
  • Purushothuman S, Nandasena C, Johnstone DM, Stone J, Mitrofanis J. The impact of near-infrared light on dopaminergic cell survival in a transgenic mouse model of parkinsonism. Brain Res. 2013;6:61–70. [PubMed]
  • Purushothuman S, Marotte L, Stowe S, Johnstone DM, Stone J. The response of cerebral cortex to haemorrhagic damage: experimental evidence from a penetrating injury model. PLoS One.2013;6:e59740. doi: 10.1371/journal.pone.0059740. [PMC free article] [PubMed] [Cross Ref]
  • Wilcock DM, Gordon MN, Morgan D. Quantification of cerebral amyloid angiopathy and parenchymal amyloid plaques with Congo red histochemical stain. Nat Protoc. 2006;6:1591–1595. doi: 10.1038/nprot.2006.277. [PubMed] [Cross Ref]
  • Yan Q, Zhang J, Liu H, Babu-Khan S, Vassar R, Biere AL, Citron M, Landreth G. Anti-inflammatory drug therapy alters beta-amyloid processing and deposition in an animal model of Alzheimer’s disease. J Neurosci. 2003;6:7504–7509. [PubMed]
  • Augustinack JC, Schneider A, Mandelkow EM, Hyman BT. Specific tau phosphorylation sites correlate with severity of neuronal cytopathology in Alzheimer’s disease. Acta Neuropathol.2002;6:26–35. doi: 10.1007/s004010100423. [PubMed] [Cross Ref]
  • Bruck W, Bitsch A, Kolenda H, Bruck Y, Stiefel M, Lassmann H. Inflammatory central nervous system demyelination: correlation of magnetic resonance imaging findings with lesion pathology. Ann Neurol. 1997;6:783–793. doi: 10.1002/ana.410420515. [PubMed] [Cross Ref]
  • Sayre LM, Zelasko DA, Harris PL, Perry G, Salomon RG, Smith MA. 4-Hydroxynonenal-derived advanced lipid peroxidation end products are increased in Alzheimer’s disease. J Neurochem. 1997;6:2092–2097. [PubMed]
  • Mecocci P, MacGarvey U, Beal MF. Oxidative damage to mitochondrial DNA is increased in Alzheimer’s disease. Ann Neurol. 1994;6:747–751. doi: 10.1002/ana.410360510. [PubMed][Cross Ref]
  • Trinchese F, Liu S, Battaglia F, Walter S, Mathews PM, Arancio O. Progressive age-related development of Alzheimer-like pathology in APP/PS1 mice. Ann Neurol. 2004;6:801–814. doi: 10.1002/ana.20101. [PubMed] [Cross Ref]
  • Oron A, Oron U, Chen J, Eilam A, Zhang C, Sadeh M, Lampl Y, Streeter J, DeTaboada L, Chopp M. Low-level laser therapy applied transcranially to rats after induction of stroke significantly reduces long-term neurological deficits. Stroke. 2006;6:2620–2624. doi: 10.1161/01.STR.0000242775.14642.b8. [PubMed] [Cross Ref]
  • Blanchard V, Moussaoui S, Czech C, Touchet N, Bonici B, Planche M, Canton T, Jedidi I, Gohin M, Wirths O, Bayer TA, Langui D, Duyckaerts C, Tremp G, Pradier L. Time sequence of maturation of dystrophic neurites associated with A? deposits in APP/PS1 transgenic mice. Exp Neurol. 2003;6:247–263. doi: 10.1016/S0014-4886(03)00252-8. [PubMed] [Cross Ref]
  • Karu T. Mitochondrial mechanisms of photobiomodulation in context of new data about multiple roles of ATP. Photomed Laser Surg. 2010;6:159–160. doi: 10.1089/pho.2010.2789.[PubMed] [Cross Ref]
  • Wilden L, Karthein R. Import of radiation phenomena of electrons and therapeutic low-level laser in regard to the mitochondrial energy transfer. J Clin Laser Med Surg. 1998;6:159–165.[PubMed]
  • Wong-Riley MT, Bai X, Buchmann E, Whelan HT. Light-emitting diode treatment reverses the effect of TTX on cytochrome oxidase in neurons. Neuroreport. 2001;6:3033–3037. doi: 10.1097/00001756-200110080-00011. [PubMed] [Cross Ref]
  • Hou JF, Zhang H, Yuan X, Li J, Wei YJ, Hu SS. In vitro effects of low-level laser irradiation for bone marrow mesenchymal stem cells: proliferation, growth factors secretion and myogenic differentiation. Lasers Surg Med. 2008;6:726–733. doi: 10.1002/lsm.20709. [PubMed][Cross Ref]
  • Tuby H, Maltz L, Oron U. Modulations of VEGF and iNOS in the rat heart by low level laser therapy are associated with cardioprotection and enhanced angiogenesis. Lasers Surg Med.2006;6:682–688. doi: 10.1002/lsm.20377. [PubMed] [Cross Ref]
  • Tuby H, Maltz L, Oron U. Induction of autologous mesenchymal stem cells in the bone marrow by low-level laser therapy has profound beneficial effects on the infarcted rat heart. Lasers Surg Med. 2011;6:401–409. doi: 10.1002/lsm.21063. [PubMed] [Cross Ref]
  • Stamer K, Vogel R, Thies E, Mandelkow E, Mandelkow EM. Tau blocks traffic of organelles, neurofilaments, and APP vesicles in neurons and enhances oxidative stress. J Cell Biol.2002;6:1051–1063. doi: 10.1083/jcb.200108057. [PMC free article] [PubMed] [Cross Ref]
  • Aliev G, Smith MA, Seyidov D, Neal ML, Lamb BT, Nunomura A, Gasimov EK, Vinters HV, Perry G, LaManna JC, Friedland RP. The role of oxidative stress in the pathophysiology of cerebrovascular lesions in Alzheimer’s disease. Brain Pathol. 2002;6:21–35. [PubMed]
  • Hamel E, Nicolakakis N, Aboulkassim T, Ongali B, Tong XK. Oxidative stress and cerebrovascular dysfunction in mouse models of Alzheimer’s disease. Exp Physiol. 2008;6:116–120. [PubMed]
  • Zhu X, Perry G, Moreira PI, Aliev G, Cash AD, Hirai K, Smith MA. Mitochondrial abnormalities and oxidative imbalance in Alzheimer disease. J Alzheimers Dis. 2006;6:147–153. [PubMed]
  • Zhang X, Le W. Pathological role of hypoxia in Alzheimer’s disease. Exp Neurol. 2010;6:299–303. doi: 10.1016/j.expneurol.2009.07.033. [PubMed] [Cross Ref]
  • Wen Y, Yang S, Liu R, Brun-Zinkernagel AM, Koulen P, Simpkins JW. Transient cerebral ischemia induces aberrant neuronal cell cycle re-entry and Alzheimer’s disease-like tauopathy in female rats. J Biol Chem. 2004;6:22684–22692. doi: 10.1074/jbc.M311768200. [PubMed][Cross Ref]
  • Uttara B, Singh AV, Zamboni P, Mahajan RT. Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Curr Neuropharmacol.2009;6:65–74. doi: 10.2174/157015909787602823. [PMC free article] [PubMed] [Cross Ref]
Rev Neurosci.  2013;24(2):205-26. doi: 10.1515/revneuro-2012-0086.

Red/near-infrared irradiation therapy for treatment of central nervous system injuries and disorders.

Fitzgerald M1, Hodgetts S, Van Den Heuvel C, Natoli R, Hart NS, Valter K, Harvey AR, Vink R, Provis J, Dunlop SA.
  • 1School of Animal Biology, The University of Western Australia, Crawley, Australia. lindy.fitzgerald@uwa.edu.au

Abstract

Irradiation in the red/near-infrared spectrum (R/NIR, 630-1000 nm) has been used to treat a wide range of clinical conditions, including disorders of the central nervous system (CNS), with several clinical trials currently underway for stroke and macular degeneration. However, R/NIR irradiation therapy (R/NIR-IT) has not been widely adopted in clinical practice for CNS injury or disease for a number of reasons, which include the following. The mechanism/s of action and implications of penetration have not been thoroughly addressed. The large range of treatment intensities, wavelengths and devices that have been assessed make comparisons difficult, and a consensus paradigm for treatment has not yet emerged. Furthermore, the lack of consistent positive outcomes in randomised controlled trials, perhaps due to sub-optimal treatment regimens, has contributed to scepticism. This review provides a balanced précis of outcomes described in the literature regarding treatment modalities and efficacy of R/NIR-IT for injury and disease in the CNS. We have addressed the important issues of specification of treatment parameters, penetration of R/NIR irradiation to CNS tissues and mechanism/s, and provided the necessary detail to demonstrate the potential of R/NIR-IT for the treatment of retinal degeneration, damage to white matter tracts of the CNS, stroke and Parkinson’s disease.

BMC Neurosci. 2013; 14: 40.
Published online Mar 27, 2013. doi:  10.1186/1471-2202-14-40
Cécile Moro,1 Napoleon Torres,1 Nabil El Massri,2 David Ratel,1 Daniel M Johnstone,3 Jonathan Stone,3 John Mitrofanis,corresponding author2 and Alim-Louis Benabid1
Author information ? Article notes ? Copyright and License information ?

Background

Parkinson’s disease is a major movement disorder characterised by the distinct signs of resting tremor, akinesia and/or lead pipe rigidity [1,2]. These arise after a substantial loss of dopaminergic cells, mainly within the substantia nigra pars compacta (SNc) of the midbrain [3,4]. The factors that generate this cell loss are not entirely clear, but there is evidence for mitochondrial dysfunction as a result of exposure to an environmental toxin (eg MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)) [5] and/or the presence of a defective gene [6].

Many previous studies have shown that some substances, such as anti-oxidants like CoQ10 (coenzyme Q10) [7] and melatonin [8], help neuroprotect dopaminergic cells in the SNc against degeneration in animal models of Parkinson’s disease. These substances are thought to reduce mitochondrial dysfunction by lessening the oxidative stress caused by free radicals generated by defective mitochondria present in Parkinson’s disease. In addition to these substances, recent studies have reported on the neuroprotective properties of low intensity light therapy, known also as photobiomodulation or near infra-red light (NIr) treatment, after parkinsonian insult. For example, NIr treatment protects neural cells in vitro against parkinsonian toxins such as MPTP and rotenone [9,10]. Further, we have shown that NIr treatment offers in vivo protection for dopaminergic cells in the SNc in an acute [11] and chronic [12] MPTP mouse (Balb/c) model. There is also a brief report indicating that NIr treatment improves the locomotor activity of mice after MPTP insult [13]. Although the mechanism of neuroprotection by NIr is not entirely clear, work on other systems indicate that NIr improves mitochondrial function and ATP synthesis in the damaged cells by increasing electron transfer in the respiratory chain and activating photoacceptors, such as cytochrome oxidase, within the mitochondria. Further, NIr has been shown to reduce the production of reactive oxygen species that are harmful to cells [14,15].

In this study, we sought to extend our earlier anatomical [11,12] and functional [16] studies by exploring the changes in locomotive behaviour of MPTP-treated mice after NIr treatment. Hitherto, this feature has not been reported extensively [13]. We undertook this behavioural analysis, together with a stereological account of SNc cell number, in two strains of mice, Balb/c (albino) and C57BL/6 (pigmented). This was done because there are reports that MPTP has differential effects on behaviour and dopamine levels in the basal ganglia in different strains of mice [17,18], as well as rats [19]. We wanted to determine whether there were mouse strain differences in the effect of NIr treatment after MPTP insult.

Methods

Subjects

Male BALB/c (albino; n=40) and C57BL/6 mice (pigmented; n=40) mice were housed on a 12 hr light/dark cycle with unlimited access to food and water. Animals were 8–10 weeks old. All experiments were approved by the Animal Ethics Committee of the University of Sydney and COMETH (Grenoble).

Experimental design

We set up four experimental groups (see Figure 1). Mice received intraperitoneal injections of either MPTP or saline, combined with simultaneous NIr treatments or not. The different groups were; (1) Saline: saline injections with no NIr (2) Saline-NIr: saline injections with NIr (3) MPTP: MPTP injections with no NIr (4) MPTP-NIr: MPTP injections with NIr. Each experimental group comprised ten mice of each strain.

Figure 1

Figure 1

Outline of the different experimental groups used in this study, namely Saline, Saline-NIr, MPTP, MPTP-NIr. The experimental time-line and behaviour time-points are shown. For the experimental time-line, there were two injections (saline or MPTP) and 

Following our previous work, we used an acute MPTP mouse model [11,16]. The acute model is a well-accepted model of the disease [20,21] and has revealed many aspects of the mechanisms of Parkinson’s disease over the years. Although it does not provide information on the chronic progressive nature of the disease, it does generate mitochondrial dysfunction, dopaminergic cell death and a reduction in locomotive activity [20,21]. The latter two issues were central in this study, making the acute model most appropriate for our use. Briefly, we made two MPTP (25 mg/kg injections; total of 50 mg/kg per mouse) or saline injections over a 24 hour period. Following each injection, mice in the MPTP-NIr and Saline-NIr groups were treated to one cycle of NIr (670 nm) of 90 seconds from a light-emitting device (LED; Quantum Devices WARP 10). This treatment equated to ~0.5 Joule/cm2 to the brain [11]. Approximately 6 hours after each injection and first NIr treatment, mice in these groups received a second NIr treatment, but no MPTP or saline injection. Hence, each mouse in these groups received four NIr treatments, equalling ~2 joules/cm2 reaching the brain. This NIr treatment regime was similar to that used by previous studies, in particular, those reporting changes after trans-cranial irradiation [11,12,1416]. For each treatment, the mouse was restrained by hand and the LED was held 1–2 cm above the head [11,12,16]. The LED generated no heat and reliable delivery of the radiation was achieved. For the Saline and MPTP groups, mice were held under the LED as described above, but the device was not turned on. After the last treatment, mice were allowed to survive for six days (Figure 1). This MPTP/NIr dose regime and survival period has been shown to furnish TH+ cell loss by MPTP and neuroprotection by NIr [8,11,16]. We also made some measurements of NIr penetration across the skin and fur of the two mouse strains. Skin was excised from the back of each mouse and positioned over a foil-coated vessel, with a calibrated light sensor at the bottom. NIr from the WARP-LED was then shone onto the skin and the penetration was recorded by the sensor (distance from WARP-LED to skin was ~4 cm and distance from skin to sensor was ~3 cm). For each strain, we compared the NIr penetration in cases where the fur was shaved from the skin to those that were unshaved. Each of the values obtained were compared to (and expressed as a percentage of) the values we recorded of NIr through the air, with no intervening skin.

Our experimental paradigm of simultaneous administration of parkinsonian insult and therapeutic application was similar to that of previous studies on animal models of Parkinson’s disease [8,11,12,16,2224]. This paradigm is unlike the clinical reality where there is cell loss prior to therapeutic intervention. However, in our experimental study we hoped to determine the maximum effect of NIr neuroprotection.

Immunocytochemistry and cell analysis

Following the survival period, mice were anaesthetised with an intraperitoneal injection of chloral hydrate (4%; 1 ml/100 g). They were then perfused transcardially with 4% buffered paraformaldehyde. The brains were removed and post-fixed overnight in the same solution. Next, brains were placed in phosphate-buffered saline (PBS) with the addition of 30% sucrose until the block sank. The midbrain was then sectioned coronally and serially (at 50 ?m) using a freezing microtome. All sections were collected in PBS and then immersed in a solution of 1% Triton (Sigma) and 10% normal goat serum (Sigma) at room temperature for ~1 hour. Sections were then incubated in anti-tyrosine hydroxylase (Sigma; 1:1000) for 48 hours (at 4°C), followed by biotinylated anti-rabbit IgG (Bioscientific; 1:200) for three hours (at room temperature) and then streptavidin-peroxidase complex (Bioscientific; 1:200) for two hours (at room temperature). To visualise the bound antibody, sections were reacted in a 3,3?– diaminobenzidine tetrahydrochloride (Sigma) – PBS solution. Sections were mounted onto gelatinised slides, air dried overnight, dehydrated in ascending alcohols, cleared in Histoclear and coverslipped using DPX. Most of our immunostained sections were counterstained lightly with neutral red as well. In order to test the specificity of the primary antibody, some sections were processed as described above, except that there was no primary antibody used. These control sections were immunonegative.

In this study, we used TH immunocytochemistry to describe patterns of cell death and protection. As with many previous studies, we interpreted a change in TH+ cell number after experimental manipulation as an index of cell survival [8,11,12,22,23,25]. If cells lose TH expression, then they are likely to undergo death subsequently [25], which then leads to a reduction in Nissl-stained (and TH+) cell number [8,23]. Notwithstanding a small number of cells that may have transient loss of TH expression [26], a key aspect of our study was whether NIr treatment saved TH expression during a period when MPTP treatment alone would have abolished it [11,12]. In terms of analysis, the number of TH+ cells within the SNc was estimated using the optical fractionator method (StereoInvestigator, MBF Science), as outlined previously [8,11,12,23]. Briefly, systematic random sampling of sites – with an unbiased counting frame (100×100 ?m) – within defined boundaries of SNc was undertaken. Counts were made from every second section, and for consistency, the right hand side of the brain was counted in all cases. All cells (nucleated only) that came into focus within the frame were counted and at least five sites were sampled per section.

Digital images were constructed using Adobe Photoshop (brightness and contrast levels were adjusted on individual images in order to achieve consistency (eg, illumination) across the entire plate) and Microsoft PowerPoint programmes.

Behavioural analysis

During the experimental period, we performed a standard open-field test [17]. Mice were placed in white boxes (~20×20×20 cm) for C57BL/6 mice and black boxes for the Balb/c mice (this was important for software detection of contrast changes). Behavioural activity was measured and videotaped using a high definition camera (25000 images/sec) that detected changes in contrast and hence movement of mice. Mice were not acclimatised to the boxes prior to testing and boxes were cleaned thoroughly to avoid olfactory clues. Animal detection was made comparing a reference image that contained no subject with the live image containing the subject; the differences between the two were identified as subject pixel. Subject pixels changes were computed (Noldus, Ethovision, XT 8.5 version) to obtain different parameters of locomotor activity, for example velocity and mobility. Velocity was the mean speed of the mouse during trials (cm/sec) measured from the centre of gravity of the animal. To avoid “jittering”, a threshold of minimal distance moved of 0.3 cm was established. Mobility calculates the duration (in sec) during which the complete area detected as animal is changing even if the centre of gravity remains the same. High mobility refers to 10% or more of changes in percentage of body area detected between two samples, and immobility refers to less than 2% of changes. Each animal was tested at four time points (Figure 1); (T1) after first MPTP or saline injection and NIr (or no) treatment; (T2) after second NIr (or no) treatment; (T3) after second MPTP or saline injection and third NIr (or no) treatment; (T4) after fourth NIr (or no) treatment. Mice were tested for ~20 minutes at each time point. We tested locomotive activity at these points, particularly T1 and T3, because we wanted to explore the effects of NIr during a time when the MPTP was most effective (eg, immediately after injections), when the mice were most immobile and “sick” [17].

For comparisons between groups in the cell analysis, a one-way ANOVA test was performed, in conjunction with a Tukey-Kramer post-hoc multiple comparison test. For the behavioural analysis, groups were compared for time (T1,T2,T3,T4), drug (MPTP or not) and light (NIr or not) conditions using a three-way ANOVA test with a Bonferroni post-hoc test (using GraphPad Prism programme).

 Results

The results that follow will consider the cell and behavioural analyses for each strain separately.

Cell analysis

Figure 2 shows the estimated number of TH+ cells in the SNc of the four groups in the Balb/c and C57BL/6 mice. Overall, the variations in number were significant for both Balb/c (ANOVA: F=4.9; p<0.001) and C57BL/6 (ANOVA: F=3.8; p<0.01) mice. For the Saline and Saline-NIr groups of both strains, the number of TH+ cells was similar; no significant differences were evident between these groups (Tukey test: p>0.05). For the MPTP groups, TH+ cell number was reduced compared to the saline control groups in both strains (~30%). These reductions were significant (Tukey test: p<0.05). In the MPTP-NIr groups, TH+ cell number was higher than in the MPTP groups of both strains, but more so in the Balb/c (~30%) compared to the C57BL/6 (~20%) mice. This increase reached statistical significance for the Balb/c group (Tukey test: p<0.05) but not the C57BL/6 group. Unlike the MPTP groups, the number of TH+ cells in the MPTP-NIr groups of both strains was not significantly different to the saline groups (Tukey test: p>0.05).

Figure 2

Figure 2

Graph showing TH+ cell number in the SNc in the four experimental groups, in either the Balb/c (grey columns) or C57BL/6 (black columns) mice. Columns show the mean ± standard error of the total number (of one side) in each group. There were 
These patterns are illustrated further in Figure 3 for both Balb/c (Figure 3A,C,E,G) and C57BL/6 (Figure 3B,D,F,H) in each of the Saline (Figure 3A,B), Saline-NIr (Figure 3C,D), MPTP (Figure 3E,F) and MPTP-NIr (Figure 3G,H) groups. Similar patterns of immunostaining were seen in both strains. Although there were fewer TH+ somata in the MPTP group (Figure 3E,F), those remaining were similar in overall appearance to those seen in the Saline (Figure 3A,B), Saline-NIr (Figure 3C,D) and MPTP-NIr (Figure 3G,H) groups. They had round or oval-shaped somata with one to two labelled dendrites.

Figure 3

Figure 3

Photomicrographs of TH+ cells in the SNc of Balb/c (A,C,E,G) and C57BL/6 (B,D,F,H) in each of the Saline (A,B), Saline-NIr (C,D), MPTP (E,F) and MPTP-NIr (G,H) groups. Similar patterns of immunostaining were seen in both strains. There were fewer TH 

Behavioural analysis

Figure 4 shows recorded values of locomotor activity in Balb/c (Figure 4A,B,C) and C57BL/6 (Figure 4A’,B’,C’) mice, in terms of velocity (Figure 4A,A’), high mobility (Figure 4B,B’) and immobility (Figure 4C,C’). Overall, there were significant interactions for time and drug conditions for velocity, high mobility and immobility in both Balb/c (ANOVA: F range=7.5-13.6; p<0.05) and C57BL/6 (ANOVA: F range=16.8-40.5; p<0.05) mice, while significant interactions for time, drug and light conditions were evident for these locomotive activities in Balb/c (ANOVA: F range=11.7-24.2; p<0.05), but not in C57BL/6 (ANOVA: F range=0.4-0.8; p>0.05) mice.

Figure 4

Figure 4

Graphs showing the results of behavioural analysis of Balb/c (A,B,C) or C57BL/6 (A’,B’,C’) mice. The behaviouralanalysis included the locomotor activities of velocity (A,A’), high mobility (B,B’) and immobility 

The patterns of locomotor activity in the Saline and Saline-NIr groups were similar in both strains of mice. There was no significant effect of the light in the different time conditions (T1-T4) in the saline-treated cases (Bonferroni test: p>0.05). Hence, for clarity, the values of these groups were pooled and are represented as a dotted line across each of the graphs. By contrast, distinct changes in locomotor activity were evident between the MPTP and MPTP-NIr groups; their values are hence represented as individual columns at each time point (Figure 4). The results for each locomotor activity in the two strains will be considered separately below.

For Balb/c mice, at T1 (after first MPTP injection and NIr treatment) and T2 (after second NIr treatment) the locomotor activities in the MPTP and MPTP-NIr groups were similar. There were no significant effects of the light in these two time conditions in the MPTP-treated cases (Bonferroni test: p>0.05; Figure 4A,B,C). The effects of MPTP were immediate; compared to the saline control groups, both groups showed less velocity (Figure 4A) and high mobility (Figure 4B) and greater immobility (Figure 4C) at T1. By T2, there was considerable recovery of each locomotor activity in both MPTP and MPTP-NIr groups, with their values returning to control levels (Figure 4A,B,C). At T3 (after second MPTP injection and third NIr treatment) and T4 (after fourth NIr treatment), unlike at T1 and T2, there were significant effects of the light in the MPTP-treated cases (Bonferroni test: p<0.05; Figure 4A,B,C). At T3 and T4, the MPTP-NIr group had greater velocity (Figure 4A) and high mobility (Figure 4B) and less immobility (Figure 4C) than the MPTP group. Compared to the saline control groups, the MPTP-NIr group had similar locomotor activities at T3 and in particular, at T4 (Figure 4A,B,C). By contrast, the MPTP group at both T3 and T4, still had considerably less velocity (Figure 4A) and high mobility (Figure 4B) and greater immobility (Figure 4C) than the saline controls.

For C57BL/6 mice, there were distinct differences in locomotor activity compared to Balb/c mice. First, in C57BL/6 mice, there were no significant effects of the light at all time conditions (T1-T4) in the MPTP-treated cases (Bonferroni test: p>0.05; Figure 4A’,B’,C’); for Balb/c mice, there was no effect of the light in the MPTP-treated cases at T1 and T2 only (Figure 4A,B,C). Second, the MPTP and MPTP-NIr groups had considerably less velocity (Figure 4A’) and high mobility (Figure 4B’) and greater immobility (Figure 4C’) than the saline controls at the majority of the time points. In contrast to Balb/c mice, there was no evidence of NIr-specific recovery of function at T3 and T4; instead MPTP-treated mice appeared to have some recovery after the second MPTP injection (T4; Figure 4A’,B’,C’) irrespective of whether or not they received NIr treatment. Finally, control C57BL/6 mice showed lower baseline velocity (Figure 4A’) and high mobility (Figure 4B’), but also less immobility (Figure 4C’), than Balb/c mice.

In order to explore whether these behavioural (and cellular) differences between the two strains was due to pigmentation, we compared the degree of NIr penetration across the skin and fur in the different strains. In the Balb/c mice, we found that NIr penetration in the unshaved cases was 16% while in the shaved cases, it was 28%. In the C57BL/6 mice, NIr penetration was less, being 19% in the shaved cases and, quite remarkably, only 0.2% in the unshaved cases. Hence, these measurements indicated that the pigmented fur of the C57BL/6 mice absorbed almost all the NIr, hence limiting severely its penetration through to the brain.

 Discussion

We have two main findings. First, the MPTP-NIr group of Balb/c mice had greater locomotor activity and, as shown previously (Shaw et al. 2010), more surviving dopaminergic cells than the MPTP group. Second, these differences in cell survival and locomotor activity between the two groups were not as clear in C57BL/6 mice. Overall, our results indicated that Balb/c mice were a better model for exploring the neuroprotective effects of NIr after MPTP treatment than C57BL/6 mice.

Comparison with previous studies

This study offers the first detailed description of changes in locomotor activity in MPTP-treated mice after NIr treatment. Whelan and colleagues [13] described briefly that NIr pre-treatment, but not post-treatment, improved locomotor activity in an acute MPTP mouse model (strain was not mentioned in that report). Our results in Balb/c mice confirms, at least in part, the results of that study.

There have been several previous reports on the behavioural and cellular changes in Balb/c and C57BL/6 mice after MPTP insult [17,18]. We confirm the findings of these reports in that there were fewer TH+ cells in the SNc of C57BL/6 mice than Balb/c mice (eg, saline controls) and that MPTP had a greater effect on locomotor activity in C57BL/6 than in Balb/c mice; further that Balb/c mice had some NIr-induced recovery of activity while C57BL/6 mice did not. Our results offered some differences to the previous studies, however. In particular, previous studies using non-stereological methods have reported a greater MPTP-induced cell loss in C57BL/6 compared to Balb/c mice [17,18]; our stereological analysis, by contrast, revealed a comparable loss in the two strains (~30%). The reason for these differences is not clear but they may reflect, for example, differences in our MPTP regimes (eg 50 mg/kg over 24 hrs vs. 60 mg/kg over 8 hrs) [17], methods of MPTP delivery (eg, intraperitoneal vs. intraventricular) [18] and methods of cell analysis (stereological vs. non-stereological) [17,18]. Finally, our control Balb/c mice had slightly better locomotor activity at baseline than the C57BL/6 mice, while Sedelis and colleagues [17] have reported the opposite. This discrepancy may reflect differences in the behavioural tests used and our measures of locomotor activity. For example, we measured velocity, high mobility and immobility using contrast changes, while the previous study recorded distance travelled with laser beam technology. Despite these differences in our studies, the key issue is that our MPTP regime was effective in generating TH+ cell loss and behavioural changes in the two strains, thereby allowing an assessment of neuroprotection by NIr treatment.

It should be noted that in this study, we did not undertake an analysis of the density of TH+ terminals in the striatum, nor of the locomotive activity of the mice after six days, the end of the experimental period. Previous studies have shown a complete recovery of TH+ terminal density in the striatum [18] and locomotive activity after six days in Balb/c mice using an acute model [19]; in C57BL/6 mice, although there are fewer TH+ terminals in the striatum of MPTP-treated animals compared to controls at this stage [18], the locomotive activity has been shown to return to control levels [19]. Hence, from these data, there would have been no point for us to explore these issues, mainly because any impact of NIr treatment – the central issue considered in the present study – would not have been elucidated.

NIr treatment improved locomotor activity after MPTP insult in Balb/c mice

Our results showed that NIr treatment improved locomotor activity after MPTP insult in Balb/c mice, hence confirming the histological findings that there were more dopaminergic cells in MPTP-NIr than in MPTP groups [11,12]. The beneficial effect of NIr treatment was not immediate. It was only after the second MPTP injection (and subsequent NIr treatments; T3 and T4) that a clear difference in locomotor activity was recorded between the MPTP-NIr and MPTP groups. Before then (T1 and T2), no differences were evident between these two groups (with the MPTP effect being similar and immediate in both groups). Hence, it appears that it takes several doses of NIr treatment to elicit a beneficial outcome. The mitochondria of the dopaminergic cells, after the third and fourth NIr treatment, may have been stimulated further to increase ATP synthesis and reduce the production of reactive oxygen species [14,15], thereby being better prepared to protect against the second MPTP insult. It is noteworthy that Whelan and colleagues [13] reported improvement of locomotor activity in MPTP-treated mice after several NIr pre-treatments, but not after a single post-treatment. Indeed, previous studies reporting beneficial results in the majority of systems have used multiple NIr treatments of ~4 J/cm2[14,15]. There may well be a therapeutic window for NIr treatment and this may vary for different animals and systems [15].

Strain differences in the effectiveness of NIr treatment after MPTP insult

Somewhat surprisingly, the beneficial effects of NIr treatment after MPTP insult were not as clear in the C57BL/6 mice. When compared to the Balb/c mice, the C57BL/6 mice had a smaller increase in dopaminergic cell number (20% vs 30%) and no clear improvement in locomotor activity in the MPTP-NIr compared to the MPTP group, at least over the later part of the survival period used in this study. Future studies may explore if there is a linear correlation between cell pathology and behavioural decline (and recovery) [28] in different strains of MPTP-treated mice after NIr treatment in the long-term; further, it would be of interest to examine if the finer details of motor disturbances in mice after MPTP treatment are improved after NIr treatment in the different mouse strains [29].

The reason for this strain difference was likely to be due to the pigmented fur of the C57BL/6 mice absorbing the majority of the NIr, preventing penetration into the brain. Our measurements indicated that in unshaved C57BL/6 mice, unlike in the shaved C57BL/6 and Balb/c (shaved and unshaved), there was very little NIr penetration (>1%). Melanin is certainly capable of absorbing the 670 nm wavelength [30] and that seemed sufficient to limit neuroprotection in the C57BL/6 mice. It is of course possible that, in addition to these penetration issues, the albino and pigmented strains have distinct cellular enzyme differences also, responsible for the different responses to NIr-induced metabolic (and therefore therapeutic) changes.

Conclusions

In summary, although our results are in an animal model of the disease, a key point is that NIr appeared to have neuroprotective effects on structures deep in the brain. Our findings that NIr treatment reduced MPTP-induced degeneration among midbrain dopaminergic cells and improved locomotor activity in Balb/c mice, due to greater NIr penetration through skin and fur, form templates for future endeavour. It remains to be determined if NIr, when applied from an external device, is able to penetrate the thicker skull and meningeal layers, together with the greater mass of brain parenchyma to reach the SNc of humans.

Abbreviations

CoQ10: Coenzyme Q10; ATP: Adenosine-5?-triphosphate; LED: Light emitting device; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NIr: Near-infrared light; PBS: Phosphate buffered saline; SNc: Substantia nigra pars compacta; SNr: Substantia nigra pars reticulata; TH: Tyrosine hydroxylase.

Competing interest

There was no conflict of interest for any of the authors: CM,NT, DR, DJ, JS, ALB and JM are full-time members of staff at their respective institutions, while CP and NEM are undergraduate students.

Authors’ contribution

All authors contributed to the analysis of the data and the writing of the manuscript. CM, NT, NEM, DR and JM contributed to the experimental work. All authors read and approved the final manuscript.

Acknowledgements

We are forever grateful to Tenix corp, Salteri family, Sir Zelman Cowen Universities Fund, Fondation Philanthropique Edmond J Safra, France Parkinson and the French National Research Agency (ANR Carnot Institute) for funding this work. We thank Sharon Spana, Vincente Di Calogero, Christophe Gaude, Caroline Meunier and Leti-DTBS staff for excellent technical assistance. We thank Sarah-Jane Leigh and Kevin Keay for their invaluable assistance with the statistics.

References

  • Blandini F, Nappi G, Tassorelli C, Martignoni E. Functional changes of the basal ganglia circuitry in Parkinson’s disease. Prog Neurobiol. 2000;14:63–88. [PubMed]
  • Bergman H, Deuschl G. Pathophysiology of Parkinson’s disease: from clinical neurology to basic neuroscience and back. Mov Disord. 2002;14:S28–S40. doi: 10.1002/mds.10140.[PubMed] [Cross Ref]
  • Rinne JO. Nigral degeneration in Parkinson’s disease. Mov Disord 8 Suppl. 1993;14:S31–S35.[PubMed]
  • McRitchie DA, Cartwright HR, Halliday GM. Specific A10 dopaminergic nuclei in the midbrain degenerate in Parkinson’s disease. Exp Neurol. 1997;14:202–213. doi: 10.1006/exnr.1997.6418.[PubMed] [Cross Ref]
  • Langston JW. The etiology of Parkinson’s disease with emphasis on the MPTP story. Neurology.1996;14:S153–S160. doi: 10.1212/WNL.47.6_Suppl_3.153S. [PubMed] [Cross Ref]
  • Kruger R, Kuhn W, Muller T, Woitalla D, Graeber M, Kosel S, Przuntek H, Epplen JT, Schols L, Riess O. Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson’s disease. Nat Genet. 1998;14:106–118. doi: 10.1038/ng0298-106. [PubMed] [Cross Ref]
  • LeWitt PA. Neuroprotection for Parkinson’s disease. J Neural Transm Suppl. 2006;14:113–122. doi: 10.1007/978-3-211-33328-0_13. [PubMed] [Cross Ref]
  • Ma J, Shaw VE, Mitrofanis J. Does melatonin help save dopaminergic cells in MPTP-treated mice? Parkinsonism Relat Disord. 2009;14:307–314. doi: 10.1016/j.parkreldis.2008.07.008.[PubMed] [Cross Ref]
  • Liang HL, Whelan HT, Eells JT, Wong-Riley MT. Near-infrared light via light-emitting diode treatment is therapeutic against rotenone- and 1-methyl-4-phenylpyridinium ion-induced neurotoxicity. Neurosci. 2008;14:963–974. doi: 10.1016/j.neuroscience.2008.03.042.[PMC free article] [PubMed] [Cross Ref]
  • Ying R, Liang HL, Whelan HT, Eells JT, Wong-Riley MT. Pretreatment with near-infrared light via light-emitting diode provides added benefit against rotenone – and MPP+– induced neurotoxicity. Brain Res. 2008;14:167–173. [PMC free article] [PubMed]
  • Shaw VE, Spana S, Ashkan K, Benabid AL, Stone J, Baker GE, Mitrofanis J. Neuroprotection of midbrain dopaminergic cells in MPTP-treated mice after near-infrared light treatment. J Comp Neurol. 2010;14:25–40. [PubMed]
  • Peoples CL, Spana S, Ashkan K, Benabid AL, Stone J, Baker GE, Mitrofanis J. Photobiomodulation enhances nigral dopaminergic cell survival in a chronic MPTP mouse model of Parkinson’s disease. Parkinsonism Relat Disord. 2012;14:469–476. doi: 10.1016/j.parkreldis.2012.01.005. [PubMed] [Cross Ref]
  • Whelan HT, DeSmet KD, Buchmann E, Henry M, Wong-Riley M, Eells JT, Verhoeve J. Harnessing the cell’s own ability to repair and prevent neurodegenerative disease. SPIE Newsroom. 2008. pp. 1–3. [PMC free article] [PubMed] [Cross Ref]
  • Desmet KD, Paz DA, Corry JJ, Eells JT, Wong-Riley MT, Henry MM, Buchmann EV, Connelly MP, Dovi JV, Liang HL, Henshel DS, Yeager RL, Millsap DS, Lim J, Gould LJ, Das R, Jett M, Hodgson BD, Margolis D, Whelan HT. Clinical and experimental applications of NIR-LED photobiomodulation. Photomed Laser Surg. 2006;14:121–128. doi: 10.1089/pho.2006.24.121.[PubMed] [Cross Ref]
  • Hamblin MR, Demidova TN. In: Mechanisms for low-light therapy. Hamblin MR, Waynart RW, Anders J, editor. San Jose, CA, USA: Proc SPIE; 2006. Mechanisms of low level light therapy; p. 6140.
  • Shaw VE, Peoples CL, Spana S, Ashkan K, Benabid AL, Stone J, Baker GE, Mitrofanis J. Patterns of cell activity in the subthalamic region associated with the neuroprotective action of near-infrared light treatment in MPTP-treated mice. Parkinson’s disease. 2012.[PMC free article] [PubMed]
  • Sedelis M, Hofele K, Auburger GW, Morgan S, Huston JP, Schwarting RKW. MPTP Susceptibility in the Mouse: Behavioural, Neurochemical, and Histological Analysis of Gender and Strain Differences. Behav Gen. 2000;14:171–182. doi: 10.1023/A:1001958023096.[PubMed] [Cross Ref]
  • Ito T, Suzuki K, Uchida K, Nakayama H. Different susceptibility to 1-methyl-4-phenylpyridium (MPP+)-induced nigro-striatal dopaminergic cell loss between C57BL/6 and BALB/c mice is not related to the difference of monoamine oxidase-B (MAO-B) Exp Toxic Path. 2011. EPub.[PubMed]
  • Riachi NJ, Behmand RA, Harik SI. Correlation of MPTP neurotoxicity in vivo with oxidation of MPTP by the brain and blood–brain barrier in vitro in five rat strains. Brain Res. 1991;14:19–24. doi: 10.1016/0006-8993(91)90854-O. [PubMed] [Cross Ref]
  • Schober A. Classic toxin-induced animal models of Parkinson’s disease: 6OHDA and MPTP. Cell Tissue Res. 2004;14:215–24. doi: 10.1007/s00441-004-0938-y. [PubMed] [Cross Ref]
  • Bové J, Perier C. Neurotoxin-based models of Parkinson’s disease. Neurosci. 2012;14:51–76.[PubMed]
  • Piallat B, Benazzouz A, Benabid AL. Subthalamic nucleus lesion in rats prevents dopaminergic nigral neuron degeneration after striatal 6-OHDA injection: behavioural and immunohistochemical studies. Eur J Neurosci. 1996;14:1408–1414. doi: 10.1111/j.1460-9568.1996.tb01603.x. [PubMed] [Cross Ref]
  • Wallace BA, Ashkan K, Heise CE, Foote KD, Torres N, Mitrofanis J, Benabid AL. Survival of midbrain dopaminergic cells after lesion or deep brain stimulation of the subthalamic nucleus in MPTP-treated monkeys. Brain. 2007;14:2129–2145. doi: 10.1093/brain/awm137. [PubMed][Cross Ref]
  • Luquin N, Mitrofanis J. Does the cerebral cortex exacerbate dopamineric cell death in the substantia nigra of 6OHDA-lesioned rats? Parkinson Related Disord. 2008;14:213–223. doi: 10.1016/j.parkreldis.2007.08.010. [PubMed] [Cross Ref]
  • Björklund A, Rosenblad C, Winkler C, Kirik D. Studies on neuroprotective and regenerative effects of GDNF in a partial lesion model of Parkinson’s disease. Neurobiol Dis. 1997;14:186–200. doi: 10.1006/nbdi.1997.0151. [PubMed] [Cross Ref]
  • Huot P, Lévesque M, Parent A. The fate of striatal dopaminergic neurons in Parkinson’s disease and Huntington’s chorea. Brain. 2007;14:222–32. [PubMed]
  • Paxinos G, Franklin BJ. The mouse brain in stereotaxic coordinates. 2. San Diego, CA, USA: Academic Press California USA; 2001.
  • Bezard E, Dovero S, Bioulac B, Gross C. Effects of different schedules of MPTP administration on dopaminergic neurodegeneration in mice. Exp Neurol. 1997;14:288–292. doi: 10.1006/exnr.1997.6648. [PubMed] [Cross Ref]
  • Goldberg NR, Haack AK, Lim NS, Janson OK, Meshul CK. Dopaminergic and behavioural correlates of progressive lesioning of the nigrostriatal pathway with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Neurosci. 2011;14:256–271. [PubMed]
  • Meredith P, Powell BJ, Riesz J, Nighswander-Rempel S, Pederson MR, Moore E. Towards structure–property-function relationships for eumelanin. Soft Matter. 2006;14:37.
Evid Based Complement Alternat Med. 2013; 2013: 594906.
Published online 2013 December 2. doi:  10.1155/2013/594906

Low-Level Laser Stimulation on Adipose-Tissue-Derived Stem Cell Treatments for Focal Cerebral Ischemia in Rats

Chiung-Chyi Shen, 1 , 2 , 3 , 4 Yi-Chin Yang, 1 Ming-Tsang Chiao, 1 Shiuh-Chuan Chan, 5 and Bai-Shuan Liu 6 ,*
Author information  Article notes  Copyright and License information
Copyright © 2013 Chiung-Chyi Shen et al.
This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

This study investigated the effects of large-area irradiation from a low-level laser on the proliferation and differentiation of i-ADSCs in neuronal cells. MTT assays indicated no significant difference between the amount of cells with (LS+) and without (LS) laser treatment (P > 0.05). However, immunofluorescent staining and western blot analysis results indicated a significant increase in the neural stem-cell marker, nestin, following exposure to low-level laser irradiation (P < 0.05). Furthermore, stem cell implantation was applied to treat rats suffering from stroke. At 28 days posttreatment, the motor functions of the rats treated using i-ADSCs (LS+) did not differ greatly from those in the sham group and HE-stained brain tissue samples exhibited near-complete recovery with nearly no brain tissue damage. However, the motor functions of the rats treated using i-ADSCs (LS?) remained somewhat dysfunctional and tissue displayed necrotic scarring and voids. The western blot analysis also revealed significant expression of oligo-2 in the rats treated using i-ADSCs (LS+) as well as in the sham group (P < 0.05). The results demonstrated that low-level laser irradiation exerts a positive effect on the differentiation of i-ADSCs and can be employed to treat rats suffering from ischemic stroke to regain motor functions.

1. Introduction

Stroke has become a common disease and has been shown to be associated with the consumption of high amounts of oil and salt. These dietary habits cause blood vessels to narrow and become prone to occlusion, which can lead to stroke. Strokes can be broadly classified into 2 categories: ischemic and hemorrhagic strokes. Differing treatment methods are used according to stroke type and lesion location. Therefore, at stroke onset, computed tomography or magnetic resonance imaging is often used for diagnosis to provide physicians with a basis of treatment [1]. The treatment methods used for the 2 types of stroke are different. (1) Ischemic stroke is caused by thrombosis or blood clots in the brain vessels, which prevent blood flow into the brain tissues. Anticoagulants or antiplatelet medications must be administered to patients as soon as possible. (2) Hemorrhagic stroke is caused by the rupture or hemorrhaging of brain vessels caused by peripheral brain trauma (subarachnoid hemorrhage). The majority of cases of hemorrhagic stroke are caused by brain trauma (e.g., car and workplace accidents). Treating these patients often requires neurosurgical interventions [2]. Ischemic strokes constitute the majority of stroke cases. When patients suffer from ischemic stroke, the brain tissues necrotize gradually because of the lack of nutrients if anticoagulants or antiplatelet medications are not administered within 3 h of the stroke. After necrosis occurs, necrotic brain tissue cannot regenerate or recover its functions even if blood-vessel reperfusion occurs [3]. In recent years, stem cell treatment has become an innovative therapy for brain tissues that cannot regenerate.

Body fat extraction (e.g., abdomen, thigh, or buttock fat) has been used for body shaping procedures in the past, and the extracted fat was discarded as medical waste. Presently, medical experts have confirmed that adipose tissues contain large amounts of mesenchymal stem cells that exhibit in vitro proliferation and multiple differentiation ability, characteristics that facilitate the repair and regeneration of damaged tissues or organs [4]. In addition, adipose-derived stem cells (ADSCs) possess several characteristics: (1) they can be easily harvested in abundant quantities without invasiveness, (2) they can proliferate in in vitro cultures, and (3) they can be applied to a wide range of body tissue types because these cells migrate to lesion sites automatically to repair damage. Studies have shown that ADSCs can differentiate into many different cell types, such as adipose, bone, cartilage, smooth muscle, cardiac muscle, endothelial, blood, liver, and even neuronal cells [5]. Because of these characteristics, ADSCs will likely be one of the major sources of autologous stem cells in the future.

The effectiveness of laser therapy on biological bodies has been confirmed, and laser therapy has been successfully applied in new technological applications such as microsurgery. Lasers can be classified as high or low power, depending on the energy levels used. High-power lasers involve using high energy levels to provoke blood coagulation, stop bleeding, cut tissues, and even damage cells, whereas low-level lasers involve using electromagnetic or photochemical processes to achieve therapeutic effects [6]. A low-level laser is defined as a laser with extremely low power and energy too low to destroy the molecular bonding capacity (e.g., hydrogen bonds and van der Waals forces) of tissues. Therefore, low-level lasers do not cause molecular structural change, protein denaturation, or cell death. Irradiation from low-level lasers on tissue does not cause an obvious temperature increase at the treatment site (less than 0.1 to 0.5°C); thus, any physiological responses of the tissues are produced by the stimulation of the laser itself, and this mechanism is described by the theory of laser biostimulation [7]. In addition, by using an appropriate energy level, low-level lasers are primarily used to stimulate biological cells to induce or strengthen physiological responses for facilitating local blood circulation, regulating cell functions, promoting immunological functions, and facilitating cell metabolism and proliferation. Using lasers to generate these physiological changes enables treatment goals such as anti-inflammation and wound-healing promotion to be met [89]. Many studies have shown that low-level laser irradiation exerts beneficial biological effects on bone, neuronal, and skin healing [1012]. However, the type, wavelength, power, and energy level of lasers used in previous studies have varied, and various effects have been observed in different cells when differing levels of laser energy were applied. Previous studies have shown that using a low-level laser with an 820–830 nm wavelength can reduce neural damage, facilitate neuronal healing, and accelerate neural recovery after an osteotomy [1314]. Using a low-level laser with a 660 nm wavelength has been demonstrated to exert healing effects on musculoskeletal injuries and inflammation [15]. In addition, many studies have indicated that a low-level laser with a 660 nm wavelength can effectively promote neural regeneration and accelerate the reinnervation of muscle fibers to promote the recovery of motor functions [1618]. Recent evidence has suggested that protein aggregates such as ?-amyloid- (A?) associated neurotoxicity and dendrite atrophy might be a consequence of brain-derived neurotrophic-factor (BDNF) deficiency. Meng et al. observed that the upregulation of BDNF caused by using low-level laser therapy (LLLT) to activate the extracellular signal-regulated kinase (ERK)/cAMP response element-binding protein (CREB) pathway can ameliorate A?-induced neuron loss and dendritic atrophy, thus identifying a novel pathway through which LLLT protects against A?-induced neurotoxicity [19].

Currently, most low-level laser therapies in practical clinical applications emphasize treatment courses covering an extensive tissue area in a relatively short period. Multichannel-laser hair treatment, which is currently available for the physical treatment of alopecia, is one such example. Therefore, we used a large-area LLLT that differs from the irradiation methods previously reported in the literature (such as single-spot low-energy laser exposure or the scanning method) to increase the local area of exposure [18]. In this study, stem cells were extracted from adipose tissues, and neural-stem-cell- (NSC-) differentiating agents were used to culture these ADSCs to transform them into induced adipose-derived stem cells (i-ADSCs), which were used as experimental cells. First, we investigated the effects of large-area irradiation from a low-level laser on the proliferation and differentiation of i-ADSCs into neuronal cells. We then investigated whether i-ADSCs treated with laser irradiation and injected via an intravenous route could integrate and survive in various locations in rat brains. We investigated if this treatment could improve the neurological dysfunctions caused by ischemic brain damage in rats and if rats could produce BDNF, using this treatment. Studies using intravenous injection to transplant i-ADSC for the treatment of ischemic stroke with protocols similar to that used in the current study are rare. In this study, we further combined the biostimulation theory from large-area LLLT with transplantation of i-ADSCs to induce neural differentiation to investigate the effectiveness of ischemic stroke treatment. In a previous study [20], we established a transient ischemia-reperfusion stroke rodent model by using right-sided middle cerebral artery occlusion (MCAO) to simulate acute clinical insults. The effectiveness of the treatment was assessed by comparing HE-staining and western blot analysis, as well as the evaluation of motor skill indices by using the rotarod and grip-strength tests. Our protocol has the potential to be developed for application in the clinical treatment of patients with ischemic stroke.

2. Materials and Methods

2.1. Isolation and Culture of ADSCs

A flowchart illustrating the experimental design of the study is shown in Figure 1. Eight-week-old male Sprague-Dawley (SD) rats were used for isolating rat ADSCs. The ADSCs were harvested from the rats’ subcutaneous anterior abdominal wall. Inguinal fat pads were excised, washed sequentially in serial dilutions of betadine, and finely minced as tissues in phosphate-buffered saline (PBS). The tissues were digested with 0.3% of Type I collagenase (Sigma) at 37°C for 60 min. The digested tissue/cell suspension was filtered through a 100-mesh filter to remove the debris, and the filtrate was centrifuged at 1000 rpm for 10 min. The cellular pellet was resuspended using DMEM/F12 (10% FBS, 1% P/S) and then cultured for 24 h at 37°C in 5% CO2. Unattached cells and debris were then removed and the adherent cells were cultured using fresh medium. The cells were cultured to 80% confluency before being released with 0.05% trypsin and then subcultured.

Figure 1

A flowchart illustrating the experimental design. Detailed procedures are described in Section 2.

2.2. ADSC Neuronal Predifferentiation

In this study, i-ADSCs obtained from culturing ADSCs by adding NSC-differentiating agents were used as experimental cells. ADSCs within 3–5 passages were detached and induced using NSC media supplementation. ADSCs were resuspended in a serum-free DMEM/F12 medium supplemented with an N2 supplement (Sigma), 20 ng/mL of epidermal growth factor (Gibco, NY, USA), and fibroblast growth factor (Gibco, NY, USA).

2.3. Setup of a Low-Level Laser Application Method

The probe of the laser irradiation device was fixed vertically on a clean, open experimental bench. The distance between the probe and the cell culture dish was 30 cm. Laser irradiation was applied in a 25°C environment by using an AlGaInP-diode laser (Konftec Co., Taipei, Taiwan) with a wavelength of 660 nm at an output power of 50 mW and frequency of 50 Hz. In the control group, the cells that did not receive laser irradiation treatment, i-ADSCs (LS) (n = 10), were compared with the experimental cells, i-ADSCs (LS+) (n = 10), which were subjected to a laser irradiation treatment of 10 min. The cells in the i-ADSCs (LS) group were cultured for 7 days, whereas the cells in the i-ADSCs (LS+) group were treated using low-level lasers on the following day for 10 min and then cultured for 6 days. The cells receiving laser irradiation were collected at various times for analysis according to the purposes of the experimental protocols. After the completion of the cultures, an optical microscope was used for observing cell morphology.

2.4. MTT Assay

The principle of the MTT (3-[4,5-dimethylthiazol-2-y1]-2,4-diphenyltetrazolium bromide) assay is that the mitochondria of living cells can transform the yellow chemical substance MTT tetrazolium into the purple non-water-soluble substance MTT formazan through the effect of succinate dehydrogenase. DMSO can be used to dissolve the purple-colored products. No such response occurs in dead cells. An optical absorbance of 570 nm was measured using an enzyme immunoassay analyzer. A higher absorbance value indicates a larger amount of cells. In this study, ADSCs neuronal predifferentiation was first distributed in a 96-well plate with approximately 10000 cells per well. The cells in the i-ADSCs (LS) group were then cultured in a 37°C environment and a 5% CO2 environment for 5 days and 7 days, respectively. The cells in the i-ADSCs (LS+) group were cultured in a 37°C environment and a 5% CO2 environment for 4 days and 6 days, respectively. After the completion of the cultures, the medium was removed by several rinses with PBS. An MTT solution of 100 ?L was added to each well of a 96-well plate in the dark (1 mL of MTT reagent was added to 9 mL of phenol-red-free, serum-free medium) and incubated in a 37°C, 5% CO2 environment for 2 h. The MTT solution was then removed and the cells were dissolved using DMSO. An optical absorbance of 570 nm was measured using an enzyme immunoassay analyzer to compare the values between the different groups.

2.5. Immunocytochemistry of i-ADSCs

After 7 days in culture, the subcultured neurospheres were washed using 0.1 M PBS 3 times and fixed with 4% paraformaldehyde for 1 h. Following the fixation, the cells were permeated with 0.1% of Triton X-100 for 10 min and then blocked with 5% nonfat milk for 30 min. The phenotypic expression of these neurospheres was examined by implementing immunocytochemical staining accompanied by antibodies against glial fibrillary acidic proteins (GFAPs) for astrocytes, mouse monoclonal antinestins for NSCs, and doublecortin (DCX), which has recently been used as a marker for neurogenesis. Briefly, the fixed cells were washed 3 times in cold PBS. After washing with PBS, the aforementioned primary antibodies were added and the slides were maintained at room temperature overnight. In the following day, the primary antibodies were removed by washing 3 times with PBS and the secondary antibodies were added before incubating the cells for 1 h. After washing off the secondary antibodies, the cells were incubated with tertiary antibodies tagged with peroxidase-antiperoxidase for 1 h. The tertiary antibodies were washed off using PBS. The cells were incubated with DAPI (Sigma, St. Louis, MO, USA) diluted with the cell culture medium for 10 min. Finally, the cells were mounted with 90% glycerol and examined using fluorescent microscopy (Olympus IX-71, Inc., Trenton, NJ, USA).

2.6. Animals and Induction of the MCAO Model

In a previous study [20], we established a transient ischemia-reperfusion stroke rodent model, using right-sided middle cerebral artery occlusion (MCAO) to simulate acute clinical insults. All of the experimental procedures were approved by the Institutional Animal Care and Use Committee of Taichung Veteran General Hospital, Taiwan. Thirty-two adult male SD rats were randomly allocated to 3 groups: the i-ADSCs (LS) therapy group (n = 12), the i-ADSCs (LS+) therapy group (n = 12), and a sham group (n = 8). The rats in all 3 groups were euthanized on the 28th day after MCAO was performed. For MCAO procedures, anesthesia was induced using 4% isoflurane (Baxter, USA) and maintained using 2% isoflurane. A midline cervical incision was made to isolate the right bilateral common carotid artery. A 25 mm-long 3-0 nylon surgical thread was then inserted into the right carotid bifurcation. In this study, 2 rounds were used to provide a more complete blockage of blood flow in the artery. When the blunted distal end met resistance, the proximal end of the thread was tightened at the carotid bifurcation. The right common, internal, and external carotid arteries were carefully separated from the adjacent vagal nerve, and the distal portions of the external and common carotid arteries were ligated. A small incision was subsequently made at the proximal portion of the external carotid artery, and a 3-0 nylon monofilament suture was gently inserted (approximately 18 mm) into the internal carotid artery. After 60 min of MCAO, the nylon surgical thread was removed to allow complete reperfusion of the ischemic area. During ischemia, rectal temperature was monitored and maintained at approximately 37°C by using a heating pad and an overhead lamp. The anesthetized rats intravenously received i-ADSCs at a concentration of 2 × 107 mL 1 via their femoral veins. The rats in the sham group underwent the same surgical procedures except that the right-sided middle cerebral artery was not occluded.

2.7. Rotarod Test

An accelerating rotarod test was performed for each rat before and on the 7th, 14th, 21st, and 28th day after cerebral ischemia-reperfusion was induced. Before the ischemia-reperfusion experiment was conducted, the animals were subjected to 3 training sessions per day for 3 days on the accelerating rotarod to obtain stable duration on the rotarod spindle. The diameter of the rotarod spindle was 7 cm. The surface of the rotarod spindle was made of knurled Perspex to provide an adequate grip, which prevented animals from slipping off the spindle. The speed of the spindle was increased from 4 to 40 rpm over a period of 5 min and the duration that the animal stayed on the device was recorded. The rats that were capable of staying on the rotarod longer than 150 s after 3 training sessions were selected for the experiments. On the testing days, the animals were tested twice, and the longest durations on the rotarod were recorded.

2.8. Grip Strength Test

Each rat was supported in a horizontal position approximately parallel to a grip bar (Model DPS-5R: range 0–5 kgf, Japan). The researcher set the rat’s forepaws on the grip bar and pulled the animal horizontally away from the bar by the base of its tail until the rat released its grip. The pulling motion was smooth and continuous. The researcher supported the rat by the abdomen when the grip was released. The reading on the strain gauge remained constant at the point of maximal value, which was recorded as the measure of forepaw grip strength. The researcher supported the rat body by both the chest and the base of the tail at an angle of ?45° down the tail. The rat was facing away from the grip bar. The rat was encouraged to grasp the bar by moving its hind paw to the bar. When the rat grasped the bar with both hind paws, establishing a “full” grip, the upper body of the rat was lowered so that the rat was in a nearly horizontal position. The rat was pulled horizontally by the base of the tail until it released its grip and was supported as previously described. The reading on the strain gauge remained constant at the point of maximal value (force was measured in grams), which was recorded as the measurement of forepaw grip strength. Three values were obtained in succession, and the median value was used as the daily score. The data were expressed as the percentage of the baseline (preischemic) value.

2.9. Hematoxylin-Eosin Staining of the Cerebellum

The SD rats were anesthetized using 10% chloral hydrate (4 ?L/kg), administered intraperitoneally, and were euthanized on the 28th day after the MCAO operation and sham treatment. For each rat, the left cerebellum was rapidly removed and postfixed in formalin for 24 h. The postfixed tissues were embedded in paraffin wax and 6-?m-thick serial coronal sections were obtained and mounted on poly-L-lysine-coated glass slices. To assess the histological changes in the MCAO and sham groups, the paraffin-embedded left cerebellum sections were stained using hematoxylin-eosin (HE), according to standard protocol before the assay was performed.

2.10. Western Blot Analysis

Proteins were extracted from the rat brains by using a cold lysis buffer (10 mM of tetra sodium pyrophosphate, 20 mM of Hepes, 1% Triton X-100, 100 mM NaCl, 2 ?g/mL of aprotinin, 2 ?g/mL of leupeptin, and 100 ?g/mL of phenylmethylsulfonyl fluoride). The protein concentrations from tissue extracts or ADSC-conditioned medium were determined using the Bradford protein assay. Equal amounts of protein were placed in a 2× sample buffer (0.125 M Tris-HCl, pH 6.8, 2% glycerol, 0.2 mg/mL of bromophenol blue dye, 2% SDS, and 10% ?-mercaptoethanol) and electrophoresed through 10% SDS-polyacrylamide gel. The proteins were then transferred onto a nitrocellulose membrane by using electroblotting. The membranes were blocked for 1 h at room temperature in a Tris-buffered saline with Tween-20 (TBST) and 5% nonfat milk. The primary antibodies (1 : 1000) with appropriate dilutions were incubated for 1 h at room temperature in TBST and 5% nonfat milk. The blots were then washed and incubated with a peroxidase-conjugated secondary antibody (1 : 2000) for 1 h in TBST. The chemiluminescent substrate for the secondary antibody was developed using the ECL detection system (Amersham, UK). The blots were exposed to film for 3–5 min and then developed.

2.11. Statistical Analysis

The data were expressed as the mean value ± standard error of the mean. The statistical significance of the differences between the groups was determined using a one-way analysis of variance followed by Tukey’s test. An alpha level of less than 0.05 (P < 0.05) was considered statistically significant.

3. Results

3.1. Effects of the Low-Level Laser on Cell Morphology

The ADSCs were passaged 3–5 times after the initial plating of the primary culture. Rat ADSCs appeared to be a monolayer of large and flat cells (Figure 2(a)). Many cells in the i-ADSCs (LS?) and i-ADSCs (LS+) groups induced a neuronal phenotype and exhibited, among one another, bipolar and multipolar elongations of neuronally induced cell-forming networks. The results show that the stem cells in both i-ADSCs (LS?) and i-ADSCs (LS+) groups developed tentacles, indicating that ADSCs were facilitating the induction of differentiation into neuronal cells. Comparative optical micrographs revealed that some attached cells exhibited a spread-out shape with a spindle-like and fibroblastic phenotype in the i-ADSCs (LS?) group (Figure 2(b)). However, most of i-ADSCs-expressing neurites extended radially, connecting like bridges with those from adjacent cells in the i-ADSCs (LS+) group (Figure 2(c)).

Figure 2

The morphology of inductions of adipose-derived stem cell (i-ADSC) differentiation into neuronal cells after low-level laser irradiation. (a) Undifferentiated ADSCs; (b) i-ADSCs (LS?); (c) i-ADSCs (LS+). The arrow denotes the neuronal-like cells. 

3.2. Effects of the Low-Level Laser on Cell Proliferation and Differentiation

In this study, MTT assays were performed on Day 5 and Day 7 to evaluate the effects of large-area low-level laser irradiation on the facilitation of cell proliferation. After analyzing the optical absorbance values, the results showed that, on Day 5, cell activity was slightly higher in the i-ADSCs (LS+) group compared with that in the i-ADSCs (LS) group. However, the difference was not statistically significant (P > 0.05). On Day 7, the cell amounts in both the i-ADSCs (LS+) and i-ADSCs (LS?) groups were larger than the amounts on Day 5. However, the cell proliferation rates were similar on Day 5 without major differences (P > 0.05) (Figure 3).

Figure 3

The cell activity of inductions of adipose-derived stem cell (i-ADSC) differentiation into neuronal cells in both the i-ADSCs (LS) and i-ADSCs (LS+) groups on Days 5 and 7 after culture.

In this study, immunofluorescent staining and western blots were used to evaluate the effects of large-area low-level laser irradiation on the facilitation of cell differentiation. Immunofluorescent staining was performed for the NSC marker, nestin, glial cell marker, GFAP antibody, and neuronal precursor-cell-marker protein, DCX. After the staining was completed, fluorescent microscopy was used to observe the amount of fluorescence expression of each antibody. The results showed that the fluorescence expression of the nestin was higher in the cells in the i-ADSCs (LS+) group than that in the cells in the i-ADSCs (LS?) group. These results indicated that ADSC differentiation into neuronal cells was facilitated after large-area low-level laser irradiation (Figure 4(a)). For GFAP, no difference was observed in the amount of fluorescence expression of GFAP between the i-ADSCs (LS?) and i-ADSCs (LS+) groups (Figure 4(b)). Furthermore, for DCX, no difference was observed between the 2 groups because the cells in both groups still exhibited stem cell morphology (Figure 4(c)).

Figure 4

The immunofluorescent staining for (a) nestin; (b) GFAP; and (c) DCX of inductions of adipose-derived stem cell (i-ADSC) differentiation into neuronal cells in both the i-ADSCs (LS?) (left) and i-ADSCs (LS+) (right) groups. The scale bar represents 

Western blot is used to quantify the expression of marker proteins. Therefore, western blot analysis was used to compare the amount of nestin expression between the i-ADSCs (LS?) and i-ADSCs (LS+) groups in this study. The results showed that cells in the i-ADSCs (LS+) group exhibited a substantially higher nestin expression compared with the cells in the i-ADSCs (LS?) group (P < 0.05) (Figure 5). Regarding the results of GFAP and DCX, no difference was observed in the fluorescence expressions of the i-ADSCs (LS?) and i-ADSCs (LS+) groups. Therefore, western blot analyses were not shown for GFAP and DCX. This result is consistent with the findings obtained using immunofluorescent staining.

Figure 5

The cell growth of inductions of adipose-derived stem cell (i-ADSC) differentiation into neuronal cells with or without laser irradiation. (a) The amount of nestin expression. (b) The figure showing the quantification. GAPDH served as the internal reference.

3.3. Evaluation of Behavior Recovery after Stroke in the Animals

In this study, treadmill and forepaw-grip tests were used to evaluate motor function recovery after stem-cell transplantation treatment in rats with ischemic stroke. The treadmill test was performed on Day 7 after stem-cell transplantation was performed on the rats with stroke. The rats from either the i-ADSCs (LS+) group or the i-ADSCs (LS) group were unable to run as quickly as the rats in the sham group. From Day 14, the rats in the i-ADSCs (LS+) group gradually recovered the ability to run. By contrast, it was observed that the rats in the i-ADSCs (LS?) group recovered slightly; however, the degree of recovery was lower than that in the i-ADSCs (LS+) group. On Day 21 after stem-cell transplantation, the recovery of running function was still more satisfactory in the i-ADSCs (LS+) group than in the i-ADSCs (LS) group. On Day 28, the motor function of the rats in the i-ADSCs (LS+) group was approaching the level of the rats in the sham group, whereas the performance of the rats in the i-ADSCs (LS?) group was still considerably weaker than that of the sham group (Figure 6).

Figure 6

The treadmill test for evaluating the recovery of the motor function of running in rats with ischemic stroke with i-ADSCs (LS?) and i-ADSCs (LS+) transplantation.

Grip-strength tests were performed on Day 7 after stem-cell transplantation. The findings were similar to the treadmill test results; the grip behavior of the rats in both the i-ADSCs (LS+) group and the i-ADSCs (LS) group was worse than that of the rats in the sham group. On Day 14, the grip strength of the rats in the i-ADSCs (LS+) group was considerably recovered. By contrast, although the rats in the i-ADSCs (LS) group showed some progress, the improvement was minimal. On Day 21, the rats in the i-ADSCs (LS+) group continued to recover, whereas it was observed that the animals in the i-ADSCs (LS?) group were not recovering as quickly. On Day 28, grip strength recovery in the i-ADSCs (LS+) group approached that of the sham group, whereas grip strength in the i-ADSCs (LS?) group was still lower (Figure 7).

Figure 7

The grip test for evaluating the recovery of grip strength in rats with ischemic stroke treated with i-ADSCs (LS) and i-ADSCs (LS+) transplantation.

 

3.4. Repair of Brain Tissues after Treatment in Animals

After euthanizing the rats, the brain specimens were treated with paraffin and then sliced. The HE-immunostaining method was used to observe the repair of brain tissues after stem-cell transplantation was performed. An upright microscope (10x) was used to macroscopically observe the brain tissue, and the results showed that brain tissue was completely repaired in the rats in the i-ADSCs (LS+) group with nearly no necrotic brain tissue. By contrast, obvious necrotic scars were observed at the ischemic sites of the brain tissue from the rats in the i-ADSCs (LS?) group (Figure 8(a)). Observed under a microscope and magnified 200 times, the results showed that the brain tissue from the rats in the i-ADSCs (LS+) group was as dense as normal brain tissues, whereas numerous cavities were observed in the brain tissue from rats in the i-ADSCs (LS?) group (Figure 8(b)).

Figure 8

The observation of brain tissue necrosis in rats with ischemic stroke treated with i-ADSCs (LS) (left) and i-ADSCs (LS+) (right) transplantation: (a) 10x; (b) 200x.

Oligodendrocytes, which are glial cells found in normal brains, form myelin in the central nervous system. Oligodendrocytes substantially decrease after brain cells are damaged, leading to myelin collapse and the loss of neural-signal conduction. Therefore, a western blot was used to analyze the amount of expression of the oligodendrocyte cell protein, oligo-2, to confirm the repair of brain tissues after stem-cell transplantation was performed in rats with stroke. The results showed that the amount of oligo-2 protein response in the brain tissues of the i-ADSCs (LS+) group was as high as that in the sham group. By contrast, the oligo-2 protein response in the i-ADSCs (LS?) group was substantially lower (P < 0.05). These results indicated that stem-cell transplantation treatments can repair brain tissues damaged by ischemia (Figure 9).

Figure 9

The observation of brain tissue repair in rats with ischemic stroke treated with i-ADSCs (LS?) and i-ADSCs (LS+) transplantation. (a) The amount oligo-2 expression. (b) Quantification. GAPDH served as the internal reference. *Significance (P < 

4. Discussion

Stem cells possess the ability to proliferate, regenerate, differentiate, and secrete cytokines. Previous studies have proven that stem-cell therapy substantially improves the damage caused by stroke. Stem cells are derived from various sites. Most previous studies have used bone marrow mesenchymal stem cells; however, we used adipose stem cells in the present study, which are easily accessible, and abundant and exhibit high differentiation and proliferation activity. Moreover, adipose stem cells do not trigger strong immune reactions (resulting in low exclusion) and rarely form teratomas. In the literature, it has been demonstrated that adipose stem cells are essential adult stem cells that differentiate into various mesoderm tissues similarly to bone marrow mesenchymal stem cells [2123]. Furthermore, adipose stem cells are more easily accessible than mesenchymal stem cells; therefore, adipose stem cells can be used as a substitute for bone marrow mesenchymal stem cells for repairing damaged tissues in the future. Adipose stem cells are generally more practical than bone marrow mesenchymal stem cells for use in research [2326].

In this study, we used large-area low-level laser irradiation to induce ADSCs to differentiate into neuronal cells. The MTT assay analysis showed that the cell activity of the i-ADSCs increased on Days 5 and 7 of culture after large-area low-level laser irradiation [2728]. However, although cell activity increased on both Days 5 and 7, the activity was also increased for the group that did not receive laser irradiation treatment. No significant difference was observed between the groups with or without laser treatment (P > 0.05). Previous studies have shown that, depending on irradiation parameters, various types of cell respond differently to laser irradiation [29]. Although the mechanism underlying this phenomenon remains obscure, several hypotheses have been proposed to explain the mechanism of laser action [30]. We speculate that the lack of significant effects might be attributable to the short duration of low-level laser irradiation, which might not provide sufficient energy to the ADSCs for them to facilitate proliferation. Alternatively, using low-level lasers on the ADSCs might not sufficiently enhance cell proliferation. Therefore, future studies should investigate the appropriate duration of low-level laser irradiation and determine how much energy is required to accelerate cell proliferation [31,32].

To understand if using large-area low-level lasers exerted a positive effect on cell differentiation, we used immunofluorescent staining and western blot analysis to evaluate whether these lasers were capable of accelerating the induction of cell differentiation [33]. The immunofluorescent staining results showed that the nestin level in the group with i-ADSCs treated using large-area low-level laser irradiation increased substantially compared with that of the group that did not receive laser treatment. This result indicated that large-area low-level laser can accelerate the differentiation of ADSCs into neuronal cells [3435]. LLLT has been demonstrated to regulate neuronal function both in vitro and in vivo. Previous studies have reported that laser treatments accelerated nerve cell sprouting and cell migration, which begin within 24 h of seeding. During the first week of cultivation, irradiated cultures contain a high number of neurons exhibiting large perikaria and branched neuronal fibers, which interconnect to form networks [36]. The possible mechanism of LLLT at the cellular level has been attributed to the acceleration of electron transfer reactions, resulting in the increase of reactive oxygen species and Ca2+as versatile second messengers [37]. Previous studies have shown that applying LLLT could influence cellular processes by altering DNA synthesis and protein expression [38], biomodulating cytoskeletal organization [39], and stimulating cellular proliferation [38]. Such properties suggest that LLLT, or interventions with similar neurobiological effects, can be used to treat neurodegeneration, a phenomenon that underlies debilitating clinical conditions.

However, no obvious differences were observed for GFAP and DCX in the test results. This can be explained by the differentiation agent used in this study, which exerts its effects primarily by inducing ADSCs to differentiate into NSCs. Therefore, although the ADSCs were treated using a low-level laser, they still did not differentiate into neural glia cells. The DCX protein can only be discovered after neurons have been formed from NSCs, which demonstrates that DCX is a late-stage protein that cannot be expressed when cells still exhibit the morphology of NSCs. Therefore, the amount of DCX expression is not affected by low-level laser exposure. Western blot analysis was used to determine the amount of nestin expression after i-ADSCs were exposed to large-area low-level laser irradiation and culture for 7 days. The results showed that the amount of nestin was higher with laser treatment. This is similar to the findings from the immunofluorescent-staining method, indicating that laser irradiation can accelerate the differentiation of ADSCs into neuronal cells. These results indicated that ADSCs can be induced to differentiate into neuronal cells after treatment by large-area laser irradiation for 10 min. Future studies should establish the precise duration of large-area LLLT required to achieve improved results.

In this study, treadmill and forepaws grip tests were used to evaluate motor function recovery after stem cell transplantation treatments in rats with stroke. The treadmill test results showed that the running function was weaker for the rats in the sham group on Day 7 after treatment. We speculate that this might have been because the brain-tissue lesion area from ischemia was too large; therefore, the transplanted stem cells did not have sufficient time to noticeably repair brain tissue. Therefore, the motor functions of the rats remained impaired on Day 7. When tested on Day 14, recoveries in motor functions were observed in both the i-ADSCs (LS?) and i-ADSCs (LS+) groups, with superior recovery in the i-ADSCs (LS+) group, indicating that the damaged brain tissue was repaired. On Day 21, the recovery of the rats’ running function was more satisfactory in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group, indicating that the brain tissue repair capability was superior to that in the i-ADSCs (LS+) group. When tested on Day 28, the running function of the rats in the i-ADSCs (LS+) group was close to that of the rats in the sham group. However, the motor function of the i-ADSCs (LS?) group remained impaired, indicating that hind-paw motor function recovery was accelerated after i-ADSCs (LS+) treatment.

The grip test results were similar to the treadmill test results. On Day 7 after stem-cell transplantation treatment of the rats with stroke, grip strength was low for both the i-ADSCs (LS+) group and i-ADSCs (LS?) groups because the damaged brain tissues were just about to be repaired; therefore, the forepaws of the rats remained weak. When tested on Day 14, no major improvement was observed in the grip strength of the rats in the i-ADSCs (LS?) group. By contrast, the grip strength improved considerably in the i-ADSCs (LS+) group, indicating that the brain tissue repair capability was more satisfactory in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group. On Days 21 and 28, the tests showed that grip strength recovery was more satisfactory in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group. On Day 28, the grip strength in the i-ADSCs (LS+) group recovered to a level close to that of the sham group, indicating that, after i-ADSCs (LS+) treatment, the damaged brain tissues of the rats with stroke were repaired quickly, enabling the recovery of forepaw-grip strength. Based on these results, we concluded that damaged brain tissues can be repaired faster and motor function can be recovered efficiently in rats with stroke after i-ADSCs (LS+) treatment.

The i-ADSCs could differentiate into neuronal cells after transplantation into the brain. As a result, they moved and repaired damaged cerebral tissue selectively and improved cerebral functions by enhancing angiogenesis, renewal of neurons, and proliferation of nerve cells [4041]. In this study, we used the HE-immunostaining method and western blot analysis to evaluate brain tissue repair on Day 28 after stem cell transplantation treatment was performed in rats with stroke. The results of immunostaining were observed using a microscope. The structures were magnified 200 times, which showed that the brain tissue in the stroke lesion was dense and similar to that of normal brain tissue in the i-ADSCs (LS+) group. By contrast, numerous cavities were observed in the ischemic lesions of the brain tissues of the rats in the i-ADSCs (LS?) group. These results indicated that necrotizing brain tissue after ischemia was quickly repaired when i-ADSCs (LS+) was used to treat the rats with stroke. We speculate that using i-ADSCs (LS+) treatment can accelerate the induction of ADSCs to differentiate into neuronal cells. Therefore, although the same number of stem cells was transplanted for treatment, a greater number of NSCs were observed in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group. Furthermore, because NSCs can protect damaged brain tissues from continuous deterioration, they can also help brain tissues to repair. Therefore, transplanting a greater amount of NSCs would likely assist in repairing brain tissues more effectively.

Regarding the results of the western blot analysis, the oligo-2 amounts after stem-cell transplantation treatment were analyzed. Because oligodendrocytes form myelin in the central nervous system, myelin levels collapse when oligodendrocytes die. Nerve conduction is delayed or interrupted after the death of oligodendrocytes, leading to limb disabilities. Therefore, the evaluation of oligo-2 could be a crucial reference in assessing the degree of brain tissue recovery after stem-cell transplantation treatments in rats with stroke. In this study, using western blot analysis showed that the amount of expression of oligo-2 in brain tissues treated with i-ADSCs (LS+) was similar to that in normal brain tissues. By contrast, brain tissues treated with i-ADSCs (LS?) exhibited a lower oligo-2 expression, indicating that using i-ADSCs (LS+) treatment in rats with stroke can repair myelin in the central nervous system, leading to the recovery of neural-signal conduction and motor function. Based on these experimental results, we concluded that using i-ADSCs (LS+) treatment in rats with stroke cannot only accelerate the repair of damaged brain tissues for the partial recovery of motor functions, but also enable the central nervous system to recover the velocity of neural-signal conduction. These results confirm that the transplantation of i-ADSCs (LS+) can accelerate repairs in rats with ischemic stroke because i-ADSCs (LS+) can more efficiently differentiate into NSCs.

5. Conclusion

In this study, we used i-ADSCs treated with large-area low-level laser irradiation to evaluate the effects of a low-level laser on cell proliferation and differentiation. The results showed that although a low-level laser cannot facilitate cell proliferation, it can accelerate the induction of ADSCs differentiating into NSCs. In this study, we successfully created large-area cell and tissue damage in rat brains by using an embolic stroke animal model. Stem-cell transplantation with either i-ADSCs (LS+) or i-ADSCs (LS?) was performed to evaluate the degree of repair after stroke in the animals. Because large-area low-level laser irradiation can accelerate the differentiation of ADSCs into NSCs, and NSCs can protect damaged brain tissues to prevent continuous deterioration from damage and to help with repair, the motor function recovery was thus superior in the rats treated using i-ADSCs (LS+) compared with that in the rats treated using i-ADSCs (LS?). From the brain tissue slices from each group of rats, we discovered that i-ADSCs (LS+) treatment more effectively repaired necrotizing brain tissues after ischemia in rat brains. Furthermore, the western blot analysis also showed that the amount of oligo-2 increased in i-ADSCs (LS+)-treated rats with stroke, confirming the repair of myelin in cerebral neurons to further assist in the recovery of neural-signal conduction in the central nervous system.

Therefore, in the present study we demonstrated that using large-area low-level lasers exerts positive effects on inducing ADSCs differentiation, and it effectively treated ischemic stroke in rats, regarding motor function recovery. In future studies, the effects of large-area low-level laser irradiation time and the appropriate dosage for the proliferation and differentiation of ADSCs should be evaluated. If the optimal irradiation time and dosage for ADSC proliferation and differentiation can be discovered in animal experiments similar to those in this study, we believe that superior experimental results can be obtained. Furthermore, if primate or canine experimental animals can be used to conduct the experimental protocols described herein, the concerns associated with individual animal differences and errors associated with motor function assessments can be minimized to obtain more reliable experimental data. Therefore, the findings of this study contribute to the development of cell therapy, which can benefit patients with stroke.

Conflict of Interests

There is no conflict of interests.

Acknowledgments

This work was supported by grants from the Taichung Veterans General Hospital and Central Taiwan University of Science and Technology (Grant. TCVGH-CTUST 1027701) and the National Science Council (Grant. NSC 102-2314-B-075A-019-MY2), Taiwan.

References
1. Chalela JA, Kidwell CS, Nentwich LM, et al. Magnetic resonance imaging and computed tomography in emergency assessment of patients with suspected acute stroke: a prospective comparison. The Lancet.2007;369(9558):293–298. [PMC free article] [PubMed]
2. Hackam DG, Spence JD. Combining multiple approaches for the secondary prevention of vascular events after stroke: a quantitative modeling study. Stroke. 2007;38(6):1881–1885. [PubMed]
3. Shen C-C, Lin C-H, Yang Y-C, Chiao M-T, Cheng W-Y, Ko J-L. Intravenous implanted neural stem cells migrate to injury site, reduce infarct volume, and improve behavior after cerebral ischemia.Current Neurovascular Research. 2010;7(3):167–179. [PubMed]
4. Yang Y-C, Liu B-S, Shen C-C, Lin C-H, Chiao M-T, Cheng H-C. Transplantation of adipose tissue-derived stem cells for treatment of focal cerebral ischemia. Current Neurovascular Research.2011;8(1):1–13. [PubMed]
5. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells.Molecular Biology of the Cell. 2002;13(12):4279–4295. [PMC free article] [PubMed]
6. Karu TI, Pyatibrat LV, Kalendo GS. Esenaliev RO. Effects of monochromatic low-intensity light and laser irradiation on adhesion of cells in vitro. Lasers in Surgery and Medicine. 1996;18:171–177.[PubMed]
7. Gaida K, Koller R, Isler C, et al. Low level laser therapy—a conservative approach to the burn scar?Burns. 2004;30(4):362–367. [PubMed]
8. Karu T. Photobiology of low-power laser effects. Health Physics. 1989;56(5):691–704. [PubMed]
9. Mester E, Mester AF, Mester A. The biomedical effects of laser application. Lasers in Surgery and Medicine. 1985;5(1):31–39. [PubMed]
10. Conlan MJ, Rapley JW, Cobb CM. Biostimulation of wound healing by low-energy laser irradiation. A review. Journal of Clinical Periodontology. 1996;3:492–496. [PubMed]
11. Yaakobi T, Maltz L, Oron U. Promotion of bone repair in the cortical bone of the tibia in rats by low energy laser (He-Ne) irradiation. Calcified Tissue International. 1996;59(4):297–300. [PubMed]
12. Ozen T, Orhan K, Gorur I, Ozturk A. Efficacy of low level laser therapy on neurosensory recovery after injury to the inferior alveolar nerve. Head & Face Medicine. 2006;2, article 3 [PMC free article][PubMed]
13. Miloro M, Halkias LE, Mallery S, Travers S, Rashid RG. Low-level laser effect on neural regeneration in Gore-Tex tubes. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics.2002;93(1):27–34. [PubMed]
14. Khullar SM, Emami B, Westermark A, Haanæs HR. Effect of low-level laser treatment on neurosensory deficits subsequent to saggittal split ramus osteotomy. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics. 1996;82(2):132–138. [PubMed]
15. Dos Reis FA, Belchior ACG, De Carvalho PDTC, et al. Effect of laser therapy (660 nm) on recovery of the sciatic nerve in rats after injury through neurotmesis followed by epineural anastomosis. Lasers in Medical Science. 2009;24(5):741–747. [PubMed]
16. Belchior ACG, Dos Reis FA, Nicolau RA, Silva IS, Perreira DM, De Carvalho PDTC. Influence of laser (660 nm) on functional recovery of the sciatic nerve in rats following crushing lesion. Lasers in Medical Science. 2009;24(6):893–899. [PubMed]
17. Barbosa RI, Marcolino AM, De Jesus Guirro RR, Mazzer N, Barbieri CH, De Cássia Registro Fonseca M. Comparative effects of wavelengths of low-power laser in regeneration of sciatic nerve in rats following crushing lesion. Lasers in Medical Science. 2010;25(3):423–430. [PubMed]
18. Gigo-Benato D, Russo TL, Tanaka EH, Assis L, Salvini TF, Parizotto NA. Effects of 660 and 780 nm low-level laser therapy on neuromuscular recovery after crush injury in rat sciatic nerve. Lasers in Surgery and Medicine. 2010;42(9):673–682. [PubMed]
19. Meng C, He Z, Xing D. Low-level laser therapy rescues dendrite atrophy via upregulating BDNF expression: implications for Alzheimer’s disease. The Journal of Neuroscience. 2013;33:13505–13517.[PubMed]
20. Shen C-C, Yang Y-C, Chiao M-T, Cheng W-Y, Tsuei Y-S, Ko J-L. Characterization of endogenous neural progenitor cells after experimental ischemic stroke. Current Neurovascular Research.2010;7(1):6–14. [PubMed]
21. Banas A, Teratani T, Yamamoto Y, et al. IFATS collection: in vivo therapeutic potential of human adipose tissue mesenchymal stem cells after transplantation into mice with liver injury. Stem Cells.2008;26(10):2705–2712. [PubMed]
22. Sanchez PL, Sanz-Ruiz R, Fernandez-Santos ME, Fernandez-Aviles F. Cultured and freshly isolated adipose tissue-derived cells: fat years for cardiac stem cell therapy. European Heart Journal.2010;31(4):394–397. [PubMed]
23. Taha MF, Hedayati V. Isolation, identification and multipotential differentiation of mouse adipose tissue-derived stem cells. Tissue and Cell. 2010;42(4):211–216. [PubMed]
24. Fraser JK, Zhu M, Wulur I, Alfonso Z. Adipose-derived stem cells. Methods in Molecular Biology.2008;449:59–67. [PubMed]
25. Ishikawa T, Banas A, Hagiwara K, Iwaguro H, Ochiya T. Stem cells for hepatic regeneration: the role of adipose tissue derived mesenchymal stem cells. Current Stem Cell Research and Therapy.2010;5(2):182–189. [PubMed]
26. Yukawa H, Noguchi H, Oishi K, et al. Cell transplantation of adipose tissue-derived stem cells in combination with heparin attenuated acute liver failure in mice. Cell Transplantation. 2009;18(5-6):611–618. [PubMed]
27. Wu JY, Chen CH, Yeh LY, Yeh ML, Ting CC, Wang YH. Low-power laser irradiation promotes the proliferation and osteogenic differentiation of human periodontal ligament cells via cyclic adenosine monophosphate. International Journal of Oral Science. 2013;5(2):85–91. [PMC free article] [PubMed]
28. Ang FY, Fukuzaki Y, Yamanoha B, Kogure S. Immunocytochemical studies on the effect of 405-nm low-power laser irradiation on human-derived A-172 glioblastoma cells. Lasers in Medical Science.2012;27(5):935–942. [PubMed]
29. Peplow PV, Chung T-Y, Baxter GD. Laser photobiomodulation of proliferation of cells in culture: a review of human and animal studies. Photomedicine and Laser Surgery. 2010;28(1):p. S3, p. S40.[PubMed]
30. Vladimirov YA, Osipov AN, Klebanov GI. Photobiological principles of therapeutic applications of laser radiation. Biochemistry. 2004;69(1):81–90. [PubMed]
31. Tuby H, Hertzberg E, Maltz L, Oron U. Long-term safety of low-level laser therapy at different power densities and single or multiple applications to the bone marrow in mice. Photomedicine and Laser Surgery. 2013;31(6):269–273. [PubMed]
32. Usumez A, Cengiz B, Oztuzcu S, Demir T, Aras MH, Gutknecht N. Effects of laser irradiation at different wavelengths (660, 810, 980, and 1.064 nm) on mucositis in an animal model of wound healing. Lasers in Medical Science. 2013 [PubMed]
33. Shen CC, Yang YC, Huang TB, Chan SC, Liu BS. Neural regeneration in a novel nerve conduit across a large gap of the transected sciatic nerve in rats with low-level laser phototherapy. Journal of Biomedical Materials Research. 2013;101(10):2763–2777. [PubMed]
34. Baratto L, Calzà L, Capra R, et al. Ultra-low-level laser therapy. Lasers in Medical Science.2011;26(1):103–112. [PubMed]
35. Rochkind S, Geuna S, Shainberg A. Chapter 25: Phototherapy in peripheral nerve injury: effects on muscle preservation and nerve regeneration. International Review of Neurobiology. 2009;87:445–464.[PubMed]
36. Rochklnd S, El-Ani D, Nevo Z, Shahar A. Increase of neuronal sprouting and migration using 780 nm laser phototherapy as procedure for cell therapy. Lasers in Surgery and Medicine. 2009;41(4):277–281. [PubMed]
37. Lan C-CE, Wu S-B, Wu C-S, et al. Induction of primitive pigment cell differentiation by visible light (helium-neon laser): a photoacceptor-specific response not replicable by UVB irradiation. Journal of Molecular Medicine. 2012;90:321–330. [PubMed]
38. Feng J, Zhang Y, Xing D. Low-power laser irradiation (LPLI) promotes VEGF expression and vascular endothelial cell proliferation through the activation of ERK/Sp1 pathway. Cellular Signalling.2012;24(6):1116–1125. [PubMed]
39. Song S, Zhou F, Chen WR. Low-level laser therapy regulates microglial function through Src-mediated signaling pathways: implications for neurodegenerative diseases. Journal of Neuroinflammation. 2012;9, article 219 [PMC free article] [PubMed]
40. Valina C, Pinkernell K, Song Y-H, et al. Intracoronary administration of autologous adipose tissue-derived stem cells improves left ventricular function, perfusion, and remodelling after acute myocardial infarction. European Heart Journal. 2007;28(21):2667–2677. [PubMed]
41. DU H-W, Liu N, Wang J-H, Zhang Y-X, Chen R-H, Xiao Y-C. The effects of adipose-derived stem cell transplantation on the expression of IL-10 and TNF-alpha after cerebral ischaemia in rats. Chinese Journal of Cellular and Molecular Immunology. 2009;25(11):998–1001. [PubMed]

J Neuroinflammation.  2012 Sep 18;9(1):219. [Epub ahead of print]

Low-level laser therapy regulates microglial function through Src-mediated signaling pathways: implications for neurodegenerative diseases.

Song S, Zhou F, Chen WR, Xing D.

Abstract

ABSTRACT:

BACKGROUND:

Activated microglial cells are an important pathological component in brains of patients with neurodegenerative diseases. The purpose of this study was to investigate the effect of He-Ne (632.8 nm, 64.6 mW/cm2) low-level laser therapy (LLLT), a non-damaging physical therapy, on activated microglia, and the subsequent signaling events of LLLT-induced neuroprotective effects and phagocytic responses.

METHODS:

To model microglial activation, we treated the microglial BV2 cells with lipopolysaccharide (LPS). For the LLLT-induced neuroprotective study, neuronal cells with activated microglial cells in a Transwell[trade mark sign] cell-culture system were used. For the phagocytosis study, fluorescence-labeled microspheres were added into the treated microglial cells to confirm the role of LLLT.

RESULTS:

Our results showed that LLLT (20 J/cm2) could attenuate toll-like receptor (TLR)-mediated proinflammatory responses in microglia, characterized by down-regulation of proinflammatory cytokine expression and nitric oxide (NO) production. LLLT-triggered TLR signaling inhibition was achieved by activating tyrosine kinases Src and Syk, which led to MyD88 tyrosine phosphorylation, thus impairing MyD88-dependent proinflammatory signaling cascade. In addition, we found that Src activation could enhance Rac1 activity and F-actin accumulation that typify microglial phagocytic activity. We also found that Src/PI3K/Akt inhibitors prevented LLLT-stimulated Akt (Ser473 and Thr308) phosphorylation and blocked Rac1 activity and actin-based microglial phagocytosis, indicating the activation of Src/PI3K/Akt/Rac1 signaling pathway.

CONCLUSIONS:

The present study underlines the importance of Src in suppressing inflammation and enhancing microglial phagocytic function in activated microglia during LLLT stimulation. We have identified a new and important neuroprotective signaling pathway that consists of regulation of microglial phagocytosis and inflammation under LLLT treatment. Our research may provide a feasible therapeutic approach to control the progression of neurodegenerative diseases.

Front Biosci (Elite Ed).  2012 Jan 1;4:818-23.

Therapeutic effect of near infrared (NIR) light on Parkinson’s disease models.

Quirk BJ, Desmet KD, Henry M, Buchmann E, Wong-Riley M, Eells JT, Whelan HT.

Source

Department of Neurology, Medical College of Wisconsin, 8701 W. Watertown Plank Rd, Milwaukee, WI 53226, USA.

Abstract

Parkinson’s disease (PD) is a neurodegenerative disorder that affects large numbers of people, particularly those of a more advanced age. Mitochondrial dysfunction plays a central role in PD, especially in the electron transport chain. This mitochondrial role allows the use of inhibitors of complex I and IV in PD models, and enhancers of complex IV activity, such as NIR light, to be used as possible therapy. PD models fall into two main categories; cell cultures and animal models. In cell cultures, primary neurons, mutant neuroblastoma cells, and cell cybrids have been studied in conjunction with NIR light. Primary neurons show protection or recovery of function and morphology by NIR light after toxic insult. Neuroblastoma cells, with a gene for mutant alpha-synuclein, show similar results. Cell cybrids, containing mtDNA from PD patients, show restoration of mitochondrial transport and complex I and IV assembly. Animal models include toxin-insulted mice, and alpha-synuclein transgenic mice. Functional recovery of the animals, chemical and histological evidence, and delayed disease progression show the potential of NIR light in treating Parkinson’s disease.

J Neurotrauma. 2012 Jan 20;29(2):401-7. doi: 10.1089/neu.2011.2062. Epub 2012 Jan 4.

Near infrared transcranial laser therapy applied at various modes to mice following traumatic brain injury significantly reduces long-term neurological deficits.

Oron A, Oron U, Streeter J, De Taboada L, Alexandrovich A, Trembovler V, Shohami E.

Source

Department of Zoology, Tel Aviv University, Faculty of Life Sciences, Tel Aviv 69978, Israel. oronu@post.tau.ac.il

Abstract

Near-infrared transcranial laser therapy (TLT) has been found to modulate various biological processes including traumatic brain injury (TBI). Following TBI in mice, in this study we assessed the possibility of various near-infrared TLT modes (pulsed versus continuous) in producing a beneficial effect on the long-term neurobehavioral outcome and brain lesions of these mice. TBI was induced by a weight-drop device, and neurobehavioral function was assessed from 1 h to 56 days post-trauma using the Neurological Severity Score (NSS). The extent of recovery is expressed as the difference in NSS (dNSS), the difference between the initial score and that at any other later time point. An 808-nm Ga-Al-As diode laser was employed transcranially 4, 6, or 8 h post-trauma to illuminate the entire cortex of the brain. Mice were divided into several groups of 6-8 mice: one control group that received a sham treatment and experimental groups that received either TLT continuous wave (CW) or pulsed wave (PW) mode transcranially. MRI was taken prior to sacrifice at 56 days post-injury. From 5-28 days post-TBI, the NSS of the laser-treated mice were significantly lower (p<0.05) than those of the non-laser-treated control mice. The percentage of surviving mice that demonstrated full recovery at 56 days post-CHI (NSS=0, as in intact mice) was the highest (63%) in the group that had received TLT in the PW mode at 100 Hz. In addition, magnetic resonance imaging (MRI) analysis demonstrated significantly smaller infarct lesion volumes in laser-treated mice compared to controls. Our data suggest that non-invasive TLT of mice post-TBI provides a significant long-term functional neurological benefit, and that the pulsed laser mode at 100 Hz is the preferred mode for such treatment.

J Neurotrauma. 2012 Jan 20;29(2):408-17. doi: 10.1089/neu.2010.1745. Epub 2011 Sep 21.

Low-level laser light therapy improves cognitive deficits and inhibits microglial activation after controlled cortical impact in mice.

Khuman J, Zhang J, Park J, Carroll JD, Donahue C, Whalen MJ.

Source

Neuroscience Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA.

Abstract

Low-level laser light therapy (LLLT) exerts beneficial effects on motor and histopathological outcomes after experimental traumatic brain injury (TBI), and coherent near-infrared light has been reported to improve cognitive function in patients with chronic TBI. However, the effects of LLLT on cognitive recovery in experimental TBI are unknown. We hypothesized that LLLT administered after controlled cortical impact (CCI) would improve post-injury Morris water maze (MWM) performance. Low-level laser light (800 nm) was applied directly to the contused parenchyma or transcranially in mice beginning 60-80 min after CCI. Injured mice treated with 60 J/cm² (500 mW/cm²×2 min) either transcranially or via an open craniotomy had modestly improved latency to the hidden platform (p<0.05 for group), and probe trial performance (p<0.01) compared to non-treated controls. The beneficial effects of LLLT in open craniotomy mice were associated with reduced microgliosis at 48 h (21.8±2.3 versus 39.2±4.2 IbA-1+ cells/200×field, p<0.05). Little or no effect of LLLT on post-injury cognitive function was observed using the other doses, a 4-h administration time point and 7-day administration of 60 J/cm². No effect of LLLT (60 J/cm² open craniotomy) was observed on post-injury motor function (days 1-7), brain edema (24 h), nitrosative stress (24 h), or lesion volume (14 days). Although further dose optimization and mechanism studies are needed, the data suggest that LLLT might be a therapeutic option to improve cognitive recovery and limit inflammation after TBI.

J Neurotrauma. 2011 Sep 21. [Epub ahead of print]

Low-Level Laser Light Therapy Improves Cognitive Deficits and Inhibits Microglial Activation after Controlled Cortical Impact in Mice.

Khuman J, Zhang J, Park J, Carroll JD, Donahue C, Whalen MJ.

Source

1 Neuroscience Center, Massachusetts General Hospital , Harvard Medical School, Charlestown, Massachusetts.

Abstract

Abstract Low-level laser light therapy (LLLT) exerts beneficial effects on motor and histopathological outcomes after experimental traumatic brain injury (TBI), and coherent near-infrared light has been reported to improve cognitive function in patients with chronic TBI. However, the effects of LLLT on cognitive recovery in experimental TBI are unknown. We hypothesized that LLLT administered after controlled cortical impact (CCI) would improve post-injury Morris water maze (MWM) performance. Low-level laser light (800?nm) was applied directly to the contused parenchyma or transcranially in mice beginning 60-80?min after CCI. Injured mice treated with 60?J/cm(2) (500?mW/cm(2)×2?min) either transcranially or via an open craniotomy had modestly improved latency to the hidden platform (p<0.05 for group), and probe trial performance (p<0.01) compared to non-treated controls. The beneficial effects of LLLT in open craniotomy mice were associated with reduced microgliosis at 48?h (21.8±2.3 versus 39.2±4.2 IbA-1+ cells/200×field, p<0.05). Little or no effect of LLLT on post-injury cognitive function was observed using the other doses, a 4-h administration time point and 7-day administration of 60?J/cm(2). No effect of LLLT (60?J/cm(2) open craniotomy) was observed on post-injury motor function (days 1-7), brain edema (24?h), nitrosative stress (24?h), or lesion volume (14 days). Although further dose optimization and mechanism studies are needed, the data suggest that LLLT might be a therapeutic option to improve cognitive recovery and limit inflammation after TBI.

Photomed Laser Surg. 2011 May;29(5):351-8. doi: 10.1089/pho.2010.2814. Epub 2010 Dec 23.

Improved cognitive function after transcranial, light-emitting diode treatments in chronic, traumatic brain injury: two case reports.

Naeser MA1, Saltmarche A, Krengel MH, Hamblin MR, Knight JA.

Author information

  • 1VA Boston Healthcare System , Boston, Massachusetts. mnaeser@bu.edu

Abstract

OBJECTIVE:

Two chronic, traumatic brain injury (TBI) cases, where cognition improved following treatment with red and near-infrared light-emitting diodes (LEDs), applied transcranially to forehead and scalp areas, are presented.

BACKGROUND:

Significant benefits have been reported following application of transcranial, low-level laser therapy (LLLT) to humans with acute stroke and mice with acute TBI. These are the first case reports documenting improved cognitive function in chronic, TBI patients treated with transcranial LED.

METHODS:

Treatments were applied bilaterally and to midline sagittal areas using LED cluster heads [2.1? diameter, 61 diodes (9?×?633?nm, 52?×?870?nm); 12-15?mW per diode; total power: 500?mW; 22.2?mW/cm(2); 13.3?J/cm(2) at scalp (estimated 0.4?J/cm(2) to cortex)].

RESULTS:

Seven years after closed-head TBI from a motor vehicle accident, Patient 1 began transcranial LED treatments. Pre-LED, her ability for sustained attention (computer work) lasted 20 min. After eight weekly LED treatments, her sustained attention time increased to 3 h. The patient performs nightly home treatments (5 years); if she stops treating for more than 2 weeks, she regresses. Patient 2 had a history of closed-head trauma (sports/military, and recent fall), and magnetic resonance imaging showed frontoparietal atrophy. Pre-LED, she was on medical disability for 5 months. After 4 months of nightly LED treatments at home, medical disability discontinued; she returned to working full-time as an executive consultant with an international technology consulting firm. Neuropsychological testing after 9 months of transcranial LED indicated significant improvement (+1, +2SD) in executive function (inhibition, inhibition accuracy) and memory, as well as reduction in post-traumatic stress disorder. If she stops treating for more than 1 week, she regresses. At the time of this report, both patients are continuing treatment.

CONCLUSIONS:

Transcranial LED may improve cognition, reduce costs in TBI treatment, and be applied at home. Controlled studies are warranted

Postepy High Med Dosw (Online).  2011 Feb 17;65:73-92.

The role of biological sciences in understanding the genesis and a new therapeutic approach to Alzheimer’s disease.

T?gowska E, Wosi?ska A.

Zak?ad Toksykologii Zwierz?t, Wydzia? Biologii i Nauk o Ziemi, Uniwersytet Miko?aja Kopernika w Toruniu.

Abstract

The paper contrasts the historical view on causal factors in Alzheimer’s disease (AD) with the modern concept of the symptoms’ origin. Biological sciences dealing with cell structure and physiology enabled comprehension of the role of mitochondrial defects in the processes of formation of neurofibrillary tangles and ?-amyloid, which in turn gives hope for developing a new, more effective therapeutic strategy for AD. It has been established that although mitochondria constantly generate free radicals, from which they are protected by their own defensive systems, in some situations these systems become deregulated, which leads to free radical-based mitochondrial defects. This causes an energetic deficit in neurons and a further increase in the free radical pool. As a result, due to compensation processes, formation of tangles and/or acceleration of ?-amyloid production takes place. The nature of these processes is initially a protective one, due to their anti-oxidative action, but as the amount of the formations increases, their beneficial effect wanes. They become a storage place for substances enhancing free radical processes, which makes them toxic themselves. It is such an approach to the primary causal factor for AD which lies at the roots of the new view on AD therapy, suggesting the use of methylene blue-based drugs, laser or intranasally applied insulin. A necessary condition, however, for these methods’ effectiveness is definitely an earlier diagnosis of the disease. Although there are numerous diagnostic methods for AD, their low specificity and high price, often accompanied by a considerable level of patient discomfort, make them unsuitable for early, prodromal screening. In this matter a promising method may be provided using an olfactory test, which is an inexpensive and non-invasive method and thus suitable for screening, although as a test of low specificity, it should be combined with other methods. Introducing new methods of AD treatment does not mean abandoning the traditional ones, based on enhancing cholinergic transmission. They are valuable as long as the therapy starts when abundant brain inclusions disturb the transmissions.

J Comp Neurol. 2010 Jan 1;518(1):25-40.

Neuroprotection of midbrain dopaminergic cells in MPTP-treated mice after near-infrared light treatment.

Shaw VE, Spana S, Ashkan K, Benabid AL, Stone J, Baker GE, Mitrofanis J.

Discipline of Anatomy & Histology F13, University of Sydney, Australia.

Abstract

This study explores whether near-infrared (NIr) light treatment neuroprotects dopaminergic cells in the substantia nigra pars compacta (SNc) and the zona incerta-hypothalamus (ZI-Hyp) from degeneration in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. BALB/c albino mice were divided into four groups: 1) Saline, 2) Saline-NIr, 3) MPTP, 4) MPTP-NIr. The injections were intraperitoneal and they were followed immediately by NIr light treatment (or not). Two doses of MPTP, mild (50 mg/kg) and strong (100 mg/kg), were used. Mice were perfused transcardially with aldehyde fixative 6 days after their MPTP treatment. Brains were processed for tyrosine hydroxylase (TH) immunochemistry. The number of TH(+) cells was estimated using the optical fractionator method. Our major finding was that in the SNc there were significantly more dopaminergic cells in the MPTP-NIr compared to the MPTP group (35%-45%). By contrast, in the ZI-Hyp there was no significant difference in the numbers of cells in these two groups. In addition, our results indicated that survival in the two regions after MPTP insult was dose-dependent. In the stronger MPTP regime, the magnitude of loss was similar in the two regions ( approximately 60%), while in the milder regime cell loss was greater in the SNc (45%) than ZI-Hyp ( approximately 30%). In summary, our results indicate that NIr light treatment offers neuroprotection against MPTP toxicity for dopaminergic cells in the SNc, but not in the ZI-Hyp.

J Photochem Photobiol B. 2009 Dec 2;97(3):145-51. Epub 2009 Sep 11.

Effect of phototherapy with low intensity laser on local and systemic immodulation following focal brain damage in rat.

Moreira MS, Velasco IT, Ferreira LS, Ariga SK, Barbeiro DF, Meneguzzo DT, Abatepaulo F, Marques MM.

LIM-51, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil.

Brain injury is responsible for significant morbidity and mortality in trauma patients, but controversy still exists over therapeutic management for these patients. The objective of this study was to analyze the effect of phototherapy with low intensity lasers on local and systemic immunomodulation following cryogenic brain injury. Laser phototherapy was applied (or not-controls) immediately after cryogenic brain injury performed in 51 adult male Wistar rats. The animals were irradiated twice (3 h interval), with continuous diode laser (gallium-aluminum-arsenide (GaAlAs), 780 nm, or indium-gallium-aluminum-phosphide (InGaAlP), 660 nm) in two points and contact mode, 40 mW, spot size 0.042 cm(2), 3 J/cm(2) and 5 J/cm(2) (3 s and 5 s, respectively). The experimental groups were: Control (non-irradiated), RL3 (visible red laser/ 3 J/cm(2)), RL5 (visible red laser/5 J/cm(2)), IRL3 (infrared laser/3 J/cm(2)), IRL5 (infrared laser/5 J/cm(2)). The production of interleukin-1IL-1beta (IL-1beta), interleukin6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-alpha) was analyzed by enzyme immunoassay technique (ELISA) test in brain and blood samples. The IL-1beta concentration in brain of the control group was significantly reduced in 24 h (p<0.01). This reduction was also observed in the RL5 and IRL3 groups. The TNF-alpha and IL-6 concentrations increased significantly (p<0.01 and p<0.05, respectively) in the blood of all groups, except by the IRL3 group. The IL-6 levels in RL3 group were significantly smaller than in control group in both experimental times. IL-10 concentration was maintained stable in all groups in brain and blood. Under the conditions of this study, it is possible to conclude that the laser phototherapy can affect TNF-alpha, IL-1beta and IL-6 levels in the brain and in circulation in the first 24 h following cryogenic brain injury.

Vopr Kurortol Fizioter Lech Fiz Kult. 2009 Nov-Dec;(6):3-11.

Many-level polysensory stimulation of brain functions by physical therapeutic agents.

[Article in Russian]

Tyshkevich TG, Ponomarenko GN.

A combination of physiotherapeutic methods for neurorehabilitative treatment has been developed and applied to the treatment of 576 patients with neurosurgical problems including the loss of brain functions as a sequel to nervous system lesions of traumatic, vascular, and other origin. Methodologically, this complex is adapted to the level and extent of the lesion and the character of regeneration of the nervous tissues. It implies many-level stimulation of neuroregeneration by syndromically and pathogenetically substantiated application of physical factors in the early post-injury and postoperative periods. The proposed approach allows the brain function to be completely restored by virtue of persistent compensatory changes in the nervous system. A combination of many-level magnetic, electrical, and laser stimulation is recommended to manage lesions in the speech, motor, and visual analyzers. Combined laser and differential electrostimulation may be prescribed to patients with nerve lesions, extremely high frequency therapy to those with epileptic syndrome, combined microwave therapy to cases with impairment of consciousness, and a variant of systemic UV irradiation with underwater shower-massaging for the treatment of vegetative and asthenic disturbances. Selected physiological aspects of the action of the above physical factors are specified. This physiotherapeutic system is protected by 20 RF patents of invention.

Mol Neurodegener. 2009 Jun 17;4:26.

Reduced axonal transport in Parkinson’s disease cybrid neurites is restored by light therapy.

Trimmer PA, Schwartz KM, Borland MK, De Taboada L, Streeter J, Oron U.

University of Virginia, Morris K Udall Parkinson’s Research Center of Excellence and Department of Neurology, Charlottesville, Virginia, USA. pat5q@virginia.edu.

ABSTRACT: BACKGROUND: It has been hypothesized that reduced axonal transport contributes to the degeneration of neuronal processes in Parkinson’s disease (PD). Mitochondria supply the adenosine triphosphate (ATP) needed to support axonal transport and contribute to many other cellular functions essential for the survival of neuronal cells. Furthermore, mitochondria in PD tissues are metabolically and functionally compromised. To address this hypothesis, we measured the velocity of mitochondrial movement in human transmitochondrial cybrid “cytoplasmic hybrid” neuronal cells bearing mitochondrial DNA from patients with sporadic PD and disease-free age-matched volunteer controls (CNT). The absorption of low level, near-infrared laser light by components of the mitochondrial electron transport chain (mtETC) enhances mitochondrial metabolism, stimulates oxidative phosphorylation and improves redox capacity. PD and CNT cybrid neuronal cells were exposed to near-infrared laser light to determine if the velocity of mitochondrial movement can be restored by low level light therapy (LLLT). Axonal transport of labeled mitochondria was documented by time lapse microscopy in dopaminergic PD and CNT cybrid neuronal cells before and after illumination with an 810 nm diode laser (50 mW/cm2) for 40 seconds. Oxygen utilization and assembly of mtETC complexes were also determined.

RESULTS: The velocity of mitochondrial movement in PD cybrid neuronal cells (0.175 +/- 0.005 SEM) was significantly reduced (p < 0.02) compared to mitochondrial movement in disease free CNT cybrid neuronal cells (0.232 +/- 0.017 SEM). For two hours after LLLT, the average velocity of mitochondrial movement in PD cybrid neurites was significantly (p < 0.003) increased (to 0.224 +/- 0.02 SEM) and restored to levels comparable to CNT. Mitochondrial movement in CNT cybrid neurites was unaltered by LLLT (0.232 +/- 0.017 SEM). Assembly of complexes in the mtETC was reduced and oxygen utilization was altered in PD cybrid neuronal cells. PD cybrid neuronal cell lines with the most dysfunctional mtETC assembly and oxygen utilization profiles were least responsive to LLLT.

CONCLUSION: The results from this study support our proposal that axonal transport is reduced in sporadic PD and that a single, brief treatment with near-infrared light can restore axonal transport to control levels. These results are the first demonstration that LLLT can increase axonal transport in model human dopaminergic neuronal cells and they suggest that LLLT could be developed as a novel treatment to improve neuronal function in patients with PD.

Lasers Surg Med. 2009 Apr;41(4):277-81.

Increase of neuronal sprouting and migration using 780 nm laser phototherapy as procedure for cell therapy.

Rochkind S, El-Ani D, Nevo Z, Shahar A.

Division of Peripheral Nerve Reconstruction, Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv 64239, Israel. rochkind@zahav.net.il

BACKGROUND AND OBJECTIVES: The present study focuses on the effect of 780 nm laser irradiation on the growth of embryonic rat brain cultures embedded in NVR-Gel (cross-linked hyaluronic acid with adhesive molecule laminin and several growth factors). Dissociated neuronal cells were first grown in suspension attached to cylindrical microcarriers (MCs). The formed floating cell-MC aggregates were subsequently transferred into stationary cultures in gel and then laser treated. The response of neuronal growth following laser irradiation was investigated.

MATERIALS AND METHODS: Whole brains were dissected from 16 days Sprague-Dawley rat embryos. Cells were mechanically dissociated, using narrow pipettes, and seeded on positively charged cylindrical MCs. After 4-14 days in suspension, the formed floating cell-MC aggregates were seeded as stationary cultures in NVR-Gel. Single cell-MC aggregates were either irradiated with near-infrared 780 nm laser beam for 1, 4, or 7 minutes, or cultured without irradiation. Laser powers were 10, 30, 50, 110, 160, 200, and 250 mW.

RESULTS: 780 nm laser irradiation accelerated fiber sprouting and neuronal cell migration from the aggregates. Furthermore, unlike control cultures, the irradiated cultures (mainly after 1 minute irradiation of 50 mW) were already established after a short time of cultivation. They contained a much higher number of large size neurons (P<0.01), which formed dense branched interconnected networks of thick neuronal fibers.

CONCLUSIONS: 780 nm laser phototherapy of embryonic rat brain cultures embedded in hyaluronic acid-laminin gel and attached to positively charged cylindrical MCs, stimulated migration and fiber sprouting of neuronal cells aggregates, developed large size neurons with dense branched interconnected network of neuronal fibers and, therefore, can be considered as potential procedure for cell therapy of neuronal injury or disease.

Lasers Surg Med. 2009 Jan;41(1):52-9.

Light therapy and supplementary Riboflavin in the SOD1 transgenic mouse model of familial amyotrophic lateral sclerosis (FALS).

Moges H, Vasconcelos OM, Campbell WW, Borke RC, McCoy JA, Kaczmarczyk L, Feng J, Anders JJ.

Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.

BACKGROUND AND OBJECTIVE: Familial amyotrophic lateral sclerosis (FALS) is a neurodegenerative disease characterized by progressive loss of motor neurons and death. Mitochondrial dysfunction and oxidative stress play an important role in motor neuron loss in ALS. Light therapy (LT) has biomodulatory effects on mitochondria. Riboflavin improves energy efficiency in mitochondria and reduces oxidative injury. The purpose of this study was to examine the synergistic effect of LT and riboflavin on the survival of motor neurons in a mouse model of FALS.

STUDY DESIGN/MATERIALS AND METHODS: G93A SOD1 transgenic mice were divided into four groups: Control, Riboflavin, Light, and Riboflavin+Light (combination). Mice were treated from 51 days of age until death. A single set of LT parameters was used: 810 nm diode laser, 140-mW output power, 1.4 cm(2) spot area, 120 seconds treatment duration, and 12 J/cm(2) energy density. Behavioral tests and weight monitoring were done weekly. At end stage of the disease, mice were euthanized, survival data was collected and immunohistochemistry and motor neuron counts were performed.

RESULTS: There was no difference in survival between groups. Motor function was not significantly improved with the exception of the rotarod test which showed significant improvement in the Light group in the early stage of the disease. Immunohistochemical expression of the astrocyte marker, glial fibrilary acidic protein, was significantly reduced in the cervical and lumbar enlargements of the spinal cord as a result of LT. There was no difference in the number of motor neurons in the anterior horn of the lumbar enlargement between groups.

CONCLUSIONS: The lack of significant improvement in survival and motor performance indicates study interventions were ineffective in altering disease progression in the G93A SOD1 mice. Our findings have potential implications for the conceptual use of light to treat other neurodegenerative diseases that have been linked to mitochondrial dysfunction.

Brain Res. 2008 Dec 3;1243:167-73. Epub 2008 Sep 30.

Pretreatment with near-infrared light via light-emitting diode provides added benefit against rotenone- and MPP+-induced neurotoxicity.

Ying R, Liang HL, Whelan HT, Eells JT, Wong-Riley MT.

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.

Parkinson’s disease (PD) is a movement disorder caused by the loss of dopaminergic neurons in the substantia nigra pars compacta, leading to nigrostriatal degeneration. The inhibition of mitochondrial respiratory chain complex I and oxidative stress-induced damage have been implicated in the pathogenesis of PD. The present study used these specific mitochondrial complex I inhibitors (rotenone and 1-methyl-4-phenylpyridinium or MPP(+)) on striatal and cortical neurons in culture. The goal was to test our hypothesis that pretreatment with near-infrared light (NIR) via light-emitting diode (LED) had a greater beneficial effect on primary neurons grown in media with rotenone or MPP(+) than those with or without LED treatment during exposure to poisons. Striatal and visual cortical neurons from newborn rats were cultured in a media with or without 200 nM of rotenone or 250 microM of MPP(+) for 48 h. They were treated with NIR-LED twice a day before, during, and both before and during the exposure to the poison. Results indicate that pretreatment with NIR-LED significantly suppressed rotenone- or MPP(+)-induced apoptosis in both striatal and cortical neurons (P<0.001), and that pretreatment plus LED treatment during neurotoxin exposure was significantly better than LED treatment alone during exposure to neurotoxins. In addition, MPP(+) induced a decrease in neuronal ATP levels (to 48% of control level) that was reversed significantly to 70% of control by NIR-LED pretreatment. These data suggest that LED pretreatment is an effective adjunct preventative therapy in rescuing neurons from neurotoxins linked to PD.

Neuroscience. 2008 Jun 2;153(4):963-74. Epub 2008 Mar 26.

Near-infrared light via light-emitting diode treatment is therapeutic against rotenone- and 1-methyl-4-phenylpyridinium ion-induced neurotoxicity.

Liang HL, Whelan HT, Eells JT, Wong-Riley MT.

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.

Parkinson’s disease is a common progressive neurodegenerative disorder characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Mitochondrial dysfunction has been strongly implicated in the pathogenesis of Parkinson’s disease. Thus, therapeutic approaches that improve mitochondrial function may prove to be beneficial. Previously, we have documented that near-infrared light via light-emitting diode (LED) treatment was therapeutic to neurons functionally inactivated by tetrodotoxin, potassium cyanide (KCN), or methanol intoxication, and LED pretreatment rescued neurons from KCN-induced apoptotic cell death. The current study tested our hypothesis that LED treatment can protect neurons from both rotenone- and MPP(+)-induced neurotoxicity. Primary cultures of postnatal rat striatal and cortical neurons served as models, and the optimal frequency of LED treatment per day was also determined. Results indicated that LED treatments twice a day significantly increased cellular adenosine triphosphate content, decreased the number of neurons undergoing cell death, and significantly reduced the expressions of reactive oxygen species and reactive nitrogen species in rotenone- or MPP(+)-exposed neurons as compared with untreated ones. These results strongly suggest that LED treatment may be therapeutic to neurons damaged by neurotoxins linked to Parkinson’s disease by energizing the cells and increasing their viability.

J Neurotrauma. 2007 Apr;24(4):651-6.

Low-level laser therapy applied transcranially to mice following traumatic brain injury significantly reduces long-term neurological deficits.

Oron A, Oron U, Streeter J, de Taboada L, Alexandrovich A, Trembovler V, Shohami E.

Department of Orthopedics, Assaf Harofeh Medical Center, Zerifin, Israel. amiroronmd@gmail.com

Low-level laser therapy (LLLT) has been evaluated in this study as a potential therapy for traumatic brain injury (TBI). LLLT has been found to modulate various biological processes. Following TBI in mice, we assessed the hypothesis that LLLT might have a beneficial effect on their neurobehavioral and histological outcome. TBI was induced by a weight-drop device, and motor function was assessed 1 h post-trauma using a neurological severity score (NSS). Mice were then divided into three groups of eight mice each: one control group that received a sham LLLT procedure and was not irradiated; and two groups that received LLLT at two different doses (10 and 20 mW/cm(2) ) transcranially. An 808-nm Ga-As diode laser was employed transcranially 4 h post-trauma to illuminate the entire cortex of the brain. Motor function was assessed up to 4 weeks, and lesion volume was measured. There were no significant changes in NSS at 24 and 48 h between the laser-treated and non-treated mice. Yet, from 5 days and up to 28 days, the NSS of the laser-treated mice were significantly lower (p < 0.05) than the traumatized control mice that were not treated with the laser. The lesion volume of the laser treated mice was significantly lower (1.4%) than the non-treated group (12.1%). Our data suggest that a non-invasive transcranial application of LLLT given 4 h following TBI provides a significant long-term functional neurological benefit. Further confirmatory trials are warranted.

Photomed Laser Surg. 2006 Aug;24(4):458-66

Effects of power densities, continuous and pulse frequencies, and number of sessions of low-level laser therapy on intact rat brain.

Ilic S, Leichliter S, Streeter J, Oron A, DeTaboada L, Oron U.

Photothera Inc., Carlsbad, California, USA.

OBJECTIVE: The aim of the present study was to investigate the possible short- and long-term adverse neurological effects of low-level laser therapy (LLLT) given at different power densities, frequencies, and modalities on the intact rat brain.

BACKGROUND DATA: LLLT has been shown to modulate biological processes depending on power density, wavelength, and frequency. To date, few well-controlled safety studies on LLLT are available. METHODS: One hundred and eighteen rats were used in the study. Diode laser (808 nm, wavelength) was used to deliver power densities of 7.5, 75, and 750 mW/cm2 transcranially to the brain cortex of mature rats, in either continuous wave (CW) or pulse (Pu) modes. Multiple doses of 7.5 mW/cm2 were also applied. Standard neurological examination of the rats was performed during the follow-up periods after laser irradiation. Histology was performed at light and electron microscopy levels.

RESULTS: Both the scores from standard neurological tests and the histopathological examination indicated that there was no long-term difference between laser-treated and control groups up to 70 days post-treatment. The only rats showing an adverse neurological effect were those in the 750 mW/cm2 (about 100-fold optimal dose), CW mode group. In Pu mode, there was much less heating, and no tissue damage was noted. CONCLUSION: Long-term safety tests lasting 30 and 70 days at optimal 10x and 100x doses, as well as at multiple doses at the same power densities, indicate that the tested laser energy doses are safe under this treatment regime. Neurological deficits and histopathological damage to 750 mW/cm2 CW laser irradiation are attributed to thermal damage and not due to tissue-photon interactions.

Zhong Xi Yi Jie He Xue Bao. 2005 Mar;3(2):128-31.

Protective effect of low-level irradiation on acupuncture points combined with iontophoresis against focal cerebral ischemia-reperfusion injury in rats.

[Article in Chinese]

Dai JY, Ge LB, Zhou YL, Wang L.

Acupuncture Clinic, Institute of Qigong, Shanghai University of Traditional Chinese Medicine, Shanghai 200030, China. djysh2002@yahoo.com.cn

OBJECTIVE: To investigate the effects of low-level laser irradiation on acupuncture points combined with iontophoresis against brain damage after middle cerebral artery occlusion (MCAO) in rats.

METHODS: Sixty-nine SD rats were randomly divided into five groups, including normal group, sham operation group, model group, electro-acupuncture group and low-level laser irradiation on acupuncture points combined with iontophoresis group (LLLI group). The cerebral ischemia-reperfusion (I/R) model was established by thread embolism of middle cerebral artery. The rats in the LLLI group, as well as the electro-acupuncture group were given treatment as soon as the occlusion finished (0 hour) and 12, 24 hours after the occlusion. We observed the changes of neurological deficit scores and the body weight of the rats at different time. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in the ratos brain tissue were tested.

RESULTS: The neurological deficit score of the LLLI group was significantly lower than that of the model group. The body weight and the activity of SOD of the rats decreased slightly, and the content of MDA decreased significantly after the treatment.

CONCLUSION: The low-level laser irradiation on acupuncture points combined with iontophoresis can prevent focal cerebral ischemia-reperfusion injury. One of its mechanisms may be increasing the activity of SOD and decreasing the damage of the oxidation products to the body.

Mitochondrion. 2004 Sep;4(5-6):559-67.

Mitochondrial signal transduction in accelerated wound and retinal healing by near-infrared light therapy.

Eells JT, Wong-Riley MT, VerHoeve J, Henry M, Buchman EV, Kane MP, Gould LJ, Das R, Jett M, Hodgson BD, Margolis D, Whelan HT.

Department of Health Sciences, College of Health Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, USA. jeells@uwm.edu

Photobiomodulation by light in the red to near infrared range (630-1000 nm) using low energy lasers or light-emitting diode (LED) arrays has been shown to accelerate wound healing, improve recovery from ischemic injury in the heart and attenuate degeneration in the injured optic nerve. Recent evidence indicates that the therapeutic effects of red to near infrared light result, in part, from intracellular signaling mechanisms triggered by the interaction of NIR light with the mitochondrial photoacceptor molecule cytochrome c oxidase. We have demonstrated that NIR-LED photo-irradiation increases the production of cytochrome oxidase in cultured primary neurons and reverses the reduction of cytochrome oxidase activity produced by metabolic inhibitors. We have also shown that NIR-LED treatment prevents the development of oral mucositis in pediatric bone marrow transplant patients. Photobiomodulation improves wound healing in genetically diabetic mice by upregulating genes important in the promotion of wound healing. More recent studies have provided evidence for the therapeutic benefit of NIR-LED treatment in the survival and functional recovery of the retina and optic nerve in vivo after acute injury by the mitochondrial toxin, formic acid generated in the course of methanol intoxication. Gene discovery studies conducted using microarray technology documented a significant upregulation of gene expression in pathways involved in mitochondrial energy production and antioxidant cellular protection. These findings provide a link between the actions of red to near infrared light on mitochondrial oxidative metabolism in vitro and cell injury in vivo. Based on these findings and the strong evidence that mitochondrial dysfunction is involved in the pathogenesis of numerous diseases processes, we propose that NIR-LED photobiomodulation represents an innovative and non-invasive therapeutic approach for the treatment of tissue injury and disease processes in which mitochondrial dysfunction is postulated to play a role including diabetic retinopathy, age-related macular degeneration, Leber’s hereditary optic neuropathy and Parkinson’s disease.

Patol Fiziol Eksp Ter. 2004 Jan-Mar;(1):15-8.

Biochemical and immunological indices of the blood in Parkinson’s disease and their correction with the help of laser therapy.

[Article in Russian]

Komel’kova LV, Vitreshchak TV, Zhirnova IG, Poleshchuk VV, Stvolinskii SL, Mikhailov VV, Gannushkina IV, Piradov MA.

The influence of laser therapy on the course of Parkinson’s disease (PD) was studied in 70 patients. This influence appeared adaptogenic both in the group with elevated and low MAO B and Cu/Zn SOD activity. Laser therapy resulted in reduction of neurological deficit, normalization of the activity of MAO B, Cu/Zn-SOD and immune indices. There was a correlation between humoral immunity and activity of the antioxidant enzymes (SOD, catalase). This justifies pathogenetically the use of laser therapy in PD.

Bull Exp Biol Med. 2003 May;135(5):430-2.

Laser modification of the blood in vitro and in vivo in patients with Parkinson’s disease.

Vitreshchak TV, Mikhailov VV, Piradov MA, Poleshchuk VV, Stvolinskii SL, Boldyrev AA.

Institute of Neurology of the Russian Academy of Medical Sciences, Moscow.

The effect of He-Ne laser radiation on activity of MAO B, Cu/Zn-SOD, Mn-SOD, and catalase in blood cells from patients with Parkinson’s disease was studied in vivo and in vitro. The effects of intravenous in vivo irradiation (intravenous laser therapy) were more pronounced than those observed in similar in vitro experiments. It is concluded that generalized effect of laser therapy involves interaction between blood cells.

Proceedings of the SPIE, Volume 5229, pp. 97-103 (2003). Laser Technology VII: Applications of Lasers. DOI: 10.1117/12.520611

Laser biostimulation of patients suffering from multiple sclerosis in respect to the biological influence of laser light.

Peszynski-Drews, Cezary; Klimek, Andrzej; Sopinski, Marek; Obrzejta, Dominik

AA (Technical Univ. of Lodz (Poland)), AB (Copernicus Hospital (Poland)), AC(Technical Univ. of Lodz (Poland)), AD (Technical Univ. of Lodz (Poland))

The authors discuss the results, obtained so far during three years’ clinical examination, of laser therapy in the treatment of patients suffering from multiple sclerosis. They regard both the results of former laboratory experiments and so far discovered mechanisms of biological influence of laser light as an objective explanation of high effectiveness of laser therapy in the case of this so far incurable disease. They discuss wide range of biological mechanisms of laser therapy, examined so far on different levels (cell, tissue, organ), allowing the explanation of beneficial influence of laser light in pathogenetically different morbidities.

Neurol Res. 2002 Jn;24(4):355-60.

Transplantation of embryonal spinal cord nerve cells cultured on biodegradable microcarriers followed by low power laser irradiation for the treatment of traumatic paraplegia in rats.

Rochkind S, Shahar A, Amon M, Nevo Z.

Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Israel. rochkind@zahav.net.il

This pilot study examined the effects of composite implants of cultured embryonal nerve cells and laser irradiation on the regeneration and repair of the completely transected spinal cord. Embryonal spinal cord nerve cells dissociated from rat fetuses and cultured on biodegradable microcarriers and embedded in hyaluronic acid were implanted in the completely transected spinal cords of 24 adult rats. For 14 consecutive post-operative days, 15 rats underwent low power laser irradiation (780 nm, 250 mW), 30 min daily. Eleven of the 15 (73%) showed different degrees of active leg movements and gait performance, compared to 4 (44%) of the 9 rats with implantation alone. In a controlgroup of seven rats with spinal cord transection and no transplantation or laser, six (86%) remained completely paralyzed. Three months after transection, implantation and laser irradiation, SSEPs were elicited in 69% of rats (p = 0.0237) compared to 37.5% in the nonirradiated group. The control group had no SSEPs response. Intensive axonal sprouting occurred in the group with implantation and laser. In the control group, the transected area contained proliferating fibroblasts and blood capillaries only. This suggests: 1. These in vitro composite implants are a regenerative and reparative source for reconstructing the transected spinal cord. 2. Post-operative low power laser irradiation enhances axonal sprouting and spinal cord repair.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2002 Jan;93(1):27-34.

Low-level laser effect on neural regeneration in Gore-Tex tubes.

Miloro M, Halkias LE, Mallery S, Travers S, Rashid RG.

Department of Surgery, Division of Oral and Maxillofacial Surgery, University of Nebraska Medical Center, Omaha 68198-5180, USA.

PURPOSE: The purpose of this investigation was to determine the effects of low-level laser (LLL) irradiation on neural regeneration in surgically created defects in the rabbit inferior alveolar nerve.

STUDY DESIGN: Five adult female New Zealand White rabbits underwent bilateral exposure of the inferior alveolar nerve. A 6-mm segment of nerve was resected, and the nerve gap was repaired via entubulation by using a Gore-Tex conduit. The experimental side received 10 postoperative LLL treatments with a 70-mW gallium-aluminum-arsenide diode at 4 sites per treatment. At 15 weeks after surgery, the nerve segments were harvested bilaterally and prepared for light microscopy. Basic fuchsin and toluidine blue were used to highlight myelinated axons. The segments were examined histomorphometrically by using computer analysis to determine mean axonal diameter, total fascicular surface area, and axonal density along the repair sites.

RESULTS: Gross examination of all nerves showed intact neural bundles with variable degrees of osseous remodeling. Light microscopic evaluation revealed organized regenerated neural tissue in both groups with more intrafascicular perineural tissue in the control group. Histomorphometric evaluation revealed increased axonal density in the laser treated group as compared with the control.

CONCLUSIONS: LLL irradiation may be a useful noninvasive adjunct to promote neuronal wound healing in surgically created defects repaired with expanded polytetrafluoroethylene entubulation.

ACTA LASER BIOLOGY SINICA Vol. 8, No.2, 1999

Vascular Low Level Laser Irradiation Therapy in Treatment of Brain Injury

WANG Yu ZHU Jing, et al

(Department of Neurosurgery, Renji Hospital Affiliated to Shanghai Second Medical University, Shanghai Medical Centre for laser Research ,200001)

Abstract: To evaluate the effect and mechanism of Vascular Low Level Laser Irradiation Therapy on brain injury. In this study thirty-eight SpragueDawley rats received Feeney’s brain impact through a left lateral craniectomy under anesthesia. Control and treatment group are set up. According to the time exposed to laser and irradiating postinjury, the treatment group is divided in four subgroups by design. Semiconductor laser was used with a power of 5mW to irridate straightly Rat’s femur venous. The Y Water maze was used to assess cognitive performance. Superoxide dismutase(SOD) activity and the level of metabolic production of free radical MDA in Brain and erythrocyte were measured to determinate the level of free radical. We find Vascular Low Level Laser Irradiation Therapy can improve posttraumatic memory deficits. SOD activity is higher in treatment groups than the control group meanwhile the level of MDA is lower. These findings suggest that Vascular Low Level Laser Irradiation produced a significant reduction in free radical’s damage to the brain postinjury.

INFARED LASER RADIATION IN THE TREATMENT OF BRAIN INJURY CONSEQUENCES

E.L. Macheret, A.O. Korkushko, T.N. Kalishchuk, M.N. Matyash

Medical Academy of Post-Diploma Education, Kiev, Ukraine

The examination of 198 patients aged 16-47 has revealed a high fre­quency of progressive pathologic states in a form of asthenia, vegeto-vascular dystonia, hypertensive, somato-vegetative, vestibular syndroms. Taking into account the changes in cortico-undercortical interrelations and expansion of pathologic process in hypothalamic area during the head trauma, we have developed effective treating methods by means of laseropuncture. Laser rays influence on acupuncture points (AP) leads to a convergence of the afferent messages upon the neurones of spinal cord, reticular formation, thalamus, hypothalamus and brain cor­tex. As a result of that a dynamic balance between the inhibition and excitation processes in the structures of central nervous system leading vegetative function and endocrine secretion recovers.  Use of infrared laser radiation is the most perspective. It docs not cause the direct photochemical reactions in biological tissues, but influences on physico-chemical structure of AP biomolecules. For laseropuncture we used an apparatus “BIOMED-01? with a wavelength of 0.89 nm. The work regime is impulsive-continuous with a modulation of frequency – from 0.1 to 1000 Hz. The middle power is up to 20 mW. The total time of the action for one sitting is till 20 min. The points selections was carried out on the grounds of the methods of acupuncture diagnosis, imagesking out the dominant clinical syndromes and including points of vascular, vegetotroimages, sedative orientation. Our clinical results, which were confirmed by paraclinical methods (EEG, dopplerography) and methods of acupuncture diagnosis have shown a high effectiveness of this therapy decreasing the drugs load and having no contradictions.

LASER-THERAPY AND ITS INFLUENCE ON HEMODYNAMICS WITH PATIENTS SUFFERED FROM GRAVE CRANIOCEREBRAL TRAUMA

Y.V. Kurako

Medical Academy, Dnepropetrovsk, Ukraine

Despite the maximal dosage of different medications taken for curing of grave craniocerebral trauma the resistance to the treatment carried out was observed. This fact stimulated the search of new methods and ways of therapy. One of the possible methods is a non-medicamental treatment based on blood irradiation with low-active helium-neon laser. The present paper presents some data concerning the laser-therapy influence in hemodynamics in the case of craniocerebral trauma. The total number of patients examined is 45. Laser-therapy was carried out through the subclavian vein (37 cases) or cubital vein (8 cases). For primary irradiation the preferable access was the central one. It was used in the acute period of craniocerebral trauma. The periferal access was used for irradiation in the posthospital period. The course of laser therapy for in-hospital patients consisted of 3-5 everyday procedures of 30 minutes each. To define the hemodynamic changes with the patients suffered from craniocerebral trauma both clinical observation and ultrasonic transcranial dopplerography were used. The last one gave the possibility to identify the type of blood flow speed disorders.

Paper received 10 May 1999; accepted after revision 23 August 1999.

Specific Effects of Laserpuncture on the Cerebral Circulation

G. Litscher (1), L. Wang (1), M. Wiesner-Zechmeister (2)(1)

Biomedical Engineering, Department of Anesthesiology and Critical Care, University of Graz, Graz, Austria(2) European Forum for Lasertherapy and Fractal Medicine

Abstract . Acupuncture is a form of traditional Chinese medicine that has developed over thousands of years. We studied the effects of laser puncture, needle acupuncture, and light stimulation on cerebral blood flow in 15 healthy volunteers (mean age 25.0±1.9 years, 5 female, 10 male) with non-invasive transcranial Doppler sonography. In addition 40-Hz stimulus-induced brain oscillations, heart rate, blood pressure, peripheral and cerebral oxygen saturation, and the bispectral index of the EEG were recorded. Stimulation with light significantly increased blood flow velocity in the posterior cerebral artery (p<0.01, ANOVA). Similar but less pronounced effects were seen after needle acupuncture (p< 0.05, ANOVA) and laserpuncture (n.s.) of vision-related acupuncture points. Furthermore both, laserpuncture and needle acupuncture, led to a significant increase in the amplitudes of 40-Hz cerebral oscillations. Stimulation of vision-related acupuncture points with laser light or needle acupuncture elicits specific effects in specific areas of the brain. The results indicate that the brain plays a key intermediate role in acupuncture. However, brain activity of itself does not explain anything about the healing power of acupuncture.

Keywords: Acupuncture; Brain; 40 Hz brain oscillations; Cerebral blood flow velocity; Laserpuncture; Light stimulation; Middle cerebral artery (MCA); Posterior cerebral artery (PCA); Transcranial Doppler sonography (TCD)

Light Therapy (LLLT) alters gene expression after acute spinal cord injury

K.R. Byrnes 1, R.W. Waynant 2, I.K. Ilev 2, B. Johnson 1, Pollard H. 1, Srivastava M. 1, Eidelman O. 1, Huang, W. 1, J.J. Anders1

1. Department of Anatomy, Physiology and Genetics, Uniformed Services University, Bethesda, MD, USA; 2. Center for Devices and Radiological Health, Food and Drug Administration, Rockville, MD, USA

Secondary injury in the spinal cord, which results in axonal degeneration, scar and cavity formation and cell death, occurs around the site of the initial trauma and is a primary cause for the lack of axonal regeneration observed after spinal cord injury (SCI). The immune response after SCI is under investigation as a potential mediator of secondary injury. Treatment of SCI with 810 nm light suppresses the immune response and improves axonal regeneration.

We hypothesize that these beneficial effects observed in the injured spinal cord are accompanied by alterations in gene expression within the spinal cord, particularly of those genes involved in secondary injury and the immune response. To test this hypothesis, a dorsal hemisection at vertebral level T9 was performed. The injured spinal cord from rat was then exposed to laser light (810nm, 150mW, 2,997 seconds, 0.3cm2 spot area, 1589 J/cm2) and spinal cord samples, including the injury site, were harvested at 6 and 48 hours and 4 days post-injury. Total RNA was extracted and purified from the lesioned spinal cord and cDNA copies were either labeled with [32P] for microarray analysis or amplified and analyzed with a polymerase chain reaction (PCR).

Microarray results revealed a suppression of genes involved in the immune response and excitotoxic cell death at 6 hours post-injury, as well as cell proliferation and scar formation at 48 hours post-injury in the light treated group. Analysis of the PCR products revealed that light treatment resulted in a significant suppression of expression of genes that normally peak between 6 and 24 hours post-injury, including the pro-inflammatory cytokine interleukin 6 (IL6), the chemokine monocyte chemoattractant protein 1 (MCP-1) and inducible nitric oxide synthase (iNOS; p<0.05). Genes expressed earlier than 6 hours post-injury, such as IL1b, tumor necrosis factor a (TNFa) and macrophage inflammatory protein 1a (MIP-1a) were not affected by light treatment.

Although the precise role some of these genes play in axonal regeneration after spinal cord injury is currently unclear, these data demonstrate that light therapy has an anti-inflammatory effect on the injured spinal cord, and may reduce secondary injury, thus providing a possible mechanism by which light therapy may result in axonal regeneration.

Laser Therapy.1997; 9 (4): 151.

An innovative approach to induce regeneration and the repair of spinal cord injury.

Rochkind S, Shahar A. Nevo Z.

An Israeli research group has investigated an innovative method of repairing injured spinal cords. In a rat model the spinal cords were transected in 31 animals (between T7/T8).  In vitro constructed composite implants were used in the transected area. These implants contained embryonal spinal cord neuronal cells dissociated from rat fetuses, cultured on biodegradable microcarriers. After being embedded in hyaluronic acid the implants were ready to be placed into the injured area. The whole lesion area was covered with a thin coagulated fibrin-based membrane. Control animals underwent the same laminectomy but did not receive any implant. In all animals the wound was closed normally. Laser therapy was started immediately after surgery. It was continued daily for two weeks using 780 nm, 200 mW, 30 minutes daily.  One group received the implant but no laser. During the 3-6 months follow up, 14 of the 15 animals that received laser (A) showed different degrees of active movements in one or both legs, compared to 4 of 9 animals in the group who had received implants but no laser (B). In the group receiving no implant and no laser (C), 1 out of 7 showed some motor movements in one leg. Somatosensory evoked potentials were elicited in 10 of the 15 rats in group A at three months, and on one side in one animal in group B. Axon sprouting was observed as soon as three days post surgery, in group A only.

Laser Therapy.1997; 9 (4): 151

New hope for patients with spinal cord injuries.

Rochkind S, Shahar A. Nevo Z.

An Israeli research group has investigated an innovative method of repairing injured spinal cords. In a rat model the spinal cords were transected in 31 animals (between T7/T8).  In vitro constructed composite implants were used in the transected area. These implants contained embryonal spinal cord neuronal cells dissociated from rat fetuses, cultured on biodegradable microcarriers. After being embedded in hyaluronic acid the implants were ready to be placed into the injured area. The whole lesion area was covered with a thin coagulated fibrin-based membrane. Control animals underwent the same laminectomy but did not receive any implant. In all animals the wound was closed normally. Laser therapy was started immediately after surgery. It was continued daily for two weeks using 780 nm, 200 mW, 30 minutes daily.  One group received the implant but no laser. During the 3-6 months follow up, 14 of the 15 animals that received laser (A) showed different degrees of active movements in one or both legs, compared to 4 of 9 animals in the group who had received implants but no laser (B). In the group receiving no implant and no laser (C), 1 out of 7 showed some motor movements in one leg. Somatosensory evoked potentials were elicited in 10 of the 15 rats in group A at three months, and on one side in one animal in group B. Axon sprouting was observed as soon as three days post surgery, in group A only.

Spine (Phila Pa 1976). 1990 Jan;15(1):6-10.

Spinal cord response to laser treatment of injured peripheral nerve.

Rochkind S, Vogler I, Barr-Nea L.

Department of Neurosurgery, Ichilov Hospital, Tel-Aviv Medical Center, Israel.

Abstract

The authors describe the changes occurring in the spinal cord of rats subjected to crush injury of the sciatic nerve followed by low-power laser irradiation of the injured nerve. Such laser treatment of the crushed peripheral nerve has been found to mitigate the degenerative changes in the corresponding neurons of the spinal cord and induce proliferation of neuroglia both in astrocytes and oligodendrocytes. This suggests a higher metabolism in neurons and a better ability for myelin production under the influence of laser treatment.

Lasers Surg Med. 1989;9(2):174-82.

Systemic effects of low-power laser irradiation on the peripheral and central nervous system, cutaneous wounds, and burns.

Rochkind S, Rousso M, Nissan M, Villarreal M, Barr-Nea L, Rees DG.

Department of Neurosurgery, Tel Aviv Medical Center, Ichilov Hospital, Israel.

Abstract

In this paper, we direct attention to the systemic effect of low-power helium-neon (HeNe) laser irradiation on the recovery of the injured peripheral and central nervous system, as well as healing of cutaneous wounds and burns. Laser irradiation on only the right side in bilaterally inflicted cutaneous wounds enhanced recovery in both sides compared to the nonirradiated control group (P less than .01). Similar results were obtained in bilateral burns: irradiating one of the burned sites also caused accelerated healing in the nonirradiated site (P less than .01). However, in the nonirradiated control group, all rats suffered advanced necrosis of the feet and bilateral gangrene. Low-power HeNe laser irradiation applied to a crushed injured sciatic nerve in the right leg in a bilaterally inflicted crush injury, significantly increased the compound action potential in the left nonirradiated leg as well. The statistical analysis shows a highly significant difference between the laser-treated group and the control nonirradiated group (P less than .001). Finally, the systemic effect was found in the spinal cord segments corresponding to the crushed sciatic nerves. The bilateral retrograde degeneration of the motor neurons of the spinal cord expected after the bilateral crush injury of the peripheral nerves was greatly reduced in the laser treated group. The systemic effects reported here are relevant in terms of the clinical application of low-power laser irradiation as well as for basic research into the possible mechanisms involved.

Health Phys. 1989 May;56(5):687-90.

New biological phenomena associated with laser radiation.

Belkin M, Schwartz M.

Goldschleger Eye Research Institute, Tel-Aviv University, Sackler School of Medicine, Tel-Hashomer, Israel.

Abstract

Low-energy laser irradiation produces significant bioeffects. These effects are manifested in biochemical, physiological and proliferative phenomena in various enzymes, cells, tissues, organs and organisms. Examples are given of the effect of He-Ne laser irradiation in preventing the post-traumatic degeneration of peripheral nerves and the postponement of degeneration of the central nervous system. The damage produced by similar radiant exposures to the corneal epithelium and endothelium is also described. It is suggested that the mechanism of laser/tissue interaction at these low levels of radiant exposure is photochemical in nature, explaining most of the characteristics of these effects. These low-energy laser bioeffects are of importance on a basic scientific level, from a laser safety aspect and as a medical therapeutic modality.

Photosensitizers

Curr Med Chem. 2015;22(18):2159-85.

Antimicrobial Photosensitizers: Drug Discovery Under the Spotlight.

Yin R, Hamblin MR1.

Author information

  • 1Wellman Center for Photomedicine, Massachusetts General Hospital, 40 Blossom Street, Boston, MA 02114, United States. Hamblin@helix.mgh.harvard.edu.

Abstract

Although photodynamic therapy (PDT) was discovered over a hundred years ago by its ability to destroy microorganisms, it has been developed mainly as a cancer therapy. In recent years, due to the inexorable rise in multi-antibiotic resistant strains of pathogens, PDT is being considered as a versatile antimicrobial approach to which microbial cells will not be able to develop resistance. The goal of this review is to survey the different classes of chemical compounds that have been tested as antimicrobial photosensitizers. Some of these compounds have been known for many years, while others have been rationally designed based on recently discovered structural principles. Tetrapyrrole-based compounds (some of which are approved as cancer therapies) that efficiently generate singlet oxygen are more efficient and broad-spectrum when they bear cationic charges, As the macrocycle structure moves from porphyrins to chlorins to phthalocyanines to bacteriochlorins the long wavelength absorption moves to the near-infrared where tissue penetration is better. Four main types of natural products have been tested: curcumin, riboflavin, hypericin and psoralens. Phenothiazinium dyes, such as methylene blue and toluidine blue, have been tested, and some are clinically approved. A variety of non-phenothiazinium dyes with xanthene, triarylmethane and indocyanine structures have also been tested. New ring structures based on BODIPY, squaraine and fullerene cages can also mediate antimicrobial PDT. Finally the process of photocatalysis using titanium dioxide can also have medical uses. Designing new antimicrobial photosensitizers is likely to keep chemists engaged for a long time to come.

J Bronchology Interv Pulmonol. 2015 Apr;22(2):99-106. doi: 10.1097/LBR.0000000000000158.

Photothermal ablation of human lung cancer by low-power near-infrared laser and topical injection of indocyanine green.

Hirohashi K1, Anayama T, Wada H, Nakajima T, Kato T, Keshavjee S, Orihashi K, Yasufuku K.

.
Author information
1*Division of Thoracic Surgery, Toronto General Hospital, University Health Network, University of Toronto, Toronto, ON, Canada †Department of Surgery, Kochi University, Kochi, Japan.

Abstract
The present study was designed to evaluate the efficacy of photothermal ablation therapy for lung cancer by low-power near-infrared laser and topical injection of indocyanine green (ICG). In vitro study 1: an 808 nm laser with 250 mW was irradiated for 10 minutes using different dilutions of ICG and the temporal thermal effect was monitored. ICG (1 mL of 0.5 g/L) was heated to a temperature of >30°C from the base temperature by laser irradiation. In vitro study 2: the cytotoxic effect of hyperthermia on human lung cancer cells was examined in different temperature and time settings. Cell viability was quantified by both an MTS assay and reculturing. Fatal conditions evaluated by reculturing were as follows: thermal treatment at 55°C for 5 minutes, 53°C for 10 minutes, and 51°C for 15 minutes. The MTS assay study suggested that thermal treatment at 59°C for 5 minutes and 57°C for 20 minutes showed a severe cytotoxic effect. In vivo study: nude mouse subcutaneous NCI-H460 human lung cancer xenograft models were used for the study. Saline or 0.5 g/L of ICG was injected topically into the tumor (n=3/group). Tumors were irradiated with a laser at 500 mW for 10 minutes. Although the tumor diameter reached 1 cm within 24 days after treatment in all 3 mice using saline/laser, tumor sizes were gradually reduced in all 3 mice using the ICG/laser. In 2 of the 3 mice using ICG/laser, tumors had disappeared macroscopically. The efficacy of the photothermal ablation therapy by low-power near-infrared laser and a topical injection of ICG was clarified using a mouse subcutaneous a lung cancer xenograft model.

.

PLoS One. 2015 Dec 17;10(12):e0145287. doi: 10.1371/journal.pone.0145287. eCollection 2015.

KillerOrange, a Genetically Encoded Photosensitizer Activated by Blue and Green Light.

Sarkisyan KS1, Zlobovskaya OA1, Gorbachev DA1,2, Bozhanova NG1, Sharonov GV1,3, Staroverov DB1, Egorov ES1,4, Ryabova AV5, Solntsev KM6, Mishin AS1,7, Lukyanov KA1,7.
Author information
1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
2Faculty of Biology, Moscow State University, Moscow, Russia.
3Faculty of Medicine, Moscow State University, Moscow, Russia.
4Pirogov Russian National Research Medical University, Moscow, Russia.
5Laser Biospectroscopy Laboratory, Prokhorov General Physics Institute, Moscow, Russia.
6School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, 30332, United States of America.
7Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia.
Abstract
Genetically encoded photosensitizers, proteins that produce reactive oxygen species when illuminated with visible light, are increasingly used as optogenetic tools. Their applications range from ablation of specific cell populations to precise optical inactivation of cellular proteins. Here, we report an orange mutant of red fluorescent protein KillerRed that becomes toxic when illuminated with blue or green light. This new protein, KillerOrange, carries a tryptophan-based chromophore that is novel for photosensitizers. We show that KillerOrange can be used simultaneously and independently from KillerRed in both bacterial and mammalian cells offering chromatic orthogonality for light-activated toxicity.

.Lasers Med Sci. 2014 Jul;29(4):1449-52. doi: 10.1007/s10103-014-1553-0. Epub 2014 Mar 4.

Laser therapy and photosensitive medication: a review of the evidence.

Kerstein RL1, Lister T, Cole R.
Author information
1Salisbury District Hospital, Odstock Road, Salisbury, SP2 8BQ, UK, ryan.kerstein@gmail.com.
Abstract
In the 2009 guidelines from the BMLA, the use of non-essential aesthetic lasers was contraindicated in patients receiving medication that causes whole-body photosensitisation as well as those causing local light sensitisation. Following this and anecdotal advice, many laser centres refuse to treat patients who are on known photosensitive medication. Therefore, specific patient cohorts that would benefit from laser therapy are being denied because of medications, such as long-term antibiotics for chronic facial acne. This article reviews the published literature on lasers and photosensitive medications, the mechanisms of photosensitivity and the role of laser in its production. The aim is to analyse the available evidence regarding adverse reactions to laser treatment related to photosensitive medication. A PubMed review of published article titles and abstracts was performed using the search term Laser with each of the following terms individually: photosensitive, photosensitiser, photosensitizer, phototoxicity, photoallergy, complications, case-report, tetracycline, minocycline, amiodarone, nitrofurantoin and medication. Four publications were identified, none of which reported any complication in the use of laser in patients taking photosensitising medication. As there are no published accounts of adverse effects of laser in patients with photosensitive medication, we performed a review of the mechanism of photosensitivity by compiling a list of photosensitive medication and the peak wavelength of radiation required to activate the drug. We recommend a national database of drugs and the wavelengths causing photosensitive reactions of each which a laser department can access prior to treatment.

Curr Pharm Des. Author manuscript; available in PMC 2012 Oct 3.
Published in final edited form as:
Curr Pharm Des. 2011; 17(13): 1303–1319.
PMCID: PMC3463379
NIHMSID: NIHMS405253

Drug Discovery of Antimicrobial Photosensitizers Using Animal Models

Sulbha K. Sharma,1 Tianhong Dai,1,2 Gitika B. Kharkwal,1,2 Ying-Ying Huang,1,2,3 Liyi Huang,1,2,4 Vida J. Bil De Arce,1George P. Tegos,1,2,5 and Michael R. Hamblin1,2,6,*
1Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA
2Department of Dermatology, Harvard Medical School, Boston, MA
3Aesthetic and Plastic Center of Guangxi Medical University, Nanning, China
4Department of Infectious Diseases, First Affiliated College & Hospital, Guangxi Medical University, Nanning, China
5Department of Pathology, University of New Mexico School of Medicine, 2325 Camino de Salud, Albuquerque, NM
6Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA
Address correspondence to this author at the Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA 02114; Tel: ????????????; ; Fax: 617-726-8566; ude.dravrah.hgm.xileh@nilbmaH
Author information ? Copyright and License information ?

Abstract

Antimicrobial photodynamic therapy (aPDT) is an emerging alternative to antibiotics motivated by growing problems with multi-drug resistant pathogens. aPDT uses non-toxic dyes or photosensitizers (PS) in combination with harmless visible of the correct wavelength to be absorbed by the PS. The excited state PS can form a long-lived triplet state that can interact with molecular oxygen to produce reactive oxygen species such as singlet oxygen and hydroxyl radical that kill the microbial cells. To obtain effective PS for treatment of infections it is necessary to use cationic PS with positive charges that are able to bind to and penetrate different classes of microbial cells. Other drug design criteria require PS with high absorption coefficients in the red/near infra-red regions of the spectrum where light penetration into tissue is maximum, high photostability to minimize photobleaching, and devising compounds that will selectively bind to microbial cells rather than host mammalian cells. Several molecular classes fulfill many of these requirements including phenothiazinium dyes, cationic tetrapyrroles such as porphyrins, phthalocyanines and bacteriochlorins, cationic fullerenes and cationic derivatives of other known PS. Larger structures such as conjugates between PS and cationic polymers, cationic nanoparticles and cationic liposomes that contain PS are also effective. In order to demonstrate in vivo efficacy it is necessary to use animal models of localized infections in which both PS and light can be effectively delivered into the infected area. This review will cover a range of mouse models we have developed using bioluminescent pathogens and a sensitive low light imaging system to non-invasively monitor the progress of the infection in real time. Effective aPDT has been demonstrated in acute lethal infections and chronic biofilm infections; in infections caused by Gram-positive, Gram-negative bacteria and fungi; in infections in wounds, third degree burns, skin abrasions and soft-tissue abscesses. This range of animal models also represents a powerful aid in antimicrobial drug discovery.

Keywords: Antimicrobial photodynamic therapy, cationic tetrapyrroles, phenothiazinium dyes, reactive oxygen species, mouse models of localized infections, bioluminescence imaging, drug discovery

1. INTRODUCTION

Photodynamic therapy (PDT) is an established modality for the treatment of cancer. It has also been extended for the treatment of noncancerous conditions such as age related macular degeneration and other dermatological applications [1, 2]. Besides these applications, there has also been a growing interest in the application of PDT for the treatment of infectious diseases [1]. PDT involves the use of non-toxic dyes that act as photoactive drugs called photosensitizers (PS) in combination with visible light of the appropriate wavelength to excite the PS. The excited state PS, in the presence of the oxygen, transfers energy or electrons to ground state molecular oxygen producing reactive oxygen species (ROS) such as singlet oxygen and hydroxyl radical which are responsible for the killing of cells [3]. When the cells to be killed are pathogenic microorganisms the procedure is termed photodynamic inactivation (PDI) or antimicrobial PDT (aPDT). A diversity of different models of infectious has been used in research for the testing the efficacy of different antimicrobial PS. Before discussing these models, a brief history and overview of antimicrobial PDT and antimicrobial PS will be discussed.

The potential of PDT as an antimicrobial therapy was recognized at the start of the twentieth century when Raab noticed the killing of the paramecia with acridine orange in presence of light [4]. However earlier results showed that the commonly used PS for cancer were poorly effective for the photodynamic killing of some well known pathogens [5]. Moreover it was assumed that the invention of antibiotics would have the lasting potential to combat infectious diseases. Quite the reverse of this, in present times effective therapy for infectious diseases is challenged by the emergence of multidrug-resistant pathogens which is leading to increased morbidity [6]. The difficulty is further aggravated due to a number of mechanisms adopted by microbes to fight against the external insults. These include, thickening of their outer wall, encoding of new proteins which prevent the penetration of drugs or actively efflux them, generation of mutants deficient in the porin channels which permit the entry of externally added chemicals, etc. As a result, it is difficult to identify a broadly applicable approach to overcome this problem [7]. In the 1990s there were reports showing that cationic PS such as phthalocyanines [8], porphyrins [9] and phenothiaziniums [10] induce a rapid and extensive light-mediated killing of typical Gram-negative bacteria, such as Escherichia coli andPseudomonas aeruginosa, in addition to the PDI of fungi and Gram-positive bacteria.

Some of the advantages of aPDT are: (A) It is broad-spectrum and can kill a wide range of microbes such as Gram-positive and Gram-negative bacteria, yeasts, fungi and parasitic protozoa as well as inactivate viruses. (B) There is a low chance of any possibility of developing photoresistant species even after multiple treatments. (C) PS and drug-light intervals can be designed that exhibit selectivity for microbes over host cells and tissue. (D) There is a low risk of inducing mutagenic effects. (E) aPDT kills microbial cells rapidly (minutes) while antibiotics can take days to work. (F) Because PS are topically delivered into infected areas, aPDT can be effective in traumatic infections where the blood supply is compromised preventing antibiotics reaching the microbes. (G) It has been demonstrated that aPDT can be effective in biofilm infections that are resistant to antibiotics. (H) Last but not the least it is inexpensive.

1.1. The General Features of PDI of Microbial cells

Due to the marked difference regarding the size and composition of various microbes there occurs a difference in the susceptibility for various organisms. In the 1990s it was found that basic differences in susceptibility to PDT exist between Gram (+) and Gram (?) bacteria. This was due to difference in morphology: the Gram (+) bacteria are surrounded by a layer of only peptidoglycan and lipoteichoic acid that is comparatively porous, while Gram (?) bacteria have a somewhat more intricate, non-porous cell wall structure consisting of an inner cytoplasmic membrane and an outer membrane, which are separated by the peptidoglycan-containing periplasm (Fig. 1). Fungal cells have intermediate permeability between Gram-positive and Gram-negative bacteria. Besides this, cysts formed by protozoa also represent challenging targets. Thus the procedure adopted for the treatment of infections cannot be focused on just one type of pathogen; rather it must be characterized by the possibility to efficiently act on microbial pathogens with very different characteristics.

Fig. (1)

Structures of the cell walls of three different classes of microbial pathogens

1.2. Photobiological Processes

The photodynamic action occurs by two mechanisms Type I and Type II. The ground state photosensitizer on absorption of a photon is converted into its long-lived triplet state via a short-lived singlet state. This triplet state is the reactive intermediate. In type I mechanism the triplet state PS transfers an electron to ground state molecular oxygen to produce reactive oxygen species (ROS) such as superoxide, hydroxyl radicals and peroxides. While in the type II mechanism the triplet state of the PS reacts undergoes energy transfer to the ground state of oxygen which is in triplet state to give another ROS very reactive species i.e. singlet oxygen (Fig. 2). This singlet oxygen then further reacts with the surrounding bio-molecules. The main molecules targeted by both the mechanisms are certain amino acids, pyrimidine and purine bases of DNA/RNA, and unsaturated lipids. The wide range of biomolecules damaged by ROS means that the spectrum of microbial targets of PDT is very broad.

Fig. (2)

Schematic mechanism of antimicrobial PDT

Some of the properties considered to be favorable for ideal antimicrobial PS: (1) The photosensitizer should have long-lived excited triplet state; and a high quantum yield for the generation of ROS on excitation with visible light. (2) It should have high extinction coefficient mainly in the red and far-red region where light transmission through tissue is maximal. Though for the treatment of superficial infections also the intensely absorbed blue light (400–420 nm) is useful. (3) A large affinity for the broadest possible range of microbial cells. (4) The PS should bind selectively to the cytoplasmic membrane, due to which the cell death will be mainly due to damage of the membrane rather than the genetic material. (5) The mechanisms involved in photodynamic inactivation should have no mutagenic effect. (6) A broad spectrum of action on bacteria, fungi, yeasts and parasitic protozoa (Figs. 2 and ?and3),3), to help the treatment of those infectious diseases which are considered to be due to the presence of a varied flora of pathogens. (7) The cell-selective binding conferred by the molecular structure should be such that there is maximum damage to the microbes with minimal damage to the host tissue.

Fig. (3)

Broad spectrum of effect of antimicrobial PDT

The advantage of the broad-spectrum exhibited by PDT (see Fig. 3) means that it could be used to treat a localized infection before the clinical microbiology laboratory identified the culprit microbe, and the appropriate antibiotic was selected. Moreover a more limited range of antimicrobial photosensitizers could be stocked in pharmacies compared to the wide range of antibiotics needed now.

2. DRUG DISCOVERY AND ANTIMICROBIAL PDT

It has been known for many decades that Gram-positive bacteria are highly susceptible to killing by traditional PS with the same molecular features as those PS used to kill cancer cells (such as porphyrins) as well as other types of photoactive dyes [11]. In the 1990s it was discovered that PS with cationic charges could kill Gram-positive bacteria, which had previously been thought to be resistant to many aPDT regimens [8, 9, 12]. Other classes of pathogens such as viruses (both enveloped and non-enveloped) [13], yeasts [14,15], filamentous fungi [16], protozoa [17], parasites [18, 19] etc, have been reported to be susceptible to aPDT mediated by cationic PS. We will give some examples of molecular structures that have been investigated as antimicrobial PS.

2.1. Phenothiazinium Dyes

Structures of members of this class are shown in Fig. 4; these compounds have a single cationic charge that is delocalized over the three-ring structure. Methylene blue (MB) 1, and toluidine blue O (TBO) 2, are probably the most-widely studied members of this class [2022]. Compounds such as these have the additional advantage that MB is clinically approved as an injectable IV therapy for methemoglobinemia [22] and both MB and TBO are generally accepted as safe for topical application to living human tissue [23]. Other members of the class that have been used as antimicrobial PS include new methylene blue 3 [24] and dimethyl-methylene blue 4 [25]. It is generally accepted that these latter compounds are more powerful antimicrobial PS than MB and TBO [26]. Interestingly we previously showed that members of this class of phenothiazinium dyes were substrates of microbial drug-efflux systems [27], and that aPDT could be potentiated by combining the phenothiazinium dye with an inhibitor of the drug-efflux pump [28]. Related structures are the benzophenoxazines and their sulfur and selenium analogs [29].

Fig. (4)

Structures of phenothiazinium dyes

2.2. Cationic Porphyrins

Porphyrins can be synthesized bearing cationic groups that are usually attached to phenyl groups substituted in the meso-position of the porphyrin macrocycle (Fig. 5). Merchat et al [9] reported that cationic meso-substituted porphyrins, namely tetra(4N-methyl-pyridyl) porphine tetraiodide (T4MPyP) 5, and tetra(4N,N,N-trimethyl-anilinium) porphine tetraiodide (T4MAP) 6, effectively mediated aPDT Gram-negative bacteria. Another cationic porphyrin, 5-phenyl-10,15,20-tris(N-methyl-4-pyridyl)-porphyrin chloride (PTMPP) or Sylsens B, 7 was shown to be to an effective and versatile antimicrobial PS that was able to kill bacteria, Candida, and the dermatophyte Trichophyton rubrum [3032]. Maisch et al reported [33] that bis-cationic porphyrins such as XF70 8 were broad spectrum antimicrobial PS, giving good photokilling of methicillin-resistant and methicillin-sensitive S. aureus strains, methicillin-resistantStaphylococcus epidermidis and E. coli. Subsequent reports suggested that these porphyrins could even be used as antibacterial compounds in the dark [34, 35]. Other reports have used a wide range of substituted cationic porphyrins to mediate a PDT of diverse species of pathogens [3640].

Fig. (5)

Structures of cationic porphyrins

2.3. Cationic Phthalocyanines

Phthalocyanines are another class of tetrapyrrole dyes that have been synthesized with cationic substituents to render them suitable for aPDT (see Fig. 6). Unlike porphyrins, phthalocyanines are usually prepared with central coordinated metal atoms to prevent aggregation and enhance photochemical properties. The diamagnetic metal ions such as zinc(II) impart a high fluorescence quantum yield, long triplet lifetimes and high triplet quantum yields which lead to a high probability of energy or electron transfer. Phthalo-cyanine zinc(II) molecules with different charges were evaluated as PS to kill bacteria by Minnock et al [8] who showed that Gram-negative bacteria could be photoinactivated when illuminated in the presence of a tetra-cationic water-soluble zinc pyridinium phthalo-cyanine, Zn-PPC, 9.

Fig. (6)

Structures of cationic phthalocyanines

Mantareva et al. [41] showed that another cationic phthalo-cyanine with four cationic groups 10 was able to photoinactivate Gram-positive Staphylococcus aureus, the Gram-negative Pseudomonas aeruginosa, and the fungal species Candida albicans.

A recent paper [42] from the same group described a Ga(III)-substituted phthalocyanine with eight cationic groups 11 that was able to mediate photoinactivation of Gram-positive, Gram-negative bacteria and Candida species in planktonic form an in biofilms.

Roncucci et al showed [43] that another tetracationic Zn-PC 12 designated as RLP068 [17] could photoinactivate S. aureus, P. aeruginosa and C. albicans and moreover did not generate any resistance even after 20 successive cycles of sub-lethal PDT and regrowth.

2.4. Cationic Bacteriochlorins

Bacteriochlorins are porphyrins with two opposed reduced pyrrole rings in the macrocycle; a molecular feature that imparts an intense absorption band in the NIR spectrum (>700-nm). In a similar manner to porphyrins they can be synthesized with peripheral cationic groups to give antimicrobial PS. Using completely synthetic methodology we prepared compounds that were rendered stable by introduction of gem-dimethyl groups that prevented adventitious re-oxidation of the reduced rings [44]. Compounds such as bis-cationic 13, tetrakis cationic 14, and hexakis cationic 15 shown in Fig. 7 were effective in killing Gram-positive, Gram-negative bacteria and fungi. Moreover they showed good selectivity for microbial cells over host

Fig. (7)

Structures of cationic bacteriochlorins

2.5. Cationic Fullerenes

Fullerenes are closed cage molecules composed entirely of carbon atoms, and the most widely studied member of this class is C60, was originally named buckminsterfullerene [45]. The extended system of conjugated double bonds present in the spherical molecule mean that these molecules absorb extensively in the visible region of the spectrum as well as the UV region. Moreover they have a triplet yield approaching unity and hence no fluorescence. The long-lived triplet state can undergo either energy transfer to produce singlet oxygen or electron transfer to produce superoxide and subsequently hydroxyl radical. Pristine C60 is highly insoluble but functionalization of the periphery with the appropriate groups imparts water solubility and if these groups are cationic, the fullerenes can act as highly effective antimicrobial PS as shown in Fig. 8. The tris-cationic fullerene 16 was shown by our laboratory [46] to be a broad spectrum PS able to mediate photokilling of Gram-positive, Gram-negative bacteria and fungi. Another study from our group [47] showed that cationic fullerenes such as 17 with 6 cationic groups was also highly effective. Other laboratories [48] have also shown that cationic fullerenes such as 18 (2 cationic groups) are also effective against Gram-negative bacteria.

Fig. (8)

Structures of cationic fullerenes

2.6. Miscellaneous Cationic PS

Nonell’s laboratory in Barcelona, Spain has developed PS based on the porphyrin-structural isomer backbone known as porphycenes [49]. By synthesizing a porphycene 19 with 3 cationic groups a broad-spectrum antimicrobial PS was obtained [50], that could kill Gram-positive and Gram-negative bacteria, as well as a fungal yeast. Moreover it was also able to effectively treat an in vivo mouse infection model using aPDT.

A group from Dublin, Ireland has developed a new class of PS based on brominated BF2 chelated tetraarylazadipyrromethane dyes [51]. By adding two cationic groups to this backbone to give compound 20a broad-spectrum antimicrobial PS was obtained [52].

Hypericin is a natural produce isolated from St John’s Wort and has been used as a PS for PDT of cancer. Hager et al [53] synthesized bis-cationic derivates of hypericin such as 21 and demonstrated these PS could be used for PDI of Propionibacterium acnes.

2.7. Conjugates Between PS and Cationic Polymers

In 1997 we formed the hypothesis [54] that by covalently attaching a non-cationic PS (chlorin(e6), ce6) to amino-groups present on a cationic polymer such as poly-L-lysine (pL) to form pL-ce6 conjugates, interesting microbial-targeted PS could be prepared (Fig. 9). Various forms of the cationic pL-ce6 22 was shown to be effective in photoinactivating both Gram-positive and Gram-negative bacteria [55, 56]. A similar synthetic approach using poly-ethylenimine (PEI) allowed the formation of PEI-ce6 243 that had the additional advantages of being protease stable and also more cost effective [57]. The concept of polycationic PS conjugates proved to be fruitful and has been extensively studied both in our laboratory [5866] and by others [6769].

Fig. (9)

Structures of miscellaneous cationic PS

2.8. PS Encapsulated Into Cationic Liposomes and Nanoparticles

It has been shown that by encapsulation of PS in cationic liposomes rather than the more usual liposomes composed of anionic or neutral lipids, the ability of non-cationic PS such as m-tetrahydroxyphenylchlorin (mTHPC) to kill microbial cells can be enhanced [70]. A similar report [71] used cationic liposomes composed with DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate) and containing either hematoporphyrin or chlorophyll a to mediated aPDT of MRSA.

Likewise the explosion of interest in nanoparticles has led to researchers preparing nanoparticles containing both PS and cationic charges for antimicrobial photoinactivation. Schwiertz et al [72] found that calcium phosphate nanoparticles containing PS and bearing cationic charges were superior PS delivery vehicles especially for Gram-negative P. aeruginosa. Ferro and coworkers [73] compared a monocationic porphyrin (5-[4-(1-dodecanoylpyridinium)]-10,15,20-triphenyl-porphine) (TDPyP) complexed into supramolecular aggregates of cationic amphiphilic beta-cyclodextrin with the same porphyrin encapsulated into cationic liposomes [74]. The cationic cyclodextrin porphyrin was more effective in mediating photoinactivation of both Gram-positive and Gram-negative bacteria than the cationic liposome formulation.

2.9. Conjugates Between PS and Antibodies

One exception to the rule that cationic groups are required to produce PS that can efficiently photoinactivate Gram-negative bacteria appears to be the use of monoclonal antibodies (mAb) conjugated to PS. A conjugate between mAb NO76 and Sn-ce6 [75] was used by Yarmush et al to kill the resistant Gram-negative speciesPseudomonas aeruginosa both in vitro [76] and in vivo [77]. The conjugate did not kill S. aureus to which the mAb did not bind. This finding (lack of cationic charge) may be explained by the tight binding between antibody and bacterial cell.

3. BIOLUMINESCENCE IMAGING OF INFECTION MODELS

3.1. Rationale for Bioluminescence Imaging of Infection Models

Animal models have become standard tools for the study a wide array of antimicrobial therapies of wound infections, including antimicrobial PDT. Mice are by far the most frequently used species in wound infection models. The principal disadvantages of mouse models relate to the small size of the animals. Hence, for example, one is limited to the number of sequential sampling of blood, other fluids, and tissues that can be performed without compromising the mouse. As a result, in vivo studies of PDT on mouse infection models suffer from difficulties in monitoring the development of an infection in mice and its response to treatment. Standard microbiological techniques used to follow infections in animal models frequently involve sacrifice of the animals, removal of the infected tissue, homogenization, serial dilution, plating and colony counting. These assays use a large number of animals, are time consuming, and often are not statistically reliable.

In order to facilitate the non-invasive monitoring of animal models of infection, we have developed a procedure that uses bioluminescent genetically-engineered bacteria and a light sensitive imaging system to allow real-time visualization of infections. When these bacteria are treated with PDT either in vitro or in vivo, the loss of luminescence parallels the loss of colony-forming ability. We have developed several mouse models of localized infections that can be followed by bioluminescence imaging (BLI) [78].

BLI can be used either to track the course of an infection or monitor the efficacy of antimicrobial therapies (see Fig. 11). Bacterial pathogenesis appeared to be unaffected by the presence of the luciferase genes, and bioluminescence can be detected throughout the study period in animals. Further, the intensity of the bioluminescence measured from the living animal correlated well with the bacterial burden subsequently determined by standard protocols [7981]. Transposon-mediated integration of the luciferase operon into the bacterial chromosome to make stable transformants means that reduction of luminescence from sites of infection in animals can be attributed to reduction of bacterial numbers rather than loss of luciferase-encoding plasmids.

Fig. (11)

Schematic illustration of bioluminescence imaging to monitor PDT response in infection models

3.2. Tracking the Course of Infections Using Bioluminescence Imaging

Different localized-infection models in various experimental animals have been used by different groups to demonstrate the utility of bioluminescence imaging using bioluminescent pathogens and repetitive imaging over the course of hours, days or weeks. A broad range of variables must be taken into account when designing these protocols. These variables are: (A) The species and strain of pathogen taking into account its rate of growth, and its specific virulence and pathogenicity characteristics that may on occasion be species specific to the animal model being employed. (B) The number of cells (CFU) innoculated into the infection together with the precise composition of the liquid in which the microbes are suspended. (C) The type of injury or trauma appplied to damage the tissue that allows entry of the pathogens. (D) The immune staus of the animals; in many cases specific immunosuppression strategies must be applied that can either be pharmacological or genetic. (E) The presence of a foreign body in the infected area; foreign bodies can dramatically potentiate infection.

When the bioluminescent microbes have been introduced into the lesion it is frequently found that a large number of the cells die in the hours following their introduction. This phenomenon is probably due to evaporation of liquid present in the innoculum and the fact that the pathogens need time to establish a source of nutrients from the tissue. When the parameters have been selected correctly it will be found that the surviving microbes then proceed to regrow and a true infection is then established. Depending on the microbes and the tissue involved a biofilm may be formed; this occurrence often leads to a chronic infection being formed that can last for several weeks. Again depending on the virulence and invasiveness of the microbes, it is possible that the microbes may reach the systemic circulation of the animal and then bacteremia or sepsis ocurs that can often be fatal. This event is easily established by culture of bioluminescent microbes from the blood or internal organs such as heart or liver. In many cases when the infection does not become systemic, as the tissue heals the bioluminescent microbes become confined in a scab or in an abscess due to the body’s natural response to wall-off infection, and this scab can eventually fall of taking the last traces of bioluminescence with it.

3.3. Bioluminescence Imaging to Demonstrate the Efficacy of PDT

To bring aPDT towards a clinical treatment it is necessary to show its effectiveness in treating actual infections rather than just inactivating pathogens in vitro. In contrast to PDT of cancer where delivery of PS is generally carried out be intravenous injection of the PS frequently associated with an appropriate delivery vehicle, adminstration of the PS for a PDT of infections is different. In this case the PS is delivered into or onto the infected area. For superficial infections this may simply be accomplished by pipetting a solution of PS onto the surface of the tissue, but for deeper infections it may involve injection or infiltration of the PS into the infected area. The delivery vehicle employed is also likely to be different for PS used for cancer and PS used for infections. For cancer the appropriate PS are much more hydrophobic and delivery vehciles such as liposomes, detergent micelles or orgaic solvent mixtures are common. For aPDT the PS employed are hydrophilic and generally water soluble. In some cases adding a proportion of a water miscible organic solvent to the aqueous solution of PS does help penetration into the infected tissue.

The drug-light interval employed in aPDT is also very different from that which is usual in PDT for cancer. In cancer applications 24 hours or even longer is a common interval, although some PS do have shorter intervals such as a few hours. By contrast in aPDT applications it has been found that drug-light intervals as short as a few minutes are optimal. The reasons for this are the fact that binding and penetration of PS to microbial cells is relatively rapid process. Uptake of the PS into mammalian cells that compose the infected tissue is a much slower process, so the desirable selectivity for microbes over host tissue is a time-dependent process and can be maximized at short drug-light intervals.

Light delivery is often as simple as shining the light as a spot that covers the infected area. One finding that impacts the methodology of carrying out aPDT is the discovery that photobleaching is an important limiting factor in the efficay of the antimicrobal PDI [82]. Photobleaching is the chemical degradation of the molecular structure of the PS by the ROS produced during PDT, that can reduce its effectiveness in killing microbes. However this limitation can be overcome by repeating the addition of the PS at intervals during the illumination [58].

Another consideration that is important in aPDT, is the realization that the relative masses of microbial cells and host cells in the infected tissue is very different. This means that the total dose of PDT (PS concentration and fluence of light) is much larger (100–1000 times larger) than the dose that is needed to kill microorganisms in vitro. A recent publication [83] provided a perspective on why this should be. Since the definition of infection is 10(5) CFU/g tissue and 10(5) bacteria have a total mass of only 1 microgram, it is evident that there is 1 million times more mass of tissue than there is mass of bacteria and this provides a real challenge for selective binding of the PS to the microbial cells rather than the host cells. Even if the infection is very severe (10(8) CFU/g tissue) the ratio is still 1000:1.

In actual practice the experimental application of aPDT for localized infections comprises the following steps. (A) Establish the infection and monitor it by BLI as described in section 3.2. (B) Apply the PS to the infected area and monitor by a second BLI procedure, any dark toxicity that may happen when the PS kills some microbial cells by its innate ability to penetrate and permeabilize the cells. (C) After a short time begin illumination and after a suitable amount of light has been delivered (for instance 10–50 J/cm2) carry out a third BLI. (D) Repeat PS addition and light delivery enough times to ensure maximum eradication of bioluminescence signal. (E) It may be necessary to deliver even more PDT than appears to be necessary judging by BLI in order to reduce the possible regrowth of microbes after the procedure. (F) Monitor any regrowth of microbes within the infection in succeeding days by daily BLI procedures. In some cases when regrowth does occur, a second aPDT procedure after some days may give a second useful reduction in bioluminescence signal. (G) When regrowth of microbes in succeeding days is problematic, it may be possible to combine aPDT with an antibiotic or other traditional antimicrobial that is microbistatic rather than microbicidal and can prevent re-growth [84].

4. PDT FOR WOUND INFECTIONS

The first mouse model of localized infection using bioluminescent bacteria to be utilized for aPDT was a model of simple excisional wounds that were superficially inoculated with bacteria. Infected wounds can be problematic in elderly patients, for some surgical patients, for non-healing leg ulcers and for battlefield combat casualties such as open fractures. Hamblin et al [66] developed a mouse model of excisional wound infections. In that model, four rectangular full-thickness excisional wounds were made in a line along the back of shaved male BALB/c mice. Wounds measured 8-mm × 12.5-mm and had at least 5 mm of unbroken skin between them. The bottom of the wound was panniculus carnosus with no visible bleeding. A suspension (50 ?L PBS) containing 5 ×106 cells of mid-log phase bioluminescent E. coli was inoculated into each wound, and the mouse was imaged with the luminescence camera to ensure equal bacterial loading into each wound. The next day, infected wounds in living mice had lost, on an average, 90% of the original luminescence signal but with considerable inter-animal variability. A rapid light dose-dependent loss of luminescence was observed as measured by image analysis after PDT with polycationic photosensitizer pL-ce6 conjugate 22. Fig. 12A shows dose response bacterial luminescence to PDT from a representative mouse in which bacteria were inoculated in all wounds, pL-ce6 22 was added to wounds 1 and 4, and wounds 3 and 4 were illuminated with red light. Therefore, wound 1 was the dark control with conjugate, wound 2 was the absolute control, wound 3 was the light-alone control and wound 4 was PDT treated. Topical application of pL-ce6 22 followed by laser illumination at 665 nm led to a 99% reduction in bacterial luminescence.

Fig. (12)

PDT for infected excisional wounds

The mouse model was also used by the same group [65] to test the efficacy of aPDT against the infections induced by a more invasive species, P. aeruginosa. In this case the highly virulent bacteria will rapidly reach the blood stream of the mice and then death will ensue. Mice with single wounds measuring 8-mm312.5-mm received 5×106 mid-log phase P. aeruginosa suspended in 50 mL of PBS. The pL-ce6 conjugate 22 was added as 50 uL of a 200-uM ce6 equivalent concentration. To allow the conjugate to bind to and penetrate the bacteria, illumination at 665 nm was commenced at 30 minutes after the inoculation of bacteria. As can be seen from a set of luminescence images from a representative mouse, shown in Fig. 12B, PDT produced a fluence-dependent loss of luminescence, until only a trace remained, after 240 J/cm2 had been delivered. Ninety % of the PDT-treated mice survived (9 out of 10), in contrast, all of non-treated control mice (n=10) died within 5 days. Furthermore the wounds treated with PDT healed better than wounds that were treated with an alternative topical antimicrobial (silver nitrate). This improvement in healing was attributed to the fact that PDTR can also destroy protease enzymes responsible for slowing down wound healing.

5. PDT FOR BURN INFECTIONS

Skin is the first line of defense providing body with a physical barrier against several pathogens including bacteria, viruses and fungi. Impairment of this important defensive function renders the skin susceptible to infections from otherwise harmless microorganisms. One of the injuries that compromise skin’s protective role is the burn injury. Not only do the burns breach the cutaneous barrier, but severely burned sites are rendered avascular, immunosuppressed, and are rich in bacterial nutrients. Consequently burns are highly susceptible to infections and large burns that occupied a high % of total body surface area often proved to be fatal in the past due to infectious complications. The microbial species responsible for these invasive burn infections include the ubiquitous pathogens P. aeruginosa, S. aureus, Candida spp., and filamentous fungi. With the use of techniques like early excision, grafting, topical antibiotics and antimicrobials, there has been a dramatic improvement in the survival rates following burn infection. However, development of microbial resistance to antibiotics and other antimicrobials has led to a renewed search for alternative approaches to prevent and combat burn infections. Antimicrobial PDT has emerged as a promising alternative to antimicrobial agents. Photodynamic inactivation of S. aureus, A. baumannii, and MRSA has been shown to be effective in animal models of infected burns in mouse and guinea pig.

aPDT of S aureus in a mouse burn model has been evaluated [85]. To create the burn injury, ends of two preheated brass rods (?95 °C) were pressed against opposite sides of a raised dorsal skin fold for 10 s (see Fig. 13). This created a third degree burn covering 2cm2 or 5% of the body surface area. Ten minutes later, burn wounds were infected with bioluminescent S. aureus and allowed to multiply in the wound for 24 h to establish an infection. At the end of 24 h, 5-phenyl-10,15, 20-tris(N-methyl-4-pyridyl)-porphyrin chloride (PTMPP, Sylsens B, 7), a cationic porphyrin that has proved to be an effective and versatile antimicrobial PS, was applied both topically and injected under the burn. The burns were illuminated directly after the application of the PTMPP with red light and periodic imaging of the mice using a sensitive camera to detect the bioluminescence signal. More than 98% of the bacteria were eradicated after a light dose of 210 J/cm2 in the presence of PTMPP. However, bacterial re-growth was observed. Collectively these data suggested that PDT had the potential to rapidly reduce the bacterial load in infected burns but treatment needed to be optimized to reduce wound damage and prevent recurrence [85].

Fig. (13)

Schematic illustration of procedures involved in carrying out PDT for burn infection

PDT of Acinetobacter baumannii in a burn wound infection was studied by Dai et al [59] using polyethylenimine chlorine (e6) (PEI-ce6) conjugate 23 and non-coherent red light at 660-nm. The burn wounds were created as described by Lambrechts et al [85]. Five minutes after the creation of the burns, a suspension of luminescent A. baumannii containing 108 cells was inoculated onto the eschar of each burn. This led to chronic infections that lasted, on average, 22 days and was characterized by a remarkably stable bacterial bioluminescence. Starting PDT on day 0 was more effective in reducing bacterial luminescence (3-log10 units) than on day 1 or day 2 (approximately 1.7-log10 reduction). Fig. 12A shows the PDT dose response of bacterial luminescence of a representative mouse burn infected with A. baumannii and treated with PDT on day 1 (24 hours) after infection. PDT induced approximately 1.8 logs reduction of bacterial luminescence from the mouse burn (Fig. 14A). Bacterial re-growth in the treated burn was observed but was generally modest. Also the PDT did not lead to inhibition of wound healing. The data suggest that PDT may be an effective new treatment for multi-drug resistant localized A. baumannii infections.

Fig. (14)

A) PDT dose response of bacterial luminescence from a representative mouse burn infected with A. baumannii and treated with PDT using PEI-ce6 conjugate 23. B) PDT dose response of bacterial luminescence from a representative mouse burn infected with

The same model was used by Ragas et al [86] to demonstrate PDT mediated by the phenothiazinium dye NMB 3. NMB was applied 30 minutes after infection followed by illumination with 180 J/cm2 of red light at 635-nm. As shown in Fig. 14B, PDT of A. baumannii led to a 3.2-log10 reduction of the bacterial luminescence after 360 J/cm2 had been delivered.

The third degree mouse burn model as described above was also used by Ragas et al to test the photodynamic efficacy of a cationic porphycene 19 against drug resistant MRSA (methicillin resistantStaphylococcus aureus). 5 minutes after creation of burns, 50 ul of bacterial suspension (108 cells) was inoculated onto the surface of each burn with a pipette tip and then smeared onto the burn surface with an inoculating loop. Porphycene 19, applied thirty minutes later followed by illumination with 180 J/cm2 of red light, led to a 2.6-log10 reduction of MRSA bioluminescence [50] as seen in Fig. 14C.

6. PDT FOR ABRASION INFECTION MODEL

Contact sports such as American football, basketball, wresting, and rugby inevitably lead to skin and soft-tissue injuries that place athletic population at increased risk for infection. Skin infections, particularly those caused by Methicillin-resistant Staphylococcus aureus (MRSA), are common among the athletes with a prevalence of over 10% [87]. The skin injuries occurred in contact sports are mostly cutaneous traumas such as cuts, abrasions, turf burns etc. In some cases, significant morbidity can occur, and in some other cases infections result in life threatening conditions [88]. Not only do infections present a public health concern, they can also disrupt or potentially eliminate a team’s chance to compete at the highest level [89].

A mouse model of skin abrasion wound infected with MRSA was developed by Dai et al. [58]. Bioluminescent strain of MRSA was used to allow the real time monitoring of the extent of infection in mouse wounds. Skin abrasions were made within defined 1×1 cm2 areas on the backs of mice using 28 gauge needles. The abrasions were made in such a manner that they only damaged the stratum corneum and upper-layer of the epidermis but not the dermis. Ten minutes after wounding, an aliquot of 50 ?L suspension containing 108 CFU of bioluminescent MRSA was inoculated to each wound. Fig. 15B shows the successive bioluminescence images of a representative abrasion wound in a mouse treated with PEI-ce6 23 in the dark. As indicated by the bacterial luminescence, the infection remained strong and stable until day 5 post-infection and detectable until day 12. Fig 15A shows the corresponding reduction in bioluminescence image intensity observed when red light was added to PEI-ce6 23. Fig. 15C quantifies the bioluminescence signals and presents the data as a time course over the whole course of the observation period until both treated and untreated abrasions healed (day 14). Fig 15D shows the Kaplan-Meier wound healing curves of the non-treated mice (n=12) and the mice treated with PDT (n=10). Statistical analysis indicated that PDT treated mice had a significant advantage in wound healing over the non-treated mice. The average wound healing times of the PDT treated mice and non-treated ones were 5.6±5.1 and 14.2±2.6 days (p=0.0002), respectively. In 6 out of 10 of the PDT treated mice, complete wound healing was achieved within 4 days post-infection. The reason for the large advantage of wound healing in the PDT group probably reflects the fact that an abrasion has unbroken skin between the scratches that can form a nucleus for wound healing. However this normal skin can be destroyed by the bacteria if the infection is untreated, leading to a uniform large wound that takes much longer to heal. Findings from this study demonstrated that PDT significantly reduced the bio-burden of MRSA in the mouse wounds, which would otherwise develop severe infections. In addition, wound healing and morbidity (body weight loss) were greatly benefited by the eradication of MRSA from the wounds. Photodynamic therapy may represent an alternative approach for the treatment of MRSA skin infections.

Fig. (15)

PDT for skin abrasion infected with MRSA

7. PDT FOR CANDIDA INFECTION MODEL

A genus of fungi, Candida spp, is a common commensal inhabitant on human mucosal surfaces and skin, yet when that outer layer of protection is compromised, Candida can cause local infection, and in immunocompromised individuals it can infect deeper layers and if it becomes systemic, can be lethal. Doyleet al. [90, 91] created C. albicans strains expressing the firefly luciferase gene under the control of the strongC. albicans ENO1 promoter and showed the fungal cells could be detected in animals with induced vulvovaginal candidiasis that had been subjected to a vaginal lavage with a solution containing luciferin. However, this in vivo reporter system only allowed limited bioluminescent detection of C. albicans in vivoprobably due to the limited permeability of hyphal cells to luciferin. This is a drawback since the yeast-to-hyphal transition is a major virulence determinant in this species [92]. D’Enfert’s laboratory recently overcame this limitation by stably transforming a synthetic codon optimized Gaussia princeps luciferase gene fused to C. albicans PGA59, which encodes a glycosylphosphatidylinositol-linked cell wall protein. Expression of the luciferase was localized at the C. albicans cell surface, allowing efficient detection of luciferase in intact cells without the necessity of the substrate, coelenterazine, penetrating into the cell.

Fungal cells prefer relatively low temperatures for growth compared to bacteria. While most pathogenic bacteria grow well at body temperature (37°C), this property is rare amongst fungi and yeasts [93]. The requirement for lower temperatures means that fungal infections on skin, mucosa, and burns tend to be superficial in nature and therefore more susceptible to aPDT.

We have tested aPDT in a mouse model of localized C. albicans infection. After screening several antimicrobial PS we settled on the phenothiazinium dye, new methylene blue (NMB) 3. Since Candida spp are eukaryotic cells, the advantage provided by PS structures with multiple cationic charges that has been clearly demonstrated in prokaryotic bacterial cells, is less pronounced, and more lipophilic less cationic molecules perform better.

We developed a new mouse model of C. albicans in a skin abrasion formed by scraping the surface of the skin with a blade. Then a suspension (40 ?L) of C. albicans in sterile phosphate buffered saline (PBS) containing 106 cells was inoculated onto the surface of the abrasion. Twenty ?L coelenterazine (Gaussia princeps luciferase substrate) was topically applied to the surface of each infected abrasion. Mice were then placed in the bioluminescence imaging camera. Twenty-four hours later topical application of NMB 3solution followed by illumination with red light produced a light-dose dependent reduction of bioluminescence as seen in Fig 16.

Fig. (16)

PDT of a Candida albicans infection

8. CONCLUSION AND FUTURE PROSPECTS

Judging by the exponential growth of published studies in antimicrobial PDT both in vitro and in vivo (see Fig. 17), the field is only going to expand further in years to come. The clinical approval in Europe and Canada of the Periowave System made by Ondine Biopharma for treating periodontitis by applying MB dye into the dental pocket followed by light delivery into the pocket using a fine fiber optic has led to over 50,000 clinical procedures being performed. The number of small companies entering this field combined with growing antibiotic resistance and the public’s tendency to distrust big pharmaceutical companies also suggests that the growth of a PDT will continue. Good animal models of localized infections suitable for testing aPDT will continue to be in demand. Bioluminescence imaging dramatically facilitates this animal testing.

Fig. (17)

Antimicrobial PDT is a rapidly growing field

?

Fig. (10)

Structures of conjugates between PS and cationic polymers

Acknowledgments

Research in the Hamblin laboratory is supported by NIH grants (R01A1050875 and R01CA/AI838801to MRH; R01CA137108 to Long Y Chiang), US Air Force MFEL Program (FA9550-04-1-0079), Center for Integration of Medicine and Innovative Technology (DAMD17-02-2-0006), CDMRP Program in TBI (W81XWH-09-1-0514).

References

1. Levy JG, Obochi M. New applications in photodynamic therapy. Introduction Photochem Photobiol.1996;64:737–9. [PubMed]
2. Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D, Korbelik M, Moan J, Peng Q. Photodynamic therapy. J Natl Cancer Inst. 1998;90:889–905. [PMC free article] [PubMed]
3. Ochsner M. Photophysical and photobiological processes in the photodynamic therapy of tumours. J Photochem Photobiol B. 1997;39:1–18. [PubMed]
4. Raab O. Über die Wirkung fluoresceinder Stoffe and Infusorien. Zeit für Biol. 1900;39:524–546.
5. Wainwright W. Photoantimicrobials – a PACT against resistance and infection. Drug Future. 2004;29:85–93.
6. Gleckman RA, Borrego F. Adverse reactions to antibiotics. Clues for recognizing, understanding, and avoiding them. Postgrad Med. 1997;101:97–8. 101–4, 107–8. [PubMed]
7. Smith TL, Pearson ML, Wilcox KR, Cruz C, Lancaster MV, Robinson-Dunn B, Tenover FC, Zervos MJ, Band JD, White E, Jarvis WR. Emergence of vancomycin resistance in Staphylococcus aureus. Glycopeptide-Intermediate Staphylococcus aureus Working Group. N Engl J Med. 1999;340:493–501.[PubMed]
8. Minnock A, Vernon DI, Schofield J, Griffiths J, Parish JH, Brown ST. Photoinactivation of bacteria. Use of a cationic water-soluble zinc phthalocyanine to photoinactivate both gram-negative and gram-positive bacteria. J Photochem Photobiol B. 1996;32:159–64. [PubMed]
9. Merchat M, Bertolini G, Giacomini P, Villanueva A, Jori G. Meso-substituted cationic porphyrins as efficient photosensitizers of gram-positive and gram-negative bacteria. J Photochem Photobiol B.1996;32:153–7. [PubMed]
10. Wilson M, Burns T, Pratten J, Pearson GJ. Bacteria in supragingival plaque samples can be killed by low-power laser light in the presence of a photosensitizer. J Appl Bacteriol. 1995;78:569–74. [PubMed]
11. Malik Z, Hanania J, Nitzan Y. Bactericidal effects of photoactivated porphyrins–an alternative approach to antimicrobial drugs. J Photochem Photobiol B. 1990;5:281–93. [PubMed]
12. Malik Z, Ladan H, Nitzan Y. Photodynamic inactivation of Gram-negative bacteria: problems and possible solutions. J Photochem Photobiol B. 1992;14:262–6. [PubMed]
13. Wainwright M. Photoinactivation of viruses. Photochem Photobiol Sci. 2004;3:406–11. [PubMed]
14. Fuchs BB, Tegos GP, Hamblin MR, Mylonakis E. Susceptibility of Cryptococcus neoformans to photodynamic inactivation is associated with cell wall integrity. Antimicrob Agents Chemother.2007;51:2929–36. [PMC free article] [PubMed]
15. Bliss JM, Bigelow CE, Foster TH, Haidaris CG. Susceptibility of Candida species to photodynamic effects of photofrin. Antimicrob Agents Chemother. 2004;48:2000–6. [PMC free article] [PubMed]
16. Gonzales FP, da Silva SH, Roberts DW, Braga GU. Photodynamic inactivation of conidia of the fungi Metarhizium anisopliae and Aspergillus nidulans with methylene blue and toluidine blue. Photochem Photobiol. 2010;86:653–61. [PubMed]
17. Kassab K, Dei D, Roncucci G, Jori G, Coppellotti O. Phthalocyanine-photosensitized inactivation of a pathogenic protozoan, Acanthamoeba palestinensis. Photochem Photobiol Sci. 2003;2:668–72. [PubMed]
18. Gottlieb P, Shen LG, Chimezie E, Bahng S, Kenney ME, Horowitz B, Ben-Hur E. Inactivation of Trypanosoma cruzi trypomastigote forms in blood components by photodynamic treatment with phthalocyanines. Photochem Photobiol. 1995;62:869–74. [PubMed]
19. Akilov OE, Kosaka S, O’Riordan K, Song X, Sherwood M, Flotte TJ, Foley JW, Hasan T. The role of photosensitizer molecular charge and structure on the efficacy of photodynamic therapy against Leishmania parasites. Chem Biol. 2006;13:839–47. [PubMed]
20. Phoenix DA, Sayed Z, Hussain S, Harris F, Wainwright M. The phototoxicity of phenothiazinium derivatives against Escherichia coli and Staphylococcus aureus. FEMS Immunol Med Microbiol.2003;39:17–22. [PubMed]
21. Wainwright M, Phoenix DA, Marland J, Wareing DR, Bolton FJ. A study of photobactericidal activity in the phenothiazinium series. FEMS Immunol Med Microbiol. 1997;19:75–80. [PubMed]
22. Wainwright M, Crossley KB. Methylene Blue–a therapeutic dye for all seasons? J Chemother.2002;14:431–43. [PubMed]
23. Wainwright M. ‘Safe’ photoantimicrobials for skin and soft-tissue infections. Int J Antimicrob Agents.2010;36:14–8. [PubMed]
24. Ragas X, Dai T, Tegos GP, Agut M, Nonell S, Hamblin MR. Photodynamic inactivation of Acinetobacter baumannii using phenothiazinium dyes: in vitro and in vivo studies. Lasers Surg Med.2010;42:384–90. [PMC free article] [PubMed]
25. O’Neill J, Wilson M, Wainwright M. Comparative antistreptococcal activity of photobactericidal agents. J Chemother. 2003;15:329–34. [PubMed]
26. Wainwright M, Phoenix DA, Rice L, Burrow SM, Waring J. Increased cytotoxicity and phototoxicity in the methylene blue series via chromophore methylation. J Photochem Photobiol B. 1997;40:233–9. [PubMed]
27. Tegos GP, Hamblin MR. Phenothiazinium antimicrobial photosensitizers are substrates of bacterial multidrug resistance pumps. Antimicrob Agents Chemother. 2006;50:196–203. [PMC free article] [PubMed]
28. Tegos GP, Masago K, Aziz F, Higginbotham A, Stermitz FR, Hamblin MR. Inhibitors of bacterial multidrug efflux pumps potentiate antimicrobial photoinactivation. Antimicrob Agents Chemother.2008;52:3202–9. [PMC free article] [PubMed]
29. Foley JW, Song X, Demidova TN, Jalil F, Hamblin MR. Synthesis and properties of benzo[a]phenoxazinium chalcogen analogues as novel broad-spectrum antimicrobial photosensitizers. J Med Chem. 2006;49:5291–9. [PMC free article] [PubMed]
30. Smijs TG, Pavel S, Talebi M, Bouwstra JA. Preclinical studies with 5,10,15-Tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride for the photodynamic treatment of superficial mycoses caused by Trichophyton rubrum. Photochem Photobiol. 2009;85:733–9. [PubMed]
31. Lambrechts SA, Aalders MC, Van Marle J. Mechanistic study of the photodynamic inactivation of Candida albicans by a cationic porphyrin. Antimicrob Agents Chemother. 2005;49:2026–34.[PMC free article] [PubMed]
32. Lambrechts SA, Aalders MC, Langeveld-Klerks DH, Khayali Y, Lagerberg JW. Effect of monovalent and divalent cations on the photoinactivation of bacteria with meso-substituted cationic porphyrins.Photochem Photobiol. 2004;79:297–302. [PubMed]
33. Maisch T, Bosl C, Szeimies RM, Lehn N, Abels C. Photodynamic effects of novel XF porphyrin derivatives on prokaryotic and eukaryotic cells. Antimicrob Agents Chemother. 2005;49:1542–52.[PMC free article] [PubMed]
34. Ooi N, Miller K, Randall C, Rhys-Williams W, Love W, Chopra I. XF-70 and XF-73, novel antibacterial agents active against slow-growing and non-dividing cultures of Staphylococcus aureus including biofilms. J Antimicrob Chemother. 2010;65:72–8. [PubMed]
35. Ooi N, Miller K, Hobbs J, Rhys-Williams W, Love W, Chopra I. XF-73, a novel antistaphylococcal membrane-active agent with rapid bactericidal activity. J Antimicrob Chemother. 2009;64:735–40. [PubMed]
36. Banfi S, Caruso E, Buccafurni L, Battini V, Zazzaron S, Barbieri P, Orlandi V. Antibacterial activity of tetraaryl-porphyrin photosensitizers: an in vitro study on Gram negative and Gram positive bacteria. J Photochem Photobiol B. 2006;85:28–38. [PubMed]
37. Alves E, Costa L, Carvalho CM, Tome JP, Faustino MA, Neves MG, Tome AC, Cavaleiro JA, Cunha A, Almeida A. Charge effect on the photoinactivation of Gram-negative and Gram-positive bacteria by cationic meso-substituted porphyrins. BMC Microbiol. 2009;9:70. [PMC free article] [PubMed]
38. Carvalho CM, Gomes AT, Fernandes SC, Prata AC, Almeida MA, Cunha MA, Tome JP, Faustino MA, Neves MG, Tome AC, Cavaleiro JA, Lin Z, Rainho JP, Rocha J. Photoinactivation of bacteria in wastewater by porphyrins: bacterial beta-galactosidase activity and leucine-uptake as methods to monitor the process. J Photochem Photobiol B. 2007;88:112–8. [PubMed]
39. Costa L, Carvalho CM, Faustino MA, Neves MG, Tome JP, Tome AC, Cavaleiro JA, Cunha A, Almeida A. Sewage bacteriophage inactivation by cationic porphyrins: influence of light parameters.Photochem Photobiol Sci. 2010;9:1126–33. [PubMed]
40. Grinholc M, Kawiak A, Kurlenda J, Graczyk A, Bielawski KP. Photodynamic effect of protoporphyrin diarginate (PPArg2) on methicillin-resistant Staphylococcus aureus and human dermal fibroblasts. Acta Biochim Pol. 2008;55:85–90. [PubMed]
41. Mantareva V, Kussovski V, Angelov I, Borisova E, Avramov L, Schnurpfeil G, Wohrle D. Photodynamic activity of water-soluble phthalocyanine zinc(II) complexes against pathogenic microorganisms. Bioorg Med Chem. 2007;15:4829–35. [PubMed]
42. Mantareva V, Kussovski V, Angelov I, Wohrle D, Dimitrov R, Popova E, Dimitrov S. Non-aggregated Ga(iii)-phthalocyanines in the photodynamic inactivation of planktonic and biofilm cultures of pathogenic microorganisms. Photochem Photobiol Sci. 2010 [PubMed]
43. Giuliani F, Martinelli M, Cocchi A, Arbia D, Fantetti L, Roncucci G. In vitro resistance selection studies of RLP068/Cl, a new Zn(II) phthalocyanine suitable for antimicrobial photodynamic therapy. Antimicrob Agents Chemother. 2010;54:637–42. [PMC free article] [PubMed]
44. Huang L, Huang YY, Mroz P, Tegos GP, Zhiyentayev T, Sharma SK, Lu Z, Balasubramanian T, Krayer M, Ruzie C, Yang E, Kee HL, Kirmaier C, Diers JR, Bocian DF, Holten D, Lindsey JS, Hamblin MR. Stable synthetic cationic bacteriochlorins as selective antimicrobial photosensitizers. Antimicrob Agents Chemother. 2010;54:3834–41. [PMC free article] [PubMed]
45. Nakamura E, Isobe H. Functionalized fullerenes in water. The first 10 years of their chemistry, biology, and nanoscience. Acc Chem Res. 2003;36:807–15. [PubMed]
46. Tegos GP, Demidova TN, Arcila-Lopez D, Lee H, Wharton T, Gali H, Hamblin MR. Cationic fullerenes are effective and selective antimicrobial photosensitizers. Chem Biol. 2005;12:1127–35. [PMC free article][PubMed]
47. Huang L, Terakawa M, Zhiyentayev T, Huang YY, Sawayama Y, Jahnke A, Tegos GP, Wharton T, Hamblin MR. Innovative cationic fullerenes as broad-spectrum light-activated antimicrobials. Nanomedicine.2010;6:442–52. [PMC free article] [PubMed]
48. Spesia MB, Milanesio ME, Durantini EN. Synthesis, properties and photodynamic inactivation of Escherichia coli by novel cationic fullerene C60 derivatives. Eur J Med Chem. 2008;43:853–61. [PubMed]
49. Stockert JC, Canete M, Juarranz A, Villanueva A, Horobin RW, Borrell JI, Teixido J, Nonell S. Porphycenes: facts and prospects in photodynamic therapy of cancer. Curr Med Chem. 2007;14:997–1026.[PubMed]
50. Ragas X, Sanchez-Garcia D, Ruiz-Gonzalez R, Dai T, Agut M, Hamblin MR, Nonell S. Cationic Porphycenes as Potential Photosensitizers for Antimicrobial Photodynamic Therapy. J Med Chem. 2010[PMC free article] [PubMed]
51. McDonnell SO, Hall MJ, Allen LT, Byrne A, Gallagher WM, O’Shea DF. Supramolecular photonic therapeutic agents. J Am Chem Soc. 2005;127:16360–1. [PubMed]
52. Frimannsson DO, Grossi M, Murtagh J, Paradisi F, O’Shea DF. Light induced antimicrobial properties of a brominated boron difluoride (BF(2)) chelated tetraarylazadipyrromethene photosensitizer. J Med Chem.2010;53:7337–43. [PubMed]
53. Hager B, Strauss WS, Falk H. Cationic hypericin derivatives as novel agents with photobactericidal activity: synthesis and photodynamic inactivation of Propionibacterium acnes. Photochem Photobiol.2009;85:1201–6. [PubMed]
54. Soukos NS, Hamblin MR, Hasan T. The effect of charge on cellular uptake and phototoxicity of polylysine chlorin(e6) conjugates. Photochem Photobiol. 1997;65:723–9. [PubMed]
55. Soukos NS, Ximenez-Fyvie LA, Hamblin MR, Socransky SS, Hasan T. Targeted antimicrobial photochemotherapy. Antimicrob Agents Chemother. 1998;42:2595–601. [PMC free article] [PubMed]
56. Hamblin MR, O’Donnell DA, Murthy N, Rajagopalan K, Michaud N, Sherwood ME, Hasan T. Polycationic photosensitizer conjugates: effects of chain length and Gram classification on the photodynamic inactivation of bacteria. J Antimicrob Chemother. 2002;49:941–951. [PubMed]
57. Tegos GP, Anbe M, Yang C, Demidova TN, Satti M, Mroz P, Janjua S, Gad F, Hamblin MR. Protease-stable polycationic photosensitizer conjugates between polyethyleneimine and chlorin(e6) for broad-spectrum antimicrobial photoinactivation. Antimicrob Agents Chemother. 2006;50:1402–10. [PMC free article][PubMed]
58. Dai T, Tegos GP, Zhiyentayev T, Mylonakis E, Hamblin MR. Photodynamic therapy for methicillin-resistant Staphylococcus aureus infection in a mouse skin abrasion model. Lasers Surg Med. 2010;42:38–44. [PMC free article] [PubMed]
59. Dai T, Tegos GP, Lu Z, Huang L, Zhiyentayev T, Franklin MJ, Baer DG, Hamblin MR. Photodynamic therapy for Acinetobacter baumannii burn infections in mice. Antimicrob Agents Chemother. 2009;53:3929–34. [PMC free article] [PubMed]
60. Garcez AS, Nunez SC, Hamblin MR, Ribeiro MS. Antimicrobial effects of photodynamic therapy on patients with necrotic pulps and periapical lesion. J Endod. 2008;34:138–42. [PMC free article] [PubMed]
61. Garcez AS, Ribeiro MS, Tegos GP, Nunez SC, Jorge AO, Hamblin MR. Antimicrobial photodynamic therapy combined with conventional endodontic treatment to eliminate root canal biofilm infection. Lasers Surg Med. 2007;39:59–66. [PMC free article] [PubMed]
62. Demidova TN, Hamblin MR. Effect of cell-photosensitizer binding and cell density on microbial photoinactivation. Antimicrob Agents Chemother. 2005;49:2329–35. [PMC free article] [PubMed]
63. Gad F, Zahra T, Hasan T, Hamblin MR. Effects of growth phase and extracellular slime on photodynamic inactivation of gram-positive pathogenic bacteria. Antimicrob Agents Chemother.2004;48:2173–8. [PMC free article] [PubMed]
64. Gad F, Zahra T, Francis KP, Hasan T, Hamblin MR. Targeted photodynamic therapy of established soft-tissue infections in mice. Photochem Photobiol Sci. 2004;3:451–8. [PMC free article] [PubMed]
65. Hamblin MR, Zahra T, Contag CH, McManus AT, Hasan T. Optical monitoring and treatment of potentially lethal wound infections in vivo. J Infect Dis. 2003;187:1717–25. [PMC free article] [PubMed]
66. Hamblin MR, O’Donnell DA, Murthy N, Contag CH, Hasan T. Rapid control of wound infections by targeted photodynamic therapy monitored by in vivo bioluminescence imaging. Photochem Photobiol.2002;75:51–7. [PubMed]
67. Polo L, Segalla A, Bertoloni G, Jori G, Schaffner K, Reddi E. Polylysine-porphycene conjugates as efficient photosensitizers for the inactivation of microbial pathogens. J Photochem Photobiol B.2000;59:152–8. [PubMed]
68. Lauro FM, Pretto P, Covolo L, Jori G, Bertoloni G. Photoinactivation of bacterial strains involved in periodontal diseases sensitized by porphycene-polylysine conjugates. Photochem Photobiol Sci. 2002;1:468–70. [PubMed]
69. Rovaldi CR, Pievsky A, Sole NA, Friden PM, Rothstein DM, Spacciapoli P. Photoactive porphyrin derivative with broad-spectrum activity against oral pathogens In vitro. Antimicrob Agents Chemother.2000;44:3364–7. [PMC free article] [PubMed]
70. Bombelli C, Bordi F, Ferro S, Giansanti L, Jori G, Mancini G, Mazzuca C, Monti D, Ricchelli F, Sennato S, Venanzi M. New cationic liposomes as vehicles of m-tetrahydroxyphenylchlorin in photodynamic therapy of infectious diseases. Mol Pharm. 2008;5:672–9. [PubMed]
71. Ferro S, Ricchelli F, Mancini G, Tognon G, Jori G. Inactivation of methicillin-resistant Staphylococcus aureus (MRSA) by liposome-delivered photosensitising agents. J Photochem Photobiol B. 2006;83:98–104.[PubMed]
72. Schwiertz J, Wiehe A, Grafe S, Gitter B, Epple M. Calcium phosphate nanoparticles as efficient carriers for photodynamic therapy against cells and bacteria. Biomaterials. 2009;30:3324–31. [PubMed]
73. Ferro S, Jori G, Sortino S, Stancanelli R, Nikolov P, Tognon G, Ricchelli F, Mazzaglia A. Inclusion of 5-[4-(1-dodecanoylpyridinium)]-10,15,20-triphenylporphine in supramolecular aggregates of cationic amphiphilic cyclodextrins: physicochemical characterization of the complexes and strengthening of the antimicrobial photosensitizing activity. Biomacromolecules. 2009;10:2592–600. [PubMed]
74. Ferro S, Ricchelli F, Monti D, Mancini G, Jori G. Efficient photoinactivation of methicillin-resistant Staphylococcus aureus by a novel porphyrin incorporated into a poly-cationic liposome. Int J Biochem Cell Biol. 2007;39:1026–34. [PubMed]
75. Lu XM, Fischman AJ, Stevens E, Lee TT, Strong L, Tompkins RG, Yarmush ML. Sn-chlorin e6 antibacterial immunoconjugates. An in vitro and in vivo analysis. J Immunol Methods. 1992;156:85–99.[PubMed]
76. Friedberg JS, Tompkins RG, Rakestraw SL, Warren SW, Fischman AJ, Yarmush ML. Antibody-targeted photolysis. Bacteriocidal effects of Sn (IV) chlorin e6-dextran-monoclonal antibody conjugates. Ann N Y Acad Sci. 1991;618:383–93. [PubMed]
77. Berthiaume F, Reiken SR, Toner M, Tompkins RG, Yarmush ML. Antibody-targeted photolysis of bacteria in vivo. Biotechnology (N Y) 1994;12:703–6. [PubMed]
78. Demidova TN, Gad F, Zahra T, Francis KP, Hamblin MR. Monitoring photodynamic therapy of localized infections by bioluminescence imaging of genetically engineered bacteria. J Photochem Photobiol B. 2005;81:15–25. [PMC free article] [PubMed]
79. Rocchetta HL, Boylan CJ, Foley JW, Iversen PW, LeTourneau DL, McMillian CL, Contag PR, Jenkins DE, Parr TR., Jr Validation of a noninvasive, real-time imaging technology using bioluminescent Escherichia coli in the neutropenic mouse thigh model of infection. Antimicrob Agents Chemother. 2001;45:129–37.[PMC free article] [PubMed]
80. Francis KP, Joh D, Bellinger-Kawahara C, Hawkinson MJ, Purchio TF, Contag PR. Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct. Infect Immun. 2000;68:3594–600. [PMC free article] [PubMed]
81. Francis KP, Yu J, Bellinger-Kawahara C, Joh D, Hawkinson MJ, Xiao G, Purchio TF, Caparon MG, Lipsitch M, Contag PR. Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon. Infect Immun.2001;69:3350–8. [PMC free article] [PubMed]
82. Latorre-Esteves E, Akilov OE, Rai P, Beverley SM, Hasan T. Monitoring the efficacy of antimicrobial photodynamic therapy in a murine model of cutaneous leishmaniasis using L. major expressing GFP. J Biophotonics. 2010;3:328–35. [PMC free article] [PubMed]
83. Hamblin MR, Dai T. Can surgical site infections be treated by photodynamic therapy? Photodiagnosis Photodyn Ther. 2010;7:134–6. [PMC free article] [PubMed]
84. Lu Z, Dai T, Huang L, Kurup DB, Tegos GP, Jahnke A, Wharton T, Hamblin MR. Photodynamic therapy with a cationic functionalized fullerene rescues mice from fatal wound infections. Nanomedicine (UK) 2010;5 in press. [PMC free article] [PubMed]
85. Lambrechts SA, Demidova TN, Aalders MC, Hasan T, Hamblin MR. Photodynamic therapy for Staphylococcus aureus infected burn wounds in mice. Photochem Photobiol Sci. 2005;4:503–9.[PMC free article] [PubMed]
86. Ragas X, Dai T, Tegos GP, Agut M, Nonell S, Hamblin MR. Photodynamic inactivation of Acinetobacter baumannii using phenothiazinium dyes: in-vitro and in-vivo studies. Lasers Surg Med. 2010 in press. [PMC free article] [PubMed]
87. Bowers AL, Huffman GR, Sennett BJ. Methicillin-resistant Staphylococcus aureus infections in collegiate football players. Med Sci Sports Exerc. 2008;40:1362–7. [PubMed]
88. Turbeville SD, Cowan LD, Greenfield RA. Infectious disease outbreaks in competitive sports: a review of the literature. Am J Sports Med. 2006;34:1860–5. [PubMed]
89. Kirkland EB, Adams BB. Methicillin-resistant Staphylococcus aureus and athletes. J Am Acad Dermatol. 2008;59:494–502. [PubMed]
90. Doyle TC, Nawotka KA, Purchio AF, Akin AR, Francis KP, Contag PR. Expression of firefly luciferase in Candida albicans and its use in the selection of stable transformants. Microb Pathog. 2006;40:69–81.[PubMed]
91. Doyle TC, Nawotka KA, Kawahara CB, Francis KP, Contag PR. Visualizing fungal infections in living mice using bioluminescent pathogenic Candida albicans strains transformed with the firefly luciferase gene.Microb Pathog. 2006;40:82–90. [PubMed]
92. Saville SP, Lazzell AL, Chaturvedi AK, Monteagudo C, Lopez-Ribot JL. Use of a genetically engineered strain to evaluate the pathogenic potential of yeast cell and filamentous forms during Candida albicans systemic infection in immunodeficient mice. Infect Immun. 2008;76:97–102. [PMC free article][PubMed]
93. Robert VA, Casadevall A. Vertebrate endothermy restricts most fungi as potential pathogens. J Infect Dis.2009;200:1623–6. [PubMed]
Mini Rev Med Chem. 2009 Jul;9(8):974-83.

A new strategy to destroy antibiotic resistant microorganisms: antimicrobial photodynamic treatment.

Maisch T1.

Author information

  • 1Antimicrobial PDT, Clinic and Polyclinic for Dermatology, Regensburg University Hospital, Germany, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany. tim.maisch@klinik.uni-regensburg.de

Abstract

Photodynamic activity of chemical compounds towards microorganisms was first published at the turn of 20th century and it is based on the concept that a chemical compound, known as the photosensitizer, is localized preferentially in the microorganism and subsequently activated by low doses of visible light of an appropriate wavelength to generate reactive oxygen species that are toxic to the target microorganisms. Processes, in which absorption of light by a photosensitizer induces chemical changes in another molecule, are defined as photosensitizing reactions. Since the middle of the last century, antibacterial photosensitizing reactions were forgotten because of the discovery and the beginning of the Golden Age of antibiotics. Certainly, in the last decades the worldwide rise in antibiotic resistance has driven research to the development of new anti-microbial strategies. Different classes of molecules including phenothiazine, porphyrines, phthalocyanines, and fullerenes have demonstrated antimicrobial efficacy against a broad spectrum of antibiotic resistant microorganisms upon illumination. Due to their extended pi-conjugated system these molecules absorb visible light, have a high triplet quantum yield and can generate reactive oxygen species upon illumination. This mini-review will focus on some major advances regarding physical and chemical properties of photosensitizers and light sources that appear to be suitable in the field of antimicrobial photodynamic therapy. Currently, topical application of a photosensitizer on infected tissues and subsequent illumination seems to be the most promising feature of antimicrobial photodynamic therapy, thereby not harming the surrounding tissue or disturbing the residual bacteria-flora of the tissue.

 

Drug Saf. 2002;25(5):345-72.

Drug-induced cutaneous photosensitivity: incidence, mechanism, prevention and management.

Moore DE1.
Author information
1Faculty of Pharmacy, The University of Sydney, Sydney, New South Wales, Australia. demoore@pharm.usyd.edu.au

Abstract
The interaction of sunlight with drug medication leads to photosensitivity responses in susceptible patients, and has the potential to increase the incidence of skin cancer. Adverse photosensitivity responses to drugs occur predominantly as a phototoxic reaction which is more immediate than photoallergy, and can be reversed by withdrawal or substitution of the drug. The bias and inaccuracy of the reporting procedure for these adverse reactions is a consequence of the difficulty in distinguishing between sunburn and a mild drug photosensitivity reaction, together with the patient being able to control the incidence by taking protective action. The drug classes that currently are eliciting a high level of adverse photosensitivity are the diuretic, antibacterial and nonsteroidal anti-inflammatory drugs (NSAIDs). Photosensitising chemicals usually have a low molecular weight (200 to 500 Daltons) and are planar, tricyclic, or polycyclic configurations, often with heteroatoms in their structures enabling resonance stabilisation. All absorb ultraviolet (UV) and/or visible radiation, a characteristic that is essential for the chemical to be regarded as a photosensitiser. The photochemical and photobiological mechanisms underlying the adverse reactions caused by the more photoactive drugs are mainly free radical in nature, but reactive oxygen species are also involved. Drugs that contain chlorine substituents in their chemical structure, such as hydrochlorthiazide, furosemide and chlorpromazine, exhibit photochemical activity that is traced to the UV-induced dissociation of the chlorine substituent leading to free radical reactions with lipids, proteins and DNA. The photochemical mechanisms for the NSAIDs that contain the 2-aryl propionic acid group involve decarboxylation as the primary step, with subsequent free radical activity. In aerated systems, the reactive excited singlet form of oxygen is produced with high efficiency. This form of oxygen is highly reactive towards lipids and proteins. NSAIDs without the 2-arylpropionic acid group are also photoactive, but with differing mechanisms leading to a less severe biological outcome. In the antibacterial drug class, the tetracyclines, fluoroquinolones and sulfonamides are the most photoactive. Photocontact dermatitis due to topically applied agents interacting with sunlight has been reported for some sunscreen and cosmetic ingredients, as well as local anaesthetic and anti-acne agents. Prevention of photosensitivity involves adequate protection from the sun with clothing and sunscreens. In concert with the preponderance of free radical mechanisms involving the photosensitising drugs, some recent studies suggest that diet supplementation with antioxidants may be beneficial in increasing the minimum erythemal UV radiation dose.

Gout

Photomed Laser Surg. 2006 Apr;24(2):140-50.

Photobiomodulation of pain and inflammation in microcrystalline arthropathies: experimental and clinical results.

Soriano F1, Campana V, Moya M, Gavotto A, Simes J, Soriano M, Soriano R, Spitale L, Palma J.

Author information

Abstract

OBJECTIVE:

This article presents the results of laser therapy in crystal (hydroxyapatite, calcium pyrophosphate, and urates) deposition-induced arthritis in rats and the clinical applications in humans.

BACKGROUND DATA:

Microcrystalline arthropathies are prevalent among geriatric patients, who are more vulnerable to the side effects of drugs. The effectiveness of laser therapy for pain relief, free of side effects, has been reported in painful conditions.

METHODS:

Two milligrams of each of the above-mentioned crystals was injected in both joints of the back limbs in three groups of rats; these groups were then treated with laser irradiation. Three other groups received no treatment after the injections. We determined the plasmatic levels of inflammatory markers (fibrinogen, prostaglandin E2, and TNF(alpha)), tissues (prostaglandin E(2)) and conducted anatomopathological studies. Twenty-five patients with acute gout arthritis were randomized into two groups and treated over 5 days: group A, diclofenac 75 mg orally, twice a day; and group B, laser irradiation once a day. Forty-nine patients with knee chronic pyrophosphate arthropathy were randomized into two groups and treated over 21 days; group A, diclofenac 50 mg orally, twice a day; and group B, laser irradiation once a day. Thirty patients with shoulder chronic hydroxyapatite arthropathy were randomized into two groups and treated over 21 days; group A, diclofenac 50 mg orally, twice a day; and group B, laser irradiation once a day.

RESULTS:

Fibrinogen, prostaglandin E(2), and TNF(alpha) concentrations in the rats injected with crystals and treated with laser decreased significantly as compared with the groups injected with crystals without treatment. Both laser therapy and diclofenac achieved rapid pain relief in patients with acute gouty arthritis without significant differences in efficacy. Laser therapy was more effective than diclofenac in patients with chronic pyrophosphate arthropathy and in patients with chronic apatite deposition disease.

CONCLUSION:

Laser therapy represents an effective treatment in the therapeutic arsenal of microcrystalline arthropathies.

Photoimmunotherapy

Oncotarget. 2015 May 13. [Epub ahead of print]

Near infrared photoimmunotherapy prevents lung cancer metastases in a murine model.

Sato K1, Nagaya T1, Nakamura Y1, Harada T1, Choyke PL1, Kobayashi H1.

Author information

  • 1Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892-1088, USA.

Abstract

Near infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of intravenously injected antibodies with the acute toxicity induced by photosensitizers after exposure to NIR-light. Herein, we evaluate the efficacy of NIR-PIT in preventing lung metastases in a mouse model. Lung is one of the most common sites for developing metastases, but it also has the deepest tissue light penetration. Thus, lung is the ideal site for treating early metastases by using a light-based strategy. In vitro NIR-PIT cytotoxicity was assessed with dead cell staining, luciferase activity, and a decrease in cytoplasmic GFP fluorescence in 3T3/HER2-luc-GFP cells incubated with an anti-HER2 antibody photosensitizer conjugate. Cell-specific killing was demonstrated in mixed 2D/3D cell cultures of 3T3/HER2-luc-GFP (target) and 3T3-RFP (non-target) cells. In vivo NIR-PIT was performed in the left lung in a mouse model of lung metastases, and the number of metastasis nodules, tumor fluorescence, and luciferase activity were all evaluated. All three evaluations demonstrated that the NIR-PIT-treated lung had significant reductions in metastatic disease (*p < 0.0001, Mann-Whitney U-test) and that NIR-PIT did not damage non-target tumors or normal lung tissue. Thus, NIR-PIT can specifically prevent early metastases and is a promising anti-metastatic therapy.

 
Ann Surg Oncol. 2015 Apr 17. [Epub ahead of print]

Photoimmunotherapy Inhibits Tumor Recurrence After Surgical Resection on a Pancreatic Cancer Patient-Derived Orthotopic Xenograft (PDOX) Nude Mouse Model.

Hiroshima Y1, Maawy A, Zhang Y, Guzman MG, Heim R, Makings L, Luiken GA, Kobayashi H, Tanaka K, Endo I, Hoffman RM, Bouvet M.

Author information

  • 1Department of Surgery, Moores Cancer Center, University of California San Diego, San Diego, CA, USA.

Abstract

BACKGROUND:

Photoimmunotherapy (PIT) uses a target-specific photosensitizer based on a near-infrared (NIR) phthalocyanine dye, IR700, to induce tumor necrosis after irradiation with NIR light to kill cancer cells, such as those that remain after surgery. The purpose of the present study was to sterilize the surgical bed after pancreatic cancer resection with PIT in carcinoembryonic antigen (CEA)-expressing, patient-derived, orthotopic xenograft (PDOX) nude mouse models.

METHODS:

After confirmation of tumor engraftment, mice were randomized to two groups: bright light surgery (BLS)-only and BLS + PIT. Each treatment arm consisted of seven tumor-bearing mice. BLS was performed under standard bright-field with an MVX10 long-working distance, high-magnification microscope on all mice. For BLS + PIT, anti-CEA antibody conjugated with IR700 (anti-CEA-IR700) (50 µg) was injected intravenously in all mice 24 h before surgery. After the surgery, the resection bed was then irradiated with a red-light-emitting diode at 690 ± 5 nm with a power density of 150 mW/cm2.

RESULTS:

Anti-CEA-IR700 labelled and illuminated the pancreatic cancer PDOX. Minimal residual cancer of the PDOX was detected by fluorescence after BLS. The local recurrence rate was 85.7 % for BLS-only and 28.6 % for BLS + PIT-treated mice (p = 0.05). The average recurrent tumor weight was 1149.0 ± 794.6 mg for BLS-only and 210.8 ± 336.9 mg for BLS + PIT-treated mice (p = 0.015).

CONCLUSION:

Anti-CEA-IR700 was able to label and illuminate a pancreatic cancer PDOX nude mouse model sufficiently for PIT. PIT reduced recurrence by eliminating remaining residual cancer cells after BLS.

PLoS One. 2015; 10(3): e0121989.
Published online 2015 Mar 23. doi:  10.1371/journal.pone.0121989

Near Infra-Red Photoimmunotherapy with Anti-CEA-IR700 Results in Extensive Tumor Lysis and a Significant Decrease in Tumor Burden in Orthotopic Mouse Models of Pancreatic Cancer

Ali A. Maawy,1 Yukihiko Hiroshima,1,2,3 Yong Zhang,2 Roger Heim,4 Lew Makings,4 Miguel Garcia-Guzman,4 George A. Luiken,5 Hisataka Kobayashi,6 Robert M. Hoffman,1,2 and Michael Bouvet1,7,*

Surinder K. Batra, Academic Editor
Competing Interests: Robert M. Hoffman is a stockholder and non-salaried associate of AntiCancer, Inc. Yukihiko Hiroshima is a non-salaried associate of AntiCancer, Inc. George A. Luiken is a stockholder and non-salaried associate of OncoFluor, Inc. The authors confirm that this does not alter the authors adherence to PLOS ONE policies on sharing data and materials.

Conceived and designed the experiments: AM YH YZ RH LM MG GL HK RH MB. Performed the experiments: AM YH YZ RH. Analyzed the data: AM YH RH MG RH MB. Contributed reagents/materials/analysis tools: RH MG RH MB. Wrote the paper: AM YH RH MG RH MB.

* E-mail: ude.dscu@tevuobm

Received 2014 Nov 30; Accepted 2015 Feb 9.

 

Introduction

Photoimmunotherapy (PIT) uses tumor specific monoclonal antibodies that are conjugated to the photosensitizer phthalocyanine dye, IR700, which is cytotoxic upon irradiation with near-infrared (NIR) light [13].

Several monoclonal antibodies (mAbs) have been used with PIT in mouse models of breast cancer including trastuzumab, a monoclonal antibody (mAb) directed against human epidermal growth factor receptor-2 (HER-2), and panitumumab, a monoclonal antibody directed against human epidermal growth factor receptor-1 (HER-1) [4,5]. Cell death was induced immediately after irradiating mAb-IR700–bound target cells with NIR light. In vivo tumor shrinkage after irradiation with NIR light was demonstrated in target cells expressing the epidermal growth factor receptor. The mAb-IR700 conjugates were effective when bound to the cell membrane and produced no phototoxicity when not bound, suggesting a different mechanism for PIT as compared to conventional photodynamic therapies [1].

Pancreatic cancer is a lethal tumor with high rates of local and distant recurrence [6,7]. In the present study, we used a chimeric monoclonal antibody against the carcinoembryonic antigen (CEA) for PIT, which is often overexpressed in pancreatic cancer and has been previously utilized by our laboratory for fluorescence-guided surgery and fluorescence laparoscopy [817]. The anti-CEA antibody was conjugated to IR700 and used for PIT treatment of human pancreatic cancer in orthotopic mouse models as well as pancreatic cancer cells in vitro.

 

Materials and Methods

Cell Culture

The human pancreatic cancer cell line BxPC-3 was stably transduced to express green fluorescent protein (GFP) as previously described [18,19]. Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), penicillin/streptomycin (Gibco-BRL, Carlsbad, CA), sodium pyruvate (Gibco-BRL), sodium bicarbonate (Cellgro, Manassas, VA), l-glutamine (Gibco-BRL), and minimal essential medium nonessential amino acids (Gibco-BRL). All cells were cultured at 37°C in a 5% CO2 incubator.

Determination of CEA antigen expression level

BxPC-3-GFP cells from a 75 cm2 flask were harvested with enzyme-free cell dissociation buffer, washed once with incubation buffer (PBS + 0.5% FBS + 0.1% sodium azide), and recovered in incubation buffer at 2 x 106 cells/ml and kept at 4°C. The cells (5 x 105) were incubated with chimeric anti-CEA antibody (Genara Biosciences LLC, Morgan Hill, CA) (10 μg/ml) in 300 μl incubation buffer for 1 hour at 4°C, washed three times with PBS and stained with an Alexa488-conjugated donkey anti human IgG (H+L) antibody (Jackson Immunoresearch, West Grove, PA) for 45 minutes, followed by three washes with PBS. A control without anti-CEA was prepared in parallel. Flow cytometry profiles from the anti-CEA antibody-treated cells and the untreated cells were established on a Guava EasyCyte Plus flow cytometer (EMD Millipore, Billerica, MA). The antibody binding capacity for anti-CEA was determined by using reference beads (Bangs Laboratories, Inc., Fishers, IN) and geometric means according to the manufacturer’s protocol.

Animals

Athymic nu/nu nude mice (AntiCancer Inc., San Diego, CA), 4–6 weeks old, were used in this study. Mice were kept in a barrier facility under HEPA filtration. Mice were fed with an autoclaved laboratory rodent diet. All mouse surgical procedures and imaging were performed with the animals anesthetized by intramuscular injection of 50% ketamine, 38% xylazine, and 12% acepromazine maleate (0.02 ml). Animals received buprenorphine (0.10 mg/kg ip) immediately prior to surgery and once a day over the next 3 days to ameliorate pain. CO2 inhalation was used for euthanasia of all animals at 5 weeks after surgery. To ensure death following CO2 asphyxiation, cervical dislocation was performed. All animal studies were conducted with an AntiCancer, Inc. Institutional Animal Care and Use Committee (IACUC)-protocol specifically approved for this study and in accordance with the principals and procedures outlined in the National Institute of Health Guide for the Care and Use of Animals under Assurance Number A3873–1.

Antibody-Dye Conjugation

A water-soluble silicon-phthalocyanine derivative, IRDye 700DX NHS ester was obtained from LI-COR Bioscience (Lincoln, NE). of Chimeric anti-CEA antibody (Genara Biosciences LLC) (2 mg [~ 14 nmol]) at a concentration of 2 mg/ml in 0.1 M Na2HPO4 (pH = 8.6) was incubated for 2 hours at room temperature with IR700dye NHS ester (135 ug, 70 nmol) prepared in anhydrous DMSO at 5 mmol/L. After the incubation period, the IR700-conjugate was buffer exchanged and purified with phosphate buffer saline (PBS, pH = 7.1) using Amicon Ultra Centrifugal Filter Units (EMD Millipore Corporation, Billerica, MA). The IR700-mAb conjugate was repeatedly diluted with 10 ml volumes of PBS and then concentrated using the filter units until less than 2% of the unconjugated IR700 dye species remained, as determined by size exclusion HPLC (SE-HPLC). Analysis of the conjugates by SE-HPLC was performed using an Agilent 1100 HPLC system fitted with a TSKgel G2000SWxl column (Tosoh Biosciences, Tokyo, Japan). The SE-HPLC elution buffer was 1X PBS (pH = 7.1) with a flow rate of 1 ml/min. UV/Vis detection at 280 nm and 690 nm was used to determine the average dye-to-antibody ratio (DAR) for each conjugates. With this sample, a purity of 97.6% with 0.5% free dye and a DAR of 4.1 was achieved.

Tumor implantation

After confluence, BxPC-3-GFP human pancreatic cancer cells (1 x 106) were injected subcutaneously into the flanks of nude mice and allowed to engraft and grow over a period of 4–6 weeks. Tumors were then harvested and tumor fragments (1 mm3) from subcutaneous tumors were sutured to the tail of the pancreas using 8–0 nylon surgical sutures (Ethilon; Ethicon Inc., Somerville, NJ). On completion, the tail of the pancreas was returned to the abdomen, and the incision was closed in one layer using 6–0 nylon surgical sutures (Ethilon) [20,21]. The tumor fragments were allowed to grow over a period of 2 weeks.

Cytotoxicity studies

BxPC-3 cells were seeded in white-wall 96-well plates (4,000/well) and allowed to attach overnight. Cells were incubated with the antibody conjugate, anti-CEA-IR700, (dye-antibody ratio of 5.1) at 10 mg/ml for 2 hours at 37°C. Four wells at a time were subjected to treatment with 690 nm light from an LED (Marubeni Corporation, Tokyo, Japan) at a power density of 50 mW/cm2 that was calibrated with a power meter equipped with a photodiode power sensor (Thorlabs Inc., Newton, NJ). After light treatment, the antibody solution was replaced with complete RPMI 1640 medium, containing CytoTox Green (Promega, Madison, WI) to monitor cell killing.

Photoimmunotherapy in vivo

Anti-CEA antibody (100μg) (Genara Biosciences LLC, Morgan Hill, CA) conjugated to IR700DX reconstituted to 100 μl was injected via tail vein in the treatment group 24 hours prior to intervention, while the control group had 100 μl of PBS similarly injected 24 hours prior to injection. Each group consisted of 10 mice with orthotopic BxPC-3-GFP tumors.

After 24 hours, the pancreatic tumors in all 10 mice in the treatment group were exposed via a left lateral incision and imaged to detect both the GFP signal and the 700 nm signal. All the mice were subsequently subjected to photoimmunotherapy by exposing the tumor to a 690 nm laser at 150 mW/cm2 for 30 minutes for a total of 270 J/cm2. The surrounding normal tissues were protected with aluminum foil during PIT. Mice were imaged at the time of therapy and weekly thereafter with tumor exposed to evaluate response to therapy. After 5 weeks the mice were sacrificed, at which point they were imaged and had their tumors resected and weighed.

Animal Imaging

Mice were imaged weekly using the Olympus OV100 small animal imaging system (Olympus Corp. Tokyo, Japan), containing an MT-20 light source (Olympus Biosystems Planegg, Germany) and DP70 CCD camera (Olympus Corp. Tokyo, Japan) [22]. All images were analyzed using Image-J (National Institute of Health Bethesda, MD) and were processed with the use of Photoshop elements-11 (Adobe Systems Inc. San Jose, CA).

Tumor size determination

The mice in both groups had weekly laparotomy to expose the pancreatic tumors via a left lateral incision. Tumors were imaged with the OV-100 by GFP expression. Tumor size was assessed using Image-J software (National Institutes of Health, Bethesda, Maryland).

Statistical Analysis

All statistical analysis was done using SPSS software version 21 (IBM, Armonk, NY). For pairwise comparisons, quantitative variables were calculated using the paired-samples Student’s t-test and confirmed with the Wilcoxon rank-sum test. A p-value ≤0.05 was considered significant. 95% confidence intervals obtained on analysis of the data were configured into the error bars of the appropriate figures and graphs.

Results and Discussion

Anti-CEA-IR700 binds to CEA-expressing pancreatic cancer cells in vitro and causes extensive cell death after light activation compared to control

The anti-CEA antibody binding capacity to BxPC3 human pancreatic cancer cells was 2,227,000 binding sites per cell by FACS analysis (Fig. 1). Cells were incubated with anti-CEA-IR700 and treated with 690 nm light. At the end of the incubation, there was 100% cell death in the anti-CEA IR700 + 690 nm light group compared to a negligible amount of cell death in the no-690 nm light group (Fig. 2). Death of 690 nm light-treated cells in absence of Anti-CEA-IR700 was negligible (data not shown).

Fig 1Fig 1

Staining of BxPC-3 cells with anti-CEA antibody and quantification of binding site

Fig 2

Fig 2

Light-induced cell death by cell-bound anti-CEA-IR700.
 
PIT results in a significant reduction in tumor size and weight in an orthotopic mouse model of pancreatic cancer

Two weeks after orthotopic implantation of BxPC-3-GFP tumors, engraftment was ensured and mice were divided into 2 groups with the treatment group receiving anti-CEA-IR700 conjugate (100 μg) and the control group receiving PBS (Fig. 3). Tumor size was assessed on a weekly basis to evaluate response to therapy and overall differences in progression. In the control group there was an initial exponential increase in tumor size that began to plateau at week 4 achieving a maximum average of 390.7 mm2 (95% CI [347.7, 433.7]). In contrast, in the treatment group there was an initial decrease in tumor size from baseline with a maximal response seen at week 1 (Figs. ?(Figs.44 and ?and5)5) with an average size of 6.65mm2 (95% CI [1.75, 11.5]). Over the course of the experiment, the tumor began to regrow, reaching a maximum average of 29.5 mm2 (95% CI [16.5, 42.5]) at 5 weeks post-treatment. The difference in tumor size between the control and the treatment groups was significant (p<0.001).

Fig 3

Fig 3

Experimental protocol for in vivo PIT.

Fig 4

Fig 4

GFP weekly imaging of pancreatic tumors.

Fig 5Fig 5

Graphical representation of tumor size over 5 weeks.
At the termination of the experiment at week 5, the tumors in both groups were excised and weighed. Complete excision was confirmed with the OV-100 by GFP fluorescence. The average tumor weight was 3872 mg (95% CI [3213, 4531]) for the control group and 239.6 mg (95% CI [81, 397]) for the treatment group (p<0.001) (Figs. (Figs.6 and and7).

Fig 6

Fig 6

Graphical representation of tumor weights.

Fig 7

Fig 7

Resected tumors.

The average body weights of the mice after one week of treatment were 26.3 grams (95% CI [25.1, 27.4]) for the PIT group and 25.1 grams for the control group (95% CI [24, 26.2]) which was not statistically different (p = 0.23). The average body weights of the mice 5 weeks after treatment were 29.2 grams (95% CI [28, 30.5]) for the PIT group and 28.7 grams (95% CI [27, 30.3]) for the control group which was also not statistically different (p = 0.64), indicating that PIT was well tolerated by the mice.

Despite the anti-tumor effects of PIT, there was however a 100% recurrence rate. Previous studies have shown that the rate and amount of tumor cell destruction is dependent on both the conjugate dose and the light dose, the product of which results in the same cytotoxic effect regardless of the method of light delivery (continuous or intermittent) [2,23]. In this regard, further investigation is needed to assess how dosing of the anti-CEA-IR700 complex and varying the mode and amount of energy delivery could increase efficacy.

A single round of treatment was employed in the present report as proof of principle in an orthotopic model of pancreatic cancer. Multiple rounds of PIT will be performed in future experiments. Repeated rounds of therapy have been shown to increase the efficacy of PIT [4]. It is expected that repeated rounds of PIT would reduce the recurrence rate. Repeated rounds of PIT should be feasible due to the low toxicity observed. PIT should be able to add the efficacy of surgery and radiation therapy when used in combination with these therapies. This will be tested in orthotopic models, including immunocompetent mice, and in experimental high metastatic models, as well as in patient-derived orthotopic xenograft (PDOX) models in future experiments.

 

Funding Statement

This work was supported by grants from the National Cancer Institute CA142669 and CA132971 (to M.B. and AntiCancer, Inc). AntiCancer, Inc. and Aspyrian Therapeutics provided support in the form of salaries for authors YZ, RH, LM, and MG-G but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

 

References

1. Mitsunaga M, Ogawa M, Kosaka N, Rosenblum LT, Choyke PL, Kobayashi H. Cancer cell-selective in vivo near infrared photoimmunotherapy targeting specific membrane molecules. Nat Med. 2011;17: 1685–1691. doi: 10.1038/nm.2554 [PMC free article] [PubMed]
2. Nakajima T, Sato K, Hanaoka H, Watanabe R, Harada T, Choyke PL, et al. The effects of conjugate and light dose on photo-immunotherapy induced cytotoxicity. BMC Cancer. 2014;14: 389 doi: 10.1186/1471-2407-14-389 [PMC free article] [PubMed]
3. Sato K, Watanabe R, Hanaoka H, Harada T, Nakajima T, Kim I, et al. Photoimmunotherapy: comparative effectiveness of two monoclonal antibodies targeting the epidermal growth factor receptor. Mol Oncol. 2014;8: 620–632. doi: 10.1016/j.molonc.2014.01.006 [PMC free article] [PubMed]
4. Mitsunaga M, Nakajima T, Sano K, Choyke PL, Kobayashi H. Near-infrared theranostic photoimmunotherapy (PIT): repeated exposure of light enhances the effect of immunoconjugate. Bioconjug Chem. 2012;23: 604–609. doi: 10.1021/bc200648m [PMC free article] [PubMed]
5. Mitsunaga M, Nakajima T, Sano K, Kramer-Marek G, Choyke PL, Kobayashi H. Immediate in vivo target-specific cancer cell death after near infrared photoimmunotherapy. BMC Cancer. 2012;12: 345 doi:10.1186/1471-2407-12-345 [PMC free article] [PubMed]
6. Bouvet M, Gamagami RA, Gilpin EA, Romeo O, Sasson A, Easter DW, et al. Factors influencing survival after resection for periampullary neoplasms. Am J Surg. 2000;180: 13–17. [PubMed]
7. Ryan DP, Hong TS, Bardeesy N. Pancreatic adenocarcinoma. N Engl J Med. 2014;371: 1039–1049. doi:10.1056/NEJMra1404198 [PubMed]
8. Hiroshima Y, Maawy A, Metildi CA, Zhang Y, Uehara F, Miwa S, et al. Successful fluorescence-guided surgery on human colon cancer patient-derived orthotopic xenograft mouse models using a fluorophore-conjugated anti-CEA antibody and a portable imaging system. J Laparoendosc Adv Surg Tech A. 2014;24: 241–247. doi: 10.1089/lap.2013.0418 [PMC free article] [PubMed]
9. Hiroshima Y, Maawy A, Sato S, Murakami T, Uehara F, Miwa S, et al. Hand-held high-resolution fluorescence imaging system for fluorescence-guided surgery of patient and cell-line pancreatic tumors growing orthotopically in nude mice. J Surg Res. 2014;187: 510–517. doi: 10.1016/j.jss.2013.11.1083[PMC free article] [PubMed]
10. Kaushal S, McElroy MK, Luiken GA, Talamini MA, Moossa AR, Hoffman RM, et al. Fluorophore-conjugated anti-CEA antibody for the intraoperative imaging of pancreatic and colorectal cancer. J Gastrointest Surg. 2008;12: 1938–1950. doi: 10.1007/s11605-008-0581-0 [PMC free article] [PubMed]
11. Maawy AA, Hiroshima Y, Kaushal S, Luiken GA, Hoffman RM, Bouvet M. Comparison of a chimeric anti-carcinoembryonic antigen antibody conjugated with visible or near-infrared fluorescent dyes for imaging pancreatic cancer in orthotopic nude mouse models. J Biomed Opt. 2013;18: 126016 doi:10.1117/1.JBO.18.12.126016 [PMC free article] [PubMed]
12. Maawy AA, Hiroshima Y, Zhang Y, Luiken GA, Hoffman RM, Bouvet M. Polyethylene glycol (PEG) linked to near infrared (NIR) dyes conjugated to chimeric anti-carcinoembryonic antigen (CEA) antibody enhances imaging of liver metastases in a nude-mouse model of human colon cancer. PLoS One. 2014;9: e97965 doi: 10.1371/journal.pone.0097965 [PMC free article] [PubMed]
13. Metildi CA, Kaushal S, Lee C, Hardamon CR, Snyder CS, Luiken GA, et al. An LED light source and novel fluorophore combinations improve fluorescence laparoscopic detection of metastatic pancreatic cancer in orthotopic mouse models. J Am Coll Surg. 2012;214: 997–1007 e1002. doi:10.1016/j.jamcollsurg.2012.02.009 [PMC free article] [PubMed]
14. Metildi CA, Kaushal S, Luiken GA, Hoffman RM, Bouvet M. Advantages of fluorescence-guided laparoscopic surgery of pancreatic cancer labeled with fluorescent anti-carcinoembryonic antigen antibodies in an orthotopic mouse model. J Am Coll Surg. 2014;219: 132–141. doi: 10.1016/j.jamcollsurg.2014.02.021[PMC free article] [PubMed]
15. Metildi CA, Kaushal S, Luiken GA, Talamini MA, Hoffman RM, Bouvet M. Fluorescently labeled chimeric anti-CEA antibody improves detection and resection of human colon cancer in a patient-derived orthotopic xenograft (PDOX) nude mouse model. J Surg Oncol. 2014;109: 451–458. doi: 10.1002/jso.23507[PMC free article] [PubMed]
16. Metildi CA, Kaushal S, Pu M, Messer KA, Luiken GA, Moossa AR, et al. Fluorescence-guided surgery with a fluorophore-conjugated antibody to carcinoembryonic antigen (CEA), that highlights the tumor, improves surgical resection and increases survival in orthotopic mouse models of human pancreatic cancer.Ann Surg Oncol. 2014;21: 1405–1411. doi: 10.1245/s10434-014-3495-y [PMC free article] [PubMed]
17. Tran Cao HS, Kaushal S, Metildi CA, Menen RS, Lee C, Snyder CS, et al. Tumor-specific fluorescence antibody imaging enables accurate staging laparoscopy in an orthotopic model of pancreatic cancer.Hepatogastroenterology. 2012;59: 1994–1999. doi: 10.5754/hge11836 [PMC free article] [PubMed]
18. Bouvet M, Wang J, Nardin SR, Nassirpour R, Yang M, Baranov E, et al. Real-time optical imaging of primary tumor growth and multiple metastatic events in a pancreatic cancer orthotopic model. Cancer Res. 2002;62: 1534–1540. [PubMed]
19. Bouvet M, Yang M, Nardin S, Wang X, Jiang P, Baranov E, et al. Chronologically-specific metastatic targeting of human pancreatic tumors in orthotopic models. Clin Exp Metastasis. 2000;18: 213–218.[PubMed]
20. Fu X, Guadagni F, Hoffman RM. A metastatic nude-mouse model of human pancreatic cancer constructed orthotopically with histologically intact patient specimens. Proc Natl Acad Sci U S A. 1992;89: 5645–5649. [PMC free article] [PubMed]
21. Furukawa T, Kubota T, Watanabe M, Kitajima M, Hoffman RM. A novel "patient-like" treatment model of human pancreatic cancer constructed using orthotopic transplantation of histologically intact human tumor tissue in nude mice. Cancer Res. 1993;53: 3070–3072. [PubMed]
22. Yamauchi K, Yang M, Jiang P, Xu M, Yamamoto N, Tsuchiya H, et al. Development of real-time subcellular dynamic multicolor imaging of cancer-cell trafficking in live mice with a variable-magnification whole-mouse imaging system. Cancer Res. 2006;66: 4208–4214. [PubMed]
23. Nakajima T, Sano K, Choyke PL, Kobayashi H. Improving the efficacy of Photoimmunotherapy (PIT) using a cocktail of antibody conjugates in a multiple antigen tumor model. Theranostics. 2013;3: 357–365. doi: 10.7150/thno.5908 [PMC free article] [PubMed]
Theranostics. 2015; 5(7): 698–709.
Published online 2015 Mar 19. doi:  10.7150/thno.11559

Near Infrared Photoimmunotherapy in the Treatment of Pleural Disseminated NSCLC: Preclinical Experience

Kazuhide Sato, Tadanobu Nagaya, Peter L. Choyke, and Hisataka Kobayashi?

? Corresponding author: Hisataka Kobayashi, MD. PhD. Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, NIH, Building 10, RoomB3B69, MSC1088, Bethesda, MD 20892-1088. Phone: 301-435-4086, Fax: 301-402-3191. E-mail:vog.hin.liam@hsayaboK.
Competing Interests: The authors have declared that no competing interest exists.

Received 2015 Jan 10; Accepted 2015 Feb 23.

 

Introduction

Lung cancer is the most common cause of cancer-related deaths worldwide. In the USA in 2014, 224,210 people were diagnosed with lung cancer and 159,260 died 1. Lung cancer is an aggressive disease with a very low 5-year survival. About 80% of lung cancers are histologically classified as non-small cell lung carcinoma (NSCLC). During the course of lung cancer, pleural spread of NSCLC, which is a lethal complication, frequently occurs in advanced patients 2. Although early stage and locally advanced NSCLC can be treated with a combination of surgery, chemotherapy, and radiation therapy, palliative chemotherapy is the only practical treatment for NSCLC with pleural metastases, resulting in only 6-9 month median survival 3. In recognition of the poor prognosis associated with pleural metastasis, such disease has recently been reclassified from T4 to M1a 4. Therefore, therapies that could treat pleural metastases without excessive collateral damage to the lungs might be predicted to prolong survival.

Intrapleural conventional photodynamic therapy (PDT) has been previously tested in patients after surgical debulking of pleural disease 5. However, this treatment (using porfimer sodium as the PDT agent) produced some toxicities due to the poor selectivity of the agent. PDT for malignant pleural mesothelioma was also performed after surgical debulking and immunochemotherapy, this phase III study for malignant pleural mesothelioma failed to show a difference in overall survival or progression free survival for the group with additional intraoperative PDT 6. More recently, a phase II trial of pleural PDT after surgery for NSCLC with pleural spread demonstrated that surgery and conventional PDT could be performed safely resulting in good local control and prolonged median survival 7 Thus, conventional PDT results in equivocal benefits for patients with metastases to the pleural. One clear problem with conventional PDT is that produces considerable damage to adjacent tissues thus, negating any potential benefit from the treatment itself.

The concept of using targeted light therapy is over three decades old 8,9. However, the original PDT agents were highly hydrophobic and therefore the pharmacokinetics of antibody conjugated PDT agents were difficult to target to tumors alone. Previous studies have attempted to target conventional PDT agents by conjugating them to antibodies. Unfortunately, these conjugates were usually trapped in the liver and could only be used in isolated body cavities such as the peritoneum 10,11. A study using a more hydrophilic phthalocyanine-based photosensitizer (Aluminum (III) Phthalocyanine Tetrasulfonate) has been published, however, no in vivo treatment response data was reported 12. The recognition that substituting a water soluble phthalocyanine-based photosensitizer (IR700) in the conjugation with an antibody and applying near infrared light has led to much higher selectivity. NIR-PIT differs from these prior PDT not only in the water-solubility of the photosensitizer, but also in its reliance on NIR light that has better tissue penetration than the lower wavelengths used for exciting PDT agents. This antibody-photosensitizer conjugates (APC) demonstrates similar intravenous pharmacokinetics to naked antibodies, resulting in highly targeted tumor accumulation with minimal non-target binding. When bound to targeted cells, APCs induce rapid, selective cytotoxicity after exposure to NIR light. In vitro studies have demonstrated that NIR-PIT is highly target cell-specific and leads to rapid and irreversible cell death due to membrane damage 1316.

One obvious limitation of NIR-PIT is that it would seem limited to tumors located relatively shallow from the surface that can be easily exposed to NIR light. However, light can be administered endoscopically and among the organs, the lungs have the best ability to transmit light because they are mostly filled with air. Thus, NIR light administered to the thoracic cavity could easily penetrate within pleural disease. In this study, we investigate the efficacy of NIR-PIT for treating pleural disease in a NSCLC mouse model.

 

Materials and methods

Reagents

Water soluble, silicon-phthalocyanine derivative, IRDye 700DX NHS ester and IRDye 800CW NHS ester were obtained from LI-COR Bioscience (Lincoln, NE, USA). Panitumumab, a fully humanized IgG2 mAb directed against EGFR, was purchased from Amgen (Thousand Oaks, CA, USA). Trastuzumab, 95% humanized IgG1 mAb directed against HER2, was purchased from Genentech (South San Francisco, CA, USA). All other chemicals were of reagent grade.

Synthesis of Antibody-dye conjugates

Conjugation of dyes with mAbs was performed according to previous reports 13,14. In brief, panitumumab or trastuzumab (1 mg, 6.8 nmol) was incubated with IR700 NHS ester (60.2 µg, 30.8 nmol) or IR800CW NHS ester (35.9 µg, 30.8 nmol) in 0.1 mol/L Na2HPO4 (pH 8.6) at room temperature for 1 hr. The mixture was purified with a Sephadex G25 column (PD-10; GE Healthcare, Piscataway, NJ, USA). The protein concentration was determined with Coomassie Plus protein assay kit (Thermo Fisher Scientific Inc, Rockford, IL, USA) by measuring the absorption at 595 nm with spectroscopy (8453 Value System; Agilent Technologies, Santa Clara, CA, USA). The concentration of IR700 or IR800 was measured by absorption at 689 nm or 774 nm respectively to confirm the number of fluorophore molecules conjugated to each mAb. The synthesis was controlled so that an average of four IR700 molecules or two IR800 molecules were bound to a single antibody. We performed SDS-PAGE as a quality control for each conjugate as previously reported 13. We abbreviate IR700 conjugated to trastuzumab as tra-IR700, to panitumumab as pan-IR700 and IR800 conjugated to trastuzumab as tra-IR800.

Cell culture

HER2 and luciferase/GFP-expressing Calu3-luc-GFP cells were established with a transfection of RediFect Red-FLuc-GFP (PerkinElmer, Waltham, MA, USA). High GFP and luciferase expression was confirmed with 10 passages of the cells. Balb/3T3 cells were transfected with RFP (EF1a)-Puro lentiviral particles (AMSBIO, Cambridge, MA, USA). High, stable RFP expression was confirmed after 10 passages in the absence of a selection agent. To evaluate specific cell killing by NIR-PIT, 3T3 cells stably expressing RFP (3T3-RFP) were used as negative controls. Cells were grown in RPMI 1640 (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies) in tissue culture flasks in a humidified incubator at 37°C at an atmosphere of 95% air and 5% carbon dioxide.

Flow Cytometry

Fluorescence from cells after incubation with pan-IR700 or tra-IR700 was measured with a flow cytometer (FACS Calibur, BD BioSciences, San Jose, CA, USA) and CellQuest software (BD BioSciences). Calu3-luc-GFP cells (1×105) were incubated with each APC for 6 hr at 37°C. To validate the specific binding of the conjugated antibody, excess antibody (50 µg) was used to block 0.5 µg of antibody-dye conjugates 16.

Fluorescence microscopy

To detect the antigen specific localization of antibody-dye conjugates, fluorescence microscopy was performed (IX61 or IX81; Olympus America, Melville, NY, USA). Ten thousand cells were seeded on cover-glass-bottomed dishes and incubated for 24 hr. Tra-IR700 was then added to the culture medium at 10 µg/mL and incubated at 37°C for 6 hr. The cells were then washed with PBS; Propidium Iodide (PI)(1:2000)(Life Technologies) and Cytox Blue (1:500)(Life Technologies), were used to detect dead cells. They were added into the media 30 min before the observation. The cells were then exposed to NIR light (2 J/cm2) and serial images were obtained. The filter was set to detect IR700 fluorescence with a 590-650 nm excitation filter, and a 665-740 nm band pass emission filter.

Analysis of the images was performed with ImageJ software (http://rsb.info.nih.gov/ij/) 17.

In vitro NIR-PIT

One hundred thousand cells were seeded into 24 well plates or ten million cells were seeded onto a 10 cm dish and incubated for 24 hr. Medium was replaced with fresh culture medium containing 10 µg/mL of tra-IR700 which was incubated for 6 hr at 37°C. After washing with PBS, phenol red free culture medium was added. Then, cells were irradiated with a NIR laser, which emits light at 685 to 695 nm wavelength (BWF5-690-8-600-0.37; B&W TEK INC., Newark, DE, USA). The actual power density of mW/cm2 was measured with an optical power meter (PM 100, Thorlabs, Newton, NJ, USA).

Cytotoxicity/ Phototoxicity assay

The cytotoxic effects of NIR-PIT with tra-IR700 were determined by the luciferase activity and flow cytometric PI staining. For luciferase activity, 150 μg/mL of D-luciferin-containing media (Gold Biotechnology, St Louis, MO, USA) was administered to PBS-washed cells 1 hr after NIR-PIT, and analyzed on a bioluminescence imaging (BLI) system (Photon Imager; Biospace Lab, Paris, France). For the flow cytometric assay, cells were trypsinized 1 hr after treatment and washed with PBS. PI was added to the cell suspension (final 2 μg/mL) and incubated at room temperature for 30 min, prior to flow cytometry.

To investigate the specificity of tra-IR700, excess trastuzumab 1,000 µg/mL added to the medium for 1 hr, and 10 µg/mL of tra-IR700 was added to the media for 6 hr. Without washing with PBS, NIR light was administered and 1 hr later PI staining was performed as above.

Estimation of GFP fluorescence intensity in vitro

Two hundred thousand cells were seeded on cover-glass-bottomed dishes and incubated for 12 hr. Tra-IR700 was then added to the culture medium (phenol red free) at 10 µg/mL and incubated at 37°C for 6 hr, followed by NIR-PIT. Cells were trypsinized 1 hr after treatment and washed with PBS, then analyzed by flow cytometry.

Animal and tumor models

All in vivo procedures were conducted in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), US National Research Council, and approved by the local Animal Care and Use Committee. Six- to eight-week-old female homozygote athymic nude mice were purchased from Charles River (NCI-Frederick). During procedures, the mice were anesthetized with inhaled isoflurane.

Six million Calu3-luc-GFP cells were injected subcutaneously in the right dorsum of the mice. The greatest longitudinal diameter (length) and the greatest transverse diameter (width) were measured with an external caliper. Tumor volumes based on caliper measurements were calculated by the following formula; tumor volume = length × width2 × 0.5. Tumors reaching approximately 100 mm3 in volume were selected for further experiments. Body weight was checked on the scale.

For BLI, D-luciferin (15 mg/mL, 200 μL) was injected intraperitoneally and the mice were analyzed with a Photon Imager for luciferase activity at day 11. Mice were selected for further study based on tumor size and bioluminescence.

In order to create a pleural disseminated NSCLC model, six million Calu3-luc-GFP NSCLC cells in PBS (total 200 μL) were injected into the thoracic cavity through a right intercostal space using a 30G needle. To avoid lung injury, the needle could only be inserted 5 mm (a foam styrol stopper prevented deeper insertion). Twenty days later, bioluminescence was performed after D-luciferin (15 mg/mL, 200 μL) was injected intraperitoneally and the mice were imaged with the Photon Imager; mice with sufficient activity were selected for further study.

In vivo fluorescence imaging

In vivo fluorescence images were obtained with a Pearl Imager (LI-COR Bioscience) for detecting IR700/ IR800 fluorescence, and a Maestro Imager (CRi, Woburn, MA, USA) for GFP. For GFP, a band-pass filter from 445 to 490 nm (excitation) and a long-pass blue filter over 515 nm (emission) were used. The tunable emission filter was automatically stepped in 10 nm increments from 500 to 600 nm for the green filter sets at a constant exposure (1000 msec). The spectral fluorescence images consist of autofluorescence spectra and the spectra from GFP (Calu3-luc-GFP tumor), which were then unmixed, based on the characteristic spectral pattern of GFP, using Maestro software (CRi).

Fluorescence thoracoscopy

A model BF XP-60 bronchoscope system was inserted by a trained bronchoscopist/thoracosciopist (KS) via an intercostal space after the animal was euthanized, and the intrathoracic cavity was observed with white light and fluorescence imaging using multi-band excitation filters. Thoracoscopic images were obtained via a dichroic splitter, in which both the excitation light images were displayed using the image processor (OTV-S7; Olympus Co., Tokyo, Japan; not commercially available), and the fluorescence images, which were filtered by in-house designed multicolor emission filters (516 to 556 nm band-pass for GFP and 680 to 710 nm band-pass for IR700) were detected with an (EM)-CCD camera (Texas Instruments, Dallas, TX, USA). Both images were displayed side by side on the PC monitor with DualView 2 software (RGB Spectrum). Real-time images of both white light and fluorescence images were recorded. Camera gain, exposure time, and binning for the fluorescence images were held constant in each fluorescent protein throughout the study. Analysis of the images was performed with ImageJ software (http://rsb.info.nih.gov/ij/).

Characterization of the pleural disseminated mouse model

Both the disseminated pleural model and the subcutaneous bilateral flank models received 100 μg of tra-IR700 or tra-IR800 intravenously (tra-IR800 was used to avoid auto-fluorescence). One day after injection, serial images were performed with a fluorescence imager (Pearl Imager) for detecting IR700/ IR800 fluorescence, with the Photon Imager for BLI, and the Olympus BF XP-60 thoracoscopy. Images of the mice were obtained with an iphone5 (Apple Inc., Cupertino, CA, USA).

In vivo NIR-PIT

Calu3-luc-GFP right dorsum tumor xenografts were randomized into 4 groups of at least 7 animals per group undergoing one of the following treatments: (repeated PIT)18: (1) no treatment (control); (2) only NIR light exposure at 50 J/cm2 on day 1 and 100 J/cm2 on day 2; (3) 100 μg of tra-IR700 i.v., no NIR light exposure; (4) 100 μg of tra-IR700 i.v., NIR light was administered at 50 J/cm2 on day 1 after injection and 100 J/cm2on day 2 after injection. These therapies were performed only once at day 14 after cell implantation. Mice were monitored daily, and tumor volumes and body weight were measured three times a week until the tumor diameter reached 2cm, whereupon the mouse was euthanized with carbon dioxide.

In vivo imaging was acquired with a fluorescence imager (Pearl Imager) for detecting IR700 fluorescence, and the Photon Imager for BLI. For analyzing BLI, ROI of similar size were placed over the entire tumor.

For evaluation of NIR-PIT effects in the pleural disseminated NSCLC mouse model, mice were randomized into 4 groups of 7 animals per group including: (1) no treatment (control); (2) only NIR light exposure at 50 J/cm2 on day 1 and 100 J/cm2 on day 2; (3) 100 μg of tra-IR700 i.v., no NIR light exposure; (4) 100 μg of tra-IR700 i.v., NIR light was administered at 50 J/cm2 on day 1 after injection and 100 J/cm2 on day 2 after injection. NIR light was applied transcutaneously followed by serial fluorescence imaging and BLI.

Histological analysis

To evaluate histological changes of lung at 1 day after PIT, microscopy was performed (BX51, Olympus America). Lungs with tumors were harvested and placed in 10% formalin. Serial 10-μm slice sections were fixed on glass slide for H-E staining.

Statistical Analysis

Data are expressed as means ± s.e.m. from a minimum of four experiments, unless otherwise indicated. Statistical analyses were carried out using a statistics program (GraphPad Prism; GraphPad Software, La Jolla, CA, USA). For multiple comparisons, a one-way analysis of variance (ANOVA) with Tukey's test was used. The cumulative probability of survival, determined herein as the tumor diameter failing to reach 2 cm, was estimated in each group with the use of the Kaplan-Meier survival curve analysis, and the results were compared with the log-rank test and Wilcoxon test. p < 0.05 was considered to indicate a statistically significant difference.

 

Results

Characterization of the cell line and the NIR-PIT effect

To monitor optically the effect of NIR-PIT, NSCLC cell line Calu3 was genetically modified to express GFP and luciferase (Calu3-luc-GFP)(Fig. ?(Calu3-luc-GFP)(Fig.1A).1A). The fluorescence signals obtained with pan-IR700 and tra-IR700 using Calu3-luc-GFP cells were evaluated by FACS. After 6 hr incubation with either pan-IR700 or tra-IR700, Calu3-luc-GFP cells showed higher brightness with tra-IR700 than with pan-IR700 consistent with the expression profile (Fig. ?(Fig.1B).1B). These signals were completely blocked by the addition of excess trastuzumab, suggesting specific binding and validating that the addition of the luciferase/ GFP gene had not altered the cell expression profile. Serial fluorescence microscopy of Calu3-luc-GFP cells performed before and after NIR-PIT (2 J/cm2) demonstrated rapidly appearing cellular swelling, bleb formation and rupture of the lysosome (Fig. ?(Fig.1C).1C). Time-lapse imaging showed acute morphologic changes in the cell membrane within 25 minutes and fluorescence of PI indicating cell death (Additional File 2: Video S1). No significant changes were observed in HER2-negative 3T3 cells after exposure to NIR light, suggesting NIR-PIT induced no damage in non-target cells (Additional File 1: Fig. S1). Based on the incorporation of PI, the cell death percentage increased in a light dose dependent manner. No significant cytotoxicity was observed with NIR light exposure alone or with tra-IR700 alone (Fig. ?(Fig.1D).1D). NIR-PIT was blocked with excess trastuzumab even in tra-IR700 containing media (Additional File 1: Fig. S2). Bioluminescence showed significant decreases of relative light units (RLU) in NIR-PIT treated cells (Fig. ?(Fig.1E).1E). BLI also showed a decrease of luciferase activity in a light dose dependent manner (Fig. ?(Fig.1F).1F). GFP fluorescence intensity was greatly reduced in dead cells (stained positive with PI), while GFP fluorescence was preserved in surviving cells (Fig. ?(Fig.1G).1G). GFP fluorescence was likely reduced after NIR-PIT because the GFP was extruded from the cytoplasm after membrane rupture leading to dilution and/or denaturation. The GFP fluorescence ratio on FACS showed decreases in a light dose dependent manner, while no decrease was detected with NIR light exposure or Pan-IR700 alone (Fig. ?(Fig.1H).1H). Collectively, these data suggested that the effects of NIR-PIT on Calu3-luc-GFP could be monitored with GFP fluorescence and bioluminescence.

Figure 1

Characterization of cell line and evaluation of NIR-PIT effect. (A) Stable expression of GFP was confirmed with FACS. (B) Expression of HER1 and HER2 in Calu3-luc-GFP cells was examined with FACS. HER2 was overexpressed while HER1 showed normal expression.

In vivo NIR-PIT reduced tumor volume and luciferase activity in a flank xenograft model

In vivo NIR-PIT experiments were first conducted on flank xenografts of Calu3-luc-GFP. The NIR-PIT regimen and imaging protocol are depicted in Fig. ?Fig.2A.2A. Both BLI and fluorescence decreased after NIR-PIT (Fig. ?(Fig.2B2B and Additional File 1: Fig. S3A). RLU of tumor in other groups showed a gradual increase due to tumor growth. In contrast, luciferase activity decreased 1 day after repeated NIR-PIT (*p = 0.002 < 0.01, PIT vs. APC, Tukey's test with ANOVA)(Fig. ?ANOVA)(Fig.2C).2C). The body weight (BW) ratio showed no remarkable acute toxicity (Fig. ?(Fig.2D).2D). Significant decreases (**p = 0.0004 < 0.001, PIT vs. APC, Tukey's test with ANOVA) in tumor volume were confirmed, which was consistent with luciferase activities (Fig. ?(Fig.2E).2E). Survival was prolonged significantly in the PIT group (***P < 0.0001, Long-rank test and Wilcoxon test)(Fig. ?test)(Fig.2F).2F). Since bioluminescence is more sensitive to tumor killing as it is based on live cells, the physical tumor volume took longer to show the effect of NIR-PIT. Collectively, these data suggest that NIR-PIT caused significant tumor reduction and prolonged survival in the in vivo flank tumor model.

Figure 2

Evaluation of NIR-PIT in flank model. (A) The regimen of NIR-PIT is shown. Images were obtained as indicated. (B) In vivo BLI and fluorescence imaging of tumor bearing mice in response to NIR-PIT. Prior to NIR-PIT, tumors were approximately the same

Characterization of the pleural disseminated NSCLC mouse model

Prior to therapy, implanted thoracic tumors were evaluated with serial fluorescence imaging, BLI and fluorescence thoracoscopy. The implanted thoracic disseminated tumors demonstrated high activity with fluorescence imaging based on IR700, IR800 and GFP, but also high activity on bioluminescence, which co-localized with each other (Fig. ?(Fig.3).3). Fluorescence thoracoscopy indicated that disseminated tumor establishment and the good contrast of IR700 between tumors and intrathoracic organs (Fig. ?(Fig.33 and Additional File 3: video S2), which confirmed pleural metastases that fluoresced preferentially with tra-IR700. These data suggest that pleural disseminated NSCLC cancer mouse model with Calu3-luc-GFP cells was successfully established; intravenously injection of agent could reach the disseminated tumors.

Figure 3

Characterization of the pleural disseminated NSCLC model. In vivo BLI and fluorescence (GFP/ IR700/ IR800) imaging of Calu3-luc-GFP tumor in flank and pleural disseminated model are shown and demonstrate colocalization of fluorescence. To avoid auto-fluorescence,

In vivo NIR-PIT effect in pleural disseminated cancer mouse model

After treatment with NIR-PIT pleural disseminated tumors decreased in bioluminescence and fluorescence (Fig. ?(Fig.4A4A and ?and4B4B and Additional File 1: Fig. S3B). While the RLU decreased in the NIR-PIT treated tumors, RLU of tumor in other groups showed a gradual increase due to tumor growth. In contrast, luciferase activity decreased 1 day after repeated NIR-PIT (*p = 0.0180 < 0.05, PIT vs. APC, Tukey's test with ANOVA)(Fig.?ANOVA)(Fig.4C).4C). The BW ratio showed no change (ns, PIT vs. APC, light, control, Tukey's test with ANOVA) (Fig. ?(Fig.4D).4D). Taken together, these data suggest that NIR-PIT caused significant tumor reduction in vivo pleural disseminated model.

Figure 4

Evaluation of NIR-PIT effects on pleural disseminated NSCLC model by bioluminescence. (A) The regimen of NIR-PIT is shown. Images were obtained as indicated. (B) In vivo BLI and fluorescence imaging of the pleural disseminated model. Prior to treatment

In vivo NIR-PIT effect assessed with GFP fluorescence imaging

Finally, to assess the effect of repeated NIR-PIT on Calu3-luc-GFP tumor in vivo, GFP fluorescence imaging was performed in both the flank model and pleural disseminated model (Fig. ?(Fig.5A).5A). With the flank model, both GFP/ IR700 fluorescence disappeared at 1 day after NIR-PIT, which was confirmed by ex vivo tumor imaging (Fig. ?(Fig.5B).5B). Using fluorescence thoracoscopy, GFP and IR700 fluorescence disappeared (Fig. ?(Fig.5C5C and videos S3 and S4 in Additional Files 4-5). A small effusion was observed with thoracoscopy (Fig. ?(Fig.5C5C arrow). Moreover, there was no apparent damage to the normal lung by NIR-PIT as observed with histological analysis (Fig. ?(Fig.66).

Figure 5

Evaluation of NIR-PIT effects on pleural metastassi model by GFP fluorescence imaging. (A) The regimen of NIR-PIT is shown. Images were obtained as indicated. (B) In vivo BLI and fluorescence imaging of the flank model in response to NIR-PIT. (C) In

Figure 6

Histological evaluation of NIR-PIT effect on lung. No apparent damage to the lung was showed by HE-staining at 1 day after NIR-PIT compared to no Tx. Bar = 50 µm.

 

Discussion

In this study, we demonstrate that an APC can be delivered to both flank and intrathoracic tumor after intravenous injection and that subsequent NIR-PIT can be successfully performed transcutaneously to the mouse thorax with acceptable morbidity. Among the imaging tools used to document tumor regression, which included BLI, fluorescence imaging and fluorescence thoracoscopy, the latter two could be used in clinical practice using the IR700 dye 19,20. BLI using firefly luciferase, although less suitable for clinical translation, was useful as a primary outcome measure as it requires both oxygen and ATP to actively transport the substrate luciferin and subsequently catalyze the photochemical reaction 21,22. Since NIR-PIT-induced necrotic cell death releases ATP, BLI is an appropriate and sensitive biomarker for NIR-PIT 16,23.In vivo GFP fluorescence imaging enables the full process of tumorigenesis, treatment, regression, metastasis, or recurrence, to be detected although this also is not translatable 24. The high level of GFP tumor fluorescence in this model permitted imaging with quantification of tumor growth and dissemination without the need for additional contrast agents. By employing cytoplasmic GFP expressing cells, antitumor effects induced by NIR-PIT could be clearly monitored as extrusion of GFP from treated cells resulted in a diminution of signal 15. The development of a mini-endoscope mimicking thoracoscopy permits intrathoracic fluorescence imaging and is the most likely method by which NIR-PIT would be administered and monitored in humans. By changing the filter sets, multicolor endoscopic imaging becomes possible to simultaneously monitor tumor regression with GFP fluorescence and accumulation of APC with IR700 fluorescence as shown in videos S3 and S4 25 (Additional Files 4-5). Both real-time color capability and direct access to the disseminated tumors, resulted in much higher resolution imaging. From the photophysical point of view, the endoscope can minimize light scattering and absorbance that is caused by overlapping tissue, resulting in more precise depiction of the lesion.

In this study, we use a pleural disseminated tumor model by simple tumor cell injection in the thoracic cavity. A variety of animal models could be used including percutaneous orthotopic injection (POI), surgical orthotopic injection (SOI), and transpleural orthotopic injection (TOI)26. Various advantages and disadvantages exist among these models. For example, SOI requires high skills and is invasive, resulting in high pre-procedure mortality, however SOI is thought to be more physiologic than others 26,27. The POI model has the advantages of simplicity and less invasiveness with a very low pre-procedure mortality rate 26. A recent study reported that implantation rates were similar among these models 26. With these considerations, we chose the POI approach. This approach had a high implantation rate (around 85%) of NSCLC pleural dissemination, which was confirmed by non-invasive BLI.

The survival of patients with NSCLC patients with pleural disseminations is only 6 to 9 months even with systemic chemotherapy 3. Surgery is not currently performed because of its morbidity and limited benefit 28. While not likely to be curative, NIR-PIT could offer the benefit of local control with minimal invasiveness. Moreover, NIR-PIT could be readily used as an adjunct to conventional surgery at the time of initial diagnosis.

There are several limitations to this study. First, not all lung cancers overexpress HER2, and therefore this particular target may not be ideal in other lung cancers. Fortunately, NIR-PIT has proven effective with almost all APCs with which it has been attempted and therefore, it is likely that the proper APC or combination of APCs could be found to treat a specific phenotype of lung cancer cell membrane expression1315,29,30. We were also unable to determine the long-term side effects of NIR-PIT in this limited model. Short-term studies of the mice demonstrated no apparent adverse events after NIR-PIT. It is possible that sudden widespread cell necrosis could cause either acute or delayed toxicity but none was observed in this model. Only small reactive pleural effusions were observed by thoracoscopy. Additionally, it is clear that NIR-PIT alone will not be sufficient to cure thoracic metastases, although the use of NIR light to activate IR700 will produce deeper tissue penetration within larger masses than the shorter wavelengths of light used in conventional PDT photoactivation or other light therapies such as those using UV light 31. Therefore, we foresee NIR-PIT as an adjuvant to surgery with an initial debulking procedure followed by NIR-PIT to “mop up” residual disease. Furthermore, it is interesting to consider the possibility that systemic chemotherapy may be more effective after NIR-PIT. Previous studies have shown that NIR-PIT causes treated tumors to exhibit increased permeability to nano-sized drugs. Therefore, current or future chemotherapies for lung cancer may benefit from prior treatment with NIR-PIT 32. Finally, in this study we irradiated transcutaneously which is difficult to translate to the clinic, however, it would be feasible to deliver light via thoracoscopy, bronchoscopy or even during open-surgery. Thus, although this particular animal model is not directly translatable, the principle of treating thoracic malignancies with light therapy is feasible.

In conclusion, this study demonstrates that NIR-PIT effectively treated pleural metastases in a mouse model of NSCLC. NIR-PIT could be a promising adjuvant for treating pleural carcinomatosis replacing or adding to existing therapies such as surgery and chemotherapy.

 

Supplementary Material

Additional File 1

Supplementary Figures S1-S3.

Additional File 2

Video S1.

Additional File 3

Video S2.

Additional File 4

Video S3.

Additional File 5

Video S4.

Acknowledgments

This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. K.S. is supported with JSPS Research Fellowship for Japanese Biomedical and Behavioral Researchers at NIH.

Contributions

K.S. mainly conducted experiments, performed analysis and wrote the manuscript; T.N. conducted thoracoscopy with K.S. P.L.C. wrote the manuscript and supervised the project; and H.K. planned and initiated the project, designed and conducted experiments, wrote the manuscript, and supervised the entire project.

 

References

1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA cancer J Clin. 2014;64:9–29.doi:10.3322/caac.21208. [PubMed]
2. Powell C a, Halmos B, Nana-Sinkam SP. Update in lung cancer and mesothelioma 2012. Am J Respir Crit Care Med. 2013;188:157–66. doi:10.1164/rccm.201304-0716UP. [PMC free article] [PubMed]
3. Reyes L, Parvez Z, Regal AM TH. Neoadjuvant chemotherapy and operations in the treatment of lung cancer with pleural effusion. J Thorac Cardiovasc Surg. 1991;101:946–7. [PubMed]
4. Goldstraw P, Crowley J. The IASLC Lung Cancer Staging Project: proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM Classification of. J Thorac Oncol.2007;2:706–14. [PubMed]
5. Pass H, DeLaney T, Tochner Z. Intrapleural photodynamic therapy: results of a phase I trial. Ann Surg Oncol. 1994;1:28–37. [PubMed]
6. Pass HI, Temeck BK, Kranda K, Thomas G, Russo A, Smith P. et al. Phase III randomized trial of surgery with or without intraoperative photodynamic therapy and postoperative immunochemotherapy for malignant pleural mesothelioma. Ann Surg Oncol. 1997;4:628–33. doi:10.1007/BF02303746. [PubMed]
7. Friedberg JS, Mick R, Stevenson JP, Zhu T, Busch TM, Shin D. et al. Phase II trial of pleural photodynamic therapy and surgery for patients with non-small-cell lung cancer with pleural spread. J Clin Oncol. 2004;22:2192–201. doi:10.1200/JCO.2004.07.097. [PubMed]
8. Mew D, Wat C, Towers GHN, Levy JG. PHOTOIMMUNOTHERAPY?: TREATMENT OF ANIMAL TUMORS WITH TUMOR-SPECIFIC. J Immunol. 1983;130:1473–7. [PubMed]
9. Oseroff a R, Ohuoha D, Hasan T, Bommer JC. Antibody-targeted photolysis: selective photodestruction of human T-cell leukemia cells using monoclonal antibody-chlorin e6 conjugates. Proc Natl Acad Sci U S A.1986;83:8744–8. [PMC free article] [PubMed]
10. Goff BA, Hermanto U, Rumbaugh J, Blake J, Bamberg M, Hasan T. Photoimmunotherapy and biodistribution with an OC125-chlorin immunoconjugate in an in vivo murine ovarian cancer model. Br J Cancer. 1994;70:474–80. [PMC free article] [PubMed]
11. Vrouenraets MB, Visser GWM, Stewart FA, Stigter M, Oppelaar H, Postmus PE. et al. Development of meta -Tetrahydroxyphenylchlorin-Monoclonal Antibody Conjugates for Photoimmunotherapy. Cancer Reserch. 1999;59:1505–13. [PubMed]
12. Vrouenraets MB, Visser GWM, Stigter M, Oppelaar H, Snow GB, van Dongen GA. Targeting of aluminum (III) phthalocyanine tetrasulfonate by use of internalizing monoclonal antibodies: Improved efficacy in photodynamic therapy. Cancer Res. 2001;61:1970–5. [PubMed]
13. Mitsunaga M, Ogawa M, Kosaka N, Rosenblum LT, Choyke PL, Kobayashi H. Cancer cell – selective in vivo near infrared photoimmunotherapy targeting specific membrane molecules. Nat Med. 2011;17:1685–91. doi:10.1038/nm.2554. [PMC free article] [PubMed]
14. Sato K, Watanabe R, Hanaoka H, Harada T, Nakajima T, Kim I. et al. Photoimmunotherapy: Comparative effectiveness of two monoclonal antibodies targeting the epidermal growth factor receptor. Mol Oncol. 2014;8:620–32. doi:10.1016/j.molonc.2014.01.006. [PMC free article] [PubMed]
15. Sato K, Choyke PL, Kobayashi H. Photoimmunotherapy of Gastric Cancer Peritoneal Carcinomatosis in a Mouse Model. PLoS One. 2014;9:e113276.. doi:10.1371/journal.pone.0113276. [PMC free article][PubMed]
16. Sato K, Hanaoka H, Watanabe R, Nakajima T, Choyke PL, Kobayashi H. Near infrared photoimmunotherapy in the treatment of disseminated peritoneal ovarian cancer. Mol Cancer Ther. 2015 Jan;14(1):141–50. doi: 10.1158/1535-7163.MCT-14-0658. Epub 2014 Nov 21. [PMC free article][PubMed]
17. Sato K, Watanabe T, Wang S, Kakeno M, Matsuzawa K, Matsui T. et al. Numb controls E-cadherin endocytosis through p120 catenin with aPKC. Mol Biol Cell. 2011;22:3103–19. doi:10.1091/mbc.E11-03-0274. [PMC free article] [PubMed]
18. Mitsunaga M, Nakajima T, Sano K, Choyke PL, Kobayashi H. Near-infrared Theranostic Photoimmunotherapy (PIT): Repeated Exposure of Light Enhances the Effect of Immunoconjugate.Bioconjug Chem. 2012;23:604–9. doi:10.1021/bc200648m. [PMC free article] [PubMed]
19. Conway JRW, Carragher NO, Timpson P. Developments in preclinical cancer imaging: innovating the discovery of therapeutics. Nat Rev Cancer. 2014;14:314–28. doi:10.1038/nrc3724. [PubMed]
20. De Jong M, Essers J, van Weerden WM. Imaging preclinical tumour models: improving translational power. Nat Rev Cancer. 2014;14:481–93. doi:10.1038/nrc3751. [PubMed]
21. Rehemtulla A, Stegman LD, Cardozo SJ, Gupta S, Hall DE, Contag CH. et al. Rapid and Quantitative Assessment of Cancer Treatment Response Using In Vivo Bioluminescence Imaging 1 Days post sham treatment. Neoplasia. 2000;2:491–5. [PMC free article] [PubMed]
22. Contag PR, Olomu IN, Stevenson DK CC. Bioluminescent indicators in living mammals. Nat Med.1998;4:245–7. [PubMed]
23. Mitsunaga M, Nakajima T, Sano K, Kramer-Marek G, Choyke PL, Kobayashi H. Immediate in vivo target-specific cancer cell death after near infrared photoimmunotherapy. BMC Cancer. 2012;12:345..doi:10.1186/1471-2407-12-345. [PMC free article] [PubMed]
24. Hoffman RM. The multiple uses of fluorescent proteins to visualize cancer in vivo. Nat Rev Cancer.2005;5:796–806. doi:10.1038/nrc1717. [PubMed]
25. Mitsunaga M, Kosaka N, Kines RC, Roberts JN, Lowy DR, Schiller JT. et al. In vivo longitudinal imaging of experimental human papillomavirus infection in mice with a multicolor fluorescence mini-endoscopy system. Cancer Prev Res (Phila) 2011;4:767–73. doi:10.1158/1940-6207.CAPR-10-0334.[PMC free article] [PubMed]
26. Mordant P, Loriot Y, Lahon B, Castier Y, Leseche G, Soria JC. et al. Bioluminescent orthotopic mouse models of human localized Non-Small cell lung cancer: Feasibility and identification of circulating tumour cells. PLoS One. 2011;6:10–2. doi:10.1371/journal.pone.0026073. [PMC free article] [PubMed]
27. Yang M, Hasegawa S, Jiang P, Wang X, Tan Y, Chishima T. et al. Widespread skeletal metastatic potential of human lung cancer revealed by green fluorescent protein expression. Cancer Res.1998;58:4217–21. [PubMed]
28. Yokoi K, Matsuguma H, Anraku M. Extrapleural pneumonectomy for lung cancer with carcinomatous pleuritis. J Thorac Cardiovasc Surg. 2002;123:184–5. doi:10.1067/mtc.2002.118039. [PubMed]
29. Shirasu N, Yamada H, Shibaguchi H, Kuroki M, Kuroki M. Potent and specific antitumor effect of CEA-targeted photoimmunotherapy. Int J Cancer. 2014;135:2697–710. doi:10.1002/ijc.28907. [PubMed]
30. Nakajima T, Sano K, Choyke PL, Kobayashi H. Improving the efficacy of Photoimmunotherapy (PIT) using a cocktail of antibody conjugates in a multiple antigen tumor model. Theranostics. 2013;3:357–65.doi:10.7150/thno.5908. [PMC free article] [PubMed]
31. Hiroshima Y, Maawy A, Zhang Y, Sato S, Murakami T, Yamamoto M. et al. Fluorescence-guided surgery in combination with UVC irradiation cures metastatic human pancreatic cancer in orthotopic mouse models. PLoS One. 2014;9:e99977.. doi:10.1371/journal.pone.0099977. [PMC free article] [PubMed]
32. Sano K, Nakajima T, Choyke PL, Kobayashi H. Markedly Enhanced Permeability and Retention Effects Induced by Photo-immunotherapy of Tumors. ACS Nano. 2013;7:717–24. doi:10.1021/nn305011p.[PMC free article] [PubMed]


Articles from Theranostics are provided here courtesy of Ivyspring International Publisher


Articles from PLoS ONE are provided here courtesy of Public Library of Science
PLoS One. 2014; 9(12): e114310.
Published online 2014 Dec 2. doi:  10.1371/journal.pone.0114310

Metastatic Recurrence in a Pancreatic Cancer Patient Derived Orthotopic Xenograft (PDOX) Nude Mouse Model Is Inhibited by Neoadjuvant Chemotherapy in Combination with Fluorescence-Guided Surgery with an Anti-CA 19-9-Conjugated Fluorophore

Yukihiko Hiroshima,#1,2,3 Ali Maawy,#2 Yong Zhang,1 Takashi Murakami,3 Masashi Momiyama,3 Ryutaro Mori,3 Ryusei Matsuyama,3 Matthew H. G. Katz,4 Jason B. Fleming,4 Takashi Chishima,3 Kuniya Tanaka,3 Yasushi Ichikawa,3 Itaru Endo,3 Robert M. Hoffman,1,2 and Michael Bouvet2,*

Shree Ram Singh, Editor
Shree Ram Singh, Editor
#Contributed equally.
* E-mail: ude.dscu@tevuobm
Competing Interests: Yukihiko Hiroshima and Yong Zhang are affiliates of AntiCancer Inc. Masashi Momiyama and Takashi Chishima were former affiliates of AntiCancer Inc. Robert M. Hoffman is a non-salaried affiliate of AntiCancer Inc. AntiCancer Inc. markets animal models of cancer. There are no other competing interests. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Conceived and designed the experiments: YH AM YZ TM MM RM RM MK JF TC KT YI IE RH MB. Performed the experiments: YH AM YZ. Analyzed the data: YH AM RH MB. Contributed reagents/materials/analysis tools: YH AM RH MB. Contributed to the writing of the manuscript: YH AM RH MB.

Received 2014 Sep 22; Accepted 2014 Nov 6.

 

Introduction

Complete tumor resection improves overall survival of pancreatic cancer patients, which is presently 5% at five years [1]. Metastatic relapse often occurs following attempted curative resection of the primary tumor as a result of invisible microscopic tumor deposits left behind. Making tumors fluoresce offers great advantages for tumor detection during surgery in order to achieve complete resection [2], [3]. We have previously shown that fluorescence-guided surgery (FGS) for pancreatic cancer decreased the residual tumor burden and improved overall and disease-free survival in mouse models using fluorescently-labeled human pancreatic cancer cell lines [4][6].

Patient-derived orthotopic xenografts (PDOX) recapitulate the biological characteristics of the disease of origin, including metastases [7][11] and are a clinically-relevant model for fluorescence-guided surgery [4],[12][14].

Recently, many studies reported positive outcomes with neoadjuvant chemotherapy (NAC) of pancreatic cancer [15][17]. NAC allows for the identification of those patients with rapidly progressive metastatic disease at the time of preoperative restaging, and can increase the R0 resection rate and reduce the risk of local tumor recurrence [15]. However, a significant number of patients still develop recurrent disease immediately after NAC treatment and subsequent surgical resection [16][18]. Therefore, new strategies in addition to NAC are needed to reduce the recurrence of pancreatic cancer. In this study, we determined the efficacy of CA19-9 conjugated with a fluorescent dye to illuminate pancreatic cancer PDOXs for FGS in combination with NAC.

 

Materials and Methods

Animals

Athymic nu/nu nude mice (AntiCancer Inc., San Diego, CA), 4–6 weeks old, were used in this study. Mice were kept in a barrier facility under HEPA filtration. Mice were fed with an autoclaved laboratory rodent diet. All mouse surgical procedures and imaging were performed with the animals anesthetized by intramuscular injection of 50% ketamine, 38% xylazine, and 12% acepromazine maleate (0.02 ml). Animals received buprenorphine (0.10 mg/kg ip) immediately prior to surgery and once a day over the next 3 days to ameliorate pain. CO2 inhalation was used for euthanasia of all animals at 90 days after surgery. To ensure death following CO2 asphyxiation, cervical dislocation was performed. All animal studies were conducted with an AntiCancer, Inc. Institutional Animal Care and Use Committee (IACUC)-protocol specifically approved for this study and in accordance with the principals and procedures outlined in the National Institute of Health Guide for the Care and Use of Animals under Assurance Number A3873-1.

Establishment of patient derived orthotopic xenograft (PDOX) of pancreatic cancer

Pancreatic cancer patient tumor tissues were obtained at surgery and cut into fragments (3-mm3) and transplanted subcutaneously in nude mice [7], [19]. The subcutaneous tumors were then passaged in nude mice both orthotopically and subcutaneously. All patients provided written informed consent and samples were procured and initially transplanted in NOD/SCID under the approval of the Institutional Review Board of MD Anderson Cancer Center.

Orthotopic tumor implantation

A small 6- to 10-mm transverse incision was made on the left flank of the mouse through the skin and peritoneum. The tail of the pancreas was exposed through this incision, and a single 3-mm3 tumor fragment from subcutaneous tumors was sutured to the tail of the pancreas using 8-0 nylon surgical sutures (Ethilon; Ethicon Inc., NJ, USA). On completion, the tail of the pancreas was returned to the abdomen, and the incision was closed in one layer using 6-0 nylon surgical sutures (Ethilon) [7], [20].

Antibody conjugation and tumor labeling

Monoclonal antibodies specific for carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) were obtained from Abcam Inc. (Cambridge, MA, USA) and (Aragen Bioscience, Inc. (Morgan Hill, CA, USA), respectively. The antibodies were labeled with the DyLight 650 Protein Labeling Kit (Thermofisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions [5], [12], [21]. To determine if the anti-CA19-9 antibody, conjugated with DyLight 650 (anti-CA19-9-650), and the anti-CEA antibody, conjugated with DyLight650 (anti-CEA-650), could label the pancreatic tumor in vivo, 50 µg of anti-CA19-9-650 or anti-CEA-650 were injected into the tail vein of the mice with subcutaneous pancreatic tumors. Twenty-four hours later, whole body images were obtained with the OV100 Small Animal Variable Magnification Imaging System (Olympus, Tokyo, Japan).

Neoadjuvant chemotherapy

After confirmation of tumor engraftment, 32 mice were randomized to 4 groups; BLS only; BLS+NAC; FGS only; and FGS+NAC. Each treatment arm involved 8 tumor-bearing mice. The mice randomized to NAC-treatment were administered 80 mg/kg gemcitabine (GEM) (Eli Lilly and Company, Indianapolis, IN, USA). GEM was injected i.p. on day 8, 15 and 22. No significant effects on body weight, morbidity, or severe toxicity were observed in NAC-treated mice.

Fluorescence-guided surgery

For fluorescence-guided surgery (FGS), a 15-mm transverse incision was made on the left flank of the mouse through the skin and peritoneum which was kept open with a retractor. The tail of the pancreas was exposed through this incision. Fifty µg of anti-CA19-9 antibody, conjugated to DyLight 650, was injected via the tail vein in the mice in the FGS group 24 hours before surgery. A MINI MAGLITE LED PRO flashlight (MAG INSTRUMENT, Ontario, CA, USA) coupled to an excitation filter (ET 640/30X, Chroma Technology Corporation, Bellows Falls, VT, USA) was used as the excitation light source. A Canon EOS 60D digital camera with an EF–S18–55 IS lens (Canon, Tokyo, Japan) coupled with an emission filter (HQ700/75M-HCAR, Chroma Technology Corporation) was used as the real-time image capturing device for FGS. BLS was performed under standard bright-field using an MVX10 microscope (Olympus, Tokyo, Japan). After completion of surgery, the incision was closed in one layer using 6-0 nylon surgical sutures, and the mice were allowed to recover in their cages.

Tissue histology

Tumor samples were removed with surrounding normal tissues at the time of resection. Fresh tissue samples were fixed in 10% formalin and embedded in paraffin before sectioning and staining. Tissue sections (3 µm) were deparaffinized in xylene and rehydrated in an ethanol series. Hematoxylin and eosin (H & E) staining was performed according to standard protocols. For immunohistochemistry, the sections were then treated for 30 min with 0.3% hydrogen peroxide to block endogenous peroxidase activity. The sections were subsequently washed with PBS and unmasked in citrate antigen unmasking solution (Mitsubishi Kagaku Iatron, Inc., Tokyo, Japan) in a water bath for 40 min at 98°C. After incubation with 10% normal goat serum, the sections were incubated with anti-CA19-9 antibody (1?100) and anti-CEA antibody (1?100) at 4°C overnight. The bound primary antibodies were detected by binding with an anti-mouse secondary antibody and an avidin/biotin/horseradish peroxidase complex (DAKO Cytomation, Kyoto, Japan) for 30 min at room temperature. The labeled antigens were visualized with a DAB kit (DAKO Cytomation). The sections were counterstained with hematoxylin and observed with a BH-2 microscope (Olympus, Tokyo, Japan) equipped with an INFINITY1 2.0 megapixel CMOS digital camera (Lumenera Corporation, Ottawa, Canada). All images were acquired using INFINITY ANALYZE software (Lumenera Corporation) without post-acquisition processing.

Evaluation of histopathological response to NAC

Histopathological response to chemotherapy drugs was defined according to Evans’s grading scheme: Grade I, little (<10%) or no tumor cell destruction is evident; Grade IIa, destruction of 10%–50% of tumor cells; Grade IIb, destruction of 51%–90% of tumor cells; Grade III, few (<10%) viable-appearing tumor cells are present; Grade IV, no viable tumor cells are present [22].

Evaluation of tumor recurrence and progression

To assess for recurrence postoperatively, animals underwent laparotomy 12 weeks after surgery, and the tumors were imaged with the Canon EOS 60D digital camera with an EF–S18–55 IS lens (Canon), excised, harvested and weighed for analysis.

Statistical analysis

PASWStatistics 18.0 (SPSS, Inc.) was used for statistical analyses. Tumor weight was expressed as mean ± SD. The two-tailed Student’s t-test was used to compare continuous variables between 2 groups. Comparisons between categorical variables were analyzed with Fisher’s exact test. A p value <0.05 was considered statistically significant for all comparisons.

 

Results

Antibody labeling

The pancreatic PDOX tumor was diagnosed as moderately differentiated adenocarcinoma with H&E staining (Figure 1A). Based on immunohistochemistry, the PDOX tumor was found to be CA19-9-positive and CEA-negative (Figures 1B and 1C). The PDOX was brightly labeled with anti-CA19-9-650 (Figure 1D), but the fluorescence signal with anti-CEA-650 was very weak (Figure 1E). The fluorescence results were consistent with the immunohistochemical results, and based on them, it was decided to use anti-CA19-9-650 to label the PDOX for FGS. Anti-CA19-9-650 was injected in the tail vein of the mice with PDOX tumors 24 hours before FGS.

Figure 1

Antibody labelling of the pancreatic cancer patient derived orthotopic xenograft.

Sensitivity of PDOX to NAC

The PDOX mice were randomized to 4 groups; BLS only; BLS+NAC; FGS only; FGS+NAC. Each treatment arm involved 8 PDOX mice. The mice randomized to the NAC group were treated with GEM on days 8, 15 and 22 (Figure 2). All animals underwent surgery on day 29 (Figures 3). The average excised PDOX tumor weight was 188.5±53.1 mg for BLS-only; 84.5±51.6 mg for BLS+NAC; 299.0±86.3 mg for FGS-only; and 141.8±48.9 mg for FGS+NAC. The average excised tumor weight in the BLS+NAC mice was significantly less than in the BLS-only mice (p?=?0.001). The average excised tumor weight in the FGS+NAC mice was also significantly less than FGS-only mice (p<0.001). Upon histological examination, over 50% of cancer cells were dead and replaced by necrotic tissue or stromal cells in the PDOX tumor treated with FGS+NAC and was judged as Evan’s grade IIb – III (Figure 4).

Figure 2

Experimental schema and FGS imaging system.

Figure 3

Representative images during FGS with or without NAC.

Figure 4

Representative gross and histological images of excised tumors in each treatment group.

Effect of NAC on tumor recurrence with BLS or FGS

With regard to the recurrent tumor weight, the average local recurrent tumor weight was 389.2±356.6 mg in BLS-only treated mice; 369.1±251.9 mg in BLS+NAC-treated mice; 73.0±77.2 mg in FGS-only treated mice; and 78.4±90.8 mg in FGS+NAC-treated mice. The average local recurrent tumor weight in FGS-only treated mice was significantly less than in BLS-only treated mice (p?=?0.041). The average metastatic recurrent tumor weight of the pancreatic cancer PDOX was 170.7±184.2 mg for BLS-only treated mice; 40.0±19.7 mg for BLS+NAC-treated mice; 31.3±37.6 mg for FGS-only mice; and 1.3±3.7 mg for FGS+NAC-treated mice. The average metastatic recurrent tumor weight in FGS+NAC was significantly less than BLS+NAC (p?=?0.001). The metastatic recurrent weight in the FGS+NAC group compared to the FGS only group was marginally significant (0.059). The average total recurrent tumor weight in FGS only was significantly less than BLS only (p?=?0.037), and that in FGS+NAC was also significantly less than BLS+NAC (p?=?0.004) (Figures 5 and ?and6).6). The recurrence rate of FGS+NAC was also significantly less than BLS+NAC (p?=?0.008). FGS+NAC significantly reduced the metastatic recurrence frequency to one of 8 mice compared to FGS only where metastasis recurred in 6 out of 8 mice and BLS+NAC where it occurred in 7 out of 8 mice (p?=?0.041) (Table 1).

Figure 5

Representative images of the recurrent PDOX tumor.

Figure 6

Recurrent tumor weights for each experimental group.

Table 1

Recurrence rate of PDOX in each treatment group.

 

Discussion

In a previous study, we conjugated a monoclonal antibody specific for the tumor-associated antigen CA19-9 with the AlexaFluor 488 green fluorophore. We were able to demonstrate in vivo binding of the antibody fluorophore conjugate to the tumor tissue in an orthotopic mouse model of human pancreatic cancer [5]. This fluorescence facilitated differentiation between normal and tumor tissue within the pancreas and also revealed microscopic foci or tumor implants within the spleen, liver, and peritoneum which were not visible under standard light microscopy. This study offered a novel technique to facilitate the intraoperative identification of both primary tumor and small metastatic lesions that may be missed at the time of surgery in those patients whose tumors express the tumor-associated antigen CA19-9.

In another study, we compared a hand-held imaging system with larger imaging systems previously used for FGS [13]. In a PDOX model labeled with Alexa Fluor 488-conjugated anti-CA 19-9 antibody, only the portable hand-held device could distinguish the residual tumor from the background, and complete resection of the residual tumor was achieved under fluorescence navigation, suggesting this system can be applied to the clinic in the near future to enable widespread application of FGS.

There are several novel aspects to the present study that should be emphasized. To the best of our knowledge, this is the first study that has utilized the combination of NAC and a CA 19-9 antibody conjugated fluorophore for FGS of pancreatic cancer. Furthermore, the present study took advantage of a longer wavelength dye, DyLight 650, which we have previous shown has better tissue penetration compared to AlexaFlour 488 [21]. In addition, the PDOX model developed in our laboratory, and used in the present study, allows for individualized therapy that is not available with pancreatic cancer cell line models [7][14],[23]. PDOX models can be helpful to determine if an individual’s tumor is sensitive to various NAC regimens. The most novel and unexpected finding was that FGS+NAC eliminated pancreatic cancer metastases in seven out of eight mice.

For bright light surgery, tumors were removed with grossly negative margins under standard bright-field using an MVX10 microscope. For fluorescence-guided surgery, tumor resection was guided by labeling the tumors with an anti-CA 19-9 antibody labeled with a 650 nm fluorophore. The pancreatic cancer PDOX used in this study had a very aggressive behavior. At FGS, we detected some tiny tumors spreading around the primary tumors, which could not be detected under normal macroscopic inspection. At the first surgery, the surgical margin was exterior to the tumor border which was recognized macroscopically. However, a larger margin provided by FGS appears insufficient to lower or prevent metastatic recurrence which required NAC in addition to FGS (Table 1). However, the larger margins afforded by FGS are necessary to lower or prevent metastatic recurrence, as the combination of BLS and NAC are ineffective to lower or prevent metastatic recurrence (Table 1).

All mice in this study were euthanized 90 days after BLS or FGS and therefore, we were not able to compare survival differences between the groups. However, as seen in Table 1, the metastatic recurrence rate in FGS+NAC was significantly less than FGS only (p?=?0.041), suggesting that FGS+NAC improves the survival of pancreatic cancer patients compared to FGS only.

In summary, we have determined the efficacy of NAC with GEM in combination with FGS on a pancreatic cancer PDOX model. The results from this study indicate that NAC in combination with FGS can reduce or even eliminate metastatic recurrence of pancreatic cancer sensitive to NAC. This is an important result for the future more effective treatment of pancreatic cancer. The present study further emphasizes the power of the PDOX model which enables metastasis to occur and thereby identify the efficacy of NAC on metastatic recurrence.

 

Funding Statement

This study was supported in part by National Cancer Institute grants CA132971 and 142669 (to MB and AntiCancer, Inc.); and Japanese Ministry of Education, Culture, Sports, Science and Technology for Fundamental Research Grant Numbers 26830081 to YH; and 26462070 to IE and 24592009 to KT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

 

References

1. Kato K, Yamada S, Sugimoto H, Kanazumi N, Nomoto S, et al. (2009) Prognostic factors for survival after extended pancreatectomy for pancreatic head cancer: influence of resection margin status on survival.Pancreas 38:605–612. [PubMed]
2. Bouvet M, Hoffman RM (2011) Glowing tumors make for better detection and resection. Sci Transl Med3:110fs110. [PubMed]
3. Rosenthal EL, Zinn KR (2013) Putting numbers to fluorescent guided surgery. Mol Imaging Biol 15:647–648. [PubMed]
4. Kaushal S, McElroy MK, Luiken GA, Talamini MA, Moossa AR, et al. (2008) Fluorophore-conjugated anti-CEA antibody for the intraoperative imaging of pancreatic and colorectal cancer. J Gastrointest Surg12:1938–1950. [PMC free article] [PubMed]
5. McElroy M, Kaushal S, Luiken GA, Talamini MA, Moossa AR, et al. (2008) Imaging of primary and metastatic pancreatic cancer using a fluorophore-conjugated anti-CA19-9 antibody for surgical navigation.World J Surg 32:1057–1066. [PMC free article] [PubMed]
6. Metildi CA, Kaushal S, Hardamon CR, Snyder CS, Pu M, et al. (2012) Fluorescence-guided surgery allows for more complete resection of pancreatic cancer, resulting in longer disease-free survival compared with standard surgery in orthotopic mouse models. J Am Coll Surg 215:126–135 discussion 135–126.[PMC free article] [PubMed]
7. Fu X, Guadagni F, Hoffman RM (1992) A metastatic nude-mouse model of human pancreatic cancer constructed orthotopically with histologically intact patient specimens. Proc Natl Acad Sci U S A 89:5645–5649. [PMC free article] [PubMed]
8. Fu X, Hoffman RM (1993) Human ovarian carcinoma metastatic models constructed in nude mice by orthotopic transplantation of histologically-intact patient specimens. Anticancer Res 13:283–286. [PubMed]
9. Fu X, Le P, Hoffman RM (1993) A metastatic orthotopic-transplant nude-mouse model of human patient breast cancer. Anticancer Res 13:901–904. [PubMed]
10. Fu XY, Besterman JM, Monosov A, Hoffman RM (1991) Models of human metastatic colon cancer in nude mice orthotopically constructed by using histologically intact patient specimens. Proc Natl Acad Sci U S A 88:9345–9349. [PMC free article] [PubMed]
11. Wang X, Fu X, Hoffman RM (1992) A new patient-like metastatic model of human lung cancer constructed orthotopically with intact tissue via thoracotomy in immunodeficient mice. Int J Cancer 51:992–995. [PubMed]
12. Hiroshima Y, Maawy A, Metildi CA, Zhang Y, Uehara F, et al. (2014) Successful fluorescence-guided surgery on human colon cancer patient-derived orthotopic xenograft mouse models using a fluorophore-conjugated anti-CEA antibody and a portable imaging system. J Laparoendosc Adv Surg Tech A 24:241–247. [PMC free article] [PubMed]
13. Hiroshima Y, Maawy A, Sato S, Murakami T, Uehara F, et al. (2014) Hand-held high-resolution fluorescence imaging system for fluorescence-guided surgery of patient and cell-line pancreatic tumors growing orthotopically in nude mice. J Surg Res 187:510–517. [PMC free article] [PubMed]
14. Metildi CA, Kaushal S, Luiken GA, Talamini MA, Hoffman RM, et al. (2014) Fluorescently labeled chimeric anti-CEA antibody improves detection and resection of human colon cancer in a patient-derived orthotopic xenograft (PDOX) nude mouse model. J Surg Oncol 109:451–458. [PMC free article] [PubMed]
15. Evans DB, Varadhachary GR, Crane CH, Sun CC, Lee JE, et al. (2008) Preoperative gemcitabine-based chemoradiation for patients with resectable adenocarcinoma of the pancreatic head. J Clin Oncol 26:3496–3502. [PubMed]
16. Katz MH, Varadhachary GR, Fleming JB, Wolff RA, Lee JE, et al. (2010) Serum CA 19-9 as a marker of resectability and survival in patients with potentially resectable pancreatic cancer treated with neoadjuvant chemoradiation. Ann Surg Oncol 17:1794–1801. [PMC free article] [PubMed]
17. Takahashi H, Ohigashi H, Ishikawa O, Eguchi H, Gotoh K, et al. (2010) Serum CA19-9 alterations during preoperative gemcitabine-based chemoradiation therapy for resectable invasive ductal carcinoma of the pancreas as an indicator for therapeutic selection and survival. Ann Surg 251:461–469. [PubMed]
18. Katz MH, Fleming JB, Bhosale P, Varadhachary G, Lee JE, et al. (2012) Response of borderline resectable pancreatic cancer to neoadjuvant therapy is not reflected by radiographic indicators. Cancer118:5749–5756. [PubMed]
19. Kim MP, Truty MJ, Choi W, Kang Y, Chopin-Lally X, et al. (2012) Molecular profiling of direct xenograft tumors established from human pancreatic adenocarcinoma after neoadjuvant therapy. Ann Surg Oncol 19Suppl 3: S395–403. [PMC free article] [PubMed]
20. Hoffman RM (1999) Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: a bridge to the clinic. Invest New Drugs 17:343–359. [PubMed]
21. Maawy AA, Hiroshima Y, Kaushal S, Luiken GA, Hoffman RM, et al. (2013) Comparison of a chimeric anti-carcinoembryonic antigen antibody conjugated with visible or near-infrared fluorescent dyes for imaging pancreatic cancer in orthotopic nude mouse models. J Biomed Opt 18:126016. [PMC free article][PubMed]
22. Evans DB, Rich TA, Byrd DR, Cleary KR, Connelly JH, et al. (1992) Preoperative chemoradiation and pancreaticoduodenectomy for adenocarcinoma of the pancreas. Arch Surg 127:1335–1339. [PubMed]
23. Hiroshima Y, Zhao M, Maawy A, Zhang Y, Katz MH, et al. (2014) Efficacy of Salmonella typhimurium A1-R versus chemotherapy on a pancreatic cancer patient-derived orthotopic xenograft (PDOX). J Cell Biochem 115:1254–1261. [PubMed]


Articles from PLoS ONE are provided here courtesy of Public Library of Science

 
Hum Vaccin Immunother. 2014 Jul 1; 10(7): 1892–1907.
Published online 2014 Apr 29. doi:  10.4161/hv.28840

Laser vaccine adjuvants

History, progress, and potential
Satoshi Kashiwagi,* Timothy Brauns, Jeffrey Gelfand, and Mark C Poznansky*

*Correspondence to: Satoshi Kashiwagi, Email: ude.dravrah.hgm@igawihsaks and Mark C Poznansky, Email: gro.srentrap@yksnanzopm

Author information  Article notes  Copyright and License information
Received 2014 Feb 14; Revised 2014 Mar 28; Accepted 2014 Apr 9.

Potential Role of Lasers as Vaccine Adjuvants

The challenge of adjuvant development for intradermal vaccines

Immunological adjuvants, (from the latin adjuvare, meaning to help), effect qualitative and quantitative changes in immune responses to a simultaneously administered vaccine antigen that result in sufficient immunological memory and protection against pathogens.1 Contemporary clinical vaccines generally use highly targeted recombinant molecules as antigens that are often poorly immunogenic in their own right and therefore require enhancement with immunologic adjuvants.26 Development of safe and potent immunologic adjuvants therefore represents an important element of current and future vaccine development.2,3 The immune potentiating effects of adjuvants are often accompanied by an increased risk of local reactogenicity or systemic toxicity. As a result, in spite of a steady proliferation of potential adjuvant candidates, the number of adjuvants that have actually been approved for use with human vaccines by regulatory agencies such as the FDA is surprisingly limited. Until the recent approval of AS04 (alum with monophosphoryl lipid A) for use in Cervarix® (GlaxoSmithKline) and AS03 (squalene-based oil-in-water emulsion) for Q-pan H5N1 influenza vaccine (GlaxoSmithKline), the FDA had only approved particulate aluminum-salt adjuvants for use with vaccines, mainly due to concerns over side effects. The European Medicines Agency (EMEA) has approved only 5.712

The tradeoff between efficacy and safety is evident in recent global experiences with influenza vaccines. Comparisons of adjuvanted to unadjuvanted vaccines in different populations consistently show that rates of seroconversion with adjuvanted vaccines is higher than with unadjuvanted vaccines, but that rates of injection site reactogenicity are also higher with the former.1315 After the release of vaccines for H1N1 in 2009, the AS03-adjuvanted influenza vaccine Pandemrix® (GlaxoSmithKline) was linked to hundreds of cases of narcolepsy in the EU.1619 In the US, where only unadjuvanted H1N1 vaccines were approved, no cases were reported.20 Since a potential link has been made between H1N1 epitopes and autoimmune narcolepsy,21 the AS03 adjuvant may have contributed to this adverse effect. The development of new vaccine adjuvants with improved safety profiles is a highly desirable factor in vaccine development.

The challenge of adjuvant development is increased when it comes to intradermal vaccines. There has been a growing interest in targeting vaccines to the skin due to the potential for the skin-based immune system to yield protective immune responses with smaller amounts of vaccine antigen.2224 Vaccination at this epithelial surface can effectively prime the body to respond to pathogens22,23,2528 and induce a robust recall immune response.2931 The intradermal route of vaccination appears to be more effective than the conventional intramuscular route for many vaccines including influenza vaccines22,23,26,28 and hepatitis B vaccines.2224 An intradermal influenza vaccine Fluzone Intradermal® (Sanofi Pasteur) was approved by the FDA in 2011 and the company has licensed such a vaccine in more than 40 countries.32

In spite of its promise, several distinct challenges have kept intradermal delivery from becoming a standard of practice in vaccination. One of these is the paucity of appropriate adjuvants.33,34 Most adjuvants used in licensed intramuscular vaccines like aluminum salt and oil-in-water adjuvants are simply too reactogenic when delivered intradermally.35,36 While some clinical trials have suggested that alum-adjuvanted vaccines are tolerable when given intradermally,37,38 reports of injection-site reactions with intradermally-delivered adjuvanted vaccines are much higher compared with non-adjuvanted formulations.39 Finally, current adjuvants may not be compatible with intradermal delivery or formulation requirements, especially with newer intradermal vaccination technologies.33,34 Not surprisingly, there is no adjuvanted intradermal vaccine licensed to date.

The use of non-destructive lasers to adjuvant vaccines

A number of studies conducted over the last 2 decades suggest that non-tissue damaging lasers can be used to modify local skin immune responses in a way that enhances systemic immune response to a vaccine introduced into the treated skin. Treating the skin with non-harmful laser light represents an adjuvanting approach that is potentially compatible with intradermal vaccination. This review focuses on the historical development, current status, and future prospect of lasers that do not breach or destroy skin tissue for this purpose. It is well established that skin injury such as scarification or burn can enhance immune responses40and lasers can be used to induce such injuries to the skin.39,4143 A number of investigators have explored the immune-stimulating effects of thermally-destructive lasers in treating cancer44,45 and a recent review has been published covering this approach.46 In addition, fractional laser devices used to enhance delivery of drugs and vaccines have been examined for their ability to alter immunologic responses in the skin to vaccines and immunotherapies.47 These lasers breach the tissue barrier by ablating tissue in small cylindrical volumes, creating a field of skin micropores whose diameter, depth, and density can be highly controlled. The tissue damage that results from the process of microporation can activate the immune system through release of damage-associated molecular patterns (DAMPs) from coagulated tissues, leading to expression of pro-inflammatory cytokines and activation of antigen presenting cells.47 This effect is enhanced by the cytokine cascades induced by the loss of cutaneous barrier integrity.48,49 The use of fractional lasers for vaccination has also been recently reviewed.50

The use of non-destructive lasers to alter tissue immune responses in a manner that can enhance systemic vaccine responses, (laser vaccine adjuvants or LVAs), is a novel approach that has just begun to be explored. LVA treatment of the skin is characterized by a combination of relatively low power densities or irradiances (0.7 to 6.0 W/cm2) combined with fairly high total fluences (energy dose supplied per unit area)—typically in the hundreds of Joules/cm2—a combination that is unique compared with most other clinical applications of lasers to the skin35,5153 (summarized in Fig. 1). LVA exposures generate moderate but non-damaging thermal responses in the tissue that are quite distinct from “athermal” low-level laser therapy (LLLT), where both laser irradiances and fluences are typically 1–2 logs lower.54,55 The fluences applied with LVAs are well beyond the range where most simulative biological responses to LLLT have been identified. Such lasers also operate at irradiances about one log greater than high fluence, low power laser treatments used to induce apoptotic effects in a variety of cancer cell lines both in vitro and in vivo.5659 With recently reported progress on a new type of LVAs, the combination of LVAs with skin-based vaccination now has the potential to yield more effective vaccine responses in a safe and cost-effective manner.

figure hvi-10-1892-g1

Figure 1. Comparison of the typical irradiances and fluences for laser dermatology procedures. A plot of irradiances vs. fluences for dermatologic applications of lasers is shown. Note that parameters of LVA are quite distinct from those of other

 

History of Laser Vaccine Adjuvants

Initial Russian studies

The concept of using non-destructive lasers as vaccine adjuvants evolved from several decades of laser research in Russia, some of which was transferred to US laboratories over the decade. The origin of this work goes back to Russian investigations of photobiomodulation that began soon after the first descriptions of the effects of low-power laser on biological processes by Endre Mester in Hungary in 1967.60 One area of clinical application that attracted early medical interest was the promotion of wound healing in various tissues.61,62 Many of these early wound healing studies utilized low-power helium-neon lasers.6366 In 1978, the copper vapor laser was clinically introduced in Russia. Unlike the continuous laser light emitted by helium-neon lasers, copper vapor (CV) lasers release very short duration pulses (10–25 ns) at very high repetition rates (5–20 kHz). CV laser light is emitted at 2 wavelengths—about 510 and 580 nm—representing the yellow-green part of the visible light spectrum. The distinctive laser emission characteristics of copper vapor lasers were of interest to Russian physicians to explore a variety of clinical applications, including wound healing.67 These explorations also gained impetus from medical research funded by the Soviet military during the Afghan conflict in the early 1980s.

While early Russian studies of CV lasers for wound healing featured either low-energy, athermal treatments at doses typical of LLLT, or high-power treatments to induce tissue coagulation,68,69 a small subset of studies in the 1980s and 1990s, many led by Dr Anatoly I Soldatov of the St. Petersburg Academy of Postgraduate Medical Education, utilized much higher irradiances and doses that induced significant photothermal and photoacoustic responses in the irradiated tissue but did not cause tissue damage typical of high power lasers. Some of these studies showed that this new type of treatment effectively promoted wound healing for specific chronic medical conditions such as gastric and duodenal ulcers, bronchitis, and bronchial hyperplasia.7072

A group of St. Petersburg investigators, led by Dr Sergei Onikienko of the St. Petersburg Military Medical Academy, hypothesized that this higher-power but non-destructive type of laser treatment might also affect immune responses in healthy tissues. These scientists began to explore the potential of CV laser to improve vaccine responses and conducted a number of studies in mice and humans in the late 1990s and early 2000s that used CV lasers to enhance responses to both prophylactic and therapeutic vaccines. These studies, the majority of which were published in Russian, non-peer reviewed journals, showed that CV laser treatment of the skin improved responses to intradermal delivery of commercial influenza and hepatitis B vaccines in documented vaccine non-responders, and also potentiated the effects of experimental therapeutic vaccines for chronic hepatitis B and cancer.

Recent US studies

In 2004, a Massachusetts General Hospital team conducting scientific assessments as part of the BioIndustry Initiative of the US State Department—a program within the cooperative nonproliferation efforts between the US and Russia73—began to meet with these investigators, explore their approach and introduce them to US investigators for potential collaborations. In 2008, a US biotechnology company initiated an effort to replicate these earlier Russian studies using a more structured approach. The first of these studies, published by the Wellman Center for Photomedicine in 2010 provided support for the Russian preclinical results by showing that a 532 nm nanosecond pulsed Nd:YAG laser could enhance antibody titer responses to a model ovalbumin vaccine and a split-virion influenza vaccine.53 Chen et al. subsequently showed that this type of laser could enhance immune responses to a nicotine vaccine35,53 and a dendritic cell vaccine.74

In parallel studies conducted at the laboratories of the Vaccine and Immunotherapy Center (VIC) at Massachusetts General Hospital, Kashiwagi et al. showed that a Nd:YVO4 laser emitting either nanosecond pulsed light at 532 nm or continuous wave, near-infrared light at 1064 nm could enhance immune responses to a model vaccine (ovalbumin, OVA) and to a live attenuated influenza vaccine. Surprisingly, the 1064 nm laser provided superior efficacy to the 532 nm laser in a lethal challenge study.51 The responses to the 532 nm laser were anticipated based on the earlier work done in Russia and at Wellman, but the responses to the 1064 nm continuous wave laser were quite unanticipated and represent a promising avenue of exploration for this approach. Taken together, the research conducted by Russian and US scientists suggests that non-destructive lasers have the potential to enhance vaccine responses and are worthy of further exploration. In addition, the recent discovery by Kashiwagi et al. supports the view that potent vaccine responses can be induced by relatively simple, low power laser systems. This finding enhances the potential and clinical applicability for the commercial development of LVAs.

 

Immunologic Effects of Laser Vaccine Adjuvants

Systemic effects on vaccine responses

Three different research groups to date have shown systemic vaccine enhancement in response to LVA treatment. Table 1 summarizes the laser types and treatment parameters used by these different groups. The initial Russian studies on LVAs, summarized in Tables 2 and ?and3,3, provide a rationale to further pursue this approach but for the most part lack adequate descriptions of methods and are published largely in non-peer review publications. In these studies, Onikienko and his colleagues used a copper vapor (CV) laser (D.V. Efremov Institute of Electrophysical Apparatus) emitting light at both 510 and 578 nm (mix of 10% 510 nm and 90% 578 nm) with a pulse width of 10–12 ns and a pulse frequency of 10–20 kHz. The laser beam had a flat top profile. Irradiances were typically between 1–6 W/cm2 and skin exposures were typically 5 mm spots.

Table thumbnail

Table 1. Comparison of laser parameters used in different LVA studies

Table thumbnail

Table 2. Preclinical study of laser adjuvant

 

Table thumbnail

Table 3. Clinical study of laser adjuvant

 

Vaccine response studies were performed in both mice and humans. A vaccination study in white mongrel mice involved a single 1- or 2-min treatment of the ear skin at an irradiance of 1–3 W/cm2 followed by an intradermal injection of 50 μL of a split inactivated influenza vaccine (Vaxigrip, Sanofi Pasteur). CV laser treatment of the skin resulted in a 54% to 86% increase in anti-influenza antibody titer at 4 wk compared with vaccine alone.75,77 Induction of cell-mediated immune response was also shown using the leukocyte migration inhibition test (LMIT).78 In a follow up study, protective immunity was examined by exposure of 2 groups of 20 of the same breed of mice to a lethal dose of H3N2 influenza (?/Aichi/2/68) via an inhalation route 14 d after vaccination. 70% of the mice receiving vaccination and laser treatment survived compared with 35% of mice receiving vaccine only.75,77

Onikienko’s team extended the vaccination approach into clinical applications with prophylactic vaccines. In one study, 42 people with documented low antibody titer responses to influenza vaccination were intradermally vaccinated with a 15 μg dose of Vaxigrip via jet injector; 22 of these received skin site exposure to CV laser at an irradiance of 1.0 W/cm2 for 2 min (120 J/cm2 total dose) right before vaccination.77 Blood was drawn from each vaccine at 4 wk and a number of immune end points, including anti-influenza antibody titration, LMIT, lymphocyte cytotoxic activity, monocyte cytokine secretory activity, and the increase in activity of the lymphocyte enzymes, were examined and compared with 22 healthy controls who were also vaccinated. Based on assessment of these assays, it was determined that 14 of the 22 laser treated non-responder subjects showed a statistically significant increase in vaccine responses compared with only 5 of 20 non-responder subjects in the vaccine-only group. In the healthy control group, significant responses were measured in 20 out of 24 subjects.

A similar study was performed in 17 people documented as hepatitis B vaccine non-responders (failure to maintain HBsAb antibody titer of greater than 10 mIU/mL 6 mo after completing the course of vaccination).77 All subjects received a course of 3 intradermal injections (0, 1, and 3 mo) of 20 µg of a recombinant hepatitis B vaccine (Combiotech) using the Mantoux technique. Nine of the subjects received a 3 min treatment of a 5 mm spot on the shoulder skin with a CV laser at 1 W/cm2 average power (180 J/cm2total dose) prior to each vaccination. Eight other subjects received hepatitis B vaccination with concomitant IL-2 injections (2 500 000 IE via subcutaneous injection) in a manner similar to Jungers et al.79 In the laser treated group, 7 out of 9 subjects reached the international standard for protection at 6 mo after the end of vaccination (10 IU/mL), while none of the IL-2 treated subjects did so.77

The St. Petersburg group also applied the laser approach to therapeutic vaccination. In one study, the laser was used to enhance responses to an investigational vaccine (recombinant HBsAg without alum, Combiotech) combined with laser treatment of the injection site. Subjects diagnosed with chronic hepatitis B for at least 2 y received either a series of 12 weekly intradermal vaccinations with or without CV laser skin pretreatment (1.0–1.5 W/cm2 on a 5 mm skin spot for 1–3 min). A control group received Lamivudine 100 mg daily for 12 wk. The effect of the treatment was evaluated by clinical indicators of disease including liver function tests, circulating HBV DNA by PCR and serum HBsAg, and immune responses using LMIT with HBsAg to measure cell-mediated immune response. At 12 wk, 5 of 9 of the laser-pretreated vaccines showed normalized ALT, HBV DNA copies below 300, and positive LMIT compared with 4 out of 11 of the Lamivudine-treated group.

The Wellman group’s initial studies at MGH similarly used a nanosecond pulsed laser operating in the green spectrum. This was a Q-switched neodymium-doped yttrium aluminum garnet (Q-Nd:YAG) laser emitting light at 532 nm with 6–7 ns pulse widths and repetition rate of 10 Hz. Their initial studies used 2 min exposures to 4 separate 6 mm spots of skin on BALB/c mice at an irradiance of 0.78 W/cm2 followed by intradermal injection of ovalbumin or inactivated influenza vaccines in each irradiated spot. The illumination of skin with the laser increased the motility of antigen presenting cells (APCs), leading to enhanced antigen uptake by APCs and helper T cell priming in the draining lymph nodes. This 2-min laser exposure increased humoral immune responses to a model vaccine (OVA) by 300 to 500% and a split-virion influenza vaccine (Fluvirin) by 400% in primary vaccination and 900% in booster vaccination compared with a non-adjuvanted group.53

In the most recently published LVA studies, the VIC group of Kashiwagi et al. at MGH used a neodymium-doped yttrium orthovanadate laser emitting light either at 532 nm in high frequency Q-switched mode (Q-Nd:YVO4) with an irradiance of 1.0 W/cm2, a pulse duration of 10 ns and a pulse repetition rate of 10 kHz, or in a continuous wave mode at the near-infrared spectrum at 1064 nm (CW-NIR) at an irradiance of 5 W/cm2. The study showed that the CW-NIR laser adjuvant induces the transient expression of a limited set of cytokines and chemokines in skin resulting in recruitment and activation of dendritic cells in skin draining lymph nodes (dLNs). Furthermore, a 1-min application of the CW-NIR laser augmented antibody response most efficiently to OVA and an influenza vaccine (whole inactivated PR8 virus) with a TH1-TH2 balanced T cell response, and conferred protection in a murine influenza lethal challenge model, whereas the 532 nm Q-Nd:YVO4 induced a TH1-skewed response with little impact on protection.51 Importantly, the protective immune responses induced by the CW-NIR were comparable to those induced by a licensed adjuvant and support the view that LVA might have utility in augmenting responses to intradermal vaccines.

Localized effects on irradiated tissues

In general, LVAs appear to work by modifying the immunologic environment within the tissue that receives the vaccine, resulting in enhancement of the vaccine response. The specific modifications in the local immune environment appear to differ depending on the type of laser used and are likely related to significant differences in the laser wavelength, pulse duration, pulse energy, and pulse frequency.

Effect on heat shock protein expression and release

A fundamental principle of vaccine adjuvant development, based on Matzinger’s danger theory of immune response80 is to trigger a danger signal to the immune system that can promote more vigorous and long-lasting responses to a vaccine antigen. These danger signals are often in the form of either DAMPs or pathogen-associated molecular patterns (PAMPs) that can trigger cytokine and chemokine cascades in the tissues, usually through Toll-like receptors (TLRs).81 Significant development work was put into developing adjuvants in the form of DAMPs or PAMPs that can trigger these TLR-mediated pathways to promote vaccine responses.7

Onikienko et al. identified an important role for heat shock protein 70 (HSP70) in mediating vaccine responses to CV laser treatment. HSPs are a family of ubiquitous intracellular molecules that function as molecular chaperones as part of numerous intracellular processes (e.g., protein folding and transport). Under conditions of stress, some of these play important roles in refolding or disposing of misfolded and denatured proteins,82 stabilizing cellular membranes and enhancing cell signaling,83 and inhibiting specific apoptotic pathways.84 The intracellular expression of many of these HSPs are significantly induced under stress conditions such as fever, radiation, infections, and neoplasia.85 Some HSPs, such as HSP70, play additional roles if and when released outside the cell. In these circumstances they can act as potent DAMP-like inducers of immunity and have been harnessed as adjuvants in experimental vaccines targeted to cancers and infections.86 HSPs expressed on the surface of stressed and damaged cells or released from necrotic cells can serve as a kind of danger signal87 and are recognized by APCs through specific receptors, such as TLRs, scavenger receptors (LOX-1), CD91, and CD14 resulting in increased antigen display by MHC class I and II molecules and priming T cells.86,88

Onikienko’s group showed that the CV laser treatment (irradiance of 1–3 W/cm2 with a pulse width of 10–12 ns and pulse repetition frequency of 10–20 kHz) to a 5 mm skin spot on a mouse ear for 3 min induced rapid, dose-related increases in extracellular HSP70 as determined by a whole-mount in vivo immunostaining of epidermal sheet of the mouse ear.52 Western blot analysis on epidermal tissue further showed an increased expression of HSP70 in the ear skin that persisted for 7–14 d.52 Since adjuvant effects in these mice similar to those caused by the CV laser could be induced by injection of exogenous by itself, Onnikienko et al. concluded that high-frequency pulsed laser treatment enhances immune responses via release and sustained expression of HSP70 by fibroblast and/or keratinocytes in the laser irradiated skin.

Cells in the skin harbor a high baseline level of intracellular HSP70 that could potentially be released under conditions of stress89 with the highest levels found in keratinocytes.90 While overexpression of heat shock proteins is a normal cellular response to stress, a number of investigators have shown that under conditions of stress a portion of constituent HSP70 is mobilized to the cell membrane and can be released from the cell through a variety of mechanisms.9193 Subsequent LVA studies performed in the US and published to date have not linked LVA skin treatment to the release or overexpression of HSP70.51,53 This difference in expression and release profiles for HSP70 may be related to the differences in the laser parameters used by the different laboratories.

Effect on immune cell migration

The use of chemical adjuvants in skin-based vaccination studies directly activate and induce migration of APCs from the skin to the proximal dLN.94,95 LVAs similarly induce APC migration to the skin and dLNs, increasing the concentration of APCs in volume of treated tissue and enhancing their ability to activate, pick up antigen, and migrate to dLNs. The means by which they accomplish these effects may be different depending on the laser type.

Onikienko et al. showed by a histological analysis with electron microscopy that CV laser treatment resulted in a significant increase of Langerhans cells at the irradiation site. Chen et al. showed by intravital microscopy that 532 nm Q-Nd:YAG laser treatment increased the motility of MHC class II-positive APCs between 0.5 and 16 h after irradiation, resulting in a larger number of antigen-positive CD11c+ dendritic cells (DCs) within the dLNs after vaccination compared with vaccination without laser treatment.53 Their analysis of tissue samples from these studies suggests that this increase in motility and migration was related to the rearrangement of extracellular matrices including enlarged perforations in the perilymphatic basement membrane, disarray of collagen fibers and disruption of cell–matrix interactions in the dermis that persisted for at least 16 h after laser treatment.74 This led to their conclusion that the facilitation of APC migration was through alteration of extracellular matrices.
Kashiwagi et al. showed that the CW-NIR laser treatment at 5.0 W/cm2 on a 5 mm spot of mouse back skin for 1 min also resulted in CD11c+ dendritic cell recruitment in the irradiated skin at 6 h after exposure.51This laser treatment did not appear to result in a significant increase in the migration of activated DCs to the draining skin-dLNs.51

Effects on inflammatory and chemokine signaling

With chemical and biological adjuvants, the activation and mobilization of APCs in the skin is a result of both autocrine and paracrine signaling through cytokines and chemokines.96 DCs are the most versatile APCs; licensed and experimental adjuvants activate DC-mediated innate immune responses that result in robust adaptive immune responses.79,97,98 Intradermal administration of adjuvants typically induces inflammatory responses including cytokine release and leukocyte infiltration.99These adjuvants are effective also because the inflammatory responses mediated by chemical adjuvant lasts for several weeks.99 Unfortunately, this persistence of inflammatory signaling may also play a role in diminishing the safety of these adjuvants.12,100

LVAs appear to function quite distinctly from chemical adjuvants in that they result in tissue signaling and the activation of APCs, but do not appear to trigger significant inflammatory responses. Chen et al. reported that the activation and migration of APCs in the skin following 532 nm Q-Nd:YAG laser treatment was not accompanied by a significant increase in inflammatory cytokines including TNF-α, IL-1β, IL-6, and CCL2.36,53 The activation picture for APCs following exposure to the Q-Nd:YAG laser-treated skin was mixed. While the overall number of DCs migrating to skin-draining LNs was significantly increased by 24 h and activation markers including CD80 and MHC class I were upregulated in skin-dLNs, critical activation markers such as CD40 and MHC class II were not upregulated.74

Kashiwagi et al. showed that the CW 1064 nm laser treatment did not result in the significant expression of proinflammatory cytokine genes such as Il1b, Il6, and Tnf and that, without introduction of a vaccine, cytokine responses and expression return to basal levels by 24 h after laser illumination.51 Nevertheless, CW-NIR laser treatment results in CD11c+ dendritic cell recruitment into the irradiated skin, increases the expression of MHC class II and co-stimulatory molecules including CD40 and CD86 on these cells, and increases the number of activated DCs in skin-dLNs 24 h after the laser irradiation.51 Since the magnitude of the CD4+ T cell responses is proportional to the number and quality of DCs that reach the dLNs,101 it is not surprising that the CW-NIR laser adjuvant enhances CD4+ T cell responses. These qualitative and quantitative DC responses correlate to the transient expression (measured 6 h after the CW-NIR laser treatment) of a set of chemokine genes including Ccl2, Ccl6, Ccl11, Ccl17, Ccl20, and Ccr7 that mediate DC migration96,102105 (Fig. 2). The migration of mature DCs to dLNs through afferent lymphatic vessels is regulated by multiple cytokines and chemokines.101,106 The transient tissue response to the CW-NIR laser illumination results in expression of chemokines sufficient to initiate DC migration and maturation in situ but may not be sufficient to provide DCs with additional guidance cues including CCL19/21 expression in lymphatic endothelial cells. Further investigations of the effects of LVA on DC migration and/or activation are needed.

figure hvi-10-1892-g2

Figure 2. Putative mechanisms of action of NIR laser adjuvant. Non-tissue damaging continuous wave (CW) near-infrared (NIR) 1064 nm laser given in short exposures to small areas of the skin is able to augment broad immunity including antibody,

 

Mechanisms of Action for Laser Vaccine Adjuvants

Photobiological basis for laser adjuvant effects

A fundamental principle of laser medicine is that emitted photons must be absorbed in order to have a biological effect. The absorbers of laser light, called chromophores, have specificity for and sensitivity to particular wavelengths of light due to how specific wavelength photons interact with electrons within the molecular structure of the chromophore. As a result, tissues preferentially absorb some wavelengths of light over others, showing greater absorption efficiency at different wavelengths.54,55 Three key chromophores in the skin are melanin, hemoglobin and water. In the ultraviolet (UV) and visible spectrum, absorption by melanin and hemoglobin dominate. The effective absorption of melanin drops off quickly beyond 700 nm and ends at around 1100 nm. Hemoglobin has a high coefficient of absorption in the visible light range with a peak at 578 nm and also falls quickly beyond 700 nm; it plays a relatively insignificant role as a chromophore beyond about 1000 nm. Water has a much lower absorption coefficient of light compared with melanin and hemoglobin until about 1000 nm, but as it makes up almost 70% of the composition of the skin, it presents a large absorption target. Beyond 1000 nm, water becomes the dominant dermal chromophore. The relatively weak absorption of the 3 main skin chromophores between 700 and 1000 nm provides an “optical window” in the skin that permits laser light to penetrate much deeper (Fig. 3). This means that the 510/578 nm CV laser, 532 nm Q-Nd:YAG and 532 nm Q-Nd:YVO4 lasers will have a much shallower effective penetration depth (depth at which the intensity of the laser energy falls to 1/e or about 37% of the incident intensity) about 1 mm, as compared to CW-NIR laser, which will be about 4 mm.107 These differences in absorption efficiency in the skin account for most of the difference in emitted irradiance between visible light and NIR systems (i.e., 1 W/cm2 in the Q-Nd:YVO4 system at 532 nm and 5 W/cm2 in the CW Nd:YVO4 system at 1064 nm).

figure hvi-10-1892-g3

Figure 3. Absorption spectrum for major skin chromophores and the optical window. Light absorption in the skin is wavelength dependent. In the UV to near infrared portion of the spectrum, the predominant tissue chromophores are hemoglobin, melanin,  

In most medical applications of lasers, much of the energy from photon absorption by chromophores is converted into heat (photothermal effect).108 In addition, some photon energy may induce chemical changes to the chromophore or surrounding molecules resulting in chemical reactions in the target tissue (photochemical effect). In living organisms these chemical alterations have biological effects, called photobiomodulation.54,55,109111 Photobiomodulation forms the basis for low-level laser therapy (LLLT) approaches. In this setting, cytochrome c oxidase is a putative receptor of light stimuli with different redox states of the molecule absorbing different wavelengths of light. Photothermal effects likely play a larger role in LVA effects when lasers with nanosecond duration pulses at high power levels are used. It is also probable that, while the irradiance and dose of adjuvanting lasers exceed the effective dose range for LLLT by 1–2 log orders, photobiomodulation plays an important role in LVA effects as well especially for the CW-NIR laser.

Photothermal effects of adjuvanting lasers

All lasers that have been used as LVAs to date induce moderate thermal effects in the treated tissues, but irradiance and fluence are specifically calibrated to remain below the level that causes skin damage. Increases in skin temperature induced by these minute-range laser exposures do not reach the pain threshold (42–43 °C)51,53 much less than the temperatures known to induce skin damage within such exposure times.112114The non-destructive nature of the LVA exposures used in the recent US mouse studies were validated by both visible skin inspection and independent analyses of biopsy for histopathological evidence of tissue damage.51,53 In addition, a clinical safety study, performed with a 1064 nm nanosecond pulse laser using irradiances and fluences equivalent to those used in the murine laser vaccine enhancement experiments, was well tolerated in humans with no subject reporting uncomfortable skin sensations or pain and no significant skin damage or changes in skin appearance noted during or as a result of any laser exposure.51

While overall increases in temperature in the laser exposed skin is modest, lasers featuring high power, nanosecond duration laser pulses are nevertheless likely to cause significant thermal stresses in the skin. This is due to a phenomenon called thermal containment. When the time over which a laser pulse is absorbed into a volume of the skin around a chromophore (tp) is significantly shorter than the time the resulting heat can be dissipated from that volume to the surrounding tissue (tr), the result is a significant increase in the temperature within that volume relative to the surrounding tissue.108 This condition is known as thermal containment and is considered to be met when tp ≈ 0.25tr. As the duration of a laser pulse decreases, the volume in which the thermal energy is contained also decreases. Thermal confinement is the basis of selective photothermolysis, a phenomenon first described by Anderson and Parrish,115117 and is the key mechanism of many medical laser applications.

In the nanosecond range, thermal containment is expected to occur at the organelle scale (e.g., 0.5 to 1.0 μm in diameter). In the visible light range where melanin is an important absorber, such targets are typically melanosomes in the basal epidermis.118,119 Given the same pulse energy, shorter pulse durations will result in larger temperature rises within more highly localized volumes around the chromophore. Eventually, sufficiently large pulse energies or sufficiently short pulse durations will result in temperature rises large enough to induce transient protein unfolding or permanent denaturation.120,121 Once these localized temperature spikes significantly exceed 100 °C, the result will be microcavitation or explosive vaporization of the water in and around the target chromophore.122124 This phase transition can cause significant damage to the tissue. Laser pulse durations in the nanosecond range with pulse powers in the kW or MW range like the CV, Q-Nd:YAG, and Q-Nd:YVO4 lasers, contain sufficient energy to induce significant temperature perturbations at the subcellular level (microhyperthermia), resulting in cellular stress from heat shock.125,126The propagation of high power (kW or MW) nanosecond-range pulses, repeated tens or thousands of times per second over a matter of minutes, likely triggers a number of stress responses in the skin that leads to enhanced immune processing of introduced vaccine antigens. As long as the combination of peak power, pulse duration and overall fluence are limited, these temperature spikes will not lead to significant irreversible damage to the tissue.

Aside from the tissue stress effects induced by highly localized generation of heat by nanosecond duration laser pulses, the modest overall increase of heat within the tissue does not appear to contribute significantly to the impact of the laser on the immune system. Chen et al. reported that skin heating did not result in a significant enhancement of immune responses.53 Kashiwagi et al. reported no correlation between measured maximal skin temperature and antibody titer in a model vaccine experiment.51

Photoacoustic effects of adjuvanting lasers

The photothermal effects generated by high energy, nanosecond pulsed lasers are accompanied by photoacoustic effects. The significant temperature discontinuities that high power, nanosecond duration laser pulses create between the absorbing target and the surrounding tissue results in different rates of thermal expansion and thus pressure differences.127,128 These pressure differences can generate an acoustic wave that propagates at a much slower rate than that of heat dissipation. When the laser pulse duration (tp) is shorter than the time required for these stress waves to propagate (tσ), a condition of acoustic containment is reached. At pulse durations below 100 ns, significant acoustic waves are generated in the tissue.129 The combination of microhyperthermia and shock wave generation from nanosecond pulse lasers can induce significant stress within cells and tissues, even when no significant damage is apparent.130132 Not surprisingly, Chen et al. reported that laser treatment of mouse skin with 6–7 ns pulses with a peak power of about 5 MW at a frequency of 10 Hz over 2 min resulted in the disruption of the dense protein network in the skin tissue, resulting in disconnected tissue with collagen fibers, as determined by electron microscopy, even while the overall tissue appearance remained normal.74 Photoacoustic effects would be expected to be reduced in the pulse lasers with much lower peak powers, which may explain why such tissue changes were not reported by Kashiwagi et al.51 Finally, these effects are not expected with the CW-NIR laser. In this case, photochemical effects are more likely to be the mechanism.

Photochemical effects

One of the key photochemical effects of laser light is the generation of reactive oxygen and reactive nitrogen species (ROS and RNS).133 Generation of oxygen and nitrogen radicals from lasers have been demonstrated at a wide range of the spectrum including UV,134 blue,135 visible,136 and near-infrared.54,55,137140Generation of these radical species form the basis for a wide variety of biological effects. Since ROS and RNS (particularly nitric oxide, NO) have been shown to stimulate cytokine production in epithelial cells via activation of MAPKs (p38, ERK), JNKs, NF-κB, AP-1, soluble guanylate cyclase (sGC)/protein kinase G (PKG)141145 and recently NLRP3 inflammasome pathways,146 it is possible that laser adjuvants can mediate activation (phosphorylation) of these pathways via ROS generation. Endogenous or exogenous ROS and NO have been shown to modulate the function of skin cells including keratinocytes147150 and mast cells.151153

The results of published LVA studies to date clearly demonstrate that these lasers result in migrational and functional changes of DCs in the skin, which may be a common pathway for laser immune enhancement. To this end, different types of lasers appear to engage distinct molecular mechanisms. Photothermal and photoacoustic stress may play a more important role in immune signaling induced by nanosecond pulsed lasers, while photobiomodulation may play a larger role in the CW-NIR laser (Fig. 4). Elucidation of the exact photoreceptors and signaling pathways that mediate these effects is warranted.

figure hvi-10-1892-g4

Figure 4. The distinctions between pulsed and continuous wave laser vaccine adjuvants. There are 2 major types of laser vaccine adjuvant (LVA). Each class of LVA has a distinct mode of laser-tissue interaction, mechanisms of action, and the effect

Advantages and Limitations of Laser Vaccine Adjuvants

Advantages of LVA over conventional adjuvants

As an adjuvanting approach, lasers have several inherent advantages over chemical and biological adjuvants. (1) Laser light does not persist in the tissue or excessively perpetuate immune signaling, so it reduces the potential for toxic adjuvant effects. (2) It does not appear to depend on conventional inflammatory pathways, similarly reducing the potential for adverse events. (3) LVAs do not cause reactogenicity at the immunization site, a common factor with nearly all current vaccine adjuvants. (4) It cannot induce anti-adjuvant antibodies since it is not a chemical or biological substance. (5) There is little risk of allergic responses from lasers. Millions of people have been treated with visible light and NIR lasers for tattoo removal, hair removal, skin tightening and regeneration. While there are several published reports of allergic response after Nd:YAG laser treatment in tattoo removal, these were essentially delayed hypersensitivity reactions against the tattoo ink in the skin.154,155 (6) LVAs are separate from the vaccine antigen and do not require formulation with the vaccine. Some adjuvants are difficult to combine with the vaccine antigen because co-formulation may cause instability in the vaccine formulation. (7) The laser has no special storage requirements such as cold-chain requirements. Finally, as work by Chen et al. has shown, the laser can be used with several other conventional adjuvants to enhance their effect.35,36

Distinctions of the continuous wave, near-infrared laser for LVA over other laser parameters

When all LVA approaches to date are considered, the CW-NIR system has some compelling advantages over the nanosecond pulsed visible light lasers used in other studies. First, the CW-NIR system is much less sensitive to differences in skin pigmentation. Laser light in the green-yellow spectrum is significantly absorbed by melanin, resulting in highly variable absorption of the same laser dose across different skin phototypes.156,157 In addition, the melanin distribution in different types of skin is not uniform.114 This means that the laser dosing in dark skin may be different than in lighter skin, requiring a more careful recalibration of dose for different skin phototypes. Under these conditions, it may prove more challenging to tolerably treat a dark-skinned recipient due to the high efficiency of light absorption. While Russian investigators conducted initial studies of CV lasers in humans (Table 3), most of their subjects were fairly light skinned. Compared with the yellow-green spectrum, at 1064 nm in the NIR spectrum the coefficient of absorption of melanin is nearly 10-fold less.156,157 Differences in thermal responses to 1064 nm laser treatment between very light skinned and very dark skinned recipients appear to be modest and these differences appear to be shaped by absorption of laser light by blood.158