Rat Retina Research Articles

Evaluation of the state of the blood-retinal barrier during the development of signs of age-related macular degeneration in OXYS rats

Age-related macular degeneration (AMD) is a multifactorial neurodegenerative disease that is becoming the leading cause of irreversible vision loss in people over 55 years of age. The development of the wet form of AMD is associated with impaired permeability of the blood- retinal barrier (BRB). It was believed that the BRB does not change in the dry form of the disease, but recently it was shown that dysfunction of the BRB may also contribute to its development; however, information about the state of the BRB at different stages of AMD, especially preclinical ones, is limited. The purpose of this study was to assess the possible contribution of changes in BRB permeability to the development of signs of AMD in OXYS rats, a model of the dry form of the disease. During the period when clinical signs of AMD in OXYS rats are absent (age 20 days), during their manifestation (~5 months) and progression (at 12 and 18 months), the permeability of the BRB for Evans blue dye and the retinal contents of the tight junction proteins occludin, claudin-5, and zonula occludens-1 (ZO-1) were assessed. Wistar rats of the same age served as controls. In OXYS rats, a decrease in the permeability of the BRB was detected, which may result in a violation of the trophic supply of the retina, as well as an increase in the level of occludin during the progression of signs of AMD. ZO-1 level decreased with age, but no interstrain differences were detected. Analysis of retinal transcriptomes (RNA-seq data) showed that in rats of both strains changes in the expression of genes included (according to KEGG) in the category of tight junctions are maximum in the period from 20 days to 3 months. In OXYS rats, the mRNA levels of the Dlg1, Cd1d1, Map3k5 and Arhgef2 genes at the age of 3 months and the Crb3, F11r, Cgn, Cd1d1 and Rap2c genes the age of 18 months are different compared to Wistar rats. Such changes in gene expression in the retina of OXYS rats as AMD signs develop indicate the activation of compensatory mechanisms.

GMFB/AKT/TGF-β3 in Müller cells mediated early retinal degeneration in a streptozotocin-induced rat diabetes model.

Retinal degeneration, characterized by Müller cell gliosis and photoreceptor apoptosis, is considered an early event in diabetic retinopathy (DR). Our previous study proposed that GMFB may mediate diabetic retinal degeneration. This study identified GMFB as a sensitive and functional gliosis marker for DR. Compared to the wild type (WT) group, Gmfb knockout (KO) significantly improved visual function, attenuated gliosis, reduced the apoptosis of neurons, and decreased the mRNA levels of tumor necrosis factor α (Tnf-α) and interleukin-1β (Il-1β) in diabetic retinas. Tgf-β3 was enriched by hub genes using RNA sequencing in primary WT and KO Müller cells. Gmfb KO significantly upregulated the transforming growth factor (TGF)-β3 protein level via the AKT pathway. The protective effect of TGF-β3 in the vitreous resulted in significantly improved visual function and decreased the number of apoptotic cells in the diabetic retina. The protection of Gmfb KO in primary Müller cells against high glucose (HG)-induced photoreceptor apoptosis was partially counteracted by TGF-β3 antibody and administration of TGFBR1/2 inhibitors. Nuclear receptor subfamily 3 group C member 1 (NR3C1) binds to the promoter region of Gmfb and regulates Gmfb mRNA at the transcriptional level. NR3C1 was increased in the retinas of early diabetic rats but decreased in the retinas of late diabetic rats. N'-[(1E)-(3-Methoxyphenyl)Methylene]-3-Methyl-1H-Pyrazole-5-Carbohydrazide (DS-5) was identified as an inhibitor of GMFB, having a protective role in DR. We demonstrated that GMFB/AKT/TGF-β3 mediated early diabetic retinal degeneration in diabetic rats. This study provides a novel therapeutic strategy for treating retinal degeneration in patients with DR.

CD40 Ligand-CD40 Interaction Is an Intermediary between Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy.

We aimed to investigate the role of the CD40-CD40 ligand (CD40L) pathway in inflammation-mediated angiogenesis in proliferative diabetic retinopathy (PDR). We analyzed vitreous fluids and epiretinal fibrovascular membranes from PDR and nondiabetic patients, cultures of human retinal microvascular endothelial cells (HRMECs) and Müller glial cells and rat retinas with ELISA, immunohistochemistry, flow cytometry and Western blot analysis. Functional tests included measurement of blood-retinal barrier breakdown, in vitro angiogenesis and assessment of monocyte-HRMEC adherence. CD40L and CD40 levels were significantly increased in PDR vitreous samples. We demonstrated CD40L and CD40 expression in vascular endothelial cells, leukocytes and myofibroblasts in epiretinal membranes. Intravitreal administration of soluble (s)CD40L in normal rats significantly increased retinal vascular permeability and induced significant upregulation of phospho-ERK1/2, VEGF, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). sCD40L induced upregulation of VEGF, MMP-9, MCP-1 and HMGB1 in cultured Müller cells and phospo-ERK1/2, p65 subunit of NF-ĸB, VCAM-1 and VEGF in cultured HRMECS. TNF-α induced significant upregulation of CD40 in HRMECs and Müller cells and VEGF induced significant upregulation of CD40 in HRMECs. sCD40L induced proliferation and migration of HRMECs. We provide experimental evidence supporting the involvement of the CD40L-CD40 pathway and how it regulates inflammatory angiogenesis in PDR.

Transcriptomic Analysis of Retinal Gene in Experimental Retinal Detachment Rats and Exploration of S100A9 and TLR4 in Human Vitreous

Purpose To screen for the differentially expressed genes in experimental retinal detachment rats, and to explore the expression of S100 calcium-binding protein A9 and Toll-like receptor 4 in the vitreous of rhegmatogenous retinal detachment patients. Methods Three rats of experimental retinal detachment and three normal rats were enrolled in the study. Transcriptomics (RNAseq) sequencing technology was used to screen differentially expressed genes in the retinas of the experimental retinal detachment group and the normal group. The selected differentially expressed genes for gene ontology and Kyoto Encyclopedia of Genes and Genomes functional enrichment analysis were performed. In addition, the vitreous of 15 patients with rhegmatogenous retinal detachment and six patients with the control group were collected. The expressions of S100 calcium-binding protein A9 and Toll-like receptor 4 were detected by Elisa, and the differences in expression levels were analyzed statistically. Results A total of 198 differentially expressed genes were screened by RNAseq sequencing, including 118 upregulated genes and 80 downregulated genes. Kyoto Encyclopedia of Genes and Genomes analysis confirmed that the most enriched pathway was the mitogen-activated protein kinase signaling pathway. Compared to the normal group, the expressions of suppressor of cytokine signaling-3, Storkhead box-2, S100 calcium-binding protein A9, Spi-1 proto-oncogene, phosphodiesterase 1B, and kinesin-light chain 1 mRNA in the retinas of the experimental retinal detachment rats were up-regulated, and the expressions of Max interacting protein 1 and the voltage-gated sodium 1 were down-regulated. Compared to the control group, the expressions of S100 calcium-binding protein A9 and Toll-like receptor 4 were upregulated by Elisa in the vitreous humor of rhegmatogenous retinal detachment patients with a statistically significant difference (p all <.05). Conclusion The differentially expressed genes of experimental retinal detachment rats were suppressor of cytokine signaling-3, Storkhead box-2, S100 calcium-binding protein A9, Spi-1 proto-oncogene, phosphodiesterase 1B, kinesin-light chain 1, Max interacting protein 1, voltage-gated sodium 1, etc. The differences of S100 calcium-binding protein A9 and Toll-like receptor 4 expressions between the rhegmatogenous retinal detachment patients and the control group were statistically significant, indicating that they may play a potential role in the inflammatory process of rhegmatogenous retinal detachment.

Human Neural Progenitors Expressing GDNF Enhance Retinal Protection in a Rodent Model of Retinal Degeneration.

Stem cell therapy for retinal degenerative diseases has been extensively tested in preclinical and clinical studies. However, preclinical studies performed in animal models at the early stage of disease do not optimally translate to patients that present to the clinic at a later stage of disease. As the retina degenerates, inflammation and oxidative stress increase and trophic factor support declines. Testing stem cell therapies in animal models at a clinically relevant stage is critical for translation to the clinic. Human neural progenitor cells (hNPC) and hNPC engineered to stably express GDNF (hNPCGDNF) were subretinally injected into the Royal College of Surgeon (RCS) rats, a well-established model for retinal degeneration, at early and later stages of the disease. hNPCGDNF treatment at the early stage of retinal degeneration provided enhanced visual function compared to hNPC alone. Treatment with both cell types resulted in preserved retinal morphology compared to controls. hNPCGDNF treatment led to significantly broader photoreceptor protection than hNPC treatment at both early and later times of intervention. The phagocytic role of hNPC appears to support RPE cell functions and the secreted GDNF offers neuroprotection and enables the extended survival of photoreceptor cells in transplanted animal eyes. Donor cells in the RCS rat retina survived with only limited proliferation, and hNPCGDNF produced GDNF in vivo. Cell treatment led to significant changes in various pathways related to cell survival, antioxidative stress, phagocytosis, and autophagy. A combined stem cell and trophic factor therapy holds great promise for treating retinal degenerative diseases including retinitis pigmentosa and age-related macular degeneration.

Histological study of the effect of hydroxychloroquine sulfate on the retina and the possible protective role of propolis in adult male albino rats

Background Hydroxychloroquine sulfate (HCS) is a widely used antimalarial drug. Recently, it garnered attention about its possible effect against COVID-19. Retinopathy is one of its dangerous side effects which may be untreated. Propolis is a substance made by honeybees and has a neuroprotective effect. Aim The study aimed to assess the impact of HCS on the retina and the possible ameliorative role of propolis in adult male albino rats. Materials and methods In all, 32 adult male albino rats were separated into four groups; group (I): the control group, group II: rats had received 100 mg/kg propolis for 12 weeks, group III: the HCS dose of 55 mg/kg/day had been administered to the rats for a period of 10 days, group IV: rats had received 55 mg/kg/day of HCS concomitant with 100 mg/kg propolis for 10 days and then propolis was continued to complete 12 weeks. Retinal specimens were obtained and processed for histological and morphometric analyses. Results Group III revealed vacuolations of photoreceptors; nuclei are widely separated in the outer and inner nuclear layers Also nuclei are darkly pigmented in the inner nuclear layer and the ganglion cell layer. Statistical analysis showed that there was a significant drop in the average number of ganglion cells when contrasted with the control group. These changes were markedly ameliorated in group HCS and propolis-treated group. Conclusion Hydroxychloroquine sulfate induced damaging effects on the rat retina and receiving propolis could attenuate these effects.

Moringa oleifera extract ameliorates diabetic retinopathy via NF-κB and VCAM-1 pathway in streptozotocin induced diabetic rats

Diabetes is a major cause of mortality and morbidity worldwide, leading to various complications, including diabetic retinopathy (DR), which can result in vision impairment and blindness. DR involves the disruption of the blood retinal barrier (BRB) and the development of neovascularization due to high blood sugar levels. Inflammatory cytokines and cell adhesion molecules play a role in this process, with NF-κB and its down regulators, such as ICAM-1 and VCAM-1, being key players. Moringa oleifera (MO), a plant with medicinal properties, is being investigated for its prospective effect in preventing and treating DR. This study explores the effects of Moringa oleifera extract on streptozotocin (STZ) induced diabetic rats with DR. Molecular docking studies indicate that components of Moringa oleifera extract, namely Quercetin and Myrecetin, strongly bind to key targets involved in DR.To induce DR, rats were given STZ, and after 8 weeks, various factors were evaluated. The results show that MO extract significantly reduces blood glucose levels compared to the diabetes group. Additionally, the extract decreases the expression of VEGF, an angiogenic mediator, and reduces the levels of TNF-α and Total Antioxidant Capacity (TAC), markers of inflammation, in the retinas of diabetic rats. Histopathological analyses reveal improvements in the retinal blood vessels of MO-treated animals compared to the diabetes group. The extract also reduces the overexpression of NF-κB and VCAM-1 in the retina. These findings suggest that MO may be useful in managing diabetic retinopathy by inhibiting angiogenic mediators and regulating NF-κB and VCAM-1.

MG132, Attenuates the Retinal Vascular Injury Through the Upregulation of Nrf2 Expression.

Purpose: This study clarifies the beneficial effects of MG132, a proteasomal inhibitor, on retinal vascular injury mediated by diabetes-induced oxidative stress through nuclear factor erythroid 2-related factor 2 (Nrf2). Methods: Diabetic rats and control animals were randomly assigned to receive MG132 or vehicle for 24 weeks, and human retinal endothelial cells (HRECs) were incubated with normal or high glucose with or without MG132. 26S proteasome activity in the rat retinas or cultured HRECs was measured using Suc-LLVY-7-amido-4-methylcoumarin. NADPH-quinone oxidoreduc-tase (NQO1), heme oxygenase (HO)-1, kelch-like ECH-associated protein 1 (Keap1) and Nrf2 were examined by Western blotting and real-time reverse transcription polymerase chain reaction. Cell apoptosis is measured through flow cytometry assay, mitochondrial reactive oxygen species (ROS) production, and retinal vascular leakage were assayed using CM-H2DCFDA fluorescent probes and Evans blue, respectively. Results: MG132 significantly inhibited the activation of 26S proteasome induced by diabetes or elevated glucose, and subsequently increased the expression of Nrf2, NQO1, and HO-1, and further reduced ROS accumulation. These changes were associated with a decrease of diabetes-induced retinal vascular leakage and retinal capillary cell apoptosis. Conclusions: MG132 decreases diabetes-induced 26S proteasome activation and exerts protective effects against retinal microvascular dysfunction in diabetic rats in association with the alleviation of retinal oxidative stress mediated by Nrf2.

Robust expression of the TRPC1 channel associated with photoreceptor loss in the rat retina

Baseline intracellular calcium levels are significantly higher in neuronal and glial cells of rat retinas with retinitis pigmentosa (RP). Although this situation could initiate multiple detrimental pathways that lead to cell death, we considered the possibility of TRPC1 being involved in maintaining calcium homeostasis in the retina by acting as a component of store-operated calcium (SOC) channels with special relevance during photoreceptor degeneration. In this study, we examined by Western blot the expression of TRPC1 in healthy control rat retinas (Sprague-Dawley, SD) and retinas with RP (P23H-1 rats). We also analyzed its specific cellular distribution by immunofluorescence to recognize changes during neurodegeneration and to determine whether its presence is consistent with high basal calcium levels and cellular survival in degenerating retinas. We found that TRPC1 immunostaining was widely distributed across the retina in both rat strains, SD and P23H, and its expression levels significantly increased in the retinas with advanced degeneration compared to the age-control SD rats. In the outer retina, TRPC1 immunoreactivity was distributed in pigment epithelium cells, the photoreceptor inner segments of older animals, and the outer plexiform layer. In the inner retina, TRPC1 labeling was detected in horizontal cells, specific somata of bipolar and amacrine cells, and cellular processes in all the strata of the inner plexiform layer. Somata and processes were also highly immunoreactive in the ganglion cell layer and astrocytes in the nerve fiber layer in all animals. In the P23H rat retinas, the TRPC1 distribution pattern changed according to advancing photoreceptor degeneration and the gliosis reaction, with TRPC1 immunoreactive Müller cells mainly in advanced stages of disease. The cellular TRPC1 immunoreactivity found in this work suggests different mechanisms of activation of these channels depending on the cell type. Furthermore, the results support the idea that photoreceptor loss due to RP is associated with robust TRPC1 protein expression in the rat inner retina and raise the possibility of TRPC1 channels contributing to maintain high basal calcium levels during neurodegeneration and/or maintenance processes of the inner retina.

Hydrogen Peroxide Affects the Electroretinogram of Isolated Perfused Rat Retina

Purpose To evaluate the effects of H2O2 as an oxidant on the electroretinogram (ERG) in isolated rat retina. Methods Retinas were isolated from rat eyes and perfused with a nutrient solution. ERGs were recorded every 3 min. Once the signal was at a steady state, H2O2 was added to the perfusion solution. Results H2O2 caused instantaneous and transient changes in amplitudes and implicit times of the ERG, followed by changes in retinal survival curves. H2O2 0.2 mM produced a rapid increase in b-wave amplitude, followed by a return to the initial value and a survival curve above the control (without H2O2). A slight increase in a-wave was observed, followed by a decrease and a recovery above the control. The slow PIII decreased and then recovered to the initial value. H2O2 0.6 mM induced a small increase in b-wave amplitude, followed by a rapid decrease without recovery. The a-wave and slow PIII decreased rapidly without recovery. The implicit times of the a-wave and b-wave increased moderately with a low dose of H2O2, whereas they significantly increased with a high dose. Whatever the dose, the slow PIII implicit time increased significantly, followed by a return to the initial value. Barium increased the a-wave and b-wave, and then H2O2 reduced the two waves with a stronger effect on the a-wave. Aspartate and barium isolated the fast PIII, which decreased after H2O2 application. Conclusions H2O2 affects retinal function as shown by ERGs in isolated rat retina. The response differs with the dose of H2O2, suggesting that mechanisms underlying the action at low doses might be different from those at high doses. Our results also suggest an effect of H2O2 on ionic currents and/or neurotransmitter releases involved in the generation of the ERG and indicate a more pronounced effect on photoreceptors than on postsynaptic cells.

Neurodegenrative changes in retina of rats with streptozotocin-induced diabetes under conditions of treatment with niacin-oxy-ethylidene-diphosphonate germanate (MIGU-4)

Background: Diabetes is accompanied by neurodegenerative changes in the retina. The determination of retinal function parameters under conditions of experimental diabetes may allow for the development of new approaches to pharmacological vision correction. Purpose: To assess changes in the metabolic (lipid peroxidation and antioxidant defence) and functional characteristics (electroretinography (ERG)) of the retina in the rat with streptozotocin (STZ)-induced diabetes, treated with niacin-oxy-ethylidene-diphosphonate germanate (MIGU-4) and a reference drug, diazepam. Material and Methods: Diabetes was induced intraperitoneally (i.p.) in Wistar male rats by streptozotocin (STZ 65 mg/kg). Four weeks thereafter, the rats received a two-week course of a daily dose of MIGU-4 (5.0 mg/kg or 25.0 mg/kg) or diazepam (0.5 mg/kg or 1.5 mg/kg). The electroretinogram (ERG) was obtained, the animals were euthanized and retinal malondialdehyde (MDA) levels and superoxide dismutase (SOD) and catalase (CAT) activity were determined. Results: In rats with untreated STZ-induced diabetes, the retinal MDA level was 3.71 times increased and CAT activity was 35% lower, compared to controls, whereas the SOD activity was half of the activity found in controls (р less 0.05). In diabetic animals treated with MIGU-4 i.p. at a 25.0-mg/kg daily dose, the retinal MDA level was 63.2% lower, whereas the CAT activity and SOD activity were 41.9% and 27.6%, respectively, higher, than in untreated diabetic rats (р less 0.05). In diabetic animals treated with diazepam i.p. at a 1.5-mg/kg daily dose, the retinal MDA level was 59.2% lower, whereas the CAT activity and SOD activity were 44.4% and 32.1%, respectively, higher, than in untreated diabetic rats (р less 0.05). In untreated diabetic rats, the ERG b-wave amplitude was 39.2% lower, ERG a-wave and b-wave latencies, 23.4% and 14.0%, respectively, higher, and the a-wave amplitude recovery rate, 38.8% lower than in controls (р less 0.05). The use of a 25.0-mg/kg daily dose of MIGU-4 resulted in a 29.7% and 33.9%, respectively, increase in the ERG b-wave amplitude and the a-wave amplitude recovery rate compared to untreated diabetic rats (р less 0.05). The use of a 1.5-mg/kg daily dose of diazepam resulted in a 25.0% and 30.0%, respectively, increase in the above ERG parameters compared to untreated diabetic rats (р less 0.05). In untreated diabetic rats, the number of squares crossed (SK) was 29.4% lower (р less 0.05); the number of central squares crossed (CSC), 52.1% lower (р less 0.05); the number of upright postures (UP), 34.2% lower; and the defecation bolus number, 32.4% higher than in controls (р less 0.05). After MIGU-4 (25-mg/kg) withdrawal, there were no more differences in SK, CSC, UP and defecation bolus numbers between treated diabetic rats and controls. In addition, the number of CSC was 4.27 times lower than in controls, and half of that in untreated diabetic animals (р less 0.05). In diabetic rats treated with diazepam, the number of UP was 2.56 times lower than in controls (р less 0.05), and 40.7% lower than in untreated diabetic rats (р less 0.05), whereas the defecation bolus number was 40.5% higher than in controls (р less 0.05). Conclusion: STZ-induced diabetes is accompanied by an impairment of the oxidant/ antioxidant balance in the retina with an increase in retinal MDA, decrease in retinal SOD and CAT activity, and ERG abnormalities such as reduced ERG amplitude and increased ERG latency. A course treatment with niacin-oxy-ethylidene-diphosphonate germanate (MIGU-4) i.p. at a daily dose of 25.0 mg/kg provides for a decrease in retinal MDA and an increase in retinal SOD and CAT activity and ERG wave amplitude The withdrawal of a two-week treatment with MIGU-4 i.p. at a daily dose of 25.0 mg/kg decreased open-field anxiety-like behaviors, whereas the withdrawal of a two-week treatment with diazepam i.p. at a daily dose of 1.5 mg/kg increased open-field anxiety-like behaviors. The corrective effect of treatment with MIGU-4 corresponds to the corrective effect of treatment with diazepam.

TRIM9 promotes Müller cell-derived retinal stem cells to differentiate into retinal ganglion cells by regulating Atoh7.

Glaucoma is a multifactorial, irreversible blinding eye disease characterized by a large number of retinal ganglion cell (RGC) deaths. Müller cell-derived retinal stem cells (RSCs) can be induced to differentiate into RGCs under certain conditions. This study aimed to explore the regulatory effect and mechanism of TRIM9 on the differentiation of Müller cell-derived stem cells into RGCs. First, episcleral vein cauterization was used to induce high intraocular pressure (IOP) rat model. Next, Müller cells were isolated from rat retina, identified and induced to dedifferentiate into RSCs. Finally, RSCs were intervened with lentivirus PGC-FU-TRIM9-GFP transfection or siRNA Atoh7 and induced to redifferentiate into RGCs. In vivo, TRIM9 was highly expressed and Müller cells proliferated abnormally in the high IOP rat model. In vitro, S-100, GFAP, vimentin, and GS were positively expressed in Müller cells isolated from rat retina, and the purity of cells was 97.17%. Under the stimulation of cytokines, the proliferative capacity of the cells and the expression of Nestin and Ki67 gradually increased with the prolongation of culture time. Furthermore, RSCs transfected with the lentiviral vector PGC-FU-TRIM9-GFP displayed a striking morphological feature of long neurites. Additionally, there was a remarkable increase in the fluorescence intensity of Brn-3b and Thy1.1, accompanied by elevated mRNA and protein expression levels of Brn-3b, Thy1.1, and Atoh7. After knocking down Atoh7, the effect of TRIM9 on the above indicators was reversed. TRIM9 might promote the differentiation of Müller cells into RGCs by regulating the expression of Atoh7.

Protective effect of evolocumab on Müller cells in the rat retina under hyperglycaemic and hypoxic conditions

AimsIn this study, rat retinal Müller cells (RMCs) were cultured in vitro to investigate the protective mechanism of evolocumab on rat RMCs in diabetes mellitus (DM) and the expression of relevant inflammatory factors. MethodsThe expression of proprotein convertase subtilisin/kexin type 9 (PCSK9) in the retinal tissues of diabetic rats was detected by immunohistochemistry. Sprague-Dawley (SD) rats at 5–7 d of life were selected as the source of RMCs and divided equally into three groups of 12 rats/24 eyes each. The effect of CoCl2 and evolocumab on the cellular activity of RMCs was determined by CCK-8 assay. The effect of CoCl2 and evolocumab on the migration level of RMCs after 72 h was measured by scratch test and the expression of various proteins after 72 h was measured by Western blot. ResultsIn STZ rats, the expression of PCSK9 was significantly upregulated in the retina, especially in the inner nuclear layer, which is mainly composed of RMCs. High glucose and CoCl2 stimulation markedly elevated PCSK9 and GFAP expression at the protein level in RMCs (P < 0.05). Evolocumab treatment (100 μg/ml) reduced the expression and secretion of inflammatory factors in stimulated RMCs (P < 0.05). Furthermore, evolocumab downregulates toll-like receptor-4 (TLR-4) levels and inhibited nuclear transcription factor-κB (NF-κB) phosphorylation in RMCs (P < 0.05). ConclusionsEvolocumab protects against inflammation in RMCs, at least in part, by negatively regulating the activation of the TLR-4/NF-κB signalling pathway. Evolocumab may be a promising anti-inflammatory therapy for ocular fundus diseases, such as DR.

Differences in Changes in the Glutamate/GABA System Activity in the Rat Retina during Aging and the Development of Retinopathy at Nighttime and Daytime

Differences in Changes in the Glutamate/GABA System Activity in the Rat Retina during Aging and the Development of Retinopathy at Nighttime and Daytime

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas.

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

Decreased retinal and choroidal endothelial surface molecules in spontaneously hypertensive rats

Hypertension is associated with changes in the retina and choroid, with resulting consequences of increased vascular permeability and microhemorrhages. To date, very little information is available regarding the changes in the retinal and choroidal endothelial surface layer. In this study, we have examined changes in protein expression of several molecules including platelet endothelial cell adhesion molecule-1 (PECAM-1), vascular endothelial cadherin (VE-cadherin), glypican-1, and syndecan-1, in spontaneously hypertensive rats (SHR) compared to control normotensive Wistar Kyoto (WKY) rats. In male SHR vs WKY rat retinas, decreases were found for VE-cadherin and syndecan-1; whereas in female retinas, decreases were found for PECAM-1, glypican-1, and syndecan-1. In male SHR vs WKY rat choroid, we found an increase in glypican-1, but choroidal syndecan-1 was decreased in SHR in both males and females. Therefore, decreases in SHR of both retinal and choroidal syndecan-1 were found in both males and females. These losses of syndecan-1 were accompanied by an increase in plasma levels of the proteoglycan, indicating possible systemic endothelial shedding. In contrast, plasma levels of glypican-1 decreased. Interestingly, in normotensive WKY rats, retinal levels of all four endothelial surface molecules were higher in females than in males, in some cases, by substantial amounts. In summary, a number of changes occur in endothelial surface molecules in SHR, with some changes being sex-dependent; it is possible that the loss of these molecules contributes to the vascular dysfunction that occurs in hypertensive retina and choroid.

Human Retinal Ganglion Cells Respond to Evolutionarily Conserved Chemotropic Cues for Intra Retinal Guidance and Regeneration.

Retinal ganglion cells (RGCs) connect the retina with the higher centers in the brain for visual perception. Their degeneration leads to irreversible vision loss in glaucoma patients. Since human RGCs (hRGCs) are born during fetal development and connections with the central targets are established before birth, the mechanism underlying their axon growth and guidance remains poorly understood. Here, using RGCs directly generated from human embryonic stem cells, we demonstrate that hRGCs express a battery of guidance receptors. These receptors allow hRGCs to read the spatially arrayed chemotropic cues in the developing rat retina for the centripetal orientation of axons toward the optic disc, suggesting that the mechanism of intra-retinal guidance is conserved in hRGCs. The centripetal orientation of hRGCs axons is not only in response to chemo-repulsion but also involves chemo-attraction, mediated by Netrin-1/DCC interaction. The spatially arrayed chemotropic cues differentially influence hRGCs physiological responses, suggesting that neural activity of hRGCs and axon growth may be coupled during inter-retinal guidance. Additionally, we demonstrate that Netrin-1/DCC interaction, besides promoting axon growth, facilitates hRGCs axon regeneration by recruiting the mTOR signaling pathway. The diverse influence of Netrin-1/DCC interaction ranging from axon growth to regeneration may involve recruitment of multiple intracellular signaling pathways as revealed by transcriptome analysis of hRGCs. From the perspective of ex-vivo stem cell approach to glaucomatous degeneration, our findings posit that ex-vivo generated hRGCs can read the intra-retinal cues for guidance toward the optic disc, the first step required for connecting with the central target to restore vision.

Successful transduction of target gene mediated by adeno-associated virus 2 into lens epithelial cells in rats

The Adeno-Associated Virus (AAV) has emerged as a promising candidate for delivery of genetic material, exhibiting significant potential in various clinical applications. Although multiple AAV serotypes have been shown to transduce ocular tissues, there have been few studies of AAV transduction of lens epithelial cells (LECs) in the ocular. In this study, we compared the efficiency of intravitreal injection of six AAV serotypes (AAV2, AAV5, AAV6, AAV8, AAV9, and AAVDJ) to transduce lens and retina in rats, The expression and localization of the reporter gene ZsGreen in the lens and retina were examined using immunofluorescence staining, and the relative expression of ZsGreen mRNA was detected using RT-qPCR. Our results demonstrated that AAV2 had the highest efficiency in transducing LECs. All six AAV serotypes could transduce the retina. To validate this observation, we further constructed an AAV2 vector with exogenous gene senescence marker protein 30 (SMP30) and performed intravitreal injection to successfully overexpress SMP30 in LECs of rats. our results provide a basis for the use of AAV vector-mediated gene therapy for lens diseases.

Retinal gliosis and phenotypic diversity of intermediate filament induction and remodeling upon acoustic blast overpressure (ABO) exposure to the rat eye

Traumatic brain injury (TBI) caused by acoustic blast overpressure (ABO) is frequently associated with chronic visual deficits in military personnel and civilians. In this study, we characterized retinal gliotic response in adult male rats following a single ABO exposure directed to one side of the head. Expression of gliosis markers and intermediate filaments was assessed at 48 h and 1 wk post-ABO exposure, in comparison to age-matched non-exposed control retina. In response to a single ABO exposure, type III IF, glial fibrillary acidic protein (GFAP) was variably induced in a subpopulation of retinal Müller glia in ipsilateral eyes. ABO-exposed eyes exhibited radial Müller glial GFAP filament extension through the inner plexiform layer (IPL) and the inner nuclear layer (INL) through the retina in both the nasal quadrant and juxta-optic nerve head (jONH) eye regions at 1 wk post-ABO. We observed an ∼6-fold increase (p ≤ 0.05) in radial glial GFAP immunolabeling in the IPL in both eye regions, in comparison to regionally matched controls. Similarly, GFAP extension through the INL into the outer retina was elevated ∼3-fold, p ≤ 0.05 in the nasal retina, but exhibited wider variability in the jONH retina. In contrast, constitutive type III IF vimentin exhibited greater remodeling in retinal Müller glia through the jONH retina compared to the nasal retina in response to ABO. We observed areas of lateral vimentin remodeling through the Müller glial end-feet, and greater mid-outer retinal radial vimentin IF extension in a subpopulation of glia at 1 wk post-ABO. We also observed a significant increase in total retinal levels of the type III IF desmin in ABO-exposed retina vs. controls (∼1.6-fold, p ≤ 0.01). In addition, ABO-exposure elicited varied glial induction of developmentally regulated type VI family IFs (nestin and synemin) in subpopulations of Müller cells at 48 h and 1 wk post-ABO. We demonstrate that multiple glial phenotypes emerge in the rat retina following a single ABO exposure, rather than a global homogeneous retinal glial response, involving less well characterized IF protein forms which warrant further investigation in the context of ABO-induced retinal gliosis.

P38 MAPK Signaling in the Retina: Effects of Aging and Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible visual impairment worldwide. Age is the greatest risk factor for AMD but the underlying mechanism remains unascertained, resulting in a lack of effective therapies. Growing evidence shows that dysregulation of the p38 MAPK signaling pathway (SP) contributes to aging and neurodegenerative diseases; however, information about its alteration in the retina with age and during AMD development is limited. To assess the contribution of alterations in p38 MAPK signaling to AMD, we compared age-associated changes in p38 MAPK SP activity in the retina between Wistar rats (control) and OXYS rats, which develop AMD-like retinopathy spontaneously. We analyzed changes in the mRNA levels of genes of this SP in the retina (data of RNA-seq) and evaluated the phosphorylation/activation of key kinases using Western blotting at different stages of AMD-like pathology including the preclinical stage. p38 MAPK SP activity increased in the retinas of healthy Wistar rats with age. The manifestation and dramatic progression of AMD-like pathology in OXYS rats was accompanied by hyperphosphorylation of p38 MAPK and MK2 as key p38 MAPK SP kinases. Retinopathy progression co-occurred with the enhancement of p38 MAPK-dependent phosphorylation of CryaB at Ser59 in the retina.