Macular Degeneration – Retinal Disease

Merry, Munk, Dotson, Walker and Devenyi et al reported functional and in anatomical improvements in dry age related, macular degeneration in this December 2016 abstract at  https://www.ncbi.nlm.nih.gov/pubmed/27989012

Eye (Lond). 2016 Dec 16. doi: 10.1038/eye.2016.259. [Epub ahead of print]

Safety and acceptability of an organic light-emitting diode sleep mask as a potential therapy for retinal disease.

Sahni JN1,2, Czanner G2,3, Gutu T1, Taylor SA1, Bennett KM4, Wuerger SM4, Grierson I2, Murray-Dunning C2, Holland MN5, Harding SP1,2; Medscape.

Author information

  • 1St Paul’s Eye Unit, Royal Liverpool University Hospitals NHS Trust, Liverpool, UK.
  • 2Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK.
  • 3Department of Biostatistics, Institute of Translational Medicine, University of Liverpool, Liverpool, UK.
  • 4Department of Psychological Sciences, Institute of Psychology, Health and Society, Liverpool, UK.
  • 5Polyphotonix Medical Ltd, Durham, UK.

Abstract

Purpose

The purpose of the study was to study the effect of an organic light-emitting diode sleep mask on daytime alertness, wellbeing, and retinal structure/function in healthy volunteers and in diabetic macular oedema (DMO).

Patients and methods

Healthy volunteers in two groups, 18-30 yrs (A), 50-70 yrs (B) and people with DMO (C) wore masks (504 nm wavelength; 80 cd/m2 luminance; <8 h) nightly for 3 months followed by a 1-month recovery period. Changes from baseline were measured for (means): psychomotor vigilance task (PVT) (number of lapses (NL), response time (RT)), sleep, depression, psychological wellbeing (PW), visual acuity, contrast sensitivity, colour, electrophysiology, microperimetry, and retinal thickness on OCT.

Results

Of 60 participants, 16 (27%) withdrew, 8 (13%) before month 1, due to sleep disturbances and mask intolerance. About 36/55 (65%) who continued beyond month 1 reported >1 adverse event. At month 3 mean PVT worsened in Group A (RT (7.65%, P<0.001), NL (43.3%, P=0.005)) and mean PW worsened in all groups (A 28.0%, P=0.01, B 21.2%, P=0.03, C 12.8%, P<0.05). No other clinically significant safety signal was detected. Cysts reduced/resolved in the OCT subfield of maximal pathology in 67% Group C eyes. Thinning was greater at 3 and 4 months for greater baseline thickness (central subfield P<0.001, maximal P<0.05).

Conclusion

Sleep masks showed no major safety signal apart from a small impairment of daytime alertness and a moderate effect on wellbeing. Masks were acceptable apart from in some healthy participants. Preliminary data suggest a beneficial effect on retinal thickness in DMO. This novel therapeutic approach is ready for large clinical trials.Eye advance online publication, 16 December 2016; doi:10.1038/eye.2016.259.

Adv Exp Med Biol. 2016;854:437-41. doi: 10.1007/978-3-319-17121-0_58.

Near-Infrared Photobiomodulation in Retinal Injury and Disease.

Eells JT1, Gopalakrishnan S2, Valter K3.
Author information
1Department of Biomedical Sciences, University of Wisconsin-Milwaukee, 2400 E. Hartford Ave., 53201, Milwaukee, WI, USA. jeells@uwm.edu.
2College of Nursing, University of Wisconsin-Milwaukee, 53201, Milwaukee, WI, USA. sandeep@uwm.edu.
3Divsion of Biomedical Sciences, Research School of Biology, Australian National University, 0200, Acton, Australia. krisztina.valter-kocsi@anu.edu.au.
Abstract
Evidence is growing that exposure of tissue to low energy photon irradiation in the far-red (FR) to near-infrared (NIR) range of the spectrum, collectively termed “photobiomodulation” (PBM) can restore the function of damaged mitochondria, upregulate the production of cytoprotective factors and prevent apoptotic cell death. PBM has been applied clinically in the treatment of soft tissue injuries and acceleration of wound healing for more than 40 years. Recent studies have demonstrated that FR/NIR photons penetrate diseased tissues including the retina. The therapeutic effects of PBM have been hypothesized to result from intracellular signaling pathways triggered when FR/NIR photons are absorbed by the mitochondrial photoacceptor molecule, cytochrome c oxidase, culminating in improved mitochondrial energy metabolism, increased cytoprotective factor production and cell survival. Investigations in rodent models of methanol-induced ocular toxicity, light damage, retinitis pigmentosa and age-related macular degeneration have demonstrated the PBM attenuates photoreceptor cell death, protects retinal function and exerts anti-inflammatory actions.
.
Mol Vis. 2015; 21: 883–892.
Published online 2015 Aug 21.
PMCID: PMC4544713

Photobiomodulation with 670 nm light increased phagocytosis in human retinal pigment epithelial cells

Shinichiro Fuma, Hiromi Murase, Yoshiki Kuse, Kazuhiro Tsuruma, Masamitsu Shimazawa, and Hideaki Haracorresponding author
Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan
corresponding authorCorresponding author.
Correspondence to: H. Hara, Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan; Phone: +81-58-230-8126; FAX: +81-58-230-8126; email: pj.ca.up-ufig@arahedih
Received 2015 Jan 29; Accepted 2015 Aug 19.

Abstract

Purpose

Photobiomodulation is the treatment with light in the far-red to near-infrared region of the spectrum and has been reported to have beneficial effects in various animal models of disease, including an age-related macular degeneration (AMD) mouse model. Previous reports have suggested that phagocytosis is reduced by age-related increased oxidative stress in AMD. Therefore, we investigated whether photobiomodulation improves phagocytosis caused by oxidative stress in the human retinal pigment epithelial (ARPE-19) cell line.

Methods

ARPE-19 cells and human primary retinal pigment epithelium (hRPE) cells were incubated and irradiated with near-infrared light (670 nm LED light, 2,500 lx, twice a day, 250 s/per time) for 4 d. Next, hydrogen peroxide (H2O2) and photoreceptor outer segments (POS) labeled using a pH-sensitive fluorescent dye were added to the cell culture, and phagocytosis was evaluated by measuring the fluorescence intensity. Furthermore, cell death was observed by double staining with Hoechst33342 and propidium iodide after photobiomodulation. CM-H2DCFDA, JC-1 dye, and CCK-8 were added to the cell culture to investigate the reactive oxygen species (ROS) production, mitochondrial membrane potential, and cell viability, respectively. We also investigated the expression of phagocytosis-related proteins, such as focal adhesion kinase (FAK) and Mer tyrosine kinase (MerTK).

Results

Oxidative stress inhibited phagocytosis, and photobiomodulation increased the oxidative stress-induced hypoactivity of phagocytosis in ARPE-19 cells and hRPE cells. Furthermore, H2O2 and photobiomodulation did not affect cell death in this experimental condition. Photobiomodulation reduced ROS production but did not affect cell viability or mitochondrial membrane potential. The expression of phosphorylated MerTK increased, but phosphorylated FAK was not affected by photobiomodulation.

Conclusions

These findings indicate that near-infrared light photobiomodulation (670 nm) may be a noninvasive, inexpensive, and easy adjunctive therapy to help inhibit the development of ocular diseases induced by the activation of phagocytosis.

Introduction

Age-related macular degeneration (AMD) is a progressive and degenerative eye disease that is a common cause of vision loss in developed countries [1]. AMD is classified into dry and wet types, and the main clinical feature common to both is the accumulation of lipofuscin in retinal pigment epithelium (RPE) cells. Wet AMD is characterized by abnormal angiogenesis, and dry AMD is characterized by atrophy of the outer retinal layers and RPE cells [2,3]. Risk factors for AMD, such as aging, light damage, smoking, genetic factors, and oxidative stress, have been reported to cause the accumulation of lipofuscin [4].

RPE cells help maintain the normal functions of photoreceptor cells by playing a role in phagocytosis, a part of the visual cycle, by forming a blood–retinal barrier that permits the exchange of waste products and nutrients between the blood and the retina [5,6]. Phagocytosis by RPE cells is an essential function of homeostasis in the retina. Photoreceptor cells are damaged by exposure to light. RPE cells can remove deteriorated photoreceptor outer segments (POS) via phagocytosis to preserve the function of photoreceptor cells [7]. It is important that the phagocytosis of RPE cells is not inhibited because the dysfunction of phagocytosis can trigger RPE damage and lysosomal disorder that prevents the breakdown of waste products, ultimately resulting in the accumulation of lipofuscin [8,9]. Thus, we believe the inhibition of lipofuscin accumulation is likely to improve AMD [10]. Phagocytosis of POS by RPE cells involves several steps, such as binding, uptake, and degradation, and each step is regulated by some proteins. Phagocytosis of POS by RPE cells requires ?v?5 integrin for binding [11]. Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase and is phosphorylated by integrin engagement. Finnemann et al. have shown that POS binding by RPE cells increases FAK complex formation with ?v?5 integrin and activates FAK [11,12]. FAK is related to the binding of POS, whereas Mer tyrosine kinase (MerTK) is not required for binding but for internalization [12,13]. Rat RPE cells expressing MerTK can bind to the surface of RPE cells but have no effect on the internalization of POS [14].

The AMD models were appeared to mitochondrial dysfunction. The accumulation of lipofuscin decreased the mitochondrial membrane potential, impaired oxidative phosphorylation in the mitochondrial respiratory chain, and decreased the activity of phagocytosis [15].

Previous reports have shown that photobiomodulation, treatment with light in the far-red to near-infrared region of the spectrum, is beneficial in treating strokes, wounds, infection, diabetic retinopathy, and AMD [1619]. Its beneficial effects include regulating cell viability by absorbing near infrared light in photosensitive molecules such as water, melanin, hemoglobin, and cytochrome c oxidase molecules. Some of the most important photosensitive molecules that respond to photobiomodulation are cytochrome c oxidase molecules, which accept electrons and are involved in producing adenosine triphosphate (ATP) in mitochondrial oxidative phosphorylation [20]. It is known that photobiomodulation activates cytochrome coxidase and increases cellular ATP, resulting in the protection of neurons [21]. It has been reported that photobiomodulation suppresses the inflammation caused by decreased mitochondrial activity [16]. Other reports have suggested that photobiomodulation increases the expression of the antioxidant enzyme MnSOD without affecting cytochrome c oxidase activity (which is related to mitochondrial activity); thus, the mechanism of photobiomodulation effects is not clear [17]. However, it is well known that photobiomodulation has effects such as the upregulation of ATP, the increase of antioxidant materials, and the prevention of inflammation in retinal neurons [17,22,23]. However, the effect of photobiomodulation on phagocytosis in RPE cells remains unclear. In the present study, we therefore investigated the effect of photobiomodulation on the oxidative stress-induced hypoactivity of phagocytes in ARPE-19 cells and primary human RPE (hRPE) cells.

Methods

Cell culture

The human retinal pigment epithelial cell line (ARPE-19) was obtained from American Type Culture Collection (Manassas, VA). The cells were maintained in Dulbecco’s Modified Eagle’s medium (DMEM)/F-12 (Wako, Osaka, Japan) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 ?g/ml streptomycin. Cultures were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The ARPE-19 cells were passaged by trypsinization every 3–4 d.

The primary hRPE cells were obtained from Lonza (Walkersville, MD). The cells were maintained in Retinal Pigment Epithelial Basal Medium (Lonza) containing 2% FBS, 4 mL L-glutamine (Lonza), 0.2 ml GA-1000 (Lonza), and 1 mL growth factor (FGF-B; Lonza) according to the manufacturer’s protocol. Cultures were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The cells were passaged by Trypsin/EDTA (Lonza), Trypsin Neutralizing Solution (Lonza), and HEPES Buffered Saline Solution (Lonza) every 3–4 d.

Isolation from porcine eyes and labeling of photoreceptor outer segments

Retinas from freshly obtained porcine eyes were homogenized with POS buffer (115 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 10 mM HEPES/KOH ph 7.5, and 1 mM dithiothreitol) containing 1.5 mM sucrose on ice. The suspension was centrifuged for 7 min at 7,510 ×g to sediment chunk pieces of retinas. A filter (BD Falcon, Franklin Lakes, NJ) was used to remove the deposits, and the filtrate was diluted with POS buffer and centrifuged again. The pellet was diluted with POS buffer containing 0.6 mM sucrose. The suspension was then added to the tube containing the continuous sucrose gradient and the whole was centrifuged for 90 min at 103,700 ×g in RP55T rotor (Hitachi Co., Ltd. Tokyo, Japan). After centrifugation, POS bands were collected and diluted with 1:3 balanced salt solution (BSS; 10 mM HEDES, 137 mM NaCl, 5.36 mM KCl, 0.81 mM MgSO4, 1.27 mM CaCl2, 0.34 mM Na2HPO4, and 0.44 mM KH2PO4). This was suspended for 7 min at 7,510 ×g to obtain a pure POS pellet, which was then stored in darkness at ?80 °C. The supernatant was removed and the POS was taken up in several milliliters of POS buffer. The media were concentrated by centrifugation at 4,000 ×g using an Amicon Ultra-15 centrifugal filter device (Millipore, Billerica, MA; molecular weight cutoff: 3,000) to combine POS with pHrodo. Unlabeled POS (5 × 107) were added to 5 mL of the BSS. POS were labeled at a final concentration of 1 mg pHrodo/10 mg protein. POS with the dye were concentrated by centrifugation at 4,000 ×g using the Amicon Ultra-15 centrifugal filter device (Millipore; molecular weight cutoff: 3,000) for 6 h at 4 °C [24].

Near-infrared photobiomodulation

For all experiments, we followed this protocol before each assay: ARPE-19 cells were plated at a density of 1.5 × 104 cells per well with DMEM/F-12 containing 10% FBS in 96-well plates. This was incubated for 4 d with or without 670 nm light emitting diode (LED; Sawa Denshi Kougyou, Saitama, Japan) treatment (250 s at 3.89 mW/cm2 twice/day). The medium was changed to DMEM/F12 containing 1% FBS, and the cells were treated with an antioxidant, N-acetylcysteine (NAC; Sigma-Aldrich, St. Louis, MO) for 1 h.

Phagocytosis assays

H2O2 at a final concentration of 0.1 mM and 1 × 105 POS/well were added to each well and incubated for 6 h. The cells were then washed five times with 1% FBS DMEM/F-12 to allow removal of non-specific POS binding to quantify specific attachement of POS by RPE cells. Images were collected using a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan) and then quantified using image processing software (Image-J, ver. 1.43 h; National Institutes of Health, Bethesda, MD). The area was then calculated.

Nuclear staining assay

Nuclear staining assays were conducted 6 h after H2O2 treatment.

Cell viability assay

Water-soluble tetrazolium salt 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5- (2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) assay kits were used to investigate the inhibitory effect of photobiomodulation on oxidative stress-induced cytotoxicity. Briefly, 10 ?l of CCK-8 (Dojindo Laboratories, Kumamoto, Japan) was added to each well, and the cells were incubated at 37 °C for 2 h. The absorbance was measured at 492 nm (reference wavelength, 660 nm) using SkanIt Re for Varioskan Flash 2.4 (ThermoFisher Scientific Inc., Waltham, MA).

Measurement of intracellular reactive oxygen species production

The measurement of intracellular reactive oxygen species (ROS) production was estimated by CM-H2DCFDA (Invitrogen). Briefly, CM-H2DCFDA was added to the medium at a final concentration of 10 ?M, followed by incubation at 37 °C for 1 h. Fluorescence was then measured using a fluorescence spectrophotometer at 488 nm excitation and 525 nm emission.

Mitochondrial membrane potential assay

The measurement of mitochondrial membrane potential was estimated using JC-1 dye (Mitochondrial Membrane Potential Probe; Invitrogen). The ARPE-19 cells (1.5 × 104 cells/well) were cultured and exposed to H2O2 for 6 h. The cells were washed and incubated with 10 ?g/ml JC-1 at 37 °C for 15 min in the dark. Images were collected using a fluorescence microscope (BZ-9000; Keyence), which detects healthy cells with JC-1 J-aggregates (excitation/emission=540/605 nm) and unhealthy cells with mostly JC-1 monomers (excitation/emission=480/510 nm).

Western blot analysis

The ARPE-19 or hRPE cells (1.5 × 104 cells/well) were seeded onto a 24-well plate and cultured at 37 °C for 4 d. After H2O2 exposure, the cells were supplemented with a 1% protease inhibitor cocktail (Sigma), 1% phosphate inhibitor cocktails 2 and 3 (Sigma), and sample buffer (Wako). The lysate was centrifuged at 12,000 ×g for 10 min, and the supernatant was collected for analysis. Protein concentration was determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL) with BSA as standard. An equal volume of protein sample and sample buffer with 10% 2-mercaptoethanol was electrophoresed with a 10% sodium dodecyl sulfate-polyacrylamide gel, and the separated proteins were then transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore). The following primary antibodies were used for immunoblotting: anti-MerTK (phospho Y749 + Y 753 + Y754; ab14921) rabbit polyclonal antibody (1: 500; abcam); anti-MerTK (ab137673) rabbit polyclonal antibody; anti-phospho-FAK (Tyr397) rabbit polyclonal antibody (1: 1000; Cell Signaling Technology, Danvers, MA); anti-FAK (C-903; sc-932) rabbit polyclonal antibody (1:1000; Santa Cruz Biotechnology, Inc. CA), and anti-?-actin mouse monoclonal antibody (1:5000; Sigma). An HRP-conjugated goat anti-rabbit antibody and an HRP-conjugated goat anti-mouse antibody (1:2000) were used as secondary antibodies. Band densities were measured using an imaging analyzer (LAS-4000 mini, Fujifilm, Tokyo, Japan), gel analysis software (Image Reader LAS-4000; Fujifilm), and detected band analysis software (Multi Gauge; Fujifilm).

Statistical analysis

Data are presented as the mean ± standard error of the mean (SEM). Statistical comparisons were made using a two-tailed paired Student t test only, where p<0.05 indicated statistical significance.

Results

Photobiomodulation enhanced the phagocytic activity in oxidative stress

To clarify the effect on phagocytic activity in near-infrared light-exposed ARPE-19 cells, the phagocytic activity in the cells was investigated after 6 h of H2O2 exposure by measuring the fluorescence intensity of intracellular POS. The quantification of fluorescence intensity was calculated as described in the Methods section. The fluorescence intensity was significantly reduced after H2O2 exposure, and NAC, an antioxidant, increased the fluorescence intensity. Moreover, photobiomodulation enhanced the fluorescence intensity of intracellular POS in comparison to the group that was only exposed to H2O2 (Figure 1 B,C). The group exposed to H2O2 and NAC group maybe also invreased the fluorescence intensity but we did not use statistical analysis between H2O2 only group and H2O2/NAC group. H2O2 or photobiomodulation did not affect cell death in this experimental condition (Figure 1D).

Figure 1

Photobiomodulation enhanced the phagocytic activity in oxidative stress. A: ARPE-19 cells were seeded at a density of 15,000 cells/well and incubated for 4 day. During this period, they were irradiated with near-infrared light. After 4 day, the medium

Photobiomodulation did not affect cell viability or mitochondrial membrane potential but reduced reactive oxygen species production

Cell viability was determined by WST-8 assay in order to clarify the other effects of photobiomodulation in ARPE-19 cells. In the group exposed only to H2O2, cell viability was reduced for 6 h compared to the control group with a reduction of 21%. Photobiomodulation did not affect cell viability (Figure 2A). CM-H2DCFH is converted to a fluorescent product (CM-H2DCF) when intracellular ROS are produced, was increased by H2O2 exposure, and 1 mM NAC significantly reduced the oxidative stress-induced ROS production in ARPE-19 cells. Daily exposure to 250 s of 670 nm photobiomodulation significantly inhibited the H2O2-induced ROS production (Figure 2B). To investigate the effect of photobiomodulation on mitochondrial activity, JC-1 dye was used. Neither H2O2 nor photobiomodulation significantly changed the red or green fluorescence (Figure 2C)

Figure 2

Photobiomodulation reduced ROS production but did not change the cell viability or mitochondrial membrane potential. A: Cell viability was measured using a WST-8 assay kit. Cell viability was reduced by oxidative stress, and photobiomodulation did not

Photobiomodulation increased the expression of phosphorylated MerTk but did not change the expression of phosphorylated FAK

We investigated the mechanism of the promotion of phagocytosis by photobiomodulation. We tested changes in the levels of phagocytosis-associated proteins, FAK and MerTK, by western blot analysis after 3 h or 6 h of H2O2 exposure to clarify the mechanism of phagocytosis. The maximum reduction of p-FAK and p-MerTK expression was observed 3 h and 6 h after H2O2 exposure, respectively (data not shown). Phosphorylated FAK was significantly reduced by H2O2 treatment, but photobiomodulation did not change the expression of FAK in comparison to the H2O2-exposed group (Figure 3A). In this point, we performed the statistical analysis between H2O2 only treated group and photobimodulation group. Although photobiomodulation did not change the expression of phosphorylated FAK, photobiomodulation improved the reduction of phosphorylated MerTK induced by the oxidative stress (Figure 3B). NAC at 1 nM increased the expression of phosphorylated FAK and MerTK.

Figure 3

Photobiomodulation increased the expression level of phosphorylated MerTK but not phosphorylated FAK. FAK and MerTK expression was measured by western blot. The quantification of the expression of phosphorylated FAK and phosphorylated MerTK was corrected

Photobiomodulation enhanced the phagocytic activity and increased the expression of phosphorylated MerTK

We investigated the phagocytosis activity and the expression of phosphorylated MerTK to validate our results concerning ARPE-19. The fluorescence intensity was significantly reduced after H2O2 exposure, and photobiomodulation enhanced the fluorescence intensity of intracellular POS in hRPE cells in comparison to the H2O2 only group (Figure 4A). In this point, we performed the statistical analysis between H2O2 only treated group and photobimodulation group. Furthermore, we investigated the expression of phosphorylated MerTK in hRPE cells. The expression of phosphorylated MerTK was increased in primary RPE cultures by photobiomodulation (Figure 4B).

Figure 4

Photobiomodulation enhanced the phagocytic activity in human retinal pigment epithelium cell cultures. A: Oxidative stress reduced the phagocytic activity, and photobiomodulation improved phagocytic activity induced by oxidative stress in hRPE cells.

Discussion

In the present study, as expected, phagocytic activity was reduced by exposure to H2O2 (Figure 1). The activity of RPE cells is reduced by oxidative stress and the auto-oxidative lipofuscin is accumulated in the lysosomes. In addition, drusen is formed in between the RPE and Bruch’s membrane. Ultimately, these things result in AMD [25,26]. The increase of oxidative stress also impairs the function of phagocytosis, and the dysfunction of phagocytosis induces the accumulation of lipofuscin [27,28].

Next, we investigated whether photobiomodulation using low-intensity and near-infrared light affects ARPE-19. Blue LED is routinely used in video display terminals and is known as an inducer of several kinds of photoreceptor cell damage in our laboratory [29]. In contrast, red LED has longer wavelengths than blue LED and has a protective effect on photoreceptors [22].

Near-infrared light photobiomodulation has a protective effect against light-induced retinal degeneration and reduced inflammation via the upregulation of mitochondrial cytochrome c oxidase expression in AMD mouse models [16,22]. Although photobiomodulation reduced the ROS production, it did not alter the cell viability or mitochondrial membrane potential (Figure 2). A previous report suggested that photobiomodulation has protective effects against high glucose-induced cell death of 661W cells (mouse photoreceptor cells) and retinal ganglion cells but not against high glucose-induced cell death of ARPE-19 cells [17]. This report also indicated that all cell lines, including ARPE-19, reduced the superoxide generation but did not change cytochrome c oxidase activity, which is related to mitochondrial activity. Furthermore, this phagocytosis assay model was set in a concentration of 0.1 mM H2O2, which did not change the cell death rate. Thus, low-intensity far-red light have no effects on cell viability or mitochondrial membrane potential.

Phagocytosis is related to FAK and MerTK expression. MerTK is an important protein in the ingestion of POS, and it has been shown that mutation of MerTK found in retinitis pigmentosa—one of the most common retinal diseases responsible for blindness—results in phagocytic dysfunction in RPE cells [30,31]. In the present study, we investigated the expression and phosphorylation of FAK and MerTK in POS exposed ARPE-19 cells to clarify the mechanism of phagocytosis enhancement with near-infrared photobiomodulation. Photobiomodulation increased phosphorylated MerTK but not phosphorylated FAK. Although photobiomodulation increased only the phosphorylated MerTK, the antioxidant NAC increased both phosphorylated FAK and MerTK. There are some difference pathway to increase the phagocytosis activity between photobiomodulation and antioxidant. A previous report suggested that mitochondrial dysfunction impairs the function of phagocytosis in retinal pigment epithelial cells [15]. Photobiomodulation may have a specific effect, which is the upregulation of phagocytosis activity through some mitochondrial pathways.

In conclusion, these findings indicate that photobiomodulation enhances phagocytosis via the MerTK-mediated upregulation of POS ingestion into RPE cells (Figure 5). Near-infrared light photobiomodulation may be a noninvasive, inexpensive, and easy adjunctive therapy to help inhibit the development of ocular diseases, such as AMD and retinitis pigmentosa. However, further experimentation and clinical studies are needed to clarify the therapeutic effects.

Figure 5

Photobiomodulation enhances phagocytosis via the upregulation of MerTK and has an anti-oxidative effect. Phagocytosis has several steps: binding to integrin, ingestion, and degradation of POS. FAK is thought to be involved in binding to integrin, and

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Articles from Molecular Vision are provided here courtesy of Emory University and the Zhongshan Ophthalmic Center, Sun Yat-sen University, P.R. China
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Br J Ophthalmol 2014 Mar 28. doi: 10.1136/bjophthalmol-2013-304477. [Epub ahead of print]

Photobiomodulation in the treatment of patients with non-center-involving diabetic macular oedema.

Tang J, Herda AA, Kern TS.

Abstract

PURPOSE:

Far-red/near-infrared phototherapy or photobiomodulation (PBM) has recently been reported to be an effective and non-invasive treatment method to inhibit lesions of diabetic retinopathy (DR) in animals. This study investigated the safety and efficacy of PBM in diabetic patients to treat non-center-involving diabetic macular oedema (NCDME).

METHODS:

This was a non-randomised, consecutive, case series, where 4 patients with type 2 diabetes with NCDME were treated for 160 s per day with PBM for 2-9 months. Demographic data including age, sex, HbA1c%, electronic ETDRS visual acuity, and retinal and macular thickness were measured using spectral domain ocular coherence tomography (SD-OCT) before and after treatment.

RESULTS:

Four eyes of 4 patients were treated, with fellow eyes serving as untreated controls. Daily PBM treatment for only 80 s per treatment twice daily caused a significant reduction in focal retinal thickening in all 4 treated eyes. No adverse effects attributable to therapy were noted by the patients or study investigators during the study period.

CONCLUSIONS:

PBM potentially offers a non-invasive and cost-effective therapeutic option for patients with NCDME. Further studies of this therapeutic option in DR are warranted.

PLoS One. 2013; 8(2): e57828.
Published online 2013 Feb 28. doi:  10.1371/journal.pone.0057828

Treatment with 670 nm Light Up Regulates Cytochrome C Oxidase Expression and Reduces Inflammation in an Age-Related Macular Degeneration Model

Rana Begum,1 Michael B. Powner,1 Natalie Hudson,1 Chris Hogg,1,2 and Glen Jeffery1,*
Alfred Lewin, Editor
1Institute of Ophthalmology, University College London, London, United Kingdom
2Moorfields Eye Hospital, London, United Kingdom
University of Florida, United States of America
Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: CH GJ. Performed the experiments: RB MBP NH. Analyzed the data: RB MBP NH. Contributed reagents/materials/analysis tools: GJ NH. Wrote the paper: RB GJ.

Author information ? Article notes ? Copyright and License information ?
Received 2012 Oct 11; Accepted 2013 Jan 26.

Introduction

Progressive ageing is associated with systemic inflammation [1][3]. This is marked in tissues with high metabolic demand such as the retina that suffers from progressive oxidative stress [4][6]. This is partly driven by excess extra-cellular deposition along an ageing Bruch’s membrane, where inflammation becomes a key feature, even in the absence of pathology [2], [7]. It is also a common feature of retinal disease whether it be age related macular degeneration (AMD), diabetic retinopathy or posterior uveitis [8][12]. Ameliorating retinal inflammation is becoming a key problem with an ageing population as this may be associated with age related retinal cell loss and declining visual function [13][15]. It is also critical in many retinal diseases [5], [10], [11].

There are multiple routes to dealing with retinal inflammation, however key to any success is the need for cheap effective therapies that are minimally invasive and do not require clinical time. Inflammation is partially driven by shifts in mitochondrial function that result in reduced adenosine triphosphate (ATP) production and increased reactive oxygen species (ROS) output [5], [7], [16]. This can be modulated by brief exposure to 670 nm light, which is absorbed by cytochrome c oxidase (COX) and increases ATP production [17], [18]. This has been shown to reduce a range of retinal inflammatory markers in normal aged mice when directly exposed to the light for brief periods [7]. It has also been used effectively to reduce damage from experimental pathology in a wide range of independent studies (see Table 1 in ref 7).

Table 1

The number of eyes used in separate experiments.

Here we ask two questions. First, is 670 nm light a potential therapeutic route in an aged mouse model of AMD? Half of AMD cases are associated with polymorphisms of the complement system including complement factor H (CFH) [19][21]. There is a mouse model of CFH?/? that has a distinct retinal phenotype with outer retinal disorganisation, elevated inflammation and reduced retinal function [22][24]that we employ here. Second, in previous studies using 670 nm light, animals have been held individually directly in front of the light source [7], [25]. Here we also ask whether 670 nm is therapeutic when given for short periods indirectly as part of the environmental lighting when animals are caged in groups and their retinae are not forcibly exposed to it individually. With both of these questions our analysis is focussed primarily on the outer retina, which is the location for disease initiation and progression in AMD, both in humans and in the mouse model [26][28].

Materials and Methods

Animals-ethics Statement

Twenty nine 16 month old CFH?/? mice were used. Thirty nine eyes from these animals were used in different procedures (Table 1). Mice had been maintained from birth in a normal animal unit with a 12[ratio]12 light cycle. None of the animals were exposed to direct lighting. All animals were used with University College London ethics committee approval and under a UK Home Office project licence (PPL 70/7036). All procedures were conducted in accordance to the United Kingdom Animal Scientific Procedures Act 1986.

Therapeutic Light Treatment and Control

The aged mice were divided randomly into two groups. These were caged separately for 14 days in adjacent compartments of a large animal cabinet. Individual cages had an internal space of 6,422 cubic centimeters, with a maximum of five animals per cage. The external walls of the cage were sprayed matt white to increase internal reflectance (Figure 1). All were exposed to low levels of room illumination on a 12[ratio]12 L/D pattern of approximately 50 Lux.

Figure 1

Cage for 670 nm exposure.

The spectrum of the room illumination was measured at 0.5 m below the light source and within the cage with an Ocean Optics (USB2000+UV-VIS-ES, Dunedin, USA), showing minimal 670 nm content (Figure 2). Room illumination was indirect. The experimental group were exposed to 670 nm light via LED sources (C.H. Electronics, UK) behind clear perspex screens at either end of the cage (Figure 1). Spectral (Ocean Optics) and intensity measurements with a radiometer (International Lights, IL 1700 SED033IF/W, Massachusetts, USA) were made directly in front of the source and behind the perspex screens. There was no spectral shift as a consequence of the perspex screen and only vary minimal attenuation in intensity.

Figure 2

Spectral composition of room light in which mice were caged.

Energy levels for 670 nm and room lighting at different parts of the cage are given in Table 2. Each source produced 20 mW/cm2 and was turned on automatically for 6 minutes at approximately 6 am and 6 pm each day for 14 days. No attempt was made to modify the animal’s behaviour during exposures. The control group were housed separately, under identical conditions and could not see the 670 nm lights.

Table 2

Energy levels with 670 nm and room lighting.

Following 14 days of light exposure all animals were killed by cervical dislocation and their eyes removed. Those for immunohistochemistry were fixed for 1 hour in 4% paraformaldehyde in phosphate buffered saline (PBS, pH 7.2) at room temperature, followed by X3 washes with PBS. Those for Quantitative real-time polymerase chain reaction (qPCR) and for Western blots were chilled on ice. Immunostaining was undertaken for a range of markers including COX, ionized calcium-binding adaptor molecule 1 (IBA-1), complement component C3, vimetin, glial fibrillary acidic protein (GFAP) and amyloid beta (A?). qPCR was used to confirm key COX and C3 labelling. Western blot analysis was undertaken additionally to confirm COX labelling as this is fundamental to the mode of action of 670 nm light.

Immunostaining- Outer Retinal Macrophages

To examine the morphology of outer retinal macrophages flat mounts were stained with IBA-1. Eyes were dissected and flat mounts of the retinal pigmented epithelial (RPE) surface were produced following method used by Hoh Kam et al [2]. These were washed with PBS X1 for 5 minutes and blocked with 5% Normal Donkey Serum (NDS) in 3% Triton X-100 PBS for 2 hours on shaker. Followed by X1 wash with PBS and a primary antibody solution prepared by diluting 1% NDS in 3% Triton X-100 and incubated with IBA-1 (rabbit polyclonal, 1[ratio]1000, A. Menarini Diagnostics, Wokingham, UK) overnight to mark macrophages. Next day the tissue was washed X3 with PBS and a secondary antibody solution prepared by diluting 2% NDS in 0.3% Triton X-100, incubated with donkey-anti rabbit 488 (1[ratio]2000, Invitrogen, Paisley, UK) for 2 hours. Tissues were washed X3 with PBS then incubated with 4?,6-diamidino-2-phenylindole (DAPI, 1[ratio]5000, Sigma Aldrich, Dorset, UK) for 1 minute in the dark to provide a counter stain. Lastly, tissues were washed several times with PBS and Tris buffered saline (TBS). The RPE flat mounts were mounted with Vectrashield (Vector laboratories, Peterborough, UK), cover slipped and sealed with nail varnish.

Immunostaining- Sections

Fixed eyes were cryo-protected in 30% sucrose in PBS and embedded in optimal cutting temperature compound (OCT, Agar scientific, Stanstead, UK). Eyes were sectioned at 10 µm and thaw-mounted onto charged slides overnight. Immunohistochemistry was performed according to Hoh Kam et al [2]. Sections were initially washed for 5 minutes with PBS and then blocked with 5% NDS in 0.3% Triton X-100 solution for 1 hour. These were washed briefly and then a primary antibody solution was prepared by diluting 1% NDS in 0.3% Triton X-100 and incubated overnight. The following day, this was washed 1X with PBS, and a secondary antibody solution was prepared as described above and incubated for 1 hour. After incubation, this was washed several times and then DAPI was applied for 1 minute as a counter stain. Sections were then washed with PBS and TBS, mounted, cover slipped and sealed as described above. The following primary antibodies were used: COX subunit VIb (rabbit monoclonal 1[ratio]200, Abcam, Cambridge, UK), C3 (rabbit polyclonal 1[ratio]20, Abcam, Cambridge, UK), vimentin (rabbit monoclonal 1[ratio]100, Abcam, Cambridge, UK), GFAP (mouse monoclonal 1[ratio]1000, Abcam, Cambridge, UK) and A? (mouse monoclonal 1[ratio]500, Covance, UK). This was followed by incubation with the appropriate Alexa fluor secondary antibody, donkey-anti rabbit 568 (1[ratio]2000, Invitrogen, Paisley, UK) and donkey anti-mouse 568 (1[ratio]2000, Invitrogen, Paisley, UK). Negative controls were done for all the above where the primary antibody was omitted.

Quantitative Real-time Polymerase Chain Reaction

RNA was extracted using TRIzol reagent (Sigma Aldrich, Dorset, UK) from whole eye cups after the removal of extra-ocular tissue, cornea, lens and ciliary body/iris. The RNA was reverse transcribed using a Qiagen QuantiTect Kit and qPCR was performed using Power SYBR PCR master mix (Applied Biosystems, Paisley, UK) with the primer pairs; COX6B1 (Fwd: ACAATCTTTAGGAGTCAGGATGG, Rev: TTCTTAGTCTGGTTCTGGTTGG) and C3 (Fwd: GCGTAGTGATTGAGGATGGTG, Rev:ACAGTGACGGAGACATACAGG). Values were normalised using beta actin mRNA levels and analysed with DART-PCR software [29]. Each group consisted of 5 mice.

Western Blot

Anterior chambers were dissected out leaving the retina and RPE-choroidal tissues, which were immediately frozen in liquid nitrogen and stored at ?80°C. The tissues were homogenized in lysis buffer containing 4% sodium dodecyl sulfate, 17.5% glycerol, 0.25 M Tris and 100 mM DTT before extracts were electrophoretically separated on sodium dodecyl sulphate polyacrylamide gels. Subsequently, immunoblotted for COX (Abcam, Cambridge, UK) and tubulin (clone DM1A, Sigma Aldrich, Dorset, UK) as a housekeeping control protein. Changes of proteins were determined by densitometric scanning of immunoblots. Values were normalised to tubulin as loading control and averaged over at least 3 independent experiments [2], [30].

Analysis

Macrophage number, morphology and distribution

Fluorescence images of the complete RPE surface were captured using a Nikon DMX1200 digital camera (Tokyo, Japan) at a magnification of X400 and saved in JPEG format to count macrophages. The numbers of IBA-1 positive cells per eye were determined using the count tool on Adobe Photoshop CS6.

To analyze macrophage morphology, images were captured at X1000 of cells that were clearly well labelled but still representative of the population. More labelled macrophages were commonly found in central regions approximately 1 mm from the optic nerve head than in other areas. The diameters of the whole mounts were approximately 5 mm with approximately 50 cells in experimental animals and about 90 cells in controls. Within each retinae 9–12 individual cells were selected for analysis on the basis of morphological clarity and a separation of more than 75 µm from any other cells analyzed. Within such constraints candidate cells were identified by scanning across the RPE surface.

For every image the total number of primary dendritic processes on each macrophage was counted by drawing a 30 µm diameter circle around the soma and then counting the processes crossing the circle, as undertaken by Lee et al [30]. The same images were used to measure primary process length from the centre of the nucleus to the tip of each dendrite. Dendritic field size was also determined using the same image. For this, the longest axis of the dendritic field was measured and that of the field orthogonal to this and the mean length calculated. Lasso tool in Adobe Photoshop CS6 was used to measure the cell body area on the same cells [30]. The distance between adjacent macrophages was calculated from nucleus of each cell to its nearest neighbour [2]. For this the images were taken at a lower magnification of X400, as was undertaken to count macrophage numbers.

Quantifying immunostaining intensity

The methods used to quantify the intensity of the immunostaining were similar to those employed previously [2], [7], [30]. Sections used for analysis had relatively uniform staining patterns. Two regions in the central retina were selected for analysis on either side of the optic nerve head that were approximately 200 µm away from the optic nerve and 150 µm wide and captured at a magnification of X400 in JPEG format. Pixel intensity was calculated in Adobe Photoshop CS6, by using the lasso tool to draw a line. No less than 10 measurements per eye were taken. The regions of interest for immune staining varied depending on the marker as they accumulate at different locations. For COX, this was at the level of the photoreceptor inner segments. For C3 it was Bruch’s membrane and photoreceptor outer segments, while for vimentin and GFAP measurements were made across the full depth of the neural retina. A? was measured along Bruch’s membrane/RPE interface. For all immunostaining sections from experimental and control groups were stained on the same day and imaged with standard microscope settings. All data were analysed with GraphPad Prism 5 and statistical analysis was undertaken using Mann-Whitney U non parametric tests.

Results

670 nm Treatment Significantly Elevates COX Expression

COX is an enzyme in the mitochondrial respiratory chain which also functions as a photoacceptor molecule activated by long wavelengths [18], [25], [31][32]. COX immunostaining was present in both groups and was largely confined to mitochondrial rich regions, including photoreceptor inner segments and the outer plexiform layer (Figure 3A–D). There were significant differences between the two groups, in the experimental group COX expression was up regulated by approximately 50% (Figure 3E). To confirm the up regulation of COX following 670 nm treatment, quantitative Western blot and qPCR analysis were undertaken. These two independent methods confirmed similar significant increases with COX expression following light exposure (Figure 3F–H).

Figure 3

Mitochondrial cytochrome c oxidase expression is enhanced in 670 nm treated aged mice.

670 nm Treatment Significantly Changes Macrophage Morphology

IBA-1 is commonly used as a marker of macrophages. IBA-1 positive cells were widely distributed in a relatively uniform pattern over the RPE surface in both experimental and control groups, with somewhat more present in central than peripheral regions (Figure 4A–B). The morphology of the macrophages in the two groups appeared to be markedly different (Figure 4C–D) and although there were less stained cells in the light treated mice, this was not statistically significant (Figure 4E). However, the morphological differences between cells in the two groups were consistently significantly different over a range of metrics.

Figure 4

IBA-1 staining showed significantly different macrophage morphology between 670 nm and control groups.

The primary processes in mice exposed to 670 nm light appeared to have wider bases and were significantly longer than those found in the control. In control mice mean primary process length was approximately 27 µm, while in experimental mice it almost doubled to approximately 54 µm (Figure 4F). Estimates for the area covered by the cells dendritic field between the two populations were consistent with this, as the longer processes in the 670 nm exposed mice covered a significantly larger area of the RPE surface (Figure 4G).

In the treated mice there was a significant increase in the distance between the cells from approximately 91 µm to approximately 151 µm (Figure 4H). Not only were the processes in treated mice longer, extending over a wider territory, but there were significantly more primary processes on them (Figure 4I). The relative expansion in the processes in 670 nm treated eyes was associated with a significant decline in the relative size of their cell bodies (Figure 4J). Hence, the dendritic architecture and size of individual macrophages in treated mice changed significantly in response to 670 nm light.

670 nm Treatment Reduces Outer Retinal Inflammation

The inflammatory marker C3 normally accumulates with age on Bruch’s membrane and on photoreceptor outer segments. C3 immunostaining was significantly lower in 670 nm treated mice at both locations than in controls (Figure 5A–D). On Bruch’s membrane it was almost halved, and on outer segments the reduction was approximately 20%. To confirm this finding C3 expression was also measured with qPCR, and again there was a significant reduction following 670 nm treatment with expression level halving (Figure 5E).

Figure 5

Outer retinal inflammation is significantly reduced following 670 nm treatment.

670 nm Treatment Reduces Retinal Stress

Retinal sections from the two groups were also stained for vimentin and GFAP. These are cytoskeletal intermediate filaments expressed in retinal Muller cells and are up regulated following retinal stress and ageing. Muller cell processes span the entire retina.

Staining was present for both vimentin and GFAP in both groups (Figure 6A–D, G–J). However expression of both was down regulated following 670 nm exposure. Vimentin labelling was much more extensive than GFAP in both experimental and control groups. With vimentin the number of Muller cell processes and their length were significantly reduced by 670 nm light (Figure 6E–F). In the untreated group (Figure 6C–D) labelling extended into the outer nuclear layer and was denser at the vitreal surface than in the treated mice. GFAP labelling was also significantly reduced following 670 nm light (Figure 6K), with more label present in the outer retina of controls (Figure 6I–J).

Figure 6

Retinal stress is significantly reduced following 670 nm treatment.

670 nm Treatment does not Alter Amyloid Beta Expression

With age, the outer retina accumulates A? mainly on Bruch’s membrane and on photoreceptor outer segments [2]. This age related deposit is pro-inflammatory. A? was measured on Bruch’s membrane in both experimental and control group (Figure 7A–B). Measurements of staining intensity made at this interface showed no difference in deposition between the two groups (Figure 7C).

Figure 7

Amyloid beta expression is unaffected by 670 nm treatment.

Discussion

This study clearly shows that brief passive exposure to 670 nm light is effective in reducing inflammation in aged CFH?/? mice in which retinal pathology has become established [23]. The retina showed marked changes after 670 nm treatment, with a significant increase in COX expression and significant reductions in C3, vimetin and GFAP expression. Further, there were significant changes in the dendritic morphology and cell body size of sub-retinal macrophages, although there number did not decline significantly. These changes occur independent of A? load, which did not differ between the two groups.

These results are similar to those that we obtained using normal aged C57BL/6 mice where retinal inflammation had become established [7]. However, in previous studies animals were individually held in front of the light in an attempt to regulate exposure [7]. This study shows that 670 nm light impacts therapeutically irrespective of the method of delivery and can be delivered by supplementing this wavelength in normal light, where it is largely absent.

While it is clear that a range of inflammatory/stress markers are reduced by 670 nm light similar to that shown by Kokkinopoulos et al [7], we did not find a statistically significant decline in macrophage numbers as found in this earlier study. However, it was obvious with other metrics that 670 nm light had a profound impact on these cells by simple observation. Another factor that significantly reduces inflammation and impacts on macrophage morphology is vitamin D. Here the number of cells is significantly reduced along with age related outer retinal inflammation. Further, the morphology of these cells also changes, but here they develop larger cell bodies and have fewer processes [30]. It is likely that reducing macrophages will be beneficial in aged tissue, but changes in their morphology in response to different therapeutic routes may be difficult to compare directly.

These results sit firmly within an expanding data set from different laboratories, consistently revealing that 670 nm exposure has a significant impact on both pathology and ageing within diverse tissues [17], [18],[25], [33][39]. Given this diversity, the mechanism of action must act upon a fundamental aspect of cell function. With ageing and many forms of pathology, mitochondrial DNA is damaged and this probably reduces ATP output, which is critical for normal metabolic function. Changes in mitochondrial function are key to theories of ageing as the driving force for ageing probably relates to mitochondrial DNA damage reducing ATP and increasing ROS output, which is inflammatory and induces tissue degradation and cell loss [40]. The impact of such damage is marked in regions were metabolic rate is great, as in the outer retina[4][6]. Age related changes here not only include increased inflammation and deposition, but also the loss of approximately 25–30% of central photoreceptors in both man and mouse [13], [41].

The exact mechanism of 670 nm has not been proven. However it has been proposed that this wavelength is selectively absorbed by COX in mitochondria [42]. COX is the rate limiting enzyme in mitochondrial respiration, which we have shown is significantly up regulated following 670 nm exposure. Its activation enhances the efficiency of oxidative phosphorylation, which may in turn lead to an increase in ATP production and a reduction in ROS [17], [18], [37], [43], [44]. This in turn may reduce oxidative stress in the outer retina, which due to very high metabolic demand is particularly susceptible to this type of damage[4][6]. Whether this impacts on transcription factors and gene expression is unknown and there remains considerable speculation regarding these issues [31], [45]. However our current in vivo studies have confirmed that retinal ATP production declines with age and that 670 nm exposure corrects this.

Dosages of 670 nm vary between studies, although they tend to be relatively brief, but efficacy is common, indicating that the actual exposure levels required may be lower than had been expected. This is consistent with our data, as exposure levels must have varied between animals because they were often asleep through exposures, while at other times they were active but not necessarily oriented towards the source of the 670 nm light. In spite of this, because of the relatively long wavelength of 670 nm, it will penetrate deeply into the tissue. Consistent with this was the observation that the light from our delivery devices could be seen through the hand in a dimly lit room. Further, in a study this wavelength has been shown to reduce the pathology from partial section of the rat optic nerve when illumination was over the top of the head and only a very small amount of the light actually penetrated skull tissues [34]. Hence, 670 nm appears to be effective relatively independent of dosage or at least over a very wide range. However, we do not know a number of key variables, including how long following an exposure the impact of 670 nm remains effective, and also whether excess exposure ultimately has a negative impact or is ineffective. Resolution of these questions remains critically important in steps towards human therapeutic treatment.

In both normal ageing and in AMD outer retinal deposits and inflammation develop, partly due to the high metabolic demand of photoreceptors and the accumulation of relatively large quantities of extra-cellular debris, including A?, the heavier elements of which are pro-inflammatory [2], [26], [46][48]. However, here we demonstrate that it is possible to disassociate some of these factors by reducing markers of inflammation/stress independent of A? load. While there is little doubt that A? is toxic [49], [50], this is mainly true of the larger molecular forms, as the smaller elements are actually critical for synaptic plasticity and normal central nervous system function [51], [52]. Eventually, it is likely that progressive A? deposition on Bruch’s membrane will significantly compromise the permeability of this interface between the outer retina and its blood supply, which will probably impact negatively on outer retinal function. However, it is unclear to what extent such changes drive neuronal degeneration compared with inflammation alone. A key strategy in fighting retinal ageing is focussed on the issue of clearing A? systemically, which in turn is hoped to impact on inflammation. However, physiological levels of A? are necessary and their removal is likely to be detrimental with a loss of function [51], [52]. An alternative route that would avoid such problems may reside in the results of the study presented here.

Therapeutic routes in AMD have already been initiated using 670 nm light. While the patient numbers have been very low, they have shown significant improvements in terms of visual function in some using devices that were held directly in front of the eye [53]. While patient numbers need to be expanded and followed over long time periods, such results linked with this study and our previous work are encouraging.

Acknowledgments

The authors would like to thank Jaimie Hoh Kam, Vivian Lee for their help and Paul Johnson for modifications to animal cages.

Funding Statement

This study was funded by the Rosetrees Trust. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Adv Exp Med Biol.  2010;664:365-74.

Near-infrared light protect the photoreceptor from light-induced damage in rats.

Qu C, Cao W, Fan Y, Lin Y.

Source

Department of Ophthalmology, Sichuan Academy of Medical Sciences, Sichuan, China.

Abstract

BACKGROUND:

A project originally developed for NASA plant growth experiments in space demonstrating the Light-Emitting Diode (LED) could promote the wound healing. Further study showed that the LED’s could protect cells by stimulating the basic energy processes in the mitochondria of each cell.OBJECTIVE:

The purpose of this study was to assess the effects of 670 nm LED to protect the photoreceptor from the light-induced damage in a rodent model.

METHODS:

SD rats were randomly assigned to one of eight groups: untreated control group, the LED-treated control group, three light-induced damage groups, and three LED-protected groups. The rats were exposed to constant light for 3 h of different illuminations of 900, 1,800 and 2,700 lux, respectively. The LED treatment (50 mW) were done for 30 min, 3 h before the light damage and 0, 24 and 48 h after the light damage. Using the electroretinogram as a sensitive indicator of retinal function, and the histopathologic change was showed as a proof of the protective effect of LED treatment.

RESULTS:

The 900 lux illumination for 3 h did not cause damage to the retina of rats, however, the 1,800 lux illumination for 3 h caused significant damage to ONL of an approximate half retina, which caused the swing of ERG b wave to be 431 muV. With the LED protection: the damage of ONL was near 1/6 of retina, which was significantly reduced than the ones without LED protection (P < 0.01); and the swing of ERG b wave was recorded to be 1,011 muV, which was increased significantly than the ones without LED protection (P < 0.01). The illumination of 2,700 lux for 3 h caused severe damage to the rats’ retinas and the LED could not protect them significantly in both of morphology and function (P > 0.05, P > 0.05).

CONCLUSIONS:

670 nm LED treatment has an evident protective effect on retinal cells against light-induced damage, which may be an innovative and non-invasive therapeutic approach to prevent or to delay age-related macular degeneration.

Exp Eye Res. 2009 Nov;89(5):791-800. Epub 2009 Jul 16.

Low power laser treatment of the retina ameliorates neovascularisation in a transgenic mouse model of retinal neovascularisation.

Yu PK, Cringle SJ, McAllister IL, Yu DY.

Centre for Ophthalmology and Visual Science and the ARC Centre of Excellence in Vision Science, The University of Western Australia, 2 Verdun Street, Nedlands, Perth, Western Australia 6009, Australia. paulakyu@cyllene.uwa.edu.au

This study was designed to determine if low power laser therapy can achieve amelioration of vasoproliferation yet preserve useful vision in the treated area in a transgenic mouse model of retinal neovascularisation. The mice were anaesthetised and the pupils dilated for ERG and fundus fluorescein angiography on postnatal day 32. The left eyes were treated with approximately 85 laser spots (532 nm, 50 ms, 300 microm diameter) at a power level of 20 mW at the cornea. The eyes were examined using ERG and fluorescein angiography, one, four and six weeks later. Flat mounts of FITC-dextran infused retinas, retinal histology and PEDF immunohistochemistry was studied one or six weeks after laser treatment. In untreated eyes the expected course of retinal neovascularisation in this model was observed. However, retinal neovascularisation in the laser treated eye was significantly reduced. The laser parameters chosen produced only mild lesions which took 10-20 s to become visible. ERG responses were comparable between the treated and untreated eyes, and histology showed only partial loss of photoreceptors in the treated eyes. PEDF intensity corresponded inversely with the extent of neovascularisation. Low power panretinal photocoagulation can inhibit retinal neovascularisation and yet preserve partial visual function in this transgenic mouse model of retinal neovascularisation.

Photomed Laser Surg. 2008 Jun;26(3):241-5

Low-level laser therapy improves vision in patients with age-related macular degeneration.

Ivandic BT, Ivandic T.

University of Heidelberg, Otto-Meyerhof Centre, Heidelberg.

Abstract Objective: The objective of this study of a case series was to examine the effects of low-level laser therapy (LLLT) in patients with age-related macular degeneration (AMD).

Background Data: AMD affects a large proportion of the elderly population; current therapeutic options for AMD are limited, however. Patients and Methods: In total, 203 patients (90 men and 113 women; mean age 63.4 +/- 5.3 y) with beginning (“dry”) or advanced (“wet”) forms of AMD (n = 348 eyes) were included in the study. One hundred ninety-three patients (mean age 64.6 +/- 4.3 y; n = 328 eyes) with cataracts (n = 182 eyes) or without cataracts (n = 146 eyes) were treated using LLLT four times (twice per week). A semiconductor laser diode (780 nm, 7.5 mW, 292 Hz, continuous emission) was used for transconjunctival irradiation of the macula for 40 sec (0.3 J/cm(2)) resulting in a total dose of 1.2 J/cm(2). Ten patients (n = 20 eyes) with AMD received mock treatment and served as controls. Visual acuity was measured at each visit. Data were analyzed retrospectively using a t-test.

Results: LLLT significantly improved visual acuity (p < 0.00001 versus baseline) in 162/182 (95%) of eyes with cataracts and 142/146 (97%) of eyes without cataracts. The prevalence of metamorphopsia, scotoma, and dyschromatopsia was reduced. In patients with wet AMD, edema and bleeding improved. The improved vision was maintained for 3-36 mo after treatment. Visual acuity in the control group remained unchanged. No adverse effects were observed in those undergoing therapy.

Conclusion: In patients with AMD, LLLT significantly improved visual acuity without adverse side effects and may thus help to prevent loss of vision

Retina. 2008 Apr;28(4):615-21.

Large-spot subthreshold infrared laser to treat diabetic macular edema.

Squirrell DM, Stewart AW, Joondeph BC, Danesh-Meyer HV, McGhee CN, Donaldson ML.

Department of Ophthalmology, Royal Hallamshire Hospital, Sheffield, United Kingdom. David.Squirrell@sth.nhs.uk

PURPOSE: To evaluate the efficacy of a large-spot subthreshold infrared laser protocol to treat diabetic maculopathy.

METHODS: In a prospective, fellow eye, controlled case series, all patients had clinically significant diabetic macular edema (DME) treated with a single application of subthreshold infrared (810 nm) laser. If bilateral disease was present, the fellow eye was treated with conventional macular laser. The study was to include 20 patients. Visual acuity and central macular thickness (CMT) measured by optical coherence tomography (OCT) were assessed in the study and fellow eyes at baseline and 6 months, and any changes were compared.

RESULTS: The 11th patient developed a choroidal infarct with subsequent profound loss of vision immediately after treatment. The study was terminated prematurely at this point. For the remaining 10 patients, there was a trend toward improvement in visual acuity in the study eye compared with the fellow eye at the 6-month follow-up (median change: +1.5 letters for study eye vs -6.5 letters for fellow eye; P = 0.08). There was also significant improvement in OCT-measured CMT in the study eye (mean decrease, 117 microm) compared with deterioration in OCT-measured CMT in the fellow eye (mean increase, 24 microm; P = 0.02).

CONCLUSION: This subthreshold infrared laser protocol led to improvement in OCT-measured CMT and stabilization of vision in most subjects. The current protocol is however unpredictable and should not be used in the treatment of DME without further modification.

Ophthalmology. 2006 Dec;113(12):2237-42. Epub 2006 Sep 25.

Subthreshold grid laser treatment of macular edema secondary to branch retinal vein occlusion with micropulse infrared (810 nanometer) diode laser.

Parodi MB, Spasse S, Iacono P, Di Stefano G, Canziani T, Ravalico G.

Eye Clinic, Azienda Ospedaliero-Universitaria di Trieste, Trieste, Italy. maubp@yahoo.it

PURPOSE: To compare the effectiveness of subthreshold grid laser treatment (SGLT) with an infrared micropulse diode laser with that of threshold grid laser treatment (TGLT) for macular edema secondary to branch retinal vein occlusion (BRVO).

DESIGN: Randomized clinical trial.

PARTICIPANTS: Thirty-six patients (36 eyes) were randomized either to infrared SGLT (17 eyes) or to krypton TGLT (19 eyes).

METHODS: Complete ophthalmic examinations, including determination of visual acuity (VA) with Early Treatment Diabetic Retinopathy Study charts, optical coherence tomography (OCT), and fluorescein angiography, were performed at the time of the study entry and at 6-month intervals, with a planned follow-up of 24 months.

MAIN OUTCOME MEASURES: Primary: decrease in mean foveal thickness (FT) on OCT. Secondary: changes of the total macular volume (TMV) over the follow-up, proportion of eyes that gained at least 10 letters (approximately > or =2 lines of VA gain) at the 12- and 24-month examinations, and timing of macular edema resolution.

RESULTS: Changes in mean FT and TMV from the initial values were statistically significant for TGLT from the 6-month examination (P<0.001) and for SGLT from the 12-month examination (P<0.001). After 1 year, there was no difference in mean FT and TMV between the 2 groups. At the 12-month examination, 10 patients of the SGLT group (59%) and 11 of the TGLT group (58%) gained at least 10 letters (2 lines) in VA. At the 24-month examination, this gain was achieved by 11 patients (65%) of the SGLT group and 11 (58%) of the TGLT group. Moreover, at the 24-month examination 59% and 26% gained 3 lines in the SGLT and TGLT groups, respectively.

CONCLUSIONS: Resolution of macular edema and VA improvement are similar to those obtained with conventional TGLT, but SGLT is not associated with biomicroscopic and angiographic signs. A multicenter randomized clinical trial would be needed to ascertain the real efficacy and the most appropriate settings of SGLT for macular edema secondary to BRVO.

Photomed Laser Surg. 2006 Apr;24(2):121-8.

Clinical and experimental applications of NIR-LED photobiomodulation.

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.

Department of Clinical Laboratory Sciences, University of Wisconsin-Milwaukee, 53226, USA.

This review presents current research on the use of far-red to near-infrared (NIR) light treatment in various in vitro and in vivo models. Low-intensity light therapy, commonly referred to as “photobiomodulation,” uses light in the far-red to near-infrared region of the spectrum (630-1000 nm) and modulates numerous cellular functions. Positive effects of NIR-light-emitting diode (LED) light treatment include acceleration of wound healing, improved recovery from ischemic injury of the heart, and attenuated degeneration of injured optic nerves by improving mitochondrial energy metabolism and production. Various in vitro and in vivo models of mitochondrial dysfunction were treated with a variety of wavelengths of NIR-LED light. These studies were performed to determine the effect of NIR-LED light treatment on physiologic and pathologic processes. NIRLED light treatment stimulates the photoacceptor cytochrome c oxidase, resulting in increased energy metabolism and production. NIR-LED light treatment accelerates wound healing in ischemic rat and murine diabetic wound healing models, attenuates the retinotoxic effects of methanol-derived formic acid in rat models, and attenuates the developmental toxicity of dioxin in chicken embryos. Furthermore, NIR-LED light treatment prevents the development of oral mucositis in pediatric bone marrow transplant patients. The experimental results demonstrate that NIR-LED light treatment stimulates mitochondrial oxidative metabolism in vitro, and accelerates cell and tissue repair in vivo. NIR-LED light represents a novel, noninvasive, therapeutic intervention for the treatment of numerous diseases linked to mitochondrial dysfunction.

Vestn Oftalmol. 2004 Nov-Dec;120(6):5-8.

Dependence of the efficiency of low-intensity laser therapy in involution chorioretinal dystrophy on a used wavelength

[Article in Russian]

Abramov MV, Egorov EA.

Seventy-five patients (75 eyes) with central involution chorioretinal dystrophy (non-exudative type at the progression stage) were followed up. All of them received low-intensity laser therapy. Irradiation of 890 nm, 644 nm and 500 nm was used in groups 1, 2 and 3, respectively. The study purpose was to compare the efficiency of wavelengths. Visual acuity and retinal sensitivity were determined. The results were evaluated immediately after treatment and in 3 months. The maximal improvement in visual acuity and retinal sensitivity was in those who received 890 nm laser therapy; 500 nm irradiation–a less pronounced effect and 640 nm–the lowest one. We attribute such distribution of efficiency to a proliferation type of each irradiation range in the macular zone.

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.

Proc Natl Acad Sci U S A. 2003 Mar 18;100(6):3439-44. Epub 2003 Mar 7.

Therapeutic photobiomodulation for methanol-induced retinal toxicity.

Eells JT, Henry MM, Summerfelt P, Wong-Riley MT, Buchmann EV, Kane M, Whelan NT, Whelan HT.

Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA. jeells@mcw.edu

Methanol intoxication produces toxic injury to the retina and optic nerve, resulting in blindness. The toxic metabolite in methanol intoxication is formic acid, a mitochondrial toxin known to inhibit the essential mitochondrial enzyme, cytochrome oxidase. Photobiomodulation by red to near-IR radiation has been demonstrated to enhance mitochondrial activity and promote cell survival in vitro by stimulation of cytochrome oxidase activity. The present studies were undertaken to test the hypothesis that exposure to monochromatic red radiation from light-emitting diode (LED) arrays would protect the retina against the toxic actions of methanol-derived formic acid in a rodent model of methanol toxicity. Using the electroretinogram as a sensitive indicator of retinal function, we demonstrated that three brief (2 min, 24 s) 670-nm LED treatments (4 J/cm(2)), delivered at 5, 25, and 50 h of methanol intoxication, attenuated the retinotoxic effects of methanol-derived formate. Our studies document a significant recovery of rod- and cone-mediated function in LED-treated, methanol-intoxicated rats. We further show that LED treatment protected the retina from the histopathologic changes induced by methanol-derived formate. These findings provide a link between the actions of monochromatic red to near-IR light on mitochondrial oxidative metabolism in vitro and retinoprotection in vivo. They also suggest that photobiomodulation may enhance recovery from retinal injury and other ocular diseases in which mitochondrial dysfunction is postulated to play a role.

Ophthalmology. 1999 Nov;106(11):2082-90.

Therapeutic benefits of infrared (810-nm) diode laser macular grid photocoagulation in prophylactic treatment of nonexudative age-related macular degeneration: two-year results of a randomized pilot study.

Olk RJ, Friberg TR, Stickney KL, Akduman L, Wong KL, Chen MC, Levy MH, Garcia CA, Morse LS.

The Retina Center of St. Louis Co., Missouri 63141, USA.

OBJECTIVE: This pilot study collected preliminary information on the effectiveness and safety of infrared (810-nm) diode laser macular grid photocoagulation in patients with nonexudative age-related macular degeneration (AMD). Results from this pilot study were used in designing a larger, multicenter, randomized clinical trial.

DESIGN: A multicenter, randomized, controlled, clinical trial.

PARTICIPANTS: A total of 229 eyes of 152 patients with AMD were enrolled in the pilot study. Seventy-five patients with 1 eye eligible (75 eyes) were enrolled in the unilateral arm of the study; 77 patients with both eyes eligible (154 eyes) were enrolled in the bilateral arm of the study. In the unilateral study arm, 32 eyes were randomized to the observation group, 27 eyes were treated with visible endpoint burns, and 16 eyes were treated with invisible endpoint (subthreshold) lesions. In the bilateral study arm, 77 eyes were in the observation group, 36 eyes were treated with visible burns, and 41 eyes were treated with subthreshold (invisible) lesions.

INTERVENTION: Eyes were treated with infrared (810-nm) diode laser macular grid photocoagulation using either visible burns or subthreshold (invisible) lesions and compared to eyes receiving no treatment.

MAIN OUTCOME MEASURES: Reduction of drusen, change in visual acuity, and rate of choroidal neovascularization (CNV) membrane formation.

RESULTS: At 12 months after treatment, 62% of eyes treated with visible burns had a clinically significant reduction in drusen, whereas this proportion (65%) was reached in 18 months for eyes treated with subthreshold lesions. At 24 months’ follow-up, treated eyes had a significant reduction in drusen compared to observation eyes (P < 0.0001). Visual acuity was significantly improved in treated eyes at 12, 18, and 24 months compared to observation eyes (P < 0.001). Choroidal neovascularization formation was similar in treated and observation eyes through 24 months’ follow-up. Complications included CNV associated with six eyes treated with visible burns and a juxtafoveal laser scar in one eye treated with visible burns.

CONCLUSIONS: Infrared (810-nm) diode laser macular grid photocoagulation in patients with nonexudative AMD significantly reduces drusen levels (P < 0.0001) and significantly improves visual acuity (P < 0.001) when either visible endpoint burns or subthreshold endpoint lesions are used. Complications were fewer using subthreshold endpoint lesions. A larger, multicenter, prospective clinical trial with longer follow-up is needed to determine the efficacy of treatment in reducing the rate of CNV formation. Data from this clinical pilot study have been used to design the Prophylactic Treatment of AMD Trial (PTAMD), a multicenter, randomized, prospective clinical trial currently in progress comparing subthreshold (invisible) treatment to observation in eyes with nonexudative AMD.

Ophthalmology. 1997 Dec;104(12):2030-8.

The treatment of macular disease using a micropulsed and continuous wave 810 nm diode laser.

Friberg TR, Karatza EC.

Eye and Ear Institute and the Department of Ophthalmology, University of Pittsburgh School of Medicine, Pennsylvania 15213, USA.

OBJECTIVE: The purpose of the study is to determine whether the 810-nm diode wavelength using a rectangular waveform is clinically effective in the treatment of choroidal neovascularization from age-related macular degeneration and to determine whether macular edema secondary to branch vein occlusion or diabetic retinopathy can be effectively treated with this laser using the micropulse waveform.

DESIGN: Review of consecutive nonrandomized patients whose eyes were treated with the diode laser over a 30-month period.

PARTICIPANTS: Fifty-three patients with an initial presentation of choroidal neovascularization located subfoveally (77%), extrafoveally (17%), and juxtafoveally (6%); 14 patients with macular edema from a branch vein occlusion; and 59 patients with diabetic macular edema, 40 of which were treated for the first time.

INTERVENTION: Ablative rectangular wave laser photocoagulation was applied to the choroidal neovascular membranes and very light threshold treatment was applied in a macular grid to treat retinal edema. Microaneurysms were not targeted.

MAIN OUTCOME MEASURES: Anatomic resolution of macular edema or choroidal neovascularization and visual acuity.

RESULTS: Sixty percent of eyes treated for choroidal neovascularization had no persistence or recurrence at 6 months, and 72% achieved visual stabilization. In 8% of eyes, some localized bleeding occurred during photocoagulation. Clinical resolution of macular edema from branch vein occlusion occurred by 6 months in 92% of eyes, and 77% had stabilization of visual acuity. At 6 months, 76% of newly treated patients with diabetic macular edema and 67% of previously treated patients had clinical resolution of their edema. Vision was improved or stabilized in 91% and 73% of newly treated and retreated patients at 6 months, respectively. CONCLUSIONS: The micropulsed 810-nm diode laser is clinically effective in the treatment of macular edema from venous occlusion and diabetic retinopathy, and the rectangular (normal) mode diode laser can be used in many eyes with choroidal neovascularization.

Vestn Oftalmol. 1997 Nov-Dec;113(6):17-9.

New method of atherosclerotic macular dystrophies treatment.

[Article in Russian]

Basinskii SN, Krasnogorskaia VN.

The authors analyze the results of treating atherosclerotic maculodystrophies by direct laser phoresis. The method consists in insertion of a collagen infusion system in Tenon’s space. Drugs (nicotinic acid or xanthinol nicotinate) are delivered to the posterior compartment of the eye through this system. Then a light guide is inserted in the tube and a 2-min session of low-intensity He-Ne laser exposure is performed at a wavelength of 630 nm, and 10 mWt/cm2 flow power density (7 to 10 sessions per course). Clinical studies showed that vision acuity increased by an average of 0.08 diopters, or by 40% of the initial level, in 72% of cases. The peripheral visual field extended by an average of 51.4 degrees for 8 meridians in 95% of patients. The index of critical frequency of flashings fusing and the frequency-contrast characteristics improved in 85% of cases. The rheography improved by 34.5% of the initial level. A stable improvement was observed for 12 months after a course of direct laser phoresis in 97.5% of patients. Hence, the new method is simple and recommended for the treatment of atherosclerotic maculodystrophies.