670 nm light mitigates oxygen-induced degeneration in C57BL/6J mouse retina

BACKGROUND Irradiation with light wavelengths from the far red (FR) to the near infrared (NIR) spectrum (600 nm -1000 nm) has been shown to have beneficial effects in several disease models. In this study, we aim to examine whether 670 nm red light pretreatment can provide protection against hyperoxia-induced damage in the C57BL/6J mouse retina. Adult mice (90-110 days) were pretreated with 9 J/cm2 of 670 nm light once daily for 5 consecutive days prior to being placed in hyperoxic environment (75% oxygen). Control groups were exposed to hyperoxia, but received no 670 nm light pretreatment. Retinas were collected after 0, 3, 7, 10 or 14 days of hyperoxia exposure (n = 12/group) and prepared either for histological analysis, or RNA extraction and quantitative polymerase chain reaction (qPCR). Photoreceptor damage and loss were quantified by counting photoreceptors undergoing cell death and measuring photoreceptor layer thickness. Localization of acrolein, and cytochrome c oxidase subunit Va (Cox Va) were identified through immunohistochemistry. Expression of heme oxygenase-1 (Hmox-1), complement component 3 (C3) and fibroblast growth factor 2 (Fgf-2) genes were quantified using qPCR. RESULTS The hyperoxia-induced photoreceptor loss was accompanied by reduction of metabolic marker, Cox Va, and increased expression of oxidative stress indicator, acrolein and Hmox-1. Pretreatment with 670 nm red light reduced expression of markers of oxidative stress and C3, and slowed, but did not prevent, photoreceptor loss over the time course of hyperoxia exposure. CONCLUSION The damaging effects of hyperoxia on photoreceptors were ameliorated following pretreatment with 670 nm light in hyperoxic mouse retinas. These results suggest that pretreatment with 670 nm light may provide stability to photoreceptors in conditions of oxidative stress.This work was supported by the Australian Research Council Centre of Excellence in Vision Science

Chemokine-mediated inflammation in the degenerating retina is coordinated by Müller cells, activated microglia, and retinal pigment epithelium

BACKGROUND Monocyte infiltration is involved in the pathogenesis of many retinal degenerative conditions. This process traditionally depends on local expression of chemokines, though the roles of many of these in the degenerating retina are unclear. Here, we investigate expression and in situ localization of the broad chemokine response in a light-induced model of retinal degeneration. METHODS Sprague-Dawley (SD) rats were exposed to 1,000 lux light damage (LD) for up to 24 hrs. At time points during (1 to 24 hrs) and following (3 and 7 days) exposure, animals were euthanized and retinas processed. Microarray analysis assessed differential expression of chemokines. Some genes were further investigated using polymerase chain reaction (PCR) and in situ hybridization and contrasted with photoreceptor apoptosis using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Recruitment of retinal CD45 (+) leukocytes was determined via fluorescence activated cell sorting (FACS), and expression of chemokine receptors determined using PCR. RESULTS Exposure to 24 hrs of LD resulted in differential expression of chemokines including Ccl3, Ccl4, Ccl7, Cxcl1, and Cxcl10. Their upregulation correlated strongly with peak photoreceptor death, at 24 hrs exposure. In situ hybridization revealed that the modulated chemokines were expressed by a combination of Müller cells, activated microglia, and retinal pigment epithelium (RPE). This preceded large increases in the number of CD45(+) cells at 3- and 7-days post exposure, which expressed a corresponding repertoire of chemokine receptors. CONCLUSIONS Our data indicate that retinal degeneration induces upregulation of a broad chemokine response whose expression is coordinated by Müller cells, microglia, and RPE. The findings inform our understanding of the processes govern the trafficking of leukocytes, which are contributors in the pathology of retinal degenerations

670-nm light treatment reduces complement propagation following retinal degeneration

AIM Complement activation is associated with the pathogenesis of age-related macular degeneration (AMD). We aimed to investigate whether 670-nm light treatment reduces the propagation of complement in a light-induced model of atrophic AMD. METHODS Sprague-Dawley (SD) rats were pretreated with 9 J/cm(2) 670-nm light for 3 minutes daily over 5 days; other animals were sham treated. Animals were exposed to white light (1,000 lux) for 24 h, after which animals were kept in dim light (5 lux) for 7 days. Expression of complement genes was assessed by quantitative polymerase chain reaction (qPCR), and immunohistochemistry. Counts were made of C3-expressing monocytes/microglia using in situ hybridization. Photoreceptor death was also assessed using outer nuclear layer (ONL) thickness measurements, and oxidative stress using immunohistochemistry for 4-hydroxynonenal (4-HNE). RESULTS Following light damage, retinas pretreated with 670-nm light had reduced immunoreactivity for the oxidative damage maker 4-HNE in the ONL and outer segments, compared to controls. In conjunction, there was significant reduction in retinal expression of complement genes C1s, C2, C3, C4b, C3aR1, and C5r1 following 670 nm treatment. In situ hybridization, coupled with immunoreactivity for the marker ionized calcium binding adaptor molecule 1 (IBA1), revealed that C3 is expressed by infiltrating microglia/monocytes in subretinal space following light damage, which were significantly reduced in number after 670 nm treatment. Additionally, immunohistochemistry for C3 revealed a decrease in C3 deposition in the ONL following 670 nm treatment. CONCLUSIONS Our data indicate that 670-nm light pretreatment reduces lipid peroxidation and complement propagation in the degenerating retina. These findings have relevance to the cellular events of complement activation underling the pathogenesis of AMD, and highlight the potential of 670-nm light as a non-invasive anti-inflammatory therapy.This work was funded by the Australian Research Council Centres of Excellence Program Grant (CE0561903)

The broad-spectrum chemokine inhibitor NR58-3.14.3 modulates macrophage-mediated inflammation in the diseased retina

Background The activity of macrophages is implicated in the progression of retinal pathologies such as atrophic age-related macular degeneration (AMD), where they accumulate among the photoreceptor layer and subretinal space. This process is aided by the local expression of chemokines, which furnish these cells with directional cues that augment their migration to areas of retinal injury. While these qualities make chemokines a potential therapeutic target in curtailing damaging retinal inflammation, their wide variety and signalling redundancy pose challenges in broadly modulating their activity. Here, we examine the efficacy of the broad-spectrum chemokine inhibitor NR58-3.14.3—a suppressor of Ccl- and Cxcl- chemokine pathways—in suppressing macrophage activity and photoreceptor death, using a light-induced model of outer retinal atrophy and inflammation. Methods Photo-oxidative damage was induced in SD rats via exposure to 1000 lux of light for 24 h, after which animals were euthanized at 0- or 7-day post-exposure time points. Prior to damage, NR58-3.14.3 was injected intravitreally. Retinas were harvested and evaluated for the effect of NR58-3.14.3 on subretinal macrophage accumulation and cytokine expression profile, as well as photoreceptor degeneration. Results We report that intravitreal administration of NR58-3.14.3 reduces the accumulation of macrophages in the outer retina following exposure to light damage, at both 0- and 7-day post-exposure time points. Injection of NR58-3.14.3 also reduced the up-regulation of inflammatory markers including of Il6, Ccl3, and Ccl4 in infiltrating macrophages, which are promoters of their pathogenic activity in the retina. Finally, NR58-3.14.3-injected retinas displayed markedly reduced photoreceptor death following light damage, at both 0 and 7 days post-exposure. Conclusions Our findings indicate that NR58-3.14.3 is effective in inhibiting subretinal macrophage accumulation in light-induced retinal degeneration and illustrate the potential of broad-spectrum chemokine inhibitors as novel therapeutic agents in thwarting retinal inflammation. Although broad-spectrum chemokine inhibitors may not be appropriate for all retinal inflammatory conditions, our results suggest that they may be beneficial for retinal dystrophies in which chemokine expression and subretinal macrophage accumulation are implicated, such as advanced AMD

670nm Photobiomodulation as a Novel Protection against Retinopathy of Prematurity: Evidence from Oxygen Induced Retinopathy Models

INTRODUCTION To investigate the validity of using 670nm red light as a preventative treatment for Retinopathy of Prematurity in two animal models of oxygen-induced retinopathy (OIR). MATERIALS AND METHODS During and post exposure to hyperoxia, C57BL/6J mice or Sprague-Dawley rats were exposed to 670 nm light for 3 minutes a day (9J/cm²). Whole mounted retinas were investigated for evidence of vascular abnormalities, while sections of neural retina were used to quantify levels of cell death using the TUNEL technique. Organs were removed, weighed and independent histopathology examination performed. RESULTS 670 nm light reduced neovascularisation, vaso-obliteration and abnormal peripheral branching patterns of retinal vessels in OIR. The neural retina was also protected against OIR by 670 nm light exposure. OIR-exposed animals had severe lung pathology, including haemorrhage and oedema, that was significantly reduced in 670 nm+OIR light-exposed animals. There were no significance differences in the organ weights of animals in the 670 nm light-exposed animals, and no adverse effects of exposure to 670 nm light were detected. DISCUSSION Low levels of exposure to 670 nm light protects against OIR and lung damage associated with exposure to high levels of oxygen, and may prove to be a non-invasive and inexpensive preventative treatment for ROP and chronic lung disease associated with prematurity.Australian Research Council Centres of Excellence Program Grant (CE0561903); Canberra Hospital Private Practice Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

670 nm light mitigates oxygen-induced degeneration in C57BL/6J mouse retina

Background: Irradiation with light wavelengths from the far red (FR) to the near infrared (NIR) spectrum (600 nm -1000 nm) has been shown to have beneficial effects in several disease models. In this study, we aim to examine whether 670 nm red light pretreatment can provide protection against hyperoxia-induced damage in the C57BL/6J mouse retina. Adult mice (90-110 days) were pretreated with 9 J/cm2 of 670 nm light once daily for 5 consecutive days prior to being placed in hyperoxic environment (75% oxygen). Control groups were exposed to hyperoxia, but received no 670 nm light pretreatment. Retinas were collected after 0, 3, 7, 10 or 14 days of hyperoxia exposure (n = 12/group) and prepared either for histological analysis, or RNA extraction and quantitative polymerase chain reaction (qPCR). Photoreceptor damage and loss were quantified by counting photoreceptors undergoing cell death and measuring photoreceptor layer thickness. Localization of acrolein, and cytochrome c oxidase subunit Va (Cox Va) were identified through immunohistochemistry. Expression of heme oxygenase-1 (Hmox-1), complement component 3 (C3) and fibroblast growth factor 2 (Fgf-2) genes were quantified using qPCR.Results: The hyperoxia-induced photoreceptor loss was accompanied by reduction of metabolic marker, Cox Va, and increased expression of oxidative stress indicator, acrolein and Hmox-1. Pretreatment with 670 nm red light reduced expression of markers of oxidative stress and C3, and slowed, but did not prevent, photoreceptor loss over the time course of hyperoxia exposure.Conclusion: The damaging effects of hyperoxia on photoreceptors were ameliorated following pretreatment with 670 nm light in hyperoxic mouse retinas. These results suggest that pretreatment with 670 nm light may provide stability to photoreceptors in conditions of oxidative stress.</p

Cone Degeneration in Aging and Age-Related Macular Degeneration

Objective: To examine the morphological features of macular photoreceptors in histologically normal retina from normal donor eyes and eyes with age-related macu- lar degeneration (AMD). Methods: The macular region was excised from 18 donor eyes (aged 22-96 years) and cryosectioned. Sections were stained with hematoxylin-eosin or double immu- nolabeled using opsin antibodies or synaptic markers. Results: Three of 8 retinas studied in detail had AMD lesions; the remainder were histologically normal. Im- munoreactivity to cone opsin was abnormal in parts of all retinas (3.5%-95.0% of each sample) and was associated with swelling of and altered immunoreactivity in the cone distal axon. In non-AMD retinas, the anomalies were mainly in nonfoveal macular locations. The nature of the anomalies was identical in non-AMD retinas and in parts of AMD retinas adjacent to overt degeneration. Conclusion: Redistribution of opsin and anomalies in the distal cone axon are common in the aging human macula and may indicate susceptibility to AMD. Clinical Relevance: The findings are consistent with tests of cone function in aging and early AMD, which suggests that integrated cone functions-including contrast sensitivity, color matching, and short wavelength- sensitive cone sensitivity-are the most reliable prognostic indicators of progression in AMD

Differential effects of TFG-β and FGF-2 on in vitro proliferation and migration of primate retinal endothelial and Müller cells

Purpose: During retinal development, the pattern of blood vessel formation depends upon the combined effects of proliferation and migration of endothelial cells, astrocytes and Müller cells. In this study, we investigated the potential for transforming