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Diabetic retinopathy (DR) causes vision loss due to sustained inflammation and vascular damage. The vascular damage is evident by fibrinogen leakage, angiogenesis, and hypoxia. Neuronal regulation of microglia via the CX3CL1 (Fractalkine or FKN)-CX3CR1 pathway plays a significant role in retinal pathology. Defects in FKN or CX3CR1 exacerbate inflammation, vascular damage, and vision impairment. However, the contribution of hypoxic astrocytes to the pathological process of DR is unclear. A hypoxic model (7 days of systemic 7.5% O 2 ) was utilized to induce retinal damage in adult mice in the absence of systemic inflammatory signals. This model induced vascular and microglial responses similar to 10 weeks of STZ-induced hyperglycemia. The goal of this study is to characterize retinal damage in WT and mice with defects in the FKN-CX3CR1 signaling axis and hence assess the impact of the microglial inflammatory responses to hypoxic retinopathy. Tissues were analyzed by immunostaining, RNA sequencing, and cytokine quantification. We found that CX3CR1 deficiency in hypoxic animals induced reactive astrogliosis and that Müller glial responses to hypoxia and systemic inflammation were dependent on FKN signaling. Exacerbated microglial reactivity to hypoxic conditions significantly altered the expression of HIF transcripts. Microglial dysregulation was found to reduce the anti-inflammatory response to hypoxic conditions, downregulate hypoxia-responsive gene expression, and restrained LPS-induced inflammatory responses. We found that microglia dysregulation alters the hypoxic response by inhibiting the upregulation of HIF2α/3α, increasing CD31 immunoreactivity, and altering the expression of ECM-associated transcripts such as type I, III, and XVIII collagens to hypoxic conditions.

Aim: The aim of this study was to evaluate the potential protective effect of Qiju Granule in a rat model of age-related macular degeneration (AMD) and investigate the underlying mechanisms involved., Methods: Rats were injected intravenously with 40 mg/kg of sodium iodate (SI) to induce a dry AMD model. The rats in the treatment group received three different doses of Qiju Granule once a day via gavage, while the rats in the control group were given an equal volume of physiological saline. On day 14 and day 28 following the intervention, various methods were employed to evaluate retinal function and structure, including electroretinography (ERG), optical coherence tomography (OCT), and histological examination. The expression of glial fibrillary acidic protein (GFAP), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF) was assessed via immunofluorescence. Beyond immunofluorescence, the mRNA levels of bFGF, BDNF, and CNTF were quantitatively determined using real-time polymerase chain reaction (qRT-PCR)., Results: Rats treated with Qiju Granule exhibited significant improvements in both retinal function and structure compared to the model group. The most noteworthy effects were observed at a high dose of Qiju Granule. Furthermore, the expression levels of bFGF, BDNF, and CNTF were significantly unregulated in the treated groups compared to the model group., Conclusions: Qiju Granule demonstrated a protective effect on the retina in the SI-induced rat model of AMD. The protective mechanism may be attributed to the upregulation of retinal neurotrophic factors expression., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests related to this study. There are no financial or personal relationships with other people or organizations that could influence the conduct or interpretation of the research., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Scope: Maternal high fructose diet (HFD) during pregnancy and lactation can initiate retinal dysfunction. However, the underlying mechanism remains largely unknown., Methods and Results: By using the rodent model of maternal HFD in this study, the results from electroretinography (ERG) indicate that b-wave amplitude, an index of inner retinal function, is significantly reduced as early as 3 months old and the deteriorated effect can be detected at 15 months old. Further, the protein expressions of CD11b (a marker of active microglia), p40 phox subunit of NADPH oxidase, GFAP (a marker of active astrocytes), and NLPR3 examined by western blot and immunofluorescence are significantly increased in the retina of the male HFD offspring at 3 months old. Treatment with omega-3 polyunsaturated fatty acids (ω-3 PUFAs) for 2 weeks (from 2.5 to 3 months old) effectively reverses the aforementioned changes., Conclusion: Together, these results indicate that the early onset and extensive retinal dysfunction may be a result of glial activation which is induced by maternal HFD to initiate an inflammatory microenvironment leading to a long-term progression of retinopathy. Short-term administration of ω-3 PUFA at a young age may be a feasible strategy to intervene in the maternal HFD-programmed retinal impairment in male offspring., (© 2024 Wiley‐VCH GmbH.)

Subretinal fibrosis is a major untreatable cause of poor outcomes in neovascular age-related macular degeneration. Mouse models of subretinal fibrosis all possess a degree of invasiveness and tissue damage not typical of fibrosis progression. This project characterises JR5558 mice as a model to study subretinal fibrosis. Fundus and optical coherence tomography (OCT) imaging was used to non-invasively track lesions. Lesion number and area were quantified with ImageJ. Retinal sections, wholemounts and Western blots were used to characterise alterations. Subretinal lesions expand between 4 and 8 weeks and become established in size and location around 12 weeks. Subretinal lesions were confirmed to be fibrotic, including various cell populations involved in fibrosis development. Müller cell processes extended from superficial retina into subretinal lesions at 8 weeks. Western blotting revealed increases in fibronectin (4 wk and 8 wk, p < 0.001), CTGF (20 wks, p < 0.001), MMP2 (12 wks and 20 wks p < 0.05), αSMA (12 wks and 20 wks p < 0.05) and GFAP (8 wk and 12 wk, p ≤ 0.01), consistent with our immunofluorescence results. Intravitreal injection of Aflibercept reduced subretinal lesion growth. Our study provides evidence JR5558 mice have subretinal fibrotic lesions that grow between 4 and 8 weeks and confirms this line to be a good model to study subretinal fibrosis development and assess treatment options., (© 2024. Crown.)

The retina is one of the highest metabolically active tissues with a high oxygen consumption, so insufficient blood supply leads to visual impairment. The incidence of related conditions is increasing; however, no effective treatment without side effects is available. Furthermore, the pathomechanism of these diseases is not fully understood. Our aim was to develop an optimal ischemic retinopathy mouse model to investigate the retinal damage in a time-dependent manner. Retinal ischemia was induced by bilateral common carotid artery occlusion (BCCAO) for 10, 13, 15 or 20 min, or by right permanent unilateral common carotid artery occlusion (UCCAO). Optical coherence tomography was used to follow the changes in retinal thickness 3, 7, 14, 21 and 28 days after surgery. The number of ganglion cells was evaluated in the central and peripheral regions on whole-mount retina preparations. Expression of glial fibrillary acidic protein (GFAP) was analyzed with immunohistochemistry and Western blot. Retinal degeneration and ganglion cell loss was observed in multiple groups. Our results suggest that the 20 min BCCAO is a good model to investigate the consequences of ischemia and reperfusion in the retina in a time-dependent manner, while the UCCAO causes more severe damage in a short time, so it can be used for testing new drugs.

Astrocytes throughout the central nervous system are heterogeneous in both structure and function. This diversity leads to tissue-specific specialization where morphology is adapted to the surrounding neuronal circuitry, as seen in Bergman glia of the cerebellum and Müller glia of the retina. Because morphology can be a differentiating factor for cellular classification, we recently developed a mouse where glial-fibrillary acidic protein (GFAP)-expressing cells stochastically label for full membranous morphology. Here we utilize this tool to investigate whether morphological and electrophysiological features separate types of mouse retinal astrocytes. In this work, we report on a novel glial population found in the inner plexiform layer and ganglion cell layer which expresses the canonical astrocyte markers GFAP, S100β, connexin-43, Sox2 and Sox9. Apart from their retinal layer localization, these cells are unique in their radial distribution. They are notably absent from the mid-retina but are heavily concentrated near the optic nerve head, and to a lesser degree the peripheral retina. Additionally, their morphology is distinct from both nerve fiber layer astrocytes and Müller glia, appearing more similar to amacrine cells. Despite this structural similarity, these cells lack protein expression of common neuronal markers. Additionally, they do not exhibit action potentials, but rather resemble astrocytes and Müller glia in their small amplitude, graded depolarization to both light onset and offset. Their structure, protein expression, physiology, and intercellular connections suggest that these cells are astrocytic, displaced from their counterparts in the nerve fiber layer. As such, we refer to these cells as displaced retinal astrocytes., (© 2024 The Authors. GLIA published by Wiley Periodicals LLC.)

Introduction: Ocular diseases pose a significant health concern for donkeys. However, studies examining the microanatomy and cell populations of the donkey retina are scarce. The current study aimed to describe the vascular pattern of the donkey retina and document its cellular components., Methods: The donkey retina specimens were obtained from different retinal regions and prepared for semithin sectioning and immunohistochemistry., Results: The donkey has a paurangiotic retina in which retinal vessels are confined to a narrow area around the optic disc. Glial cells coexist with the blood vessels being very numerous in the vascular region and become scanty in the avascular ones. S-100-positive astrocytes could be observed in these avascular areas. Ganglion cells are organized in a single layer with the least population existing in the peripheral retina. Acidic fibroblast growth factor (AFGF) is immunoreactive in amacrine and ganglion cells. A subpopulation of amacrine cells reacted strongly to tyrosine hydroxylase (TH), and others reacted positively to S-100 protein. Ganglion cell nuclei exhibited a strong immunoreactivity to S-100 protein as well. Furthermore, glial fibrillary acidic protein (GFAP) is used to identify Müller cells that extend their processes across the retina from the inner to the outer limiting membrane., Conclusions: In conclusion, our findings provide novel insights into the normal retinal organization. The donkey retina shows the characteristic expression of immunohistochemical markers for the major cell types. In addition, the distribution of glial cells is comparable between the vascular and avascular regions., (© 2024 S. Karger AG, Basel.)

The impact of maternal undernutrition on morphological changes of the retina was assessed in two generations of aged offspring. Wistar 18 rats (9 of each generation of 20-month-old female offspring; in total - 27 eyes) were analyzed. The first generation offspring were born to mothers who: (a) were restricted to food only before pregnancy (pre-pregnancy); (b) whose food was restricted before and during pregnancy. The control group and all the offspring were fed normally. After enucleating the eyes, paraffin sections were stained with hematoxylin and eosin. The thickness of retina layers was measured. Cryosections were immunostained using glial fibrillary acidic protein, ionized calcium-binding adaptor molecule1, RNA-binding protein with multiple splicing for evaluation of macroglia, microglia and retinal ganglion cells by digital image analysis tools. Our data have shown atrophy of photoreceptor layer and degeneration of outer nuclear layer in all investigated groups, but less damage was found in the control group. Higher Müller cell activity and greater number of microglial cells was observed in the second generation offspring born from both restricted diet groups. Higher numbers of microglial and retinal ganglion cells were observed in the second generation in comparison to the first generation offspring. Malnutrition of the mother may be one of the possible causes of degeneration of the outer layers of the retina and activation of Müller cells in the second generation offspring. The effect of maternal nutritional restriction on the number of microglial and retinal ganglion cells is unclear., (©The Author(s) 2023. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

The purpose of this study was to investigate Müller cells during the fetal development of the human eye. Müller cells in eyes of 39 human fetuses (11-38 weeks of gestation, WOG) and 6 infants (5 died of abusive head trauma, AHT, aged 1-9 months) were immunohistochemically stained and investigated for spatial and temporal immunoreaction of nestin, CD44, collagen IX and GFAP, which are stem cell markers or markers of intermediate filaments, respectively, in one of the hitherto largest cohorts of fetal eyes. Müller cells could be detected immunohistochemically as early as 12 WOG by immunohistochemical staining with nestin. Nestin was more strongly expressed in Müller cells of the peripheral retina and a centroperipheral gradient of immunoreaction over time was observed. CD44 was predominantly expressed in fetal eyes of the late second and early third trimester between (23 and 27 WOG) and significantly stronger in the infant eyes. Collagen IX labeling in the central retina was significantly stronger than in more peripheral sectors and increased with fetal age. GFAP staining in Müller cells was seen in the eye of a fetus of 38 WOG who died postnatally and in the infant eyes with and without history of AHT. Additionally, GFAP staining was present in the astrocytes of fetal and infant eyes. All examined markers were expressed by Müller cells at different developmental stages highlighting the plasticity of Müller cells during the development of the human eye. GFAP should be cautiously used as a marker for AHT as it was also expressed in fetal astrocytes and Müller cells in eyes without history of AHT., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Age-related disorders, such as Alzheimer's disease (AD) and age-related macular degeneration (AMD) share common features such as amyloid-β (Aβ) protein accumulation. Retinal deposition of Aβ aggregates in AMD patients has suggested a potential link between AMD and AD. In the present study, we analyzed the expression pattern of a focused set of miRNAs, previously found to be involved in both AD and AMD, in the retina of a triple transgenic mouse model of AD (3xTg-AD) at different time-points. Several miRNAs were differentially expressed in the retina of 3xTg-AD mice, compared to the retina of age-matched wild-type (WT) mice. In particular, bioinformatic analysis revealed that miR-155 had a central role in miRNA-gene network stability, regulating several pathways, including apoptotic and inflammatory signaling pathways modulated by TNF-related apoptosis-inducing ligand (TNFSF10). We showed that chronic treatment of 3xTg-AD mice with an anti-TNFSF10 monoclonal antibody was able to inhibit the retinal expression of miR-155, which inversely correlated with the expression of its molecular target SOCS-1. Moreover, the fine-tuned mechanism related to TNFSF10 immunoneutralization was tightly linked to modulation of TNFSF10 itself and its death receptor TNFRSF10B, along with cytokine production by microglia, reactive gliosis, and specific AD-related neuropathological hallmarks (i.e., Aβ deposition and Tau phosphorylation) in the retina of 3xTg-AD mice. In conclusion, immunoneutralization of TNFSF10 significantly preserved the retinal tissue in 3xTg-AD mice, suggesting its potential therapeutic application in retinal degenerative disorders., (© 2021. The Author(s).)

Purpose: Migration and integration remain critical challenges for stem cell replacement therapy. Glial barriers play an important role in preventing cell migration and integration. The purpose of this study was to investigate the effect and mechanisms of chondroitinase ABC on the migration of murine retinal progenitor cells (mRPCs) transplanted into the subretinal space of B6 mice., Methods: mRPCs were harvested from the neural retinas of P1 enhanced green fluorescent protein (GFP) B6 mice. Two μl containing 2 × 10 5 expanded RPCs alone or combined with chondroitinase ABC in suspension were injected into the subretinal space of the recipient B6 mice. Immunohistochemistry was performed on the recipient B6 retinas to evaluate the glial barrier formation and migration of the mRPCs. Western blotting was also used to check the expression of the glial barriers., Results: Glial fibrillary acidic protein (GFAP) and vimentin could be seen around the transplanted mRPCs in the B6 mice. Formation of glial barriers prevented the migration of donor cells into the retinal layers. Chondroitinase ABC promoted the migration and survival rates of the engrafted retinal progenitor cells in the retinal layers of recipient B6 mice. Injection induced upregulation of GFAP, chondroitin, and CD44 expression. Chondroitinase ABC disrupted the glial barriers. The CD44 around the mRPCs was much lower in the chondroitinase group. However, the CD44 in the retinal layers was considerably higher in the chondroitinase group. With the employment of chondroitinase ABC, more cells migrated into the outer nuclear layer or inner nuclear layer. The chondroitin and CD44 expression decreased 3 weeks after transplantation in the chondroitinase ABC group., Conclusions: Chondroitinase ABC degraded glial barriers and enhanced the migration of transplanted mouse retinal progenitor cells. Chondroitinase ABC may also have induced activation of the CD44 signaling pathway to exert the effect., (Copyright © 2021 Molecular Vision.)

Diabetic retinopathy (DR) is the most common diabetic neurovascular complication, and the leading cause of preventable blindness among working-age individuals. Recently, agmatine, the endogenous decarboxylated L-arginine, has gained attention as a pleiotropic agent that modulates the diabetes-associated decline in quality of life, and exhibited varied protective biological effects. Diabetes was induced by a single streptozotocin (STZ, 50 mg/kg, i.p.) injection. When diabetes was verified, the animals were randomly allocated into three groups (16 rat each); diabetic, agmatine-treated diabetic (1 mg/kg, daily, for 12 weeks), and control group. Blood glucose homeostasis, retinal redox status, apoptotic parameters, nitric oxide synthase (NOS), nitric oxide (NO), vascular endothelial growth factor (VEGF), glutamate, glutamine, glutamine synthase (GS) activity, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and mitogen-activated protein kinase (MAPKs) pathways were assayed biochemically. Retinal vascular permeability was measured. Retinal morphology was evaluated by hematoxylin and eosin staining. Retinal N-methyl-D-aspartic acid receptor1 (NMDAR1) and glutamate aspartate transporter (GLAST) mRNA were quantified. Glucose transporter 1, pro-caspase3, and glial fibrillary acidic protein (GFAP) expression were quantified by immunohistochemistry. Chronic agmatine treatment abrogated STZ-induced retinal neurodegeneration features including gliosis, and neuronal apoptosis, restored retinal vascular permeability, mostly through antioxidant, anti-apoptotic capacity, abolishing glutamate excitotoxicity, modulating the activity of NMDARs, MAPKs/NFκB, and NOS/NO pathways. By restoring the molecular and functional background of retinal neurovascular homeostatic balance, agmatine would be appropriate therapeutic option acting upstream of the DR, impeding its progression.

Ionising radiation interacts with lenses and retinae differently. In human lenses, posterior subcapsular cataracts are the predominant observation, whereas retinae of adults are comparably resistant to even relatively high doses. In this study, we demonstrate the effects of 2 Gy of low linear energy transfer ionising radiation on eyes of B6C3F1 mice aged postnatal day 2. Optical coherence tomography and Scheimpflug imaging were utilised for the first time to monitor murine lenses and retinae in vivo. The visual acuity of the mice was determined and histological analysis was conducted. Our results demonstrated that visual acuity was reduced by as much as 50 % approximately 9 months after irradiation in irradiated mice. Vision impairment was caused by retinal atrophy and inner cortical cataracts. These results help to further our understanding of the risk of ionising radiation for human foeti (∼ 8 mo), which follow the same eye development stages as neonatal mice., (Copyright © 2020. Published by Elsevier Ltd.)

Determine the impact of the mTOR inhibitor, rapamycin, on the hyperglycemia-induced expression of vascular endothelial growth factor (VEGF) and the production of reactive oxygen species (ROS) in retinal cells. Rats made hyperglycemic for 8 weeks by streptozotocin, as well as control rats, received i.p. rapamycin (1 mg/kg) for 3 days prior to immunostaining of their retinas with anti-VEGF and anti-glial fibrillary acidic protein (GFAP) and measuring retinal protein levels of VEGF and GFAP by Western blotting. In other experiments, flow cytometry analysis of ethidium fluorescence determined intracellular ROS levels in the absence or presence of rapamycin (1 μM) under normoglycemic (5.5 mM) and hyperglycemic (25 mM) conditions in a rat retinal Müller cell line (TR-MUL5) and primary human retinal microvascular endothelial cells (HRMECs). In the diabetic retina, VEGF was elevated and colocalized with the glial marker, GFAP, whose level was also elevated. Treatment with rapamycin inhibited the diabetes-induced VEGF and GFAP increases. We also found that raising extracellular glucose from 5.5 mM to 25 mM resulted in significant rapamycin-sensitive increases in the ROS levels of TR-MUL5 cells and HRMECs. In rat retina, rapamycin attenuates the diabetes-induced VEGF overexpression, and in cultured Müller cells and HRMECs, inhibits the hyperglycemia-induced boost ROS.

Introduction: Research in to the pathophysiology of the complex layers of retinal and sub-retinal cells is hampered by inadequate recognition of particular cells and tissues. A comprehensive panel of antibodies recognising retinal tissues is lacking. Our purpose was to determine the value of a panel of antibodies labelling various cells in the human retina., Method: Five groups of antibodies labelled frozen sections of retinas: (1) protein kinase C-α, Glutamine Synthetase (GS) and ionized calcium-binding adapter molecule 1 (Iba1); (2) Parvalbumin, Calretinin and glial fibrillary acidic protein (GFAP); (3) Thy1, GS and Iba1; (4) Rhodopsin, GS and Iba1; and (5) Brn3a, Rhodopsin and protein kinase C-α. The distribution of these antigens were determined by confocal microscopy and calculated grey value of each antibody in each layer of the retina by Image J., Results: Different antibodies determined certain retinal layers. Thy 1 is a good determinant of the ganglion cell layer, whilst GS is present in all layers except the photoreceptor layer. Brn3a is specific for the ganglion cell layer whilst parvalbumin marks the ganglion cell layer and the outer plexiform layer. Rhodopsin strongly marks the photoreceptor layer, but this is also marked weakly by GFAP., Conclusion: The multiple labelling of human retinal cells brings further understanding of the biological characteristics and functions of these cells, and provides a theoretical basis for their possible role in diseases. In the growing field of human retina research, our data may provide a point of reference for future studies of the human retina.

Amyloid-β (Aβ), reported as a significant constituent of drusen, was implicated in the pathophysiology of age-related macular degeneration (AMD), yet the identity of the major pathogenic Aβ species in the retina has remained hitherto unclear. Here, we examined the in-vivo retinal impact of distinct supramolecular assemblies of Aβ. Fibrillar (Aβ40, Aβ42) and oligomeric (Aβ42) preparations showed clear biophysical hallmarks of amyloid assemblies. Measures of retinal structure and function were studied longitudinally following intravitreal administration of the various Aβ assemblies in rats. Electroretinography (ERG) delineated differential retinal neurotoxicity of Aβ species. Oligomeric Aβ42 inflicted the major toxic effect, exerting diminished ERG responses through 30 days post injection. A lesser degree of retinal dysfunction was noted following treatment with fibrillar Aβ42, whereas no retinal compromise was recorded in response to Aβ40 fibrils. The toxic effect of Aβ42 architectures was further reflected by retinal glial response. Fluorescence labelling of Aβ42 species was used to detect their accumulation into the retinal tissue. These results provide conceptual evidence of the differential toxicity of particular Aβ species in-vivo, and promote the mechanistic understanding of their retinal pathogenicity. Stratifying the impact of pathological Aβ aggregation in the retina may merit further investigation to decipher the pathophysiological relevance of processes of molecular self-assembly in retinal disorders.

Purpose: Dysregulation of the complement cascade contributes to a variety of retinal dystrophies, including age-related macular degeneration (AMD). The central component of complement, C3, is expressed in abundance by macrophages in the outer retina, and its ablation suppresses photoreceptor death in experimental photo-oxidative damage. Whether this also influences macrophage reactivity in this model system, however, is unknown. We investigate the effect of C3 ablation on macrophage activity and phagocytosis by outer retinal macrophages during photo-oxidative damage., Methods: Age-matched C3 knockout (KO) mice and wild-type (WT) C57/Bl6 mice were subjected to photo-oxidative damage. Measurements of the outer nuclear layer (ONL) thickness and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to assess pathology and photoreceptor apoptosis, respectively. Macrophage abundance and phagocytosis were assessed with immunolabeling for pan-macrophage and phagocytic markers, in conjunction with TUNEL staining in cohorts of C3 KO and WT mice., Results: The C3 KO mice exhibited protection against photoreceptor cell death following photo-oxidative damage, which was associated with a reduction in immunoreactivity for the stress-related factor GFAP. In conjunction, there was a reduction in IBA1-positive macrophages in the outer retina compared to the WT mice and a decrease in the number of CD68-positive cells in the outer nuclear layer and the subretinal space. In addition, the engulfment of TUNEL-positive and -negative photoreceptors by macrophages was significantly lower in the C3 KO mice cohort following photo-oxidative damage compared to the WT cohort., Conclusions: The results show that the absence of C3 mitigates the phagocytosis of photoreceptors by macrophages in the outer retina, and the net impact of C3 depletion is neuroprotective in the context of photo-oxidative damage. These data improve our understanding of the impact of C3 inhibition in subretinal inflammation and inform the development of treatments for targeting complement activation in diseases such as AMD., (Copyright © 2020 Molecular Vision.)

Light insult causes photoreceptor death. Few studies reported that continuous exposure to light affects horizontal, Müller and ganglion cells. We aimed to see the effect of constant light exposure on bipolar and amacrine cells. Adult Sprague-Dawley rats were exposed to 300 or 3000 lux for 7 days in 12-h light: 12-h dark cycles (12L:12D). The latter group was then exposed to 24L:0D for 48 h to induce significant damage. The same animals were reverted to 300 lux and reared for 15 days in 12L:12D cycles. They were sacrificed on different days to find the degree of retinal recovery, if any, from light injury. Besides photoreceptor death, continuous light for 48 h resulted in downregulation of parvalbumin in amacrine cells and recoverin in cone bipolar cells (CBC). Rod bipolar cells (RBC) maintained an unaltered pattern of PKC-α expression. Upon reversal, there were increased expressions of parvalbumin in amacrine cells and recoverin in CBC, while RBC showed an increasing trend of PKC-α expression. The data show that damage in bipolar and amacrine cells after exposure to intense, continuous light can be ameliorated upon reversal to normal LD cycles to which the animals were initially acclimated to., (© 2020 American Society for Photobiology.)

The present study was aimed to reveal the function of extracellular RNAs (exRNAs) in retinal ischemia reperfusion (I/R) injury, and evaluate whether RNase administration can effectivelyreduce I/Rinjury. A retinal I/R injury C57BL/6J wild-type mice model was established by elevating intraocular pressure for 1 h. All mice received 3 doses of RNase or the same dose of normal saline at different time points. After 7 days of reperfusion, retinal damage was quantified by counting retinal ganglion cells and measuring retinal layer thickness. The apoptotic retinal cells were detected by the TUNEL experiment, and the expressions of caspase-3, proinflammatory cytokines in retinal tissues, and glial fibrillary acidic protein (GFAP) protein and mRNA were detected to determine the underlying mechanism. It was found that RNase administration (1) reduced the significant loss of retinal morphology caused by I/R injury; (2) down-regulated the expression of NF-κBp65, IL-6 and GFAP relative to the I/R mice; (3) decreased the apoptosis of retinal cells and the levels of caspase-3; (4) attenuated exRNAs levels in retinal tissues on day 7 after retinal I/R. In short, increased exRNAs may contribute to retinal I/R damages in mice, and RNase therapy can effectively attenuate retinal damage by reducing inflammatory response and apoptosis., Competing Interests: Declaration of Competing Interest Without conflict of interest in the submission of our manuscript., (Copyright © 2020. Published by Elsevier B.V.)

Purpose: Pathological neovascularization and fibrosis are common pathological changes of many retinal diseases, such as proliferative retinopathy (PR) and age-related macular degeneration (AMD). Treatment modalities for these pathological changes are limited. The purpose of the present study was to test the effects of palmitoylethanolamide (PEA), an endocannabinoid mimetic amide, on retinal neovascularization and fibrosis and to determine its molecular mechanism of action., Methods: A rat Müller cell line (rMC-1), a mouse model of oxygen-induced retinopathy (OIR), and the very-low-density lipoprotein receptor (VLDLR) knockout mouse model were used. PEA was intraperitoneally injected or orally administrated in animal models. Inflammation and profibrotic changes were evaluated by western blot analysis. Glial fibrillary acidic protein (GFAP) and peroxisome proliferator-activated receptor alpha (PPARα) were measured by RT-PCR and western blot analysis., Results: Profibrotic changes were present in OIR and Vldlr-/- retinas. PEA significantly alleviated inflammation and inhibited neovascularization in OIR and Vldlr-/- retinas and suppressed profibrotic changes in OIR and Vldlr-/- retinas. Moreover, PEA potently suppressed Müller gliosis in these retinas. In rMC-1 cells, PEA suppressed Müller gliosis, reduced inflammatory cytokines, and attenuated profibrotic changes. Further, both mRNA and protein levels of PPARα were elevated in the retina under PEA treatment, and the effects of PEA were abolished in Pparα-/- OIR mice., Conclusions: PEA reduced retinal neovascularization and fibrotic changes and suppressed Müller gliosis in experimental PR and neovascular AMD by activating PPARα. PEA may be a potential treatment for retinopathies with pathological neovascularization and fibrosis.

Purpose: To investigate the effect of a selective aquaporin 4 (AQP4) inhibitor, 2-(nicotinamide)-1,3,4-thiadiazole (TGN-020), on the expression of vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS) production, as well as on the retinal edema in diabetic retina., Methods: Intravitreal injections of bevacizumab, TGN-020, or phosphate-buffered saline (PBS) were performed on streptozotocin-induced diabetic rats. Retinal sections were immunostained for anti-glial fibrillary acidic protein (GFAP), anti-AQP4, and anti-VEGF. Protein levels of VEGF from collected retinas were determined by Western blot analysis. In addition, retinal vascular leakage of Evans Blue was observed in the flat-mounted retina from the diabetic rats in the presence or absence of TGN-020. Volumetric changes of rat retinal Müller cells (TR-MUL5; transgenic rat Müller cells) and intracellular levels of ROS were determined using flow cytometry analysis of ethidium fluorescence in the presence or absence of TGN-020 or bevacizumab under physiological and high glucose conditions., Results: In the diabetic retina, the immunoreactivity and protein levels of VEGF were suppressed by TGN-020. AQP4 immunoreactivity was higher than in the control retinas and the expressions of AQP4 were co-localized with GFAP. Similarly to VEGF, AQP4 and GFAP were also suppressed by TGN-020. In the Evans Blue assay, TGN-020 decreased leakage in the diabetic retinas. In the cultured Müller cells, the increase in cell volumes and intracellular ROS production under high glucose condition were suppressed by exposure to TGN-020 as much as by exposure to bevacizumab., Conclusion: TGN-020 may have an inhibitory effect on diabetic retinal edema.

Purpose: Permanent loss of visual function after rhegmatogenous retinal detachment can occur despite successful surgical reattachment in humans. New treatment modalities could be explored in a detachment model with loss of retinal function. In previous porcine models, retinal function has returned after reattachment, regardless of height and duration of detachment. Difference in retinal tension between the models and the disease might explain these different outcomes. This study investigates, for the first time in an in vivo porcine model, another characteristic of rhegmatogenous retinal detachment - the loss of retinal tension., Methods: Left eyes (n = 12) of 3-month-old domestic pigs were included. Baseline multifocal electroretinogram (mfERG) and a fundus photograph were obtained following anaesthesia (isoflurane). The pigs were vitrectomized, saline was injected subretinally, and the RPE was removed. The eyes were evaluated at 2, 4 and 6 weeks after surgery. Four eyes were enucleated at each evaluation for histologic examinations., Results: A retinal detachment structurally resembling rhegmatogenous retinal detachment was induced in 11 out of 12 pigs. MfERG amplitudes were significantly decreased despite partial reattachment four and 6 weeks after detachment. The retinal thickness decreased with 27%, the inner nuclear layer degenerated, Müller cells hypertrophied, and outer segments were lost. In the ganglion cell layer, cellularity increased and there was cytoplasmic staining with Cyclin D1. Vimentin and GFAP staining for glial cells increased. After 2 weeks of detachment, the ganglion cells had lost their nucleus and nucleolus., Conclusions: Loss of retinal tension in the detached retina seems to induce permanent damage with loss of retinal function. Death of ganglion cells, observed as soon as 2 weeks after detachment, explains the permanent loss of retinal function. The new model enables investigations of time-relationship between retinal detachment and lasting damage in addition to exploration of novel treatment modalities., (© 2019 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation.)

Purpose: To investigate the function and expression of the PGE2 receptors EP1-4 in rat retinal ischemia-reperfusion (I/R) injury and to determine the regulatory role of resveratrol (RES) in this process., Methods: In vitro, we stimulated primary astrocytes extracted from the optic disc of rats with epidermal growth factor (EGF) and RES, and detected the location of EP1-4 expression with immunofluorescence. The expression of antiglial fibrillary acidic protein (GFAP), EGF receptor (EGFR), inducible NOS (iNOS), and EP1-4 in astrocytes was detected with western blotting. In vivo, we established an I/R injury model and RES treatment model with Sprague-Dawley rats. Changes in the thickness of the inner retina were observed with hematoxylin and eosin (H&E) staining. EP1-4 localization in the retina was observed with immunohistochemistry. The expression of COX-2, iNOS, and EP1-4 in the control and model groups was detected with western blotting., Results: In this study, immunofluorescence and immunohistochemistry showed that EP1-4 are expressed in astrocytes and the rat retina. EGF stimulation increased the expression of EGFR, iNOS, EP1, EP2, and EP4 in astrocytes. The expression of EP1-4 was statistically significantly increased on the third day after model induction, and EP1-4 expression decreased to normal levels on day 7. EGF and RES mediated the decrease in the expression of EP2. RES treatment significantly reduced retinal damage and RGC loss, as demonstrated by the relatively intact tissue structure on day 7 observed with H&E staining. Moreover, inflammation was associated with this I/R injury model, as demonstrated by the early induction of proinflammatory mediators, and this inflammation was significantly attenuated after RES treatment., Conclusions: These results indicate that the COX-2/PGE2/EPs pathway is involved in retinal damage and astrocyte inflammation. In addition, the results suggest that the neuroprotective effects of RES may be associated with decreased production of inflammatory mediators. These results suggest that the PGE2 receptor may be a key factor in the treatment of neurodegenerative diseases, and that RES may be used as a possible therapeutic strategy for glaucoma., (Copyright © 2020 Molecular Vision.)

Objectives: To testify that endothelial cells (ECs) induce astrocyte maturation by leukaemia inhibitory factor (LIF) secretion., Materials and Methods: In vivo experiments, mice bearing floxed alleles of YAP were crossed with mice expressing a Cre recombinase driven by the endothelial Tek promoter (Tek-Cre) to finally obtain the following three genotypes: YAP f/f , Tek-Cre; YAP f/w , Tek-Cre; and YAP f/f . Retinal vascularization and astrocyte network were evaluated by whole-mount fluorescence and Western blotting. In vitro, experiments were performed in an astrocyte and human microvascular endothelial cell (HMEC-1) coculture model to analyse the mechanisms underlying the effect of endothelial YAP on astrocytes., Results: In vivo, YAP f/f ;Tek-Cre mice showed delayed angiogenesis, sparse vessels and decreased glial fibrillary acidic protein (GFAP)+ astrocytes but aberrant growth of endothelial networks and immature astrocytes (platelet-derived growth factor A, PDGFRA+ astrocytes) overgrowth. In vitro, Yap deletion attenuated the LIF release that delayed the maturation of retinal astrocyte which was consistent with the results of HMEC-1-astrocyte coculture. The effect of YAP overexpression on LIF-LIFR axis in HMEC-1 interferes the GFAP expression of astrocyte. In contrast, LIF protein rescues the astrocytic GFAP expression when EC YAP was inhibited by siRNAs., Conclusions: We show that EC yes-associated protein (YAP) is not only a critical coactivator of Hippo signalling in retinal vessel development but also plays an essential role in retinal astrocyte maturation by regulating LIF production., (© 2020 The Authors. Cell Proliferation published by John Wiley & Sons Ltd.)

Injured optic nerves induce death in almost all retinal ganglion cells (RGC) and cause a loss of axons. To date, we have studied injured RGC axon regeneration by using a traumatic optic nerve injury (TONI) rodent model, and we revealed that axonal regeneration is induced by the graft of an autologous peripheral nerve. The efficient approach to the regeneration of axons thus needs an environmental adjustment of RGC. However, the RGC environment induced by TONI remains unknown. Here, we analyzed female and male C57BL/6 mouse retinal tissue alterations in detail after TONI and focused on the major phospholipid species that are enriched in the whole retina. Reactive astrocyte accumulation, glia scar formation, and demyelination were observed in the injured optic nerve area, while RGC cell death, astrocyte accumulation, and Glial fibrillary acidic protein (GFAP) positive Müller cell increases were detected in the retinal layer. Furthermore, phosphatidylinositol (PI) 18:0/20:4 was localized to three nuclear layer structures: the ganglion cell layer (GCL), the inner nuclear layer (INL), and the outer nuclear layer (ONL) in control retina; however, the localization of 18:0/20:4 PI in TONI was disturbed. Meanwhile, phosphatidylserine (PS) 18:0/22:6 showed that the expression was specifically in the inner plexiform layer (IPL) with similar signal intensity in both cases. Other PS species and phosphatidylethanolamine (PE) were differentially localized in the retinal layer; however, the expressions of PE including docosahexaenoic acid (DHA) were affected by TONI. These results suggest that not only GCL but also other retinal layers were influenced by TONI., (© 2019 Wiley Periodicals, Inc.)

Purpose: To investigate whether reduced Sox9 function exerts neuroprotection in light-induced retinal damage in rats and to explore the potential mechanism behind it., Methods: Retinal light damage was used as a model for retinal degeneration. Two weeks before light damage in adult Sprague Dawley (SD) rats, the Sox9-shRNA lentiviral vector was intravitreally injected. On days 3, 7, and 14, retinal function was assessed using electroretinography (ERG), and the thickness of the outer nuclear layer (ONL) was measured in hematoxylin and eosin (HE) stained sections. The protein levels of glial fibrillary acidic protein (GFAP), vimentin, nestin, and chondroitin sulfate proteoglycans (Cspgs), which are related to gliosis and extracellular matrix (ECM) remodeling, were observed using western blot analysis. The expression of GFAP was further evaluated by immunohistochemistry., Results: On days 3, 7, and 14 after light damage, the thickness of the ONL and the amplitudes of the ERG waves were significantly better preserved in the Sox9-shRNA group when compared with the control group. The protein levels of GFAP, vimentin, nestin, and Cspgs were significantly downregulated in the Sox9-shRNA group. Furthermore, the staining intensity and the spatial distribution of GFAP in the retinas were also obviously attenuated at every studied time point., Conclusions: Intravitreal injection of the Sox9-shRNA lentiviral vector preserved rat retinal morphology and function after light damage and downregulated GFAP, vimentin, nestin, and Cspgs, which are related to Müller cell gliosis and ECM remodeling. The results indicate that Sox9 might be a potential therapeutic target for retinal degenerative diseases., (Copyright © 2019 Molecular Vision.)

Naturally occurring cell death is a fundamental developmental mechanism for regulating cell numbers and sculpting developing organs. This is particularly true in the nervous system, where large numbers of neurons and oligodendrocytes are eliminated via apoptosis during normal development. Given the profound impact of death upon these two major cell populations, it is surprising that developmental death of another major cell type-the astrocyte-has rarely been studied. It is presently unclear whether astrocytes are subject to significant developmental death, and if so, how it occurs. Here, we address these questions using mouse retinal astrocytes as our model system. We show that the total number of retinal astrocytes declines by over 3-fold during a death period spanning postnatal days 5-14. Surprisingly, these astrocytes do not die by apoptosis, the canonical mechanism underlying the vast majority of developmental cell death. Instead, we find that microglia engulf astrocytes during the death period to promote their developmental removal. Genetic ablation of microglia inhibits astrocyte death, leading to a larger astrocyte population size at the end of the death period. However, astrocyte death is not completely blocked in the absence of microglia, apparently due to the ability of astrocytes to engulf each other. Nevertheless, mice lacking microglia showed significant anatomical changes to the retinal astrocyte network, with functional consequences for the astrocyte-associated vasculature leading to retinal hemorrhage. These results establish a novel modality for naturally occurring cell death and demonstrate its importance for the formation and integrity of the retinal gliovascular network., Competing Interests: The authors have declared that no competing interests exist.

The current study was undertaken to investigate whether histone deacetylases (HDACs) can modulate the viability of retinal ganglion cells (RGCs) and the activity of glial cells in a mouse model of retinal ischemia-reperfusion (IR) injury. C57BL/6J mice were subjected to constant elevation of intraocular pressure for 60 min to induce retinal IR injury. Expression of macroglial and microglial cell markers (GFAP and Iba1), hypoxia inducing factor (HIF)-1α, and histone acetylation was analyzed after IR injury. To investigate the role of HDACs in the activation of glial cells, overexpression of HDAC1 and HDAC2 isoforms was performed. To determine the effect of HDAC inhibition on RGC survival, trichostatin-A (TSA, 2.5 mg/kg) was injected intraperitoneally. After IR injury, retinal GFAP, Iba1, and HIF-1α were upregulated. Conversely, retinal histone acetylation was downregulated. Notably, adenoviral-induced overexpression of HDAC2 enhanced glial activation following IR injury, whereas overexpression of HDAC1 did not significantly affect glial activation. TSA treatment significantly increased RGC survival after IR injury. Our results suggest that increased activity of HDAC2 is closely related to glial activation in a mouse model of retinal IR injury and inhibition of HDACs by TSA showed neuroprotective potential in retinas with IR injuries.

Objective: The Spontaneously Diabetic Torii (SDT) fatty rat, established by introducing the fa allele (obesity gene) of the Zucker fatty rat into the SDT rat genome, is a new model of obese type 2 diabetes. We studied the pathologic features of diabetic retinopathy (DR) in this animal., Methods: The eyes of SDT fatty, SDT (controls), and Sprague Dawley (SD) rats (normal controls) were enucleated at 8, 16, 24, 32, and 40 weeks of age ( n = 5-6 for each rat type at each age). The retinal thicknesses, numbers of retinal folds, and choroidal thicknesses were evaluated. Immunostaining for glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF) was performed. Quantitative analyses of the immunopositive regions were performed using a cell-counting algorithm., Results: The retinas tended to be thicker in the SDT fatty rats and SDT rats than in the SD rats; the choroids tended to be thicker in the SDT fatty rats than in the SD rats. The retinal folds in the SDT fatty rats developed earlier and were more severe than in the SDT rats. Quantitative analyses showed that the GFAP- and VEGF-positive regions in the retinas of the SDT fatty rats were significantly larger than those of the SDT rats., Conclusions: SDT fatty rats developed more severe DR earlier than the SDT rats. The SDT fatty rats might be useful as a type 2 diabetes animal model to study DR., Competing Interests: All authors declare that there are no conflicts of interest regarding the publication of this paper. Drs. Ohta and Sasase are employees of Japan Tobacco Inc., (Copyright © 2019 Yoshiaki Tanaka et al.)

Diabetic retinopathy (DR) is recognized as one of the leading causes of blindness worldwide. Searching and validation for a novel therapeutic strategy to prevent its progress are promising. This work aimed to assess the retinal protective effects of duloxetine (DLX) in Alloxan-induced diabetic mice model. Animals were equally and randomly divided to four groups (eight mice per group); group 1: is the control group, 2: diabetic group, 3&4: diabetic and after 9 weeks received DLX for 4 weeks (15 mg/kg and 30 mg/kg), respectively. Quantitative real-time PCR (qPCR) analysis revealed nerve growth factor (NGF), inducible nitric oxide synthase (iNOS) and transforming growth factor beta (TGF-β) genes upregulation in the diabetic group compared to controls. Also, increased retinal malondialdehyde (MDA) and the decline of reduced glutathione (GSH) levels were observed. The morphometric analysis of diabetic retina revealed a significant reduction in total retinal thickness compared to control. Diabetic retinal immunostaining and Western blot analyses displayed glial fibrillary acidic protein (GFAP) and vascular endothelial cell growth factor (VEGF) proteins expression upregulation as well as glucose transporter-1 (GLUT-1) downregulation comparing to controls. However, DLX-treated groups showed downregulated NGF, iNOS, and TGF-β that was more obviously seen in the DLX-30 mg/kg group than DLX-15 mg/kg group. Furthermore, these groups showed amelioration of the oxidative markers; MDA and GSH, retaining the total retinal thickness nearly to control, GFAP and VEGF downregulation, and GLUT-1 upregulation compared to diabetic group. Taken together, it could be summarized that duloxetine can attenuate DR via the anti-inflammatory and the anti-oxidative properties as well as modulating the angiogenic and the neurotrophic factors expressions. This could hopefully pave the road to be included in the novel list of the therapeutic regimen for DR after validation in the clinic., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: In the earliest stages of diabetic retinopathy (DR), a dysfunction of Müller cells, characterized by high levels of glial fibrillary acidic protein (GFAP), and aquaporins (AQP), has been observed. Although chronic hyperglycemia causes the activation of Müller cells, the effect of glycemic fluctuations is yet unknown. The aim of the study was to analyze the impact of glucose variability on rat retinal Müller cells (rMC-1) adapted to either normal (5 mM) or high (25 mM) glucose levels., Methods: rMC-1 were cultured in a medium containing either 5 mM (N cells) or 25 mM of glucose (H cells) and then incubated for 96 h in a medium containing (a) low glucose (either 1-3 or 5 mM), (b) basal glucose (either 5 or 25 mM), (c) high glucose (either 25 or 45 mM), (d) basal and high glucose alternated every 24 h; (e) low- and high glucose alternated every 24 h; (f) basal glucose with episodes of low glucose for 30 min twice a day. Müller cells activation was evaluated by measuring the levels of GFAP, AQP4, and phospho-active extracellular signal-regulated kinase (pERK)., Results: Under both basal and high glucose concentrations rMC-1 were viable, but their response to glucose excursions was different. In N cells kept under normal (5 mM) glucose, a significant glial activation was measured not only in response to constant high glucose but also to alternating low/high glucose. In H cells, adapted to 25 mM glucose, a significant response was observed only after exposition to a lower (5 mM) glucose concentration., Conclusion: Our results highlight Müller cells activation in response to glucose variability and a different susceptibility depending on the basal glucose conditions.

Previous studies have reported that topical exposure to the toll-like receptor (TLR) 9 ligand CpG-ODN causes widespread ocular inflammation, including retinal microglial activation and posterior segment inflammation. Here we sought to determine the effects of systemic exposure to CpG-ODN in the retina and whether this inflammatory response was altered with Cx 3 cr1 deficiency or hyperglycemia. Male non-diabetic Cx 3 cr1 +/gfp and Cx 3 cr1 gfp/gfp littermates (normoglycemic controls) and Cx 3 cr1 +/gfp Ins2 Akita and Cx 3 cr1 gfp/gfp Ins2 Akita diabetic mice were injected intraperitoneally with 40 μg CpG-ODN. Immunofluorescence staining was performed 1 week later to assess the expression of MHC Class II and glial fibrillary acidic protein (GFAP), as well as to identify morphological changes to microglia and changes in retinal macrophage cell density. Systemic exposure to CpG-ODN induced the upregulated expression of both GFAP on retinal Müller cells and MHC Class II on the retinal vasculature. Additionally, there was an increased accumulation of macrophages in the subretinal space 1 week after exposure to systemic CpG-ODN as well as characteristic morphological changes to microglia indicative of an activated phenotype. These preliminary studies demonstrate that low-grade inflammatory changes were not enhanced in Cx 3 cr1-deficient or diabetic mice, indicating that the inflammatory response to systemic CpG-ODN in the retina is unaltered in the context of Cx 3 cr1 deficiency or prolonged hyperglycemia., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Primary open-angle glaucoma (POAG) is one of the most common causes for blindness worldwide. Although an elevated intraocular pressure (IOP) is the main risk factor, the exact pathology remained indistinguishable. Therefore, it is necessary to have appropriate models to investigate these mechanisms. Here, we analysed a transgenic glaucoma mouse model (βB1-CTGF) to elucidate new possible mechanisms of the disease. Therefore, IOP was measured in βB1-CTGF and wildtype mice at 5, 10 and 15 weeks of age. At 5 and 10 weeks, the IOP in both groups were comparable (P > 0.05). After 15 weeks, a significant elevated IOP was measured in βB1-CTGF mice (P < 0.001). At 15 weeks, electroretinogram measurements were performed and both the a- and b-wave amplitudes were significantly decreased in βB1-CTGF retinae (both P < 0.01). Significantly fewer Brn-3a + retinal ganglion cells (RGCs) were observed in the βB1-CTGF group on flatmounts (P = 0.02), cross-sections (P < 0.001) and also via quantitative real-time PCR (P = 0.02). Additionally, significantly more cleaved caspase 3 + RGCs were seen in the βB1-CTGF group (P = 0.002). Furthermore, a decrease in recoverin + cells was observable in the βB1-CTGF animals (P = 0.004). Accordingly, a significant down-regulation of Recoverin mRNA levels were noted (P < 0.001). Gfap expression, on the other hand, was higher in βB1-CTGF retinae (P = 0.023). Additionally, more glutamine synthetase signal was noted (P = 0.04). Although no alterations were observed regarding photoreceptors via immunohistology, a significant decrease of Rhodopsin (P = 0.003) and Opsin mRNA (P = 0.03) was noted. We therefore assume that the βB1-CTGF mouse could serve as an excellent model for better understanding the pathomechanisms in POAG., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Pregabalin is the first drug to receive FDA approval for treating diabetic neuropathic pain. This study investigated the neuroprotective effect of pregabalin in an experimental model of diabetic retinopathy and tested some possible mechanisms underlying the putative neuroprotective effect. Male Wistar rats received streptozotocin (45 mg/kg) to induce type 1 diabetes mellitus. After two weeks, a course of pregabalin (3, 10 and 30 mg/kg) has been launched for five consecutive weeks. Retinal expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) was estimated by real-time PCR and retinal glutamate content was also estimated. Further, retinal caspase-3 immunoblotting and DNA fragmentation assays determined the degree of apoptosis. Pregabalin improved histopathological abnormalities in diabetic retinas and suppressed the diabetes-enhanced retinal expression of IL-1β, TNF-α, CD 11 b (a surface marker for microglia) while attenuated expression of caspase-3 and DNA fragmentation versus the diabetic group. In addition, diabetic rats treated with pregabalin displayed reductions in retinal glutamate, nitric oxide and malondialdehyde (MDA) and enhanced reduced glutathione (GSH) content versus the diabetic controls. Furthermore, pregabalin enhanced the histopathological picture and reduced fibrosis in the optic nerve of diabetic rats in addition to suppression of the content of the glia fibrillary acidic protein. The findings provide the first evidence demonstrating that pregabalin alleviates retinal neuroinflammation, apoptosis and oxidative stress in an experimental type 1 diabetes mellitus. Therefore, pregabalin might serve as a potential therapy for retinopathy after adequate clinical research., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose : Most complex gangliosides in vertebrates are formed from ganglioside GM3. GM3 deficiency in humans can result in epilepsy and visual impairment. To investigate whether a deficiency of GM3 is involved in visual function, ST3GAL5 -/- mice with mutations in the ST3GAL5 gene-coded GM3 synthase were employed. Materials and Methods : Sixty mice were employed in this study. The glycosphingolipids of mice retinas were analyzed through high performance thin layer chromatography. The morphology of the optic nerves and retinas were evaluated by hematoxylin and eosin staining and immunohistochemical analysis using an anti-glial fibrillary acidic protein (GFAP) antibody. An electroretinogram (ERG) was applied on the eyes of 4, 9, 12, and 14-month-old mice. Also, visual evoked potential (VEP) was applied on 13-month-old mice. Results : The GM3 in the retinas was detected in ST3GAL5 +/+ mice but not ST3GAL5 -/- mice. Also, GM1b and GD1α expressions and lactosylceramide accumulation were found in the ST3GAL5 -/- mouse retinas. There was no significant difference in GFAP expression in the retinas or optic discs between ST3GAL5 +/+ and ST3GAL5 -/- mice. Furthermore, the outcome of ERG and VEP analysis showed no disparity between the two strains in 13 and 14-month-old mice. Conclusion : In the eye, neither histopathological abnormalities nor abnormal functions of the retina were found in GM3-deficient mice. Differing from the situation in patients with GM3 deficiency, the lack of GM3 in mice did not lead to optic nerve atrophy.

The fovea centralis, an anatomically concave pit located at the center of the macula, is avascular, hypoxic, and characteristic of stem-cell niches of other tissues. We hypothesized that in the fovea, undifferentiated retinal-stem-cell-like cells may exist, and that neurogenesis may occur. Hence, we performed an immunohistological study using cynomolgus monkey retinas. After preparing frozen tissue sections of the retina including the foveal pit, immunostaining was performed for glial fibrillary acidic protein (GFAP), nestin, vimentin, neuron-specific class III β-tubulin (Tuj-1), arrestin 4, neurofilament, CD117, CD44, Ki67, and cellular retinaldehyde-binding protein (CRALBP), followed by fluorescence and/or confocal microscopy examinations. Immunostaining of the tissue sections enabled clear observation of strongly GFAP-positive cells that corresponded to the inner-half layer of the foveolar Müller cell cone. The surface layer of the foveal slope was partially costained with GFAP and vimentin. Tuj-1-positive cells were observed in the innermost layer of the foveolar retina, which spanned to the surrounding ganglion cell layer. Moreover, colocalization of Tuj-1 and GFAP was observed at the foveal pit. The coexpression of CD117 and CD44 was found in the interphotoreceptor matrix of the fovea. The foveolar cone stained positive for both nestin and arrestin 4, however, the photoreceptor layer outside of the foveola displayed weak staining for nestin. Colocalization of nestin and vimentin was observed in the inner half of the Henle layer, while colocalization of nestin and neurofilament was observed in the outer half, predominantly. Scattered Ki67-positive cells were observed in the cellular processes of the outer plexiform layer and the ganglion cell layer around the foveola. Immunostaining for CRALBP was negative in most parts of the GFAP-positive area. The Müller cell cone was divided into GFAP-strongly positive cells, presumably astrocytes, in the inner layer and nestin-positive/GFAP-weakly positive radial glia-like cells in the outer layer. These findings indicated that groups of such undifferentiated cells in the foveola might be involved in maintaining morphology and regeneration.

Purpose: Subretinal fibroneovascularization is one of the most common causes of vision loss in neovascular AMD (nAMD). Anti-VEGF therapy effectively inhibits vascular leak and neovascularization but has little effect on fibrosis. This study aimed to identify a combination therapy to concurrently inhibit subretinal neovascularization and prevent fibrosis., Methods: We generated transgenic mice in which induced disruption of Müller cells leads to subretinal neovascularization, which is reliably accompanied by subretinal fibrosis. We conducted Western blots and immunohistochemistry to study changes in transforming growth factor-β (TGFβ) signaling including endoglin, a coreceptor essential for TGFβ signaling, and then tested the effects of monthly intravitreal injection of anti-VEGF-A and anti-endoglin, either alone or in combination, on the development of subretinal fibroneovascularization in our transgenic mice., Results: Müller cell disruption increased expression of TGFβ1, TGFβ type 1 receptor, and phosphorylated-Smad3. Endoglin was strongly expressed in subretinal fibroneovascular tissue. Fluorescein angiography and measurements of retinal vascular permeability indicated that intravitreal anti-VEGF-A in combination with anti-endoglin treatment more efficiently inhibited vascular leak compared with either monotherapy. Immunostaining of retinal wholemounts with antibodies against glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1 indicated that the combination therapy also effectively prevented subretinal fibrosis and inhibited microglial activation. Luminex cytokine assays indicated that intravitreal anti-VEGF-A and anti-endoglin treatment, either alone or in combination, reduced the production of IL33 and macrophage inflammatory protein-3α., Conclusions: Our findings offer a potentially novel combination approach to concurrently managing subretinal neovascularization and fibrosis in nAMD.

Apolipoprotein E (APOE) is a component of lipid-transporting particles and a recognition ligand for receptors, which bind these particles. The APOE isoform ε2 is a risk factor for age-related macular degeneration; nevertheless, APOE absence in humans and mice does not significantly affect the retina. We found that retinal cholesterol biosynthesis and the levels of retinal cholesterol were increased in Apoe -/- mice, whereas cholesterol elimination by metabolism was decreased. No focal cholesterol deposits were observed in the Apoe -/- retina. Retinal proteomics identified the most abundant cholesterol-related proteins in WT mice and revealed that, of these cholesterol-related proteins, only APOA4 had increased expression in the Apoe -/- retina. In addition, there were changes in retinal abundance of proteins involved in proinflammatory and antiinflammatory responses, cellular cytoskeleton maintenance, vesicular traffic, and retinal iron homeostasis. The data obtained indicate that when APOE is absent, particles containing APOA1, APOA4, and APOJ still transport cholesterol in the intraretinal space, but these particles are not taken up by retinal cells. Therefore, cholesterol biosynthesis inside retinal cells increase, whereas metabolism to oxysterols decreases to prevent cells from cholesterol depletion. These and other compensatory changes underlie only a minor retinal phenotype in Apoe -/- mice., (Copyright © 2018 Saadane et al.)

Exposure to ethanol in utero leads to several brain development disorders including retinal abnormalities whose underlying cellular pathogenesis remains elusive. We recently reported that fetal alcohol exposure (FAE) in vervet monkeys induces anomalies of full-field electroretinogram (ERG) waveforms that suggest premature aging of the retina. The goal of this study is to characterize the anatomo-functional mechanisms underlying the retinal changes observed in fetal alcohol exposed (FAE) monkeys, and age- and sex-matched normals. First, we examined in vivo the fundus of the eyes, measured intraocular pressure (IOP) and assessed cone activity using flicker ERG. Second, we investigated ex vivo, protein expression and anatomical organization of the retina using Western blotting, classical histology and immunohistochemistry. Our results indicated that the fundus of the eyes showed both, increased vascularization (tessellated fundus) and IOP in FAE monkeys. Furthermore, light-adapted flicker responses above 15 Hz were also significantly higher in FAE monkeys. Although there were no obvious changes in the overall anatomy in the FAE retina, Glial Fibrillary Acidic Protein (GFAP, a potent marker of astrocytes) immunoreactivity was increased in the FAE retinal ganglion cell layer indicating a strong astrogliosis. These alterations were present in juvenile (2 years old) monkeys and persist in adults (8 years old). Moreover, using specific cell type markers, no significant modifications in the morphology of the photoreceptors, horizontal cells, bipolar cells, and amacrine cells were observed. Our data indicate that FAE does indeed induce anatomical changes within the retinal ganglion cell layer that are reflected in the increased photosensitivity of the cone photoreceptors., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Antibiotics such as gentamicin (an aminoglycoside) and penicillin (a beta-lactam antibiotic) are routinely used in retinal cell and explant cultures. In many cases, these in vitro systems are testing parameters regarding photoreceptor transplantation or preparing cells for transplantation. In vivo, milligram doses of gentamicin are neurotoxic to the retina. However, little is known about the effects of antibiotics to retina in vitro and whether smaller doses of gentamicin are toxic to retinal cells. To test toxicity, retinal cells were dissociated from tiger salamander, placed in culture, and treated with either 20 μg/ml gentamicin, 100 μg/ml streptomycin, 100 U/ml antibiotic/antimycotic, 0.25 μg/ml amphotericin B, or 100 U/ml penicillin G. All dosages were within manufacturer's recommended levels. Control cultures had defined medium only. Cells were fixed at 2 h or 7 days. Three criteria were used to assess toxicity: (1) survival of retinal neurons, (2) neuritic growth of photoreceptors assessed by the development of presynaptic varicosities, and (3) survival and morphology of Mueller cells. Rod cells were immunolabeled for rod opsin, Mueller cells for glial fibrillary acidic protein, and varicosities for synaptophysin. Neuronal cell density was reduced with all pharmacological treatments. The number of presynaptic varicosities was also significantly lower in both rod and cone photoreceptors in treated compared to control cultures; further, rods were more sensitive to gentamicin than cones. Penicillin G (100 U/ml) was overall the least inhibitory and amphotericin B the most toxic of all the agents to photoreceptors. Mueller cell survival was reduced with all treatments; reduced survival was accompanied by the appearance of proportionally fewer stellate and more rounded glial morphologies. These findings suggest that even microgram doses of antibiotic and antimycotic drugs can be neurotoxic to retinal cells and reduce neuritic regeneration in cell culture systems.

Photobiomodulation (PBM) with 670 nm light has been shown to accelerate wound healing in soft tissue injuries, and also to protect neuronal tissues. However, little data exist on its effects on the non-neuronal components of the retina, such as Müller cells (MCs), which are the principal macroglia of the retina that play a role in maintaining retinal homeostasis. The aim of this study was to explore the effects of 670 nm light on activated MCs using in vivo and in vitro stress models. Adult Sprague-Dawley rats were exposed to photo-oxidative damage (PD) for 24 h and treated with 670 nm light at 0, 3 and 14 days after PD. Tissue was collected at 30 days post-PD for analysis. Using the in vitro scratch model with a human MC line (MIO-M1), area coverage and cellular stress were analysed following treatment with 670 nm light. We showed that early treatment with 670 nm light after PD reduced MC activation, lowering the retinal expression of GFAP and FGF-2. 670 nm light treatment mitigated the production of MC-related pro-inflammatory cytokines (including IL-1β), and reduced microglia/macrophage (MG/MΦ) recruitment into the outer retina following PD. This subsequently decreased photoreceptor loss, slowing the progression of retinal degeneration. In vitro, we showed that 670 nm light directly modulated MC activation, reducing rates of area coverage by suppressing cellular proliferation and spreading. This study indicates that 670 nm light treatment post-injury may have therapeutic benefit when administered shortly after retinal damage, and could be useful for retinal degenerations where MC gliosis is a feature of disease progression., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose: Geographic atrophy (GA) is the late stage of non-neovascular age-related macular degeneration. A lack of animal models for GA has hampered treatment efforts. Presented herein is a rat model for GA using subretinal injection of sodium iodate (NaIO3)., Methods: Rats were given subretinal injections of NaIO3 (5 μg/μL) using a pico-injector. Fundus photographs and spectral domain optical coherent tomography scans were collected at 1, 3, 7, 14, and 28 days after injection, at which time rats were euthanized and eyes were enucleated. Eyes were either cryopreserved or dissected into retinal and choroidal flatmounts. Fluorescence immunohistochemistry was performed for retinal glial fibrillary acidic protein (activated Müller cells and astrocytes) and vimentin (Müller cells), as well as peanut agglutin lectin (photoreceptors) labeling. RPE/choroids were labeled for RPE65 and CD34. Images were collected on Zeiss confocal microscopes., Results: Fundus photos, spectral domain optical coherent tomography, and RPE65 staining revealed well-demarcated areas with focal loss of RPE and photoreceptors in NaIO3-treated rats. At 1 day after injection, RPE cells appeared normal. By 3 days, there was patchy RPE and photoreceptor loss in the injected area. RPE and photoreceptors were completely degenerated in the injected area by 7 days. A large subretinal glial membrane occupied the degenerated area. Choriocapillaris was highly attenuated in the injected area at 14 and 28 days., Conclusions: The rat model reported herein mimics the photoreceptor cell loss, RPE atrophy, glial membrane formation, and choriocapillaris degeneration seen in GA. This model will be valuable for developing and testing drugs and progenitor cell regenerative therapies for GA.

Sox2 is a transcriptional regulator that is highly expressed in retinal astrocytes, yet its function in these cells has not previously been examined. To understand its role, we conditionally deleted Sox2 from the population of astrocytes and examined the consequences on retinal development. We found that Sox2 deletion does not alter the migration of astrocytes, but it impairs their maturation, evidenced by the delayed upregulation of glial fibrillary acidic protein (GFAP) across the retina. The centro-peripheral gradient of angiogenesis is also delayed in Sox2-CKO retinas. In the mature retina, we observed lasting abnormalities in the astrocytic population evidenced by the sporadic loss of GFAP immunoreactivity in the peripheral retina as well as by the aberrant extension of processes into the inner retina. Blood vessels in the adult retina are also under-developed and show a decrease in the frequency of branch points and in total vessel length. The developmental relationship between maturing astrocytes and angiogenesis suggests a causal relationship between the astrocytic loss of Sox2 and the vascular architecture in maturity. We suggest that the delay in astrocytic maturation and vascular invasion may render the retina hypoxic, thereby causing the abnormalities we observe in adulthood. These studies uncover a novel role for Sox2 in the development of retinal astrocytes and indicate that its removal can lead to lasting changes to retinal homeostasis., (© 2017 Wiley Periodicals, Inc.)

Purpose: To assess the retinal toxicity of an intravitreal injection of infliximab, a monoclonal antibody to tumor necrosis factor α, in a rabbit model., Materials and Methods: Two groups of adult albino rabbits (n = 5) received intravitreal injections of infliximab (0.1 ml) in the study eye and balanced salt solution (BSS, 0.1 ml) in the control eye at baseline. Group 1 was administered with 1.5 mg/0.1 ml, and group 2 was injected with 7.5 mg/0.1 ml of infliximab solution. Electroretinography (ERG) was performed at baseline and at 1, 7, 30, and 45 days after the injection. Visual evoked potentials (VEPs) were recorded at 7 and 45 days after the injection. After the last electrophysiological assessment, the rabbits were euthanized and retinal histopathology and immunhistochemistry for glial fibrillary acidic protein (GFAP) were performed., Results: ERG responses demonstrated no significant deficit in retinal function in eyes injected with infliximab. Mean dark-adapted a-wave and b-wave maximal amplitude and semi-saturation constant values at baseline and throughout the 45 days of follow-up after the injection indicated no remarkable difference in outer retinal function between the control and experimental eyes. VEP responses were similar at each time point (7 and 45 days). No difference was seen in retinal histopathology and immunocytochemistry sections in eyes receiving the 1.5 mg/0.1 ml dose compared to the control eyes. However, increased GFAP labeling in retinal Müller cells was detected in rabbit eyes treated with the 7.5 mg/0.1 ml dose., Conclusions: Intravitreal injection of 1.5 mg/0.1 ml infliximab dose has no toxic effect on the integrity (functional or structural) of the retina in rabbits. A higher dose of 7.5 mg/0.1 ml may be slightly toxic as suggested by positive Müller cell GFAP expression. Additional studies of retinal toxicity at higher doses and after multiple injections are needed to establish the retinal safety of intravitreal infliximab therapy in humans.

Diabetic retinopathy (DR) develops due to hyperglycemia and inflammation-induced vascular disruptions in the retina with connexin43 expression patterns in the disease still debated. Here, the effects of hyperglycemia and inflammation on connexin43 expression in vitro in a mouse model of DR and in human donor tissues were evaluated. Primary human retinal microvascular endothelial cells (hRMECs) were exposed to high glucose (HG; 25 mM) or pro-inflammatory cytokines IL-1β and TNF-α (10 ng/mL each) or both before assessing connexin43 expression. Additionally, connexin43, glial fibrillary acidic protein (GFAP), and plasmalemma vesicular associated protein (PLVAP) were labeled in wild-type (C57BL/6), Akita (diabetic), and Akimba (DR) mouse retinas. Finally, connexin43 and GFAP expression in donor retinas with confirmed DR was compared to age-matched controls. Co-application of HG and cytokines increased connexin43 expression in hRMECs in line with results seen in mice, with no significant difference in connexin43 or GFAP expression in Akita but higher expression in Akimba compared to wild-type mice. On PLVAP-positive vessels, connexin43 was higher in Akimba but unchanged in Akita compared to wild-type mice. Connexin43 expression appeared higher in donor retinas with confirmed DR compared to age-matched controls, similar to the distribution seen in Akimba mice and correlating with the in vitro results. Although connexin43 expression seems reduced in diabetes, hyperglycemia and inflammation present in the pathology of DR seem to increase connexin43 expression, suggesting a causal role of connexin43 channels in the disease progression., Competing Interests: Colin R. Green is a founding scientist of OcuNexus Therapeutics, Inc. (USA) (and previously CoDa Therapeutics, Inc.), which has intellectual property related to the regulation of connexin channels in the treatment of ocular and other diseases.

Immature astrocytes and blood vessels enter the developing mammalian retina at the optic nerve head and migrate peripherally to colonize the entire retinal nerve fiber layer (RNFL). Retinal vascularization is arrested in retinopathy of prematurity (ROP), a major cause of bilateral blindness in children. Despite their importance in normal development and ROP, the factors that control vascularization of the retina remain poorly understood. Because astrocytes form a reticular network that appears to provide a substrate for migrating endothelial cells, they have long been proposed to guide angiogenesis. However, whether astrocytes do in fact impose a spatial pattern on developing vessels remains unclear, and how astrocytes themselves are guided is unknown. Here we explore the cellular mechanisms that ensure complete retinal coverage by astrocytes and blood vessels in mouse. We find that migrating astrocytes associate closely with the axons of retinal ganglion cells (RGCs), their neighbors in the RNFL. Analysis of Robo1; Robo2 mutants, in which RGC axon guidance is disrupted, and Math5 (Atoh7) mutants, which lack RGCs, reveals that RGCs provide directional information to migrating astrocytes that sets them on a centrifugal trajectory. Without this guidance, astrocytes exhibit polarization defects, fail to colonize the peripheral retina, and display abnormal fine-scale spatial patterning. Furthermore, using cell type-specific chemical-genetic tools to selectively ablate astrocytes, we show that the astrocyte template is required for angiogenesis and vessel patterning. Our results are consistent with a model whereby RGC axons guide formation of an astrocytic network that subsequently directs vessel development., (© 2017 Wiley Periodicals, Inc.)

Purpose: The purpose of this study was to characterize retinal degenerative morphologic modifications in Zucker Diabetic Fatty (ZDF) rats, a genetic model of type 2 diabetes, by histologic and immunohistochemical evaluation., Methods: Male lean (?/+; n = 10) and ZDF (fa/fa; n = 20) rats were used. At 24 weeks of age, body weights and blood glucose levels were determined. Eyes were removed and processed for paraffin wax embedding. Sections through the optic disc were stained for hematoxylin and eosin or immunostained for TUNEL, advanced glycation end products (AGEs), glial fibrillary acidic protein (GFAP), glutamate/aspartate transporter (GLAST), isolectin B4, recoverin, retinal pigment epithelium-specific 65-kDa protein, rhodopsin, vimentin, and zonula occludens protein 1. Retinal morphometry, cell counts, glial activation degree and immunoreactivity of AGEs and GLAST were also determined., Results: ZDF rats were observed to be diabetic from week 9 and by week 24. These animals showed retinal morphologic degenerative changes, increased neuroretinal thickness, and decreased number of nuclei. Glial cells activation with massive GFAP upregulation was present. Cellular morphologic modifications were also observed. GLAST immunofluorescence was decreased, whereas AGEs were increased in comparison with lean rats., Conclusions: Spontaneous development of diabetes in ZDF rats results in neuroglia morphologic degenerative changes at 24 weeks of age. This animal model may be useful to understand the pathogenesis of diabetic retinopathy and to screen neuroprotective drugs in diabetes.

Background: Retinal ischaemia is a common feature shared by numerous eye diseases. Ischaemic insult leads to retinal dysfunction and neuronal death. Lycium barbarum polysaccharides are well known for eyesight preservation. We have previously reported the effect of Lycium barbarum polysaccharides on cell death, blood ocular barrier and oxidative stress within 24 h retinal ischaemia. This study focuses on retinal function and looks for ultrastructural and cellular correlates after a relatively long period of reperfusion for 7 days., Methods: Two-hour ischaemia was induced by intraluminal occlusion of the internal carotid artery. Either Lycium barbarum polysaccharides or phosphate-buffered saline was orally pre-administered daily for 7 days before ischaemia and continued for 1, 3 and 7 days after reperfusion. Electroretinogram was performed to evaluate visual function. Paraffin-embedded retinal sections were prepared 7 days after reperfusion and utilized for histological and immunohistochemical analyses., Results: Ischaemia led to sustained inhibition of b-wave amplitude and oscillatory potentials. Lycium barbarum polysaccharide-treated mice exhibited greater b-wave and oscillatory potential responses from days 1 to 7 after reperfusion. In addition, increased number of viable cells and calretinin-positive cells, as well as enhanced immunoreactivity of protein kinase C alpha and attenuated glial fibrillary acidic protein expression, was noted in Lycium barbarum polysaccharide-treated retina., Conclusions: Daily consumption of Lycium barbarum polysaccharides effectively alleviated ischaemia-induced retinal dysfunction as well as reduced correlated neuronal death and glial activation. This prolonged effect could last at least 7 days. It suggested that Lycium barbarum polysaccharides might serve as a neuroprotective agent in ischaemic retinopathies., (© 2017 Royal Australian and New Zealand College of Ophthalmologists.)

Moderate to intense light is reported to damage the chick retina, which is cone dominated. Light damage alters neurotransmitter pools, such as those of glutamate. Glutamate level in the retina is regulated by glutamate-aspartate transporter (GLAST) and glutamine synthetase (GS). We examined immunolocalization patterns and the expression levels of both markers and of glial fibrillary acidic protein (GFAP, a marker of neuronal stress) in chick retina exposed to 2000 lux under 12-h light:12-h dark (12L:12D; normal photoperiod), 18L:6D (prolonged photoperiod), and 24L:0D (constant light) at post-hatch day 30. Retinal damage (increased death of photoreceptors and inner retinal neurons and Müller cell hypertrophy) and GFAP expression in Müller cells were maximal in 24L:0D condition compared to that seen in 12L:12D and 18L:6D conditions. GS was present in Müller cells and GLAST expressed in Müller cell processes and photoreceptor inner segments. GLAST expression was decreased in 24L:0D condition, and the expression levels between 12L:12D and 18L:6D, though increased marginally, were statistically insignificant. Similar was the case with GS expression that significantly decreased in 24L:0D condition. Our previous study with chicks exposed to 2000 lux reported increased retinal glutamate level in 24L:0D condition. The present results indicate that constant light induces decreased expressions of GLAST and GS, a condition that might aggravate glutamate-mediated neurotoxicity and delay neuroprotection in a cone-dominated retina.

Purpose: Diabetic retinopathy (DR) is the most frequently occurring complication of diabetes. Alterations in ubiquitin-proteasome system (UPS) have been associated with several degenerative disorders. Hence, in this study, we investigated the status and role of UPS and ER stress in the retina of diabetic rats., Methods: Diabetes was induced in rats by streptozotocin. Retinal markers, ER stress markers, components of UPS, ERAD, and autophagy were analyzed after 2- and 4-months of diabetes. Apoptosis was analyzed by TUNEL Assay., Results: There were increased acellular capillaries and pericyte loss in diabetic rat retina. Decreased protein expression of UPS components - ubiquitin activating enzyme (E1), deubiquitinating enzymes (UCHL1 and UCHL5), SIAH1 (E3 ligase) and free ubiquitin were observed in the diabetic rats. Increased ER stress markers (ATF6, XBP1, and CHOP), decreased expression of HRD1, declined autophagy (LC3B) and increased apoptosis were observed in diabetic rats. Interestingly, treatment of diabetic rats with a chemical chaperone (4-PBA) restored the levels of DUBs and ameliorated ER stress-induced retinal cell death in type 1 diabetic rats., Conclusion: The declined UPS components: E1 and HRD1 in the retina of diabetic rats could elicit ER stress, and the prolonged ER stress may trigger CHOP-mediated neuronal apoptosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)