Spinal Cord – Cerebral Injury

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.

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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

Lasers Med Sci. 2014 Nov;29(6):1849-59. doi: 10.1007/s10103-014-1586-4. Epub 2014 May 24.

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

Paula AA1, Nicolau RA, Lima Mde O, Salgado MA, Cogo JC.

Author information
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 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.

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.

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.

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 Jun;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.