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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. References

Glial cells play an important role in the maintenance of normal structure and function of the neural components of the central nervous system. The Müller cells are one of the macroglial elements in the retina and their wide-ranging roles are responsible for the protection and proper functioning of the photoreceptors. In the present study, we aimed to test the effects of pretreatment with 670 nm red light on Müller cells in the light-induced model of retinal degeneration. Adult Sprague–Dawley albino rats were treated with 670 nm red light, from an LED source prior to exposure to bright (1000 lux) continuous light for 24 h. Müller cell-specific markers were used to assess structural and functional changes in this cell type 1 week after contact with damaging light. Changes in gene (Edn2, LIF, TNF-α) and protein (S100β, Vimentin, LIF, iNOS, GS, Cyclin-D1) levels and localization were evaluated using RT-qPCR, and immunohistochemistry. Our results showed that 670 nm light pretreatment ameliorates the light-induced alterations in the expression of Müller-cell specific markers for structure, stress, metabolism and inflammation. This suggests that 670 nm light preconditioning may promote neuroprotective effects in the retina from light-induced damage, possibly through pathways regulating the roles of Müller cells in maintaining retinal homeostasis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. References

Müller cells are the principal glial cells in the vertebrate retina, positioned radially, spanning the full thickness of the tissue, from the ganglion cell layer to the photoreceptors in the outer nuclear layer. Although these cells are not directly involved in the process of visual transduction, they serve a variety of roles that are crucial in the maintenance of homeostasis in the retina. Müller cells provide architectural stability to the retina, structural and metabolic support to photoreceptors. Considering their strategic location within the tissue, Müller cells not only foster an anatomical link between different retinal cell types but also contribute to neuronal signaling, by providing an optimal environment for photoreceptor function and neuroprotection during stress. Like other glial cells in the central nervous system (CNS), Müller cells are rich in ion channels and aquaporins, ligand receptors, enzymes and transporters through which they are able to regulate blood flow, actively remove toxic metabolic wastes, detect changes in cellular stress and produce neuroprotective factors (1–3). Furthermore, a recent study has suggested that Müller cells possess the ability to enhance visual acuity in humans (4).

In pathological conditions, Müller cells are known to respond by up-regulating cytokines, which in turn can initiate inflammatory processes and/or the production of neuroprotective factors to limit potentially devastating effects on the retina. In conditions where photoreceptors are stressed, the injury or the damage to the cells induces the activation of signal transduction pathways leading to the up-regulation of neuroprotective factors, such as CNTF and FGF2 in glial cells (5–10). The initiation of these neuroprotective processes relies on the active communication between Müller glia and photoreceptors (7,11–14).

Furthermore, Müller cells display stem cell properties and regenerative capacity (15,16). During retinal degeneration, activated Müller cells proliferate, form glial scars, that consequently lead to the remodeling of the retina at the later stages of degeneration (17–21). However, the formation of a subretinal glial scar is detrimental to the surviving photoreceptors, by forming a barrier between the blood supply from the choroid and the outer retina.

The light-induced damage in rodent retina became an established model of retinal degeneration, owing to its capacity to induce photoreceptor-specific cell death, breakdown of the blood–retina barrier, oxidative stress, inflammation and consequently, loss of functional vision (22–24), some of the hallmarks of human forms of retinal degenerations, such as age-related macular degeneration and retinitis pigmentosa (20,25–27). In these animal models, initially, the damage is focal, most prominent in the superior retina. However, it becomes progressive in the later stages, extending into the inferior region involving a large area of the retina (28,29), and undergoes remodeling and reprogramming of its neural circuitry (17,19,20). Following the onset of injury to the photoreceptors, immediate activation of proteins associated with immune response, retinal stress and intermediate filament proteins occur in Müller cells (18,30). Subsequently, Müller cells undergo hypertrophy, proliferation of cell processes and migration to fill in the spaces of the lost photoreceptors. These Müller cell-mediated processes contribute to the formation of subretinal glial scars and rewiring the neuronal circuits that lead to functional loss and can hinder any restorative therapeutic approaches.

A large body of evidence is accumulating that supports the beneficial effects of tissue exposure to light wavelengths from the far red (FR) to near-infrared (NIR) spectrum from 600 to 1000 nm. This process, which is also known as photobiomodulation or NIR phototherapy, has been used in treating various disease conditions in humans and in animal models. NIR phototherapy has been shown to promote wound healing (31), improve recovery rates of soft tissue injuries and myocardial infarction (32,33). Recent microarray studies revealed significant changes in the expression of genes associated with stress response, oxidative stress, cell death, inflammation, neuroprotection and mitochondrial respiration (34,35). The underlying mechanism of its therapeutic effect is thought to be mediated by the action of photoacceptors found in tissues, which upon absorption of specific light wavelength, stimulate biological responses. Several studies have proposed that cytochrome oxidase (COX), the rate-limiting enzyme in terminal phosphorylation in the mitochondrial respiratory chain, and a photoacceptor molecule of light waves in the range of 600–1000 nm, as the primary mediator of photobiomodulation (36–39). Mitochondrial COX activity has been shown to be activated upon the absorption of the 670 nm light wavelength, and consequently, a peak in ATP production has been observed (40).

We have previously demonstrated that 670 nm light treatment protected the rodent retina from damaging effects of bright white light exposure (41). The commonly observed therapeutic effects of 670 nm light treatment include its capacity to prevent cell death, reduce oxidative stress, mitigate inflammatory response and restore cellular metabolic function. Considering that the Müller cells are also known to mediate the aforementioned pathological features in retina compromised by bright light, we hypothesized that pretreatment with 670 nm light is likely to reduce the ensuing damaging effects. In our current study, we assess the effects of pretreatment with 670 nm light on the retinal Müller glia, using the bright white light-induced retinal damage model.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. References

Animals.  All procedures were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and with the requirements of The Australian National University Animal Experimentation Ethics Committee. Albino Sprague–Dawley (SD) rats were reared in low (5 lx) light levels with a 12 h light, 12 h dark cycle. Food and water were available ad libitum. Once animals reached P100–P120, they were divided into four groups. Animals in the first group were not exposed to either 670 nm red light or damaging white light (CONTROL group, n = 8). In the second group, animals were exposed to damaging white light only (LD group, n = 8) and in the third group animals were exposed to 670 nm red light only (NIR CONTROL group, n = 8). Animals in the fourth group were treated with 670 nm red light prior to exposure to bright continuous white light (PRECON group, n = 12). Some animals in the LD group were “sham-treated” and had undergone the same procedures as the animals in the PRECON group, but the LED array was not switched on. Based on our previous study, there was no significant difference between the sham-treated LD retinas and the control LD group (41). In the current study, we pooled the analysis of the sham-treated and nonhandled animals in the LD group.

Light damage.  Animals were transferred to individual transparent cages with food placed on the cage floor and water was provided in transparent bottles to avoid shading of the light entering the cage. Fluorescent light tubes (18 W; Cool White) were placed 200 mm above the bottom of the cage, so that the light intensity reached 1000 lx at the cage floor. Prior to light exposure, animals were dark adapted overnight. Light exposure started at 9:00 A.M. in all experimental paradigms. Animals were exposed to bright light for 24 h and then returned to low light level environment (5 lux) to recover for 1 week, except for some animals from the Precon group that were sacrificed straight after bright continuous light (BL) exposure for TdT-mediated dUTP nick end labeling (TUNEL) assay.

670 nm pretreatment paradigms.  Animals were restrained in a cloth to aid manual handling, and placed under the 670 nm LED array (Quantum Devices, Barneveld, WI). Animals were positioned so that their eye level was approximately 2.5 cm away from the light source and were exposed to the light for 3 min at 60 mW cm−2. This treatment protocol produced an energy fluence of 9 J cm−2 at eye level. Animals were treated with NIR one time daily on five consecutive days prior to BL (PRECON group, n = 8). In the NIR Control group, animals were treated with 670 nm red light once daily for 5 days but were not exposed to BL (NIR Control group, n = 8).

Tissue collection and preparation.  Tissue was collected 1 week following BL exposure. Eyes were enucleated and processed for immunohistochemical analysis and retinas were isolated for RT-qPCR. Animals were euthanized with an overdose of sodium pentobarbital (>60 mg kg−1, intraperitoneal). Retinas from the right eye of each animal were collected and stored in RNAlater® (Ambion, Applied Biosystems, Foster City, CA) overnight at 4°C. Left eyes were marked at the superior aspect of the limbus for orientation, enucleated and immersion-fixed in 4% paraformaldehyde in 0.1 m phosphate-buffered saline (PBS) at pH 7.4 for 2 h. Tissue was rinsed thrice in 0.1 m PBS and left in a 15% sucrose solution overnight for cryoprotection. The next day, eyes were embedded in Tissue-Tek OCT Compound (Sakura Finetek, Tokyo, Japan), and snap frozen in liquid nitrogen. They were cryosectioned at 16 μm thickness, in the sagittal plane, to allow a dorsal to ventral observation of the retina using Leica CM1850 cryostat (Leica Microsystems, Nossloch, Germany). Sections were mounted on gelatin and poly-l-lysine-coated slides and dried overnight at 50°C before being stored at −20°C.

Detection of cell death using TUNEL assay.  Cryosections of SD rat retina were immunolabeled with the TUNEL technique to visualize the fragmentation of DNA, a hallmark feature of apoptosis (42), using an established protocol (43). To identify the layers of the retina, DNA-specific dye bisbenzamide Hoechst (1:1000; Calbiochem) was used.

Immunohistochemical and confocal analyses on retinal sections.  For all immunohistochemistry (IHC) experiments, we followed the established protocol previously described with minor modifications (44). Frozen sections of the retina were labeled with antibodies against inducible nitric oxide synthase (iNOS; 1:200 rabbit polyclonal) (Santa Cruz Biotechnology, Santa Cruz, CA), glutamine synthetase (GS, mouse monoclonal) (1:200; Upstate Biotechnology), anti-rat vimentin, (1:200; Abcam, Cambridge, UK) and cyclin D1 (1:200, rabbit polyclonal) (Santa Cruz Biotechnology, Santa Cruz, CA). Briefly, sections were permeabilized with an antigen retrieval solution (Immunosolution Pty Ltd, Everton Park, Australia) for 45 min at 37°C, followed by blocking with 10% normal goat serum (Sigma Aldrich, St. Louis, MO) for 1 h before being incubated with the primary antibody for 24 h at 4°C. Following rinses with 0.1 m PBS, sections were treated with a secondary antibody of either rabbit IgG conjugated with Alexa Fluor 594 or mouse IgG conjugated with Alexa Fluor 488 (1:1000; Molecular Probes, Eugene, OR) for 24 h at 4°C before incubation with the DNA-specific dye bisbenzamide Hoescht (1:10 000) for 2 min. Slides were coverslipped using a mixture of glycerol gelatin (Sigma, St. Louis, MO) and water. Sections were examined, scanned and analyzed using Carl Zeiss LSM 5 Pascal confocal microscope (Germany) and LM Zeiss Apotome (Germany). Only samples that were processed and imaged concurrently were used for analysis. During image collection, the photomultiplier settings were kept constant to allow comparison of protein levels.

RNA isolation and cDNA synthesis.  Total cellular RNA was extracted from individual retinas and purified using the RNA extraction kit protocol (RNAqueous-Micro kit; Ambion). The purified RNA was quantified on a spectrophotometer (ND-1000; Nanodrop Technologies, Wilmington, DE). The integrity of the samples was assessed using a bioanalyzer (2100 Bioanalyzer; Agilent Technologies, Santa Clara, CA). The cDNA was synthesized by reverse transcription using reverse transcriptase or the first-strand cDNA synthesis kit following the manufacturer’s protocols (Superscript III; Invitrogen, Carlsbad, CA).

Real-time quantitative polymerase chain reaction (RT-qPCR).  Quantitative analysis of expression levels for genes Edn2 (Rn00561135_m1), LIF (Rn00573491_g1) and Tnf-α (Rn00562055) was determined by RT-qPCR using Taqman® probes that were combined with the Gene Expression Master-Mix (Applied Biosystems, Foster City, CA) with the use of StepOne Plus qPCR machine with the StepOne software v2.1 (Applied Biosystems). Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as a reference gene. The variabilities were accounted for by performing the Taqman amplification assay in duplicates (individual sample variability) with triplicate biological samples to account for individual animal differences. The fold changes were determined using comparative cycle threshold (Ct; delta–delta ct).

Statistical analyses.  Data were analyzed using nonparametric tests: Kruskal–Wallis followed by Mann–Whitney U. P < 0.05 was considered significant.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. References

In order to assess the effects of pretreatment with 670 nm light on Müller cells, qualitative immunohistochemical and quantitative gene expression analyses of a variety of pathological indicators were performed. In the current study, we have demonstrated that treatment with 670 nm light ameliorates the damaging effects of bright continuous light. In the nontreated retina, in the area of severe photoreceptor damage, marked alterations in the expression of Müller-cell specific structural and stress markers were observed. These changes became less marked when retinas were treated with 670 nm light. In addition, the up-regulation of the photoreceptor-specific stress and Müller cell-associated inflammatory genes and proteins was also significantly lower in the 670 nm-treated groups compared with the nontreated, LD-stressed retinas. Treatment with 670 nm light also prevented Müller cell proliferation.

670 nm light reduced photoreceptor cell death and maintained Müller cell integrity

In this study, we used the TUNEL assay to identify photoreceptors undergoing cell death and the results are summarized in Fig. 1a. As shown in the top panels, no TUNEL+ cells were detected in retinas from the Control and NIR groups. TUNEL+ nuclei were observed following exposure of the retina to 24 h bright light in the nontreated, LD group (Fig. 1a, bottom left). The distribution of TUNEL+ cells was nonuniform; they were localized along the outer nuclear layer (ONL) and were more concentrated in the superior region of the retina, close to the optic disk, previously identified as the “hotspot” (29,41). In contrast, the amount of TUNEL+ photoreceptor cells was reduced in retinas pretreated with 670 nm red light (Fig. 1a, bottom right). It is important to note that 670 nm light treatment alone did not cause damage to the retina (Fig. 1a, top right).

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Figure 1.  The effects of 670 nm red light pretreatment on the photoreceptors and the structure of retinal Müller cells 1 week after BL exposure. Representative images of retinal sections, from the superior retina, labeled with (a) TUNEL (red) (b–f) anti-S100β (green). (a) In Control and NIR retinae, TUNEL+ cells were not found in the INL and ONL (top panels). Following the exposure of the retina to 24 h of bright light, TUNEL+ nuclei were detected along the ONL of the animals from the LD group (bottom left panel). The number of TUNEL+ cells was reduced in retinas pretreated with 670 nm light from the Precon group (bottom right panel). (b, c) In Control retina, S100β labeled the entire Müller cell from the inner limiting membrane to the outer limiting membrane. (d, e) The S100β staining of the Müller cells shows that in the hotspot and penumbra areas cells were severely disrupted and swollen (white arrowheads). There was also strong labeling in the subretinal space (white arrows), suggesting the presence of proliferative Müller cell processes. (f) Changes in the 670 nm red light-treated retinae from the Precon group only showed mild changes in the labeling pattern. Bisbenzamide is used as a nuclear stain to identify the layers of the retina (blue). Scale bar, 25 μm.

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The structure of Müller cells was visualized using anti-S100β immunolabeling. As previously reported, S100β protein has been identified along the cell bodies and processes of Müller cells (45,46). In the normal control group, S100β staining showed fine and slender Müller cell processes vertically spanning the entire retina, from the inner limiting membrane to the outer limiting membrane (Fig. 1b, green). Treatment with 670 nm light of undamaged retinae did not alter the structure of the Müller cell (Fig. 1c). Following exposure to bright damaging white light for 24 h, the immunoreactivity to S100β showed mottled labeling along the processes of Müller cells in the hotspot area, and its immediate surroundings, the penumbra. Processes positive for S100β appeared thickened and disrupted, suggesting that Müller cells were undergoing swelling or hypertrophy (Fig. 1d, arrowhead). Patches of strong S100β staining were detected, suggesting the formation of subretinal glial scar in the area, where DNA-specific labeling showed severe disruption of the photoreceptor layer (ONL, blue) (Fig. 1d, white arrow). In the “penumbra” (Fig. 1e), S100β labeling appeared discontinuous and disorganized along the Müller processes, demonstrating thickening and tortuosity of these cells (Fig. 1e, white arrowheads). Similar to the hotspot, the subretinal space displayed intense aggregate of S100β labeling, suggesting the presence of gliosis (Fig. 1e, white arrow).

In retinae treated with 670 nm light, S100β labeling showed a slight swelling of the processes and endfeet, but the Müller cells were largely intact and showed normal organization (Fig. 1f, red arrow). Moreover, there was no indication of gliosis in the subretinal space.

The TUNEL profiles of the photoreceptors and the severe alterations in Müller cells, as displayed by the disrupted pattern of S100β in the BL-exposed, nontreated LD group, indicated that concurrent with photoreceptor cell death, these glial cells are structurally compromised. Present data suggest that preconditioning the retina with 670 nm light was able to preserve the structural integrity of the Müller cells. Given that there was no apparent change in the labeling pattern in the retina from the NIR group compared with the normal control, 670 nm light pretreatment alone did not alter Müller cell integrity. Overall, this supports previous findings that treatment with 670 nm light can not only prevent photoreceptor death but also help maintain the integrity of the general structure of the retina following exposure to lethal doses of white light (41,47).

670 nm red light pretreatment mitigated retinal stress

In pathological conditions of the retina, Müller cells are known to alter the expression of cytoskeletal intermediate filament proteins such as glial fibrillary acidic protein (GFAP) and vimentin (21,48–50). As such, these proteins are known to indicate the presence of stress in the retina. We used vimentin labeling to assess retinal stress. In normal control retina, vimentin labeling was detected in the inner retinal surface, in astrocytes and in the inner processes of Müller cells, as thin slender streaks vertically oriented through the inner plexiform layer to the outer plexiform layer (Fig. 2a). Exposure to bright white light induced a strong increase of immunoreactivity in the inner retina, as well as disruption of the typical vertical pattern of Müller cells. Their processes appeared thickened distally, while were retracted and fragmented proximal to the outer retina. In the penumbra, vimentin immunoreactivity was also up-regulated (Fig. 2c). Some Müller cells in this area showed continuous vimentin labeling, demonstrating hypertrophy. Moreover, deposition of clumps of vimentin was also found in the subretinal space indicating the presence of gliosis. Figure 2d shows the effects of 670 nm pretreatment in the LD retinae, demonstrating a mild increase in vimentin immunoreactivity with modest swelling of Müller cells, though still showing close to normal cell organization. No vimentin labeling was observed beyond the ONL layer, suggesting the absence of gliosis (data not shown).

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Figure 2.  The effects of 670 nm red light pretreatment in the expression of the Müller cell stress marker, vimentin BL after exposure. (a–d) Representative images of cryosectioned retina from the superior region stained with antibody against vimentin (green). (a) The immunoreactivity of vimentin in the Control retina appeared as thin vertically oriented streaks, labeling the entire Müller cell in an organized pattern. After exposing the retina to continuous bright light, strong up-regulation and severe disturbance of vimentin immunolabeling were observed in the hotspot (b) and penumbra (c) of the nontreated LD retinae. Increase in vimentin immunoreactivity, but normal organization, was detected in the Müller cells in the Precon group (d). Bisbenzamide is used as a nuclear stain to identify the layers of the retina (blue). Scale bar, 25 μm.

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The current observation that the expression of the cytoskeletal vimentin protein was altered following exposure to BL indicates that Müller cells suffer stress from photic injury. 670 nm light treatment resulted in significantly more moderate changes, suggesting a reduced stress level in these retinae. Previously, we showed that light-induced up-regulation of GFAP, another intermediate filament protein, was reduced in 670 nm-treated retinae (41), which agrees with our present finding. This implies that 670 nm red light is able to reduce LD-induced stress and injury to the retina.

Effects of 670 nm light treatment on the initiation of neuroprotection

It has been suggested that the initiation of neuroprotection requires a close communication between photoreceptors and Müller cells during retinal stress. A signaling pathway involving endothelin-mediated activation of Müller cells has been suggested to play a role in this process (51). Previous studies have shown the existence of a common signaling pathway between the Müller cells and photoreceptors following BL-exposure, which involves induction of Endothelin 2 (Edn2) and leukemia inhibitory factor (LIF) genes (51–53). Edn2 is expressed in photoreceptors in response to stress, sending signals to the Müller cells, that produce LIF, a cytokine, in response to the stress signals from their immediate surroundings. Preconditioning the retina with sublethal doses of light has been shown to induce Edn2 expression in photoreceptors and LIF in Müller cells (52). This signaling pathway is believed to play a role in the up-regulation of neuroprotective factors (FGF-2) and has been demonstrated to promote photoreceptor protection (11–14,51,54).

In the current study, quantitative analysis using RT-qPCR showed significant up-regulation in Edn2 gene expression following exposure to BL as illustrated in Fig. 3a. The nontreated LD group showed a 21.7-fold increase of Edn2 expression (< 0.05), while only a two-fold increase was detected in the retinae treated with 670 nm light, showing no significant change compared to control, but demonstrating a significant reduction compared to the LD group (< 0.05).

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Figure 3.  The effects of 670 nm red light pretreatment in the regulation of Edn2 and LIF genes (a, b) and protein expression of LIF and GS in BL-exposed retinae (c–f). (a, b) Fold change of 1 (red line) means no change in gene expression, fold change of >1 denotes up-regulation, while <1 indicates down-regulation. Bars are mean ± SEM. *Statistically significant differences (< 0.05) compared with the LD group. (c) Representative images of retinae from all experimental groups immunostained with LIF (red) and GS (green). LIF immunolabeling was not detected in the retina from Control group, while GS staining is abundant in the entire Müller cell and its processes and endfeet. In LD animals, LIF immunolabeling increased and GS decreased. In this group, a strong LIF staining was observed in the processes and cell bodies of the Müller cells both in the hotspot (d) and penumbra (e). The GS immunoreactivity was reduced with patches of labeling detected in the outer retina. (f) In the Precon group, LIF staining was muted (arrowhead), while the level of GS immunoreactivity was similar to that of Control levels. Scale bar: 25 μm.

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Joly et al. (10) reported that a subset of Müller cells produced the cytokine LIF upon retinal stress and demonstrated that this molecule is essential to the initiation of the Edn2 signaling pathway. Therefore, we assessed changes in LIF gene regulation and localization of LIF protein in the retina in all experimental groups.

The changes in LIF gene expression are depicted in Fig. 3b. Compared to the control, retinas from the LD group showed a 37-fold increase in LIF gene expression. The 670 nm-pretreated retinae from the Precon group revealed a significantly reduced fold change of 3.2 (< 0.05).

In the normal control retina, LIF immunolabeling was not detectable (Fig. 3c). 670 nm treatment alone did not alter LIF protein expression (data not shown). Following the exposure to damaging bright light, positive LIF immunostaining was observed along the entire Müller cell as punctate labeling (Fig. 3d, red). These speckles of LIF staining were found in the inner and outer processes of Müller cells, and showed clumping in the outer retina along the area of the collapsed ONL in the hotspot (Fig. 3d), and clustering of puncta was observed in the disrupted ONL of the penumbra (Fig. 3e). Retinas treated with 670 nm light only showed muted LIF labeling localized in the inner processes of the Müller cell (Fig. 3f, arrowhead).

Our current findings support previous reports of a close correlation between LIF and Edn2 regulation following exposure to damaging light (51). In this study, pretreatment with 670 nm red light showed significant down-regulation of Edn2 and LIF genes, as well as reduction in LIF protein levels following exposure to damaging light, suggesting a significant reduction in retinal stress levels. Our current results suggest that 670 nm light pretreatment is able to modulate the expression of the Edn2 gene in the photoreceptors and the Müller cell-specific LIF following BL-exposure, which validates our previous findings from a microarray study showing reduced fold changes of both genes in the 670 nm-pretreated retinas compared with the nontreated BL-exposed retina (35).

670 nm light maintains Müller cell capacity to recycle neurotransmitters

One important role of Müller cells is to clear excess toxic glutamate that is constantly released by the neighboring photoreceptors and to recycle it into nontoxic glutamine. GS is the key enzyme produced by Müller cells that serve this role in the retina, by forming nontoxic glutamine from glutamate using ammonia (55,56). By utilizing ammonia, this process also plays a role in the control of tissue ammonia levels and the regulation of the acid/base balance in the retina. In normal control retinae, GS was expressed along the entire Müller cell, present in both the vertical and transverse processes (Fig. 3c, green). Similar pattern of labeling is observed in the NIR control retina (data not shown). In the hotspot, the general pattern was similar; however, labeling became patchy, especially in the inner retina, while in the depleted ONL layer, labeling became clumpy (Fig. 3d, green). In the penumbra, the inner retina still showed patchy and reduced labeling, while in the outer retina, GS labeling demonstrated the proliferating glial processes (Fig. 3e, green). In retinae treated with 670 nm light, the intensity and pattern of GS staining were comparable to that of the Control (Fig. 3f, green). The present results agreed with earlier reports that light-induced damage of photoreceptors causes a down-regulation of GS production in Müller cells (30). Here we demonstrated that the level of GS staining in the 670 nm-treated retina was almost identical to that of the normal control, suggesting that 670 nm light pretreatment maintains Müller cell metabolic activity.

Effects of the 670 nm light pretreatment on inflammatory processes

A common feature of many pathological conditions in the CNS is inflammation, a reaction of the tissue to local injury that can often lead to the progression of the disease. Early inflammatory changes have been detected in the light-induced photoreceptor degeneration model, triggering an immune response resulting in further damage (57,58). Studies have shown the involvement of Müller cells in mediating inflammatory response in degenerative conditions by secreting pro-inflammatory cytokines such as tumor necrosis factor (TNF-α), interleukin (IL-1) and producing free radicals (59–62).

In the presence of TNF-α, expression of the inducible isoform of nitric oxide synthase (iNOS) is initiated. This enzyme has been shown to be present in the retinal pigment epithelium, macrophages, microglia and in Müller cells. It produces nitric oxide (NO), a reactive radical gas (63–65). NO production in Müller cells may play a protective role (66) as part of the retinal immune response, as has been reported in macrophages (67). However, it can also be detrimental to retinal neurons, by disrupting their metabolism (68). In certain forms of retinal dystrophies such as diabetic retinopathy and glaucoma, iNOS has been implicated to mediate the pro-inflammatory response (69–72). Elevated levels of iNOS-produced NO molecules can indirectly accelerate tissue damage by causing the formation of reactive nitrogen species, known potent pro-inflammatory molecules (73,74).

In the current work, we assessed the expression changes in the Tnf-α gene and the iNOS protein in the retina following BL exposure. Our gene expression analysis showed that exposure of the retina to BL stimulated an up-regulation of the inflammatory Tnf-α gene in the nontreated LD retinas, exhibiting a 2.8-fold increase compared with the controls (Fig. 4a), while in the Precon group, BL exposure caused only a 1.2-fold up-regulation of this gene. NIR treatment alone did not cause significant change in Tnf-α expression, compared with control.

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Figure 4.  The effects of pretreatment with 670 nm red light and BL exposure in the expression of inflammation-related Tnf-α gene and iNOS protein in the retina assessed by RT-qPCR and immunohistochemical labeling. (a) The level of expression of Tnf-α gene showing all experimental groups is analyzed and presented as described in a previous section (see Figure 3a,b). Exposure to BL caused a 2.8-fold up-regulation in the nontreated LD group and this level of expression was reduced in the pretreated retina from the Precon group to 1.2-fold. (b–e) Representative micrographs of sections of the retina from the superior region stained with anti-iNOS (red). The retina was double-labeled with S100β (green) to identify the Müller cells. In Control retina, iNOS staining was not detected (b). A strong iNOS immunostaining was detected in the hotspot and penumbra areas in the LD group (c, d). The labeling was distributed in the retinal vessel (arrowhead), inner retina and the subretinal region appeared to be co-labeling with the proliferating Müller cell processes (arrow). The iNOS immunostaining was also present in the retina from the Precon group, but with sparse appearance and only limited to the vessels (e). Bisbenzamide is used as a nuclear stain to identify the layers of the retina (blue). Scale bar: 25 μm.

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Immunohistochemical labeling of iNOS was undetectable in the retinas from the normal control group (Fig. 4b). The immunoreactivity of iNOS became visible as bright red spots following exposure to damaging light in the hotspot and the penumbra regions of nontreated retinae (Fig. 4c,d). In the hotspot region, iNOS was present in the ganglion cell layer and inner nuclear layer. In the severely disrupted outer retina they appeared as aggregates, possibly marking invading macrophages (Fig. 4c). In the penumbra region, iNOS labeling was detected in the nuclear layers and in the superficial (Fig. 4d, arrowhead) and deep retinal vessels. In the subretinal region, strong iNOS labeling was present along the proliferating Müller cells (Fig. 4d, arrow). In retinas pretreated with 670 nm red light, iNOS staining was sparse and limited to the vessels (Fig. 4e).

Our findings are in agreement with previous reports showing that there is an up-regulation of pro-inflammatory cytokine and iNOS production in Müller cells in light-evoked photoreceptor degeneration (70,75,76) that has been found to coincide with an increased presence of NO (76). During the initial stages of the damage, iNOS is produced by the activated Müller cells in the presence of cytokines (TNF-α). In addition, monocytes and macrophages gathering in the choroidal vasculature, and infiltrating the subretinal space and the retina, recruited to the site of damaged photoreceptors also produce iNOS (58,77), contributing further to the progression of the damage (78). The reduction in TNF-α production and the consequent absence of iNOS in retinas treated with the 670 nm light provides evidence that this treatment has an anti-inflammatory effect in this model. These results are in agreement with previous findings that 670 nm light pretreatment modulates the inflammatory response in mammalian tissues (35,39,41,79,80).

Treatment with 670 nm light prevents Müller cell proliferation

It has been shown that following injury, a subset of Müller cells display stem cell properties. They re-enter the cell cycle and differentiate into other cell types, especially those experiencing severe damage such as the photoreceptors (16,81,82). Cyclin D1 is a proto-oncogene used as a marker for proliferating cells. Figure 5a shows that in normal control retinae, cyclin D1 was not detectable. NIR treatment on its own did not induce cell proliferation (Fig. 5b). Following BL exposure in the hotspot, strong cyclin D1 immunoreactivity was detected in the INL, in the severely depleted ONL, the subretinal debris layer and the choroid (Fig. 5c, in red). In the penumbra, the labeling pattern was similar to that of the neighboring hotspot (Fig. 5e). Here, labeling in the ONL appeared punctate along Müller cell processes within the retina and in the subretinal space, where glial scarring occurred (Fig. 5e,f). In retinae pretreated with 670 nm light, no cyclin D1 immunolabeling was observed (Fig. 5d).

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Figure 5.  Visualization of the cell cycle marker protein, cyclin D1. Representative images of retinal sections from the superior area stained with cyclin D1 antibody (red). (a, b) Immunolabeling of cyclin D1 was not present in the Control and NIR Control groups. (c, e) Bright light exposure induced strong immunoreactivity of cyclin D1 in the hotspot and penumbra areas of the retina from the nontreated LD group, localizing in the INL and subretinal space. (f) Section of the retina from the LD penumbra, double-stained with cyclin D1 (red) and vimentin (green) to demonstrate that cyclin D1 was localized to Müller cells. (d) Cyclin D1 was not detected in retinas treated with 670 nm red light from the Precon group. Bisbenzamide is used as a nuclear stain to identify the layers of the retina (blue). Scale bar: 25 μm.

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Our current observation of the presence of the cell cycle marker cyclin D1 in the nontreated BL-exposed retinas indicates that Müller cells are undergoing proliferative changes. This is in agreement with previous findings that injury or damage leading to photoreceptor loss, including light-induced photoreceptor degeneration, stimulates Müller cell proliferation (17,19). Proliferation of Müller cells serves to restore retinal structure, similar to the role of fibroblasts that heal skin injuries (83,84). However, the formation of scar tissue in the retina has irreversible consequences. Subretinal glial scars create a barrier between the retina and its metabolic source, the choroid, as well as seal ion channels in Müller cells. This mechanical barrier thereby hinders the delivery of nutrients (1) and disrupts tissue homeostasis (2) and thus leads to further photoreceptor death (85). In late stages of retinal degenerations, following the substantial loss of photoreceptors, retinal re-modeling takes place, where proliferating Müller cells play a pivotal role. This re-organization involves a rearrangement of neural circuitry of the remaining neuronal cells in the retina and can impede any restorative therapeutic attempts (18,86). This study has demonstrated that 670 nm red light pretreatment can prevent the initiation of proliferative response of Müller cells.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusion
  7. References

In the current work, we demonstrated that treatment with 670 nm light prior to exposure to damaging white light was protective to the retina, significantly reducing photoreceptor loss and mitigating pathological changes in the Müller cells. The observed impact of damaging light exposure on stress levels in photoreceptors and Müller cells was also reduced. Most importantly, Müller cell-mediated immune response was significantly mitigated at both gene and protein levels. These results are in agreement with previous reports that 670 nm light pretreatment promotes cell survival and modulates inflammatory response. Taken together, 670 nm light preconditioning may harness the inherent ability of the Müller cells to modulate not only transient but possibly long-term photoreceptor protection.

Studies have demonstrated that 670 nm light is able to reverse damage caused by COX-specific toxins in various neuronal cells, including the photoreceptors (39,79). These studies seem to support the theory that 670 nm light acting through COX, the rate-limiting enzyme in the mitochondrial respiration. Considering that Müller cells and the adjacent photoreceptors are rich in mitochondria (87), it is likely that, at least in part, 670 nm may exert its beneficial effects by enhancing the activity of COX in these cells. This activation enhances the efficacy of the oxidative phosphorylation leading to an increase in ATP production and the reduction of free radicals. Consequently, it decreases oxidative stress in the outer retina, thereby ameliorating the bright light-induced damage and maintaining retinal homeostasis. Furthermore, the interaction of 670 nm light and COX can indirectly or directly activate the transcription factors and corresponding gene expression through downstream regulation of the cellular signaling networks in the retina (see Fig. 6).

image

Figure 6.  Schematic representation of the proposed mechanism of 670 nm light pretreatment in a mammalian cell.

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References

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  6. Conclusion
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