Eye Disorders

Int J Ophthalmol. 2016 Jan 18;9(1):145-52. doi: 10.18240/ijo.2016.01.24. eCollection 2016.

Photobiomodulation for the treatment of retinal diseases: a review.

Geneva II1.
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Author information
1Department of Ophthalmology, State University of New York, Upstate Medical University, Center for Vision Research, Syracuse, NY 13202, USA.
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Abstract
Photobiomodulation (PBM), also known as low level laser therapy, has recently risen to the attention of the ophthalmology community as a promising new approach to treat a variety of retinal conditions including age-related macular degeneration, retinopathy of prematurity, diabetic retinopathy, Leber’s hereditary optic neuropathy, amblyopia, methanol-induced retinal damage, and possibly others. This review evaluates the existing research pertaining to PBM applications in the retina, with a focus on the mechanisms of action and clinical outcomes. All available literature until April 2015 was reviewed using PubMed and the following keywords: “photobiomodulation AND retina”, “low level light therapy AND retina”, “low level laser therapy AND retina”, and “FR/NIR therapy AND retina”. In addition, the relevant references listed within the papers identified through PubMed were incorporated. The literature supports the conclusion that the low-cost and non-invasive nature of PBM, coupled with the first promising clinical reports and the numerous preclinical-studies in animal models, make PBM well-poised to become an important player in the treatment of a wide range of retinal disorders. Nevertheless, large-scale clinical trials will be necessary to establish the PBM therapeutic ranges for the various retinal diseases, as well as to gain a deeper understanding of its mechanisms of action.
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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.
PLoS One. 2015; 10(10): e0139003.
Published online 2015 Oct 1. doi:  10.1371/journal.pone.0139003
PMCID: PMC4591336

Photobiomodulation Mitigates Diabetes-Induced Retinopathy by Direct and Indirect Mechanisms: Evidence from Intervention Studies in Pigmented Mice

Alexandra Saliba,#1,2 Yunpeng Du,#1 Haitao Liu,1 Shyam Patel,3 Robin Roberts,3 Bruce A. Berkowitz,3,4 and Timothy S. Kern1,5,*
Bang V Bui, Editor
1Case Western Reserve University, Cleveland, Ohio, United States of America
2Catholic University of Brasilia, Brasilia, Brazil
3Department of Anatomy and Cell Biology, Wayne State University, Detroit, Michigan, United States of America
4Department of Ophthalmology, Wayne State University, Detroit, Michigan, United States of America
5Cleveland Veteran’s Affairs Medical Center, Research Service 151, Cleveland, Ohio, United States of America
University of Melbourne, AUSTRALIA
#Contributed equally.
Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: TK BAB. Performed the experiments: AS YD HL SP RR. Analyzed the data: TK YD. Contributed reagents/materials/analysis tools: BAB. Wrote the paper: TK.

* E-mail: ude.esac@kst
Author information ? Article notes ? Copyright and License information ?
Received 2015 Jun 23; Accepted 2015 Sep 8.

Abstract

Objective

Daily application of far-red light from the onset of diabetes mitigated diabetes-induced abnormalities in retinas of albino rats. Here, we test the hypothesis that photobiomodulation (PBM) is effective in diabetic, pigmented mice, even when delayed until weeks after onset of diabetes. Direct and indirect effects of PBM on the retina also were studied.

Methods

Diabetes was induced in C57Bl/6J mice using streptozotocin. Some diabetics were exposed to PBM therapy (4 min/day; 670 nm) daily. In one study, mice were diabetic for 4 weeks before initiation of PBM for an additional 10 weeks. Retinal oxidative stress, inflammation, and retinal function were measured. In some mice, heads were covered with a lead shield during PBM to prevent direct illumination of the eye, or animals were treated with an inhibitor of heme oxygenase-1. In a second study, PBM was initiated immediately after onset of diabetes, and administered daily for 2 months. These mice were examined using manganese-enhanced MRI to assess effects of PBM on transretinal calcium channel function in vivo.

Results

PBM intervention improved diabetes-induced changes in superoxide generation, leukostasis, expression of ICAM-1, and visual performance. PBM acted in part remotely from the retina because the beneficial effects were achieved even with the head shielded from the light therapy, and because leukocyte-mediated cytotoxicity of retinal endothelial cells was less in diabetics treated with PBM. SnPP+PBM significantly reduced iNOS expression compared to PBM alone, but significantly exacerbated leukostasis. In study 2, PBM largely mitigated diabetes-induced retinal calcium channel dysfunction in all retinal layers.

Conclusions

PBM induces retinal protection against abnormalities induced by diabetes in pigmented animals, and even as an intervention. Beneficial effects on the retina likely are mediated by both direct and indirect mechanisms. PBM is a novel non-pharmacologic treatment strategy to inhibit early changes of diabetic retinopathy.

Introduction

Diabetes is a major cause of visual impairment, and there is considerable clinical and research interest in diabetic retinopathy. Proven therapeutic approaches, such as good glycemic control, high energy laser photocoagulation, or intravitreal injections of anti-VEGF therapies or triamcinolone, are invasive, damaging, or require direct involvement by health-care professionals, and not all patients respond to these approaches. Supplemental therapeutic approaches are needed.

Photobiomodulation (PBM) is the application of low-level light that has a biological effect, such as to relieve pain or heal wounds. Numerous studies have shown that light in the far-red to near-infrared region of the spectrum (630–1000 nm) can have beneficial effects in vitro and in vivo to heal existing tissue damage and to inhibit the development of tissue pathology. Medical PBM using coherent (lasers) or noncoherent (Light Emitting Diodes; LEDs) light has been found to have beneficial effects in a variety of conditions, including accelerated healing of wounds and ulcers, cardiac ischemia, stroke, Parkinson’s disease, and optic nerve degeneration [112]. Studies related to the retina likewise have demonstrated that the low intensity light treatment mitigates pathology in retinal degeneration models [4,1316], and recently, also in diabetic retinopathy [17,18]. Our previous study in diabetic albino rats showed that whole-body exposure to far-red light (670 nm) for only 4 minutes per day from the onset of diabetes mitigated abnormalities that are believed to contribute to diabetic retinopathy, including increased generation of superoxide, induction of a local pro-inflammatory environment, and dysfunction or degeneration of retinal neurons [18].

The purpose of the present study was to extend those studies to determine if PBM would have similar beneficial effects under the following different conditions: (i) in another species (mice), (ii) in the presence of heavy pigmentation (C57Bl/6J), (iii) as an intervention therapy, (iv) when direct exposure of the eyes to the PBM was blocked, and (v) when activity of the antioxidant enzyme, heme oxygenase 1 (HO-1), was inhibited. Our results suggest that the PBM has both neuronal and vascular beneficial effects on pigmented diabetic mice, and that this effect is mediated at least in part systemically.

Materials and Methods

This study was performed in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and with authorization of the Institutional Animal and Care Use Committee (IACUC) at Case Western Reserve University and Wayne State University. Animals were housed and maintained in normal 12h:12h light-dark cycle laboratory lighting.

Animals

Male C57BL/6J mice were obtained from the Jackson Laboratory, and were housed in ventilated microisolator with free access to water and food. Diabetes was induced at 2–3 months of age by intraperitoneal injection of a freshly prepared solution of streptozotocin in citrate buffer (55 mg/Kg of body weight for five consecutive days). Insulin was given as 0–0.2 units subcutaneously between 0–3 times a week to inhibit weight loss, while still allowing hyperglycemia. To allow the animals to stabilize somewhat after induction of diabetes, blood glucose concentration was not measured until at least 7 days after the final administration of streptozotocin. Blood glucose was determined with a portable glucose meter, using blood collected from the tail vein under nonfasting conditions. The onset of diabetes was defined as three consecutive measures of blood glucose over than 275 mg/dl. HbA1c was measured as reported previously (Study 1 [19,20]; Study 2 [21].

There were two parts to this work. In the first, five groups with n = 12 animals per group were assigned as:(i) non-diabetic controls, (ii) diabetic controls, (iii) diabetic exposed to PBM starting 4 weeks after the induction of diabetes, (iv) diabetic exposed to PBM while the head was shielded from the light by a lead covering, and (v) diabetic treated with a heme oxygenase-1 (HO-1) inhibitor (tin protoporphyrin, SnPP; Frontier Scientific Inc, Logan, UT) [22], starting 4 weeks after the induction of diabetes. All animals were euthanized at 14 weeks of diabetes (5–6 months of age). In the second study, three groups were studied: (1) non-diabetic untreated control (n = 9), (2) diabetic untreated control (n = 3), and (3) diabetic treated with PBM from the onset of diabetes (n = 5). These animals were humanely euthanized at 8 weeks of diabetes.

Photobiomodulation

The far-red light was generated by LEDs (SpectraLife™; Quantum Devices, WI). This device was determined to deliver 670nm light at a power of 20.25 mW/cm2 at the 2–3 cm distance used between the device and the animal (measured with Spectro-radiometer; specbos 1211UV, Dataoptics, Inc, Ypsilanti MI). The mice were exposed to this radiation for 240 sec each day for the 10 weeks. The daily radiant exposure was thus 240 x 20.25 = 4860 mJ/cm2 (or ~5 J/cm2). In study 1, treated mice were placed in a DecapiCone™ restrainer bag (Braintree Scientific, Braintree, MA), and then in an open-top polypropylene holder using a Velcro strap to secure the animals. To ensure that the polypropylene bag would not impair passage of the red light, transmittance through the bag was tested; compared to the air, one layer of the plastic used to restrain the animals absorbed 8.2% of the light (0.082 Abs) at 670 nm. Intervention with PBM treatment was started four weeks after the onset of diabetes, and was continued for ten additional weeks. To minimize handling, animals in study 2 were allowed free movement in a small space during PBM. PBM was started immediately after confirmation of diabetes in this experimental study.

Retinal Superoxide

Fresh retinas from animals were analyzed for superoxide production as previously described [18,23,24]. Briefly, retinas were placed in 0.2 ml of Krebs/HEPES buffer and allowed to equilibrate in the dark at 37°C under 95% O2/5% CO2 conditions for 20 min. To each tube, 0.5 mM lucigenin (Sigma Chemical Company, St. Louis, MO) was added and incubated for an additional 10 minutes before having the photon emission detected by a luminometer (Analytical Luminescence Laboratory, San Diego, CA). Retinal protein was quantified (Bio-Rad), and the luminescence expressed per mg of protein.

HO-1 inhibition

Activity of HO-1 was inhibited systemically with SnPP in one of the diabetic groups getting the PBM treatment. The SnPP was freshly made dissolved in 0.2 N NaOH, adjusted to physiological pH 7.4 with 1N HCl, and protected from light. Mice received 100 ?mol/kg once per week by intraperitoneal injection. This dose of SnPP has been reported to inhibit HO-1 activity in vivo [25].

Leukostasis

Blood was removed from the vasculature of anesthetized animals by perfusion with phosphate-buffered saline (PBS; 10 mM phosphate buffer, 2.7 mM potassium chloride and 137 mM sodium chloride, pH 7.4; Sigma-Aldrich, St. Louis, MO) via a heart catheter. Animals were then perfused with fluorescein-coupled concanavalin A lectin (20 ?g/ml in PBS; Vector Laboratories, Burlingame, CA), as described previously [20,23,26]. Flat-mounted retinas were imaged via fluorescence microscopy, and the number of leukocytes adherent to the vascular wall was counted.

Visual Function

The virtual optokinetic system (OptoMetry; CerebralMechanics, Inc,) was used as described previously [19,27]. At 14 weeks of diabetes, mice were allowed to move freely on the platform of the visual OptoMotor system while the visual stimulus was projected around the platform. Spatial frequency threshold was measured at maximum contrast. For contrast sensitivity, only a single value (0.064 c/d; the maximal point in a full contrast sensitivity curve) was measured. These tests are psychophysical measures that assess function of both retinal and central visual pathways. Each measurement was repeated several times to evaluate the reproducibility of responses. The grader was masked with respect to the animals’ experimental group.

Immunoblots

Immunoblots of ICAM-1 (intercellular adhesion molecule-1; 1:500 dilution, R&D Systems, MN), iNOS (1:1000 dilution, Santa Cruz, CA) and HO-1 (1:1000 dilution, Cell Signaling Technology, Inc., Danvers, MA) were performed using the respective antibodies. The retina or cells was isolated and placed into 80 ?l of lysis buffer supplemented with protease inhibitor (1:1000 dilution), sonicated and centrifuged. The supernatant was collected and each sample containing the same amount of protein was fractionated by SDS-PAGE and electroblotted to PVDF membranes. After blocking nonspecific binding with 5% BSA, the membranes were incubated with rabbit polyclonal antibodies followed by incubation with antibody against rabbit IgG. Densities were normalized to that of ß-actin in the same lane (1:3000 dilution, Abcam, Inc., Cambridge, MA).

Leukocyte-mediated killing of retinal endothelial cells [20,23,28]

Transformed mouse retinal endothelial cells (mREC; generated from Immortomice [29]) were grown in DMEM containing 10% FBS and 5 or 25 mM glucose. Cells were cultured at 37°C in 5% CO2 and 95% air, and the media was changed every other day for 5 days. When mRECs were 80% confluent (approx. 500,000 cells), leukocytes (100,000; enriched from blood with RBC lysis buffer) from animals in the nondiabetic, diabetic, and diabetic treated with PBM groups were added to the mREC and incubated for 24 hrs [23]. After incubation, the leukocytes were carefully removed by gentle washing, and viability of remaining retinal endothelial cells was measured by trypan blue exclusion. Briefly, an aliquot of the endothelial cell suspension was diluted 1:1 (vol/vol) with 0.1% trypan blue, and the cells were counted with a hemocytometer. Cell death was reported as the percentage of blue-stained cells (dead cells) of the total number of cells. Approximately 200–400 cells were counted in each sample.

MEMRI

MEMRI is the imaging modality of choice for non-invasively studying the function of voltage-gated Ca(2+) channels on excitable cells, such as rod cell L-type calcium channels [30,31]. The MEMRI procedure in mice has been described previously [3235]. MEMRI data from the central retinal (± 1 mm from the center of the optic nerve) were analyzed using the region-of-interest following our prior procedure [3235]. The resolution obtained in the central retina is sufficient for extracting meaningful layer-specific anatomical and functional data, as previously discussed [36,37]. In the present studies, whole-retinal thicknesses in nondiabetic mice was ~ 220 ?m studied with an axial pixel size of 25 ?m [36]. Thus, the spatial uncertainty is ~ ½ pixel thick (~12.5 ?m) [36]. We rely on the well-defined laminar structure of the retina to distinguish, for example, inner retinal uptake from outer retinal uptake.

Statistical Analyses

Data were expressed as means ±SD, unless otherwise noted. Groups were compared using analysis-of-variance followed by Fischer’s test, with p ? 0.05 being considered statistically significant. MEMRI data (mean ± SEM) were analyzed using a generalized estimating equation (GEE) approach which performs a general linear regression analysis using contiguous locations or measurements in each subject and accounts for the within-subject correlation between contiguous locations or measurements [37,38]. Comparisons of MEMRI data between groups were performed using individual t-tests at different locations of the intraretinal profiles; those selected regions identified from the t-tests as significant were then analyzed using GEE [37,38]

Results

Animals

After injection with STZ, the presence of diabetes was confirmed based on blood glucose levels and body weight. Over the weeks of diabetes, the mean blood glucose level, HbA1c and body weight were significantly different between STZ-treated mice and control mice (Tables ?(Tables11 and ?and2).2). PBM treatment had no significant effect on body weight, non-fasting blood glucose or HbA1c compared to the other diabetic groups.

Table 1

Animal Data.
Table 2

Average body weight and HbA1c of nondiabetics (N), diabetics (D), and diabetics receiving PBM therapy (D+ PBM) in Trial 2.

Study 1

Intervention with PBM mitigated diabetes-induced molecular abnormalities in retinas from pigmented mice

In wildtype C57Bl/6J mice, diabetes caused metabolic and physiologic abnormalities in the retina, including increased superoxide production, leukostasis (Fig 1), increased expression of ICAM-1 and iNOS, and subnormal expression of HO-1 (Fig 2). Diabetes of 2 months duration also significantly impaired visual function, as assessed from spatial frequency threshold and contrast sensitivity (measured at a single point; 0.064 c/d) (not shown; both p<0.0001). Although initiation of the PBM therapy in these pigmented animals was delayed for 1 month of untreated diabetes, the subsequent intervention with PBM therapy nevertheless significantly mitigated many of these defects. Intervention with daily exposure to the PBM totally inhibited the increase in retinal superoxide, and significantly inhibited diabetes-induced abnormalities in leukostasis, retinal ICAM-1 expression, and spatial frequency threshold (p<0.0001). The light therapy had no significant effect on the diabetes-induced changes in retinal iNOS or HO-1 or defect in contrast sensitivity measured in these samples.

Fig 1

Intervention with PBM mitigated diabetes-induced (a) generation of superoxide by retina and (b) leukostasis in the retinal vasculature.
Fig 2

Effect of PBM intervention on retinal (a) ICAM-1, (b) iNOS and (c) HO-1 in diabetic mice.

HO-1

In vitro studies previously showed that incubation of retinal cells in 30 mM glucose caused a significant reduction in HO-1 expression, and that PBM significantly inhibited this reduction [18]. To determine if this effect on HO-1 contributed to the observed beneficial effects of PBM in vivo, we simultaneously administered PBM along with a pharmacologic inhibitor of HO-1 (SnPP) for the 10 weeks with the light treatment. Pharmacologic inhibition of HO-1 in vivo completely prevented the benefits of PBM in the diabetes-induced increase in retinal leukostasis (Fig 1), suggesting that the beneficial effect of PBM on leukocyte adhesion required HO-1. In contrast, inhibition of HO-1 did not counteract the effect of PBM on the diabetes-induced generation of superoxide or expression of ICAM-1, iNOS or HO-1. In fact, inhibition of HO-1 during PBM suppressed the diabetes-induced induction of iNOS expression better than that observed with PBM alone. Surprisingly, both the head shield and SNPP increased HO-1 expression. Administration of SnPP to PBM-treated diabetics had no significant effect on spatial frequency threshold or single point contrast sensitivity compared to animals treated with PBM only (not shown).

Beneficial effects of PBM are mediated in part via systemic effects

To test if some of the observed beneficial effects of PBM on retina were mediated systemically, an opaque (lead) head shield was used to prevent direct irradiation to the eye. Neither retinal superoxide production, leukostasis, iNOS (Figs ?(Figs11 and ?and2),2), nor the tests of visual function differed appreciably between the diabetics getting PBM with or without the head shield, suggesting that the beneficial effects of PBM on the retina in diabetes did not require direct illumination of the head or eyes for these parameters. As an additional test of PBM’s systemic effects, we assessed the ability of PBM to exert beneficial effects on leukocytes flowing in the systemic circulation. We previously have found that leukocytes play a major role in diabetes-induced degeneration of retinal capillaries [23,39]. Consistent with our prior reports, leukocytes from control diabetics (not treated with PBM) caused significantly more endothelial cytotoxicity in co-cultures than did leukocytes from nondiabetic animals (Fig 3). Intervention with PBM for only 4 min per day totally suppressed the diabetes-induced killing of retinal endothelial cells by leukocytes from diabetic mice.

Fig 3

Intervention with PBM mitigates leukocyte-mediated cytotoxicity of endothelial cells caused by leukocytes from diabetic animals.

Study 2

PBM mitigated diabetes-induced calcium channel dysfunction across all retinal layers

As previously reported [21,33], diabetes reduced manganese uptake across the retina in the dark compared with non-diabetic mice (Fig 4). PBM-treated diabetic mice exhibited largely normal calcium channel function in all layers of the retina, except in the presumptive rod inner segment layer.

Fig 4

Preventive PBM treatment corrects diabetes-induced calcium channel dysfunction across the retina.

Discussion

PBM is a novel therapeutic approach to mitigate the development of diabetic retinopathy, and preliminary studies in diabetic rodents and patients have shown promise [17,18]. Of note, the therapy is easy to administer, and the beneficial effects were detected at a low amount of daily radiant exposure. We demonstrate herein that the beneficial effects of PBM are mediated at least in part via indirect effects, and occur even if the initiation of therapy is delayed (post-conditioning).

We used a head shield to determine if observed beneficial effects of PBM on the retina required direct illumination of the retina. The diabetic mice that had their heads covered to prevent light from reaching the eyes for the 4 min of illumination nevertheless showed beneficial effects of the PBM treatment with respect to superoxide generation, leukostasis, expression of inflammatory proteins, and visual function. Previously, others [11,12,41] reported that beneficial effects of PBM in a model of Parkinson’s disease were not inhibited by a head shield, likewise suggesting that the neuroprotective effects of PBM in that model were mediated at least partly by indirect (systemic) means. Indirect effects of PBM detected also in other studies include where treatment of one side of the body had beneficial effects also on the untreated side with regard to skin blood flow, temperature in the feet of diabetic patients [42], or healing of skin wounds and burns [43,44]. Previously reported beneficial effects of PBM in kidney and heart [7,8] of diabetic animals are consistent with PBM exerting at least some effects via systemic mediators, but far-red light is known to penetrate deeply into tissues, and thus might directly irradiate those tissues.

The molecular and cellular changes initiated by PBM which mediate observed beneficial effects are under investigation. Mitochondrial cytochrome c oxidase is often considered to be a target of PBM therapy [15,4547], although a recent study from our group did not find evidence to support this in the retina of diabetic rats [18]. Intriguingly, MEMRI studies showed that PBM did not inhibit diabetes-induced defects in ion movement in the inner segment layer of retinal photoreceptors, the region that contains the majority of retinal mitochondria. Thus our findings provide suggest that PBM exerts beneficial effects that are independent of photoreceptor mitochondria.

In addition, several studies have demonstrated that PBM can activate stem cells to proliferate [4850]. This might be relevant, since release of stem cells and progenitor cells from bone marrow is impaired in diabetes [5154], and administration of stem cells to diabetic animals inhibits lesions of the retinopathy [55].

PBM imparts significant anti-oxidant benefits in retinas of diabetic albino rats and in pigmented mice. Whether this is mediated via direct or indirect mechanisms is not known, but it is clear that increased retinal oxidative stress is a major contributor to the pathogenesis of diabetic retinopathy. Recent studies suggest that this oxidative stress is generated in large part by rod cells [24]. Oxidative stress is a known modulator of calcium channel function [56], so we assessed effects of PBM on diabetes-induced alterations in retinal ion movement using MEMRI. Previous studies have shown that the diabetes-induced impairment in intraretinal manganese uptake are corrected by antioxidants (including lipoic acid and targeted overexpression of peroxisomal catalase) or overexpression of the antioxidant enzyme, superoxide dismutase [33,57]. We now show that PBM improves both inner and outer retinal uptake of manganese, consistent with the hypothesis that retinal oxidative stress in diabetes impairs ion channel function, and that mitigation of the ion channel defects by PBM occurs secondary to inhibition of the oxidative stress. How PBM affects calcium channel function is unclear at present.

HO-1 is a highly inducible enzyme that mediates cytoprotective responses to toxic insults, including inflammation and oxidative stress. The enzyme or its products have important cardiovascular protective effects, such as vasodilator, anti-inflammatory, antihypertensive, antioxidant and anti-apoptotic actions [5861]. To determine if HO-1 played a role in the observed beneficial effects of PBM in retinas of diabetic animals, some diabetics treated with PBM were also given the HO-1 inhibitor, SnPP, with the expectation that parameters made worse by the inclusion of the HO-1 inhibitor would indicate possible roles of HO-1 in beneficial effects of PBM. Compared to diabetics with PBM alone, leukostasis was greatly exacerbated in retinal vessels of diabetics treated with PBM and SNPP, suggesting that HO-1 acts to mitigate the leukostasis in diabetes. Since (i) leukostasis involves binding of an activated leukocyte to an adhesion molecule (like ICAM-1), and (ii) SnPP did not alter the beneficial effect of PBM on ICAM-1 expression, these results imply that the effects of SnPP on leukostasis are mediated within the leukocytes. Likewise, SnPP inhibited the diabetes-induced reduction in HO-1 expression, apparently acting on a feed-back mechanism. Inhibition of HO-1 during PBM suppressed the diabetes-induced induction of iNOS expression better than that observed with PBM alone, suggesting that HO-1 activity somehow is involved in regulation of iNOS expression in diabetes.

Beneficial effects of PBM occur despite pigmentation, and are apparent when administered from either the onset of diabetes (prevention) [18] or after a period of untreated diabetes (intervention; present study and [17]). These findings provide evidence of beneficial actions of far-red light on important early changes of diabetic retinopathy, and show that PBM can inhibit development of diabetes-induced molecular abnormalities in the retina, as well as mitigating existing abnormalities. PBM is non-invasive, inexpensive, and easily to administer, and offers a non-pharmacologic approach to help inhibit lesions of diabetic retinopathy. Importantly, the beneficial effects are apparent with exposure to the light for only a few minutes per day.

Funding Statement

This work was supported by grants from the National Eye Institute (EY00300, EY022938, R24EY024864 to TSK and EY021619 to BAB), the Medical Research Service of the Department of Veteran Affairs (TSK), and the Brazilian Institution CAPES (AS). Support was also provided by the CWRU Visual Science Research Center core facility (P30EY11373), and Research to Prevent Blindness to Kresge Eye Institute (BAB).

Data Availability

All relevant data are within the paper.

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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
PLoS One. 2014 Jun 23;9(6):e100389. doi: 10.1371/journal.pone.0100389. eCollection 2014.
Combining Neuroprotectants in a Model of Retinal Degeneration: No Additive Benefit.

Di Marco F1, Di Paolo M1, Romeo S1, Colecchi L1, Fiorani L1, Spana S2, Stone J3, Bisti S4.

Author information

  • 1Department of Biotechnology and Applied Clinical Science, University of L’Aquila, L’Aquila, Italy.
  • 2Discipline of Physiology and Bosch Institute, University of Sydney, Sydney, New South Wales, Australia.
  • 3Discipline of Physiology and Bosch Institute, University of Sydney, Sydney, New South Wales, Australia; ARC Centre of Excellence in Vision Science, The Australian National University, Canberra, Australia.
  • 4Department of Biotechnology and Applied Clinical Science, University of L’Aquila, L’Aquila, Italy; ARC Centre of Excellence in Vision Science, The Australian National University, Canberra, Australia.

Abstract

The central nervous system undergoing degeneration can be stabilized, and in some models can be restored to function, by neuroprotective treatments. Photobiomodulation (PBM) and dietary saffron are distinctive as neuroprotectants in that they upregulate protective mechanisms, without causing measurable tissue damage. This study reports a first attempt to combine the actions of PBM and saffron. Our working hypothesis was that the actions of PBM and saffron in protecting retinal photoreceptors, in a rat light damage model, would be additive. Results confirmed the neuroprotective potential of each used separately, but gave no evidence that their effects are additive. Detailed analysis suggests that there is actually a negative interaction between PBM and saffron when given simultaneously, with a consequent reduction of the neuroprotection. Specific testing will be required to understand the mechanisms involved and to establish whether there is clinical potential in combining neuroprotectants, to improve the quality of life of people affected by retinal pathology, such as age-related macular degeneration, the major cause of blindness and visual impairment in older adults.

Invest Ophthalmol Vis Sci.  2013 May 1;54(5):3681-90. doi: 10.1167/iovs.12-11018.

Low-intensity far-red light inhibits early lesions that contribute to diabetic retinopathy: in vivo and in vitro.

Tang J, Du Y, Lee CA, Talahalli R, Eells JT, Kern TS.

Source

Case Western Reserve University, Cleveland, OH 44106, USA.

Abstract

PURPOSE:

Treatment with light in the far-red to near-infrared region of the spectrum (photobiomodulation [PBM]) has beneficial effects in tissue injury. We investigated the therapeutic efficacy of 670-nm PBM in rodent and cultured cell models of diabetic retinopathy.

METHODS:

Studies were conducted in streptozotocin-induced diabetic rats and in cultured retinal cells. Diabetes-induced retinal abnormalities were assessed functionally, biochemically, and histologically in vivo and in vitro.

RESULTS:

We observed beneficial effects of PBM on the neural and vascular elements of retina. Daily 670-nm PBM treatment (6 J/cm(2)) resulted in significant inhibition in the diabetes-induced death of retinal ganglion cells, as well as a 50% improvement of the ERG amplitude (photopic b wave responses) (both P < 0.01). To explore the mechanism for these beneficial effects, we examined physiologic and molecular changes related to cell survival, oxidative stress, and inflammation. PBM did not alter cytochrome oxidase activity in the retina or in cultured retinal cells. PBM inhibited diabetes-induced superoxide production and preserved MnSOD expression in vivo. Diabetes significantly increased both leukostasis and expression of ICAM-1, and PBM essentially prevented both of these abnormalities. In cultured retinal cells, 30-mM glucose exposure increased superoxide production, inflammatory biomarker expression, and cell death. PBM inhibited all of these abnormalities.

CONCLUSIONS:

PBM ameliorated lesions of diabetic retinopathy in vivo and reduced oxidative stress and cell death in vitro. PBM has been documented to have minimal risk. PBM is noninvasive, inexpensive, and easy to administer. We conclude that PBM is a simple adjunct therapy to attenuate the development of diabetic retinopathy.

Laser Med Sci.  2012 May;27(3):585-91. doi: 10.1007/s10103-011-0991-1. Epub 2011 Sep 27.

Antiinflammatory effect of low-level laser therapy on Staphylococcus epidermidis endophthalmitis in rabbits.

Ma WJ, Li XR, Li YX, Xue ZX, Yin HJ, Ma H.

Source

Tianjin Medical University Eye Centre, Tianjin, 300070, China.

Abstract

A rabbit model of endophthalmitis was established to evaluate the antiinflammatory effect of low-level laser therapy (LLLT) as an adjunct to treatment for Staphylococcus epidermidis endophthalmitis. Rabbits were randomly divided into three groups to receive intravitreal injections into their left eye: group A received 0.5 mg vancomycin (100 ul), group B received 0.5 mg vancomycin + 0.2 mg dexamethasone (100 ul), and group C received 0.5 mg vancomycin (100 ul) and continuous wave semiconductor laser irradiation (10 mW, wavelength = 632 nm) focused on the pupil. Slit lamp examination and B-mode ultrasonography were conducted to evaluate the symptoms of endophthalmitis. Polymorphonuclear cells and tumour necrosis factor alpha (TNF-a) in aqueous fluid were measured at 0 h, and 1, 2, 3, 7 and 15 days. A histology test was conducted at 15 days. B-mode ultrasonography and histology revealed that groups B and C had less inflammation than group A at 15 days. Groups B and C had fewer polymorphonuclear cells and lower levels of TNF-a in aqueous fluid than group A at 2, 3 and 7 days (P < 0.05). There was no significant difference between groups B and C (P > 0.05). There was no significant difference between groups A, B and C at 15 days (P > 0.05). As an adjunct to vancomycin therapy to treat S. epidermidis endophthalmitis, LLLT has an antiinflammatory effect similar to that of dexamethasone.

Invest Ophthalmol Vis Sci.  2011 Jun 1;52(6):3582-92. Print 2011 May.

Photobiomodulation protects the retina from light-induced photoreceptor degeneration.

Albarracin R, Eells J, Valter K.

Source

Research School of Biology, National University, Canberra, Australia.

Abstract

PURPOSE:

In this study, the hypothesis that near-infrared (NIR) light treatment (photobiomodulation) attenuates bright-light damage in the albino rat retina was tested.

METHODS:

Young adult Sprague-Dawley (SD) albino rats were raised in dim (5 lux), cyclic light and then exposed to bright (1000 lux), continuous light for 24 hours. The animals were treated with 670-nm light (9 J/cm(2)) in an LED array before, during, or after exposure to light. The retinas were examined for function, structural changes, cell loss, and markers of stress and inflammation at 1 week and 1 month after exposure to damaging white light.

RESULTS:

Bright light caused photoreceptor-specific cell death in control retinas. Significant upregulation of stress and neuroprotective factors and the presence of activated microglia were also noted after light-induced damage. Photobiomodulation profoundly attenuated histopathologic alterations in all three treatment groups. NIR treatment also abolished microglial invasion of the retina and significantly reduced the presence of stress and neuroprotectant molecules. Bright-light-induced reductions in photoreceptor function were significantly ameliorated by photobiomodulation in animals treated before and during exposure to damaging light. Photoreceptor function was initially reduced in animals treated after bright-light-induced damage, but recovered by 1 month after exposure.

CONCLUSIONS:

NIR photobiomodulation is protective against bright-light-induced retinal degeneration, even when NIR treatment is applied after exposure to light. This protective effect appears to involve a reduction of cell death and inflammation. Photobiomodulation has the potential to become an important treatment modality for the prevention or treatment of light-induced stress in the retina. More generally, it could be beneficial in the prevention and treatment of retinal conditions involving inflammatory mechanisms.

Eur J Ophthalmol 2011; 00(00)
Article Type:
DOI:10.5301/EJO.2011.8175

T. Ivandic

Abstract

Purpose: Retinitis pigmentosa (RP) is an inherited disorder of the retina, which ultimately leads to blindness. No therapy is available. This case report examined whether low-level laser therapy (LLLT) can help treat RP.

.Methods: A 55-year-old patient presented at the ambulatory with difficulties in reading. Diagnosis revealed RP with typical symptomatology including hemeralopy and tritan-dyschromatopsy. Best visual acuity was 20/50 in both eyes. Visual fields were concentric and reduced to 5°. Electroretinography: b-wave abolished. Biomicroscopic examination showed an optic atrophy, attenuated retinal vessels with typical retinal pigmentation. After the patient had given his informed consent, LLLT was applied in compliance with the Declaration of Helsinki using a continuous-wave laser diode (780 nm, 10 mW, 292 Hz modulation, 3 mm2 spot size). The retina was irradiated from the exterior through the conjunctiva-sclera by guiding the beam exit at 1 cm distance above the eye ball with perpendicular anterior-posterior movements. The treatment lasted 40 sec (0.4 W/cm2) and was repeated 2 times per week for a total of 4 treatments (1.6 W/cm2). After 5 years without any therapy, the same treatment was repeated. Thereafter, during the subsequent 2 years, 17 additional treatments were performed at irregular time points.
.
Results: Visual acuity in both eyes increased from 20/50 at baseline to 20/20 after LLLT (4 initial treatments). Visual fields showed normalized outer limits with a mid-peripheral circular scotoma. After 5 years without any therapy, a relapse to the original condition was observed. After repeating LLLT (4 additional treatments), the condition improved again. By further sporadic individual treatments, the improved condition could be maintained for 2 years.
.
Conclusions: LLLT was shown to improve and maintain vision in a case with RP, and may thereby contribute to slowing down blindness.

Ophthalmic Surg Lasers Imaging.  2011 Jan 1;42(1):59-63. doi: 10.3928/15428877-20101124-07. Epub 2010 Dec 1.

The efficacy of low-energy selective laser trabeculoplasty.

Tang M, Fu Y, Fu MS, Fan Y, Zou HD, Sun XD, Xu X.
Source

Shanghai Jiaotong University Affliated First People’s Hospital, Ophthalmology, Shanghai, China.

Abstract

BACKGROUND AND OBJECTIVE:

to analyze the efficacy of low-energy selective laser trabeculoplasty (SLT) in patients.

PATIENTS AND METHODS:

in 74 patients (74 eyes) with ocular hypertension, suspected glaucoma, or primary open-angle glaucoma, SLT was the first-choice treatment. Thirty-nine patients in the low-energy group received treatment using half of conventional laser energy over 360° of the trabecular meshwork (at 100 points). Thirty-five patients in the control group received conventional laser energy. Patients were observed for 1 year. Complications and intraocular pressure (IOP) were observed.

RESULTS:

postoperative transient IOP spike (? 3 mm Hg) occurred in three eyes on the day of treatment and partial peripheral anterior synechiae occurred in one eye 1 month after treatment only in the control group. Effective rates of treatment (? 20% IOP reduction) at week 2 and month 1, 3, 6, and 12 after treatment were 69.23%, 64.10%, 61.54%, 53.85%, and 48.72% in the low-energy group and 71.43%, 71.43%, 60%, 51.43%, and 48.57% in the control group, respectively. There was no statistically significant difference between the two groups at various time points (P = .836, .501, .892, .835, .990).

CONCLUSION:

compared with SLT using conventional laser energy, low-energy SLT lowers IOP with fewer complications, making it a safe and effective option.

Vopr Kurortol Fizioter Lech Fiz Kult. 2009 Sep-Oct;(5):37-9.

Effect of infrared low-intensity laser therapy on orbital blood circulation in children with progressive short sightedness

 [Article in Russian]

Shurygina IP, Khadzhieva MR.

This work was designed to study effect of low-intensity infrared laser radiation (LIIRR) on orbital circulation in children with progressive progressive short-handedness. Parameters of the blood flow in the orbital artery, central retinal artery, and posterior ciliary artery were evaluated in a total of 88 patients at the age from 6 to 14 years. The study group (group 1) comprised 66 children with moderately severe progressive myopia (112 eyes), the control one (group 2) included 22 patients without visual disturbances (44 eyes). Patients of group 1 received physiotherapy for the treatment of significantly deteriorated hemodynamics in the orbital region. Indirect action of LIIRR on the orbital region promoted stabilization of short-handedness in 78.8% of the treated patients. It is concluded that the method described in this paper is clinically efficient and may be recommended for the prevention of progressive myopia in children.

Vopr Kurortol Fizioter Lech Fiz Kult. 2009 Nov-Dec;(6):27-9.

[Comparative hemodynamic characteristics of patients with the organ of vision subjected to low-intensity laser radiation of the green and red wavelengths]

[Article in Russian]

Kamenskikh TG, Ra?gorodski? IuM, Kolbenev OI, Galanzha A, Orlova AS, Kamenskikh ID.

This comparative study was designed to evaluate effect of low-intensity speckle laser radiation in the green and red wavelengths on intraocular hemodynamics in 202 patients (202 eyes) with retinal vascular problems and without them. Irradiation was performed using “Izumrud” and “Rubin” adapters for an AMO-ATOS unit. Peak systolic velocity and peripheral resistance index in posterior short ciliary arteries and central retinal artery were measured. It was shown for the first time that green speckle laser radiation (wavelength 535 nm) has more pronounced (compared with the red light) effect on intraocular hemodynamics; thereby, it makes a greater contribution to the improvement of the visual function (e.g. in the case of glaucoma) and to the widening of the fields of vision.

Vestn Oftalmol. 2009 Sep-Oct;125(5):57-60.

[Use of plasmapheresis-based extracorporeal hemocorrection in the treatment of endogenic uveitis and autoimmune eye diseases]

[Article in Russian]

Frolov AB, Grechany? MP, Chentsova OB.

The paper gives the methods of plasmapheresis-based extracorporeal exposure to blood and its components to correct hemostatic disorders. Afferent methods, as well as quantum hemotherapy methods are outlined. The immunomodulating mechanism of their action, which favors a prompter elimination of inflammation, increases ocular functions, and reduces recurrences, is revealed. The mechanisms of action of plasmapheresis as a technique used alone and in combination with laser and ultraviolet blood irradiation and indications for and contraindications to the use of various hemocorrection methods are described.

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.

Aviakosm Ekolog Med. 2009 Jul-Aug;43(4):51-5.

Influence of infrared cold laser radiation emission on free radical processes in various tissues of rats with circulatory cerebral hypoxia

 [Article in Russian]

Shurygina IP, Miliutina NP, Pokudina IO, Prokof’ev VN, Kussmaul’ AR, Shkurat TP.

Influence of infrared cold laser emission (IRCL) on the dynamic equilibrium between lipid peroxidation and tension of the antioxidant defense system in rat’s tissues (blood, brain, retina, cornea) was evaluated in animals with circulatory cerebral hypoxia induced by occlusion of the left carotid artery. Tissues of white rats were examined for IRCL effects on hemiluminescence, malonic dialdehyde, SOD and catalase activities on the background of circulatory cerebral hypoxia. Data of the experiment evidenced an antioxidant effect of posthypoxic IRCL therapy as it reduces intensity of the free radical processes in plasma, cerebral tissues and retina. The experiment demonstrated the IRCL ability to modulate LPO, to stiffen the antioxidant defense system in the event of eye diseases originated from circulatory hypoxia of the ocular analyzer.

Photomed Laser Surg. 2009 Jun 10. [Epub ahead of print]

Early Diagnosis of Ocular Hypertension Using a Low-Intensity Laser Irradiation Test.

Ivandic BT, Hoque NN, Ivandic T.

1 University of Heidelberg, Otto-Meyerhof Centre , Heidelberg, Germany .

Abstract Objective: We investigated the potential use of low-intensity laser irradiation (LILI) as a diagnostic tool for identifying hypertensive eyes at risk of glaucoma.

Background data: The diagnosis of early-stage ocular hypertension is particularly difficult to establish. Methods: This study of a case series included 123 healthy subjects with normal vision. The intraocular pressure (IOP) was determined before (baseline) and 30 min after a 30-sec irradiation of the limbus area with laser light (780 nm; 7.5 mW; 292 Hz modulation).

Results: Baseline IOP was >21 mm Hg in 44 of 211 eyes (20.9%), consistent with ocular hypertension. LILI decreased the mean IOP by 6.2 mm Hg (-25.7%; p < 0.001; paired t test) in these eyes. The remaining 167 eyes (79.1%) exhibited a normotensive IOP </=21 mm Hg. LILI reduced the mean IOP by 2.9 mm Hg (-17.1%; p < 0.001) in these eyes, but there were different response patterns: 1) the IOP did not change (27.0%); 2) the IOP was reduced by the same extent in both eyes (32.3%); 3) initial IOP differences between left and right eyes became level and the absolute IOP was reduced to a lower level that was identical in both eyes (18.0%); and 4) the initial difference in IOP between the left and right eye persisted despite LILI (22.7%).

Conclusion: LILI lowers IOP, even in normotensive eyes. This effect may be useful to determine the individual physiological IOP and to diagnose latent ocular hypertension in eyes with presumably normotensive IOP.

Aviakosm Ekolog Med. 2009 May-Jun;43(3):60-4.

Infrared cold laser radiation as an antimutagen

[Article in Russian]

Shurygina IP, Belichenko NI, Mashkina EV, Pavlov NB, Bogacheva MA, Shkurat TP.

Abstract

Effects of infrared cold laser radiation (IRCLR) on mutagenesis and proliferation of the corneal epithelium were studied with laboratory white mice subjected to instigated circulatory hypoxia of the brain. The experiment was to reveal whether IRCLR influences the frequency of chromosomal rearrangements and to allow calculation of the corneal cells mitotic index for circulatory brain hypoxia. Laser radiation was shown to reconstitute the normal frequency of chromosomal aberrations as well as the mitotic cycle in epithelial cells of the mice cornea. Data of the experiment are promising from the standpoint of antihypoxic use of IRCLR in ophthalmology.

Vestn Oftalmol. 2009 Mar-Apr;125(2):24-6.

Effect of infrared low-intensity laser radiation on a mutation process and proliferative corneal activity in experimental cerebral hypoxia

 [Article in Russian]

Shurygina IP, Galenkina NM, Shkurat TP.

The paper deals with the impact of infrared low-intensity laser radiation (IRLILR) on a mutation process and the proliferative activity of the animal cornea during stimulation of circulatory brain hypoxia. During an experiment on laboratory albino rats, IRLILR was studied for its impact on the level of chromosomal rearrangements and the mitotic index in the corneal cells was calculated in circulatory brain hypoxia. Laser exposure during stimulation of circulatory brain hypoxia favors normalization of the level of chromosomal aberrations and a mitotic cycle in the rat corneal epithelial cells. The experimental findings suggest that IRLILR may be used in ophthalmological care for antihypoxic purposes.

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.

Neuroscience. 2006 May 12;139(2):639-49. Epub 2006 Feb 7.

Photobiomodulation partially rescues visual cortical neurons from cyanide-induced apoptosis.

Liang HL, Whelan HT, Eells JT, Meng H, Buchmann E, Lerch-Gaggl A, Wong-Riley M.

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

Near-infrared light via light-emitting diode treatment has documented therapeutic effects on neurons functionally inactivated by tetrodotoxin or methanol intoxication. Light-emitting diode pretreatment also reduced potassium cyanide-induced cell death, but the mode of death via the apoptotic or necrotic pathway was unclear. The current study tested our hypothesis that light-emitting diode rescues neurons from apoptotic cell death. Primary neuronal cultures from postnatal rat visual cortex were pretreated with light-emitting diode for 10 min at a total energy density of 30 J/cm2 before exposing to potassium cyanide for 28 h. With 100 or 300 microM potassium cyanide, neurons died mainly via the apoptotic pathway, as confirmed by electron microscopy, Hoechst 33258, single-stranded DNA, Bax, and active caspase-3. In the presence of caspase inhibitor I, the percentage of apoptotic cells in 300microM potassium cyanide was significantly decreased. Light-emitting diode pretreatment reduced apoptosis from 36% to 17.9% (100 microM potassium cyanide) and from 58.9% to 39.6% (300 microM potassium cyanide), representing a 50.3% and 32.8% reduction, respectively. Light-emitting diode pretreatment significantly decreased the expression of caspase-3 elicited by potassium cyanide. It also reversed the potassium cyanide-induced increased expression of Bax and decreased expression of Bcl-2 to control levels. Moreover, light-emitting diode decreased the intensity of 5-(and -6) chloromethy-2′, 7-dichlorodihydrofluorescein diacetate acetyl ester, a marker of reactive oxygen species, in neurons exposed to 300 microM potassium cyanide. These results indicate that light-emitting diode pretreatment partially protects neurons against cyanide-induced caspase-mediated apoptosis, most likely by decreasing reactive oxygen species production, down-regulating pro-apoptotic proteins and activating anti-apoptotic proteins, as well as increasing energy metabolism in neurons as reported previously.

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. 2005 Mar-Apr;121(2):5-8.

Progressing myopia in children: does it need treatment or not?

 [Article in Russian]
 Tarutta EP, Iomdina EN, Akhmedzhanova EV.

The purpose of the case study was to evaluate the remote consequences of a complex of laser and repeated surgical sclerorestorative procedures made in progressing myopia and its complications. Three hundred and forty-six children, aged 8-10, with rapidly progressing uncomplicated myopia of 4.25 to 9.5 D were shared between 2 groups. Two hundred and forty patients of the experimental group were made sclerorestorative procedures and transscleral low-energy laser stimulation of the ciliary muscle by means of infrared laser MACDEL-09. No such treatment was applied to patients of the control group. When indicated, preventive laser coagulation of the retina was made in both groups. The dynamic 10-year follow-up over the status of refraction and eye bottom showed that the complex scheme of repeated sclerorestorative procedures and low-energy laser treatment combined with preventive peripheral laser coagulation of the retina cut the rate of progressing myopia and prevented peripheral vitreoretinal dystrophy and retinal detachment in children and teenagers with progressing myopia.

J. Biol. Chem., Vol. 280, 4761-4771, February 11, 2005

Photobiomodulation Directly Benefits Primary Neurons Functionally Inactivated by Toxins

ROLE OF CYTOCHROME c OXIDASE*

Margaret T. T. Wong-Riley, Huan Ling Liang, Janis T. Eells¶, Britton Chance||, Michele M. Henry**, Ellen Buchmann, Mary Kane, and Harry T. Whelan

From the Departments of Cell Biology, Neurobiology and Anatomy, Neurology, and **Ophthalmology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, Department of Health Sciences, University of Wisconsin, Milwaukee, Wisconsin 53201, and ||Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6059

Far red and near infrared (NIR) light promotes wound healing,but the mechanism is poorly understood. Our previous studiesusing 670 nm light-emitting diode (LED) arrays suggest thatcytochrome c oxidase, a photoacceptor in the NIR range, playsan important role in therapeutic photobiomodulation. If thisis true, then an irreversible inhibitor of cytochrome c oxidase,potassium cyanide (KCN), should compete with LED and reduceits beneficial effects. This hypothesis was tested on primarycultured neurons. LED treatment partially restored enzyme activityblocked by 10–100 µM KCN. It significantly reducedneuronal cell death induced by 300 µM KCN from 83.6 to43.5%. However, at 1–100 mM KCN, the protective effectsof LED decreased, and neuronal deaths increased. LED significantlyrestored neuronal ATP content only at 10 µM KCN but notat higher concentrations of KCN tested. Pretreatment with LEDenhanced efficacy of LED during exposure to 10 or 100 µMKCN but did not restore enzyme activity to control levels. Incontrast, LED was able to completely reverse the detrimentaleffect of tetrodotoxin, which only indirectly down-regulatedenzyme levels. Among the wavelengths tested (670, 728, 770,830, and 880 nm), the most effective ones (830 nm, 670 nm) paralleledthe NIR absorption spectrum of oxidized cytochrome c oxidase,whereas the least effective wavelength, 728 nm, did not. Theresults are consistent with our hypothesis that the mechanismof photobiomodulation involves the up-regulation of cytochromec oxidase, leading to increased energy metabolism in neuronsfunctionally inactivated by toxins.


Received for publication, August 23, 2004 , and in revised form, November 10, 2004.

* This work was supported by Defense Advanced Research ProjectsAgency Grants DARPA N66001-01-1-8969 and DARPA N66001-03-1-8906(to H. T. W., J. T. E., and M. T. T. W.-R.), National Eye InstituteGrants EY05439 (to M. T. T. W.-R.) and EY11396 (to J. T. E.),National Institute of Environmental Health Grant ES06648 (toJ. T. E.), National Eye Institute Core Grant P30-EY01931 (toJ. T. E. and M. T. T. W.-R., Core Investigators), and the BleserFoundation Endowed Professorship (to H. T. W.). The costs ofpublication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked”advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact.

To whom correspondence should be addressed: Dept. of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-8467; Fax: 414-456-6517; E-mail: mwr@mcw.edu

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.

Proceedings of the National Academy of Science

PNAS | March 18, 2003 | vol. 100 | no. 6 | 3439-3444

Therapeutic photobiomodulation for methanol-induced retinal toxicity

J. T. Eells * , M. M. Henry *, P. Summerfelt, M. T. T. Wong-Riley, E. V. Buchmann, M. Kane, N. T. Whelan, and H. T. Whelan

Departments of *Pharmacology and Toxicology, Cell Biology, Neurobiology, and Anatomy, and Neurology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226

Edited by Britton Chance, University of Pennsylvania School of Medicine, Philadelphia, PA, and approved January 10, 2003 (received for review August 5, 2002)

Methanol intoxication produces toxic injury to the retina andoptic nerve, resulting in blindness. The toxic metabolite inmethanol intoxication is formic acid, a mitochondrial toxinknown to inhibit the essential mitochondrial enzyme, cytochromeoxidase. Photobiomodulation by red to near-IR radiation hasbeen demonstrated to enhance mitochondrial activity and promotecell survival in vitro by stimulation of cytochrome oxidaseactivity. The present studies were undertaken to test the hypothesisthat exposure to monochromatic red radiation from light-emittingdiode (LED) arrays would protect the retina against the toxicactions of methanol-derived formic acid in a rodent model ofmethanol toxicity. Using the electroretinogram as a sensitiveindicator of retinal function, we demonstrated that three brief(2 min, 24 s) 670-nm LED treatments (4 J/cm2), delivered at5, 25, and 50 h of methanol intoxication, attenuated the retinotoxiceffects of methanol-derived formate. Our studies document asignificant recovery of rod- and cone-mediated function in LED-treated,methanol-intoxicated rats. We further show that LED treatmentprotected the retina from the histopathologic changes induced bymethanol-derived formate. These findings provide a link betweenthe actions of monochromatic red to near-IR light on mitochondrial oxidativemetabolism in vitro and retinoprotection in vivo. They alsosuggest that photobiomodulation may enhance recovery from retinalinjury and other ocular diseases in which mitochondrial dysfunctionis postulated to play a role.

Vestn Oftalmol. 2002 Mar-Apr;118(2):15-7.

Treatment of lacrimal duct diseases using low energy helium-neon laser

Dushin NV, Azibekian AB, Ali Akhsan M.

Treatment of diseases of the lacrimal duct remains a pressing problem in ophthalmology. These diseases are responsible for 71-85.4% diseases in capable people. Sixty patients with chronic dacryocystitis with partially retained patency of the lacrimal duct were treated by He-Ne low-frequency laser ULF-01 (output power 6.10 mcWt, laser beam wave length 0.632 nm).

The patients received 3-5 min sessions twice a week, 5-8 sessions per course. Positive effect was attained in 56 patients: complete cessation of excessive lacrimal discharge in 38 patients and subjective improvement in 18. He-Ne laser exposure brings about a good antiinflammatory effect; in combination with antibiotic therapy it promotes rapid sanitization of the lacrimal duct, removes edema, and rapidly normalizes lacrimal discharge.

Vestn Oftalmol. 2005 Jan-Feb;121(1):35-7.

 Quantum therapy and the composition of the moister of the eye chamber (an experimental study).

[Article in Russian]

Pavliuk EIu, Sherkhoeva DTs, Pavliuk AIu, Khristoforov VN.

We examined 12 rabbits, 6 of whom (12 eyes) were exposed to magneto-infrared laser radiation (MILR) and another 6 (12 eyes) were controls. The parameters of pulse and continuous infrared LED radiation were as follows: wavelength–860 nm, pulse capacity–2 W, mean radiation capacity–10 mW, magnetic field strength–up to 17 mTl. A study of the moister of the anterior chamber showed a MILR-induced activated metabolism, i.e. a better acid-base balance (ABB), more intense oxygenation in the ocular tissues and decreased acidosis. Higher concentrations of buffer bases (ABEe and SBEc) cause shifts in ABB towards metabolic alkalosis. A lower concentration of glucose denotes intensified processes related with its utilization. A lack of changes in the quantity of salts in the moister of the anterior chamber rules out the possibility of that the content of glucose would go down due to its dissolution with a big volume of newly produced moister. A lack of an increase in the concentration of whole protein, as observed after MILR, can be regarded as indirect evidence to absence of any adverse effect on the vascular wall.

Vestn Oftalmol. 2002 May-Jun;118(3):15-7.

 Laser magnetotherapy after cataract extraction with implantation of intraocular lens

[Article in Russian]

Maksimov VIu, Zakharova NV, Maksimova IS, Golushkov GA, Evseev SIu.

Effects of low-intensive laser and alternating magnetic field on the course of the postoperative period were studied in patients with exudative reaction after extracapsular cataract extraction with implantation of intraocular lens (IOL). The results are analyzed for 148 eyes with early exudative reaction after IOL implantation (136 patients aged 42-75 years). The patients were observed for up to 6 months. The treatment efficiency was evaluated by the clinical picture of inflammatory reaction, visual acuity, and results of biochemical analysis of the lacrimal fluid (the ratio of lipid peroxidation products to antioxidants in cell membrane). The course of the postoperative period was more benign and recovery sooner in patients of the main group in comparison with the control.

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.

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.

Vestn Oftalmol. 1996 Apr-Jun;112(2):25-6.

 Laser puncture combined with drug therapy in the treatment of primary corneal stromal dystrophy

[Article in Russian]

Maichuk IuF, Orlovskaia LE, Mal’-Alla DR.

A protocol of multiple-modality treatment has been developed, making use of laser puncture by stimulating infrared laser and drugs-aqueous 4% taufon and polygluquine taufon and artificial tears. The treatment proved to be highly effective: vision acuity stabilized, lacrimal production increased, and erosive cornea epithelialized.

Lasers Med Sci. 2008 Jan 29 [Epub ahead of print

Experimental study on low pulse energy processing with femtosecond lasers for glaucoma treatment.

Hou DX, Butler DL, He LM, Zheng HY.

Precision Engineering and Nanotechnology Centre, School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore, 639798, Singapore, dongxia@pmail.ntu.edu.sg

The feasibility of low energy processing in ocular tissues with femtosecond laser sources was investigated in this research. One laser source was a femtosecond amplifier, and the other was a femtosecond oscillator. The amplifier used in this experiment was a CPA-2001 (Clark-MXR, Inc), with 150 fs pulse duration and 1 kHz repetition rate. The femtosecond oscillator (model 900-B Mira) produced a 200 fs pulse duration and a 76 MHz repetition rate. Both these two laser systems operated at 800 nm wavelengths. Firstly, the pulse intensity thresholds in water produced by the two laser sources were compared. The optical breakdown probability analysis shows that the pulse energy threshold achieved by the oscillator was less than 10% of that achieved by the amplifier. Then, the non-linear propagation of the femtosecond pulses in the ocular tissues was studied with the femtosecond oscillator. The results showed a potential for pulse energy processing at the nanojoule level with a femtosecond oscillator in glaucoma treatment.

The comparative analysis of using lowpower laser radiation, magnetic therapy and electrical stimulation in stabilization of visual functions in primary open-angle glaucoma

Listopadove N A et al.

127 men with a primary open-angle glaucoma(POAG) were treated with either L/LT, magnetic therapy or electrical stimulation. The examination included visus, visocontrastometry and automatic static perimetry. The field of sight at an initial stage of POAG was 56% of laser, 52 for magnetic therapy and 27 for electrical stimulation. In the advanced stage the figures were 39, 37 and 18, respectively.

Vestn Oftalmol. 1998 Nov-Dec;114(6):6-9.

Heparin inhalations and laser exposure of blood in treatment patients with open-angle glaucoma

 [Article in Russian]

Balashova LM, Listopadova NA, Zaitseva NS, Teplinaskaia LE, Efimov VS, Grishin VL, Kantarzhi EP.

Comparative assessment of methods aimed at amelioration of the immunohemostatic processes in patients with open-angle glaucoma suffering from chronic vascular diseases showed that the most remarkable improvement of the visual functions and decrease of the level of circulating immune complexes in the blood were attained in the patients treated by heparin inhalations combined with intravenous laser exposure of the blood as against patients treated by one of these methods alone or traditionally.

Treatment of myopia with helium-neon laser stimulation.

Rabadanova M G et al.

A new technique of stimulating the ciliary muscle in cases of progressing myopia is described. The positive results have been confirmed through measurement of the intraocular pressure, refraction reduction and increase of visual acuity.

Vestnik oftalmologii. 1999;115 5): 20-21.

The treatment of postraumatic uveitis with low-intensity laser Radiation.

Inkova G A, Ionin A P, Ionina G I.

Eighty-two patients with severe posttraumatic uveitis (eye inflammation) which could not be treated by traditional antiinflammatory therapy were exposed to LLLT. The patients were divided into 3 groups: – infrared laser exposure semiconductor pulsed laser, – intravenous exposure of the blood to a He-Ne laser and – both treatments. The treatment efficacy was monitored by measuring lipid  peroxides and superoxide dismutase in the lacrimal fluid. The treatment proved to be effective. The best results were attained by applying both methods of exposure, as was shown by sooner normalization of the content of lipid peroxidation products and activity of superoxide dismutase.

Vestn Oftalmol. 2001 May-Jun;117(3):29-31.
 

Ultraviolet irradiation of blood in combined treatment of traumatic endophthalmitis

[Article in Russian]

Aznabaev MT, Babushkin AE, Karabanova IV.

Thirty-five patients (35 eyes) with traumatic endophthalmitis were treated. Ultraviolet exposure of autoblood was used in 16 patients, the rest 19 were treated routinely (antibiotics, etc.). Use of ultraviolet exposure of the blood in combined therapy of traumatic endophthalmitis more rapidly (12.6 vs. 22.1 days) and effectively (93.7 vs. 68.4%) arrested intraocular infection and more often preserved the objective vision (31.3 vs. 10.5%).

Vestn-Oftalmol. 1996; 112 (1): 31-32

Effects of low-intensity infrared laser irradiation on the eye: An experimental study.

 Prokofeva G L, Kravchenko E V, Mozherenkov-V P.

Prokofeva evaluated the doses of infrared laser exposure for the structures of the eye in rabbit experiments, and the potentials of such lasers in ophthalmology were assessed. Wavelength was 890 nm and doses varied from 0.0001 to 1.0 J/cm2, corresponding to exposure duration of 0.3 to 45 min. Experiments were carried out on 20 animals. The right eyes were exposed, and the left ones were control. An increase of intraocular pressure was recorded at a dose of 0.1 J /cm2 (4.5 min) and higher. Morphological examination showed dilated, well filled and newly formed vessels in the ciliary body and iris, as well as oedema and destruction of the external layers of the retina. Exposure to a dose of 0.05 J/cm2 and lower did not lead to destruction of any ocular structures or increase of intraocular pressure. The maximal dose causing no side effects for the organ of vision was established at 0.05 J/cm2.

Vestn Oftalmol. 2001 Sep-Oct;117(5):11-4.

Comparative effectiveness of d

Article in Russian]ifferent methods of quantum hemotherapy in the treatment of juvenile diabetic retinopathy

Nedzvetskaia OV.

Effects of ultraviolet exposure of the blood (UVEB), intravenous laser exposure of the blood (IVLEB), and transcutaneous magnetic laser exposure of the blood (TMLEB) on ocular functions, microcirculation, and hemodynamics were studied in 79 patients with juvenile diabetic retinopathy. All these treatments had a nonspecific positive effect on the spatial contrast sensitivity, microcirculation, and choroid hemodynamics of the eye. Correcting mainly intravascular changes in the microcirculatory bed, quantum hemotherapy methods are pathogenetically justified in the treatment and prevention of tissue ischemia in diabetic involvement of the organ of vision. Results of noninvasive TMLEB with generalized and local effects were statistically similar to results of invasive UVEB and IVLEB.

Ofthalmol Zh.  1990;(1):39-42.

Laser puncture in the combined treatment of a weak degree of myopia in schoolchildren].

[Article in Russian]
Tsikova TD.

Abstract

To increase effectiveness of treatment, the influence of laser puncture on some functional indices of myopic eyes has been studied. Comparative analysis of results after laser puncture used as an independent method in 45 children (85 eyes)–group I, and in a complex accommodation-convergence system, of the eye in 66 children (130 eyes)–group II, has shown that laser puncture has a positive influence on visual acuity and accommodation-convergence system. As compared with group I, in group II normalization of functional indices (reserves of accommodation, convergence and divergence) begins earlier and stability of the results obtained is higher.