Retinal Astrocytes Research Articles

Transient ocular hypertension remodels astrocytes through S100B.

Glaucoma is a series of irreversible and progressive optic nerve degenerations, often accompanied by astrocyte remodeling as the disease progresses, a process that is insufficiently understood. Here, we investigated the morphology of retinal and optic nerve head (ONH) astrocytes under mechanical stress, and explored whether a specific phase is present that precedes astrocyte remodeling. A mouse model of transient ocular hypertension (OHT) and an in vitro cell stretch model were established to mimic the pathological conditions of increased intraocular pressure and mechanical stress on cultured cells. Glial fibrillary acidic protein (GFAP), S100B, and actin staining were used to characterize astrocyte morphology and cytoskeleton, with qPCR used to measure mRNA expression. We also silenced S100B expression and conduct RNA sequencing on ONH astrocytes. Astrocytes displayed weaker GFAP intensity (p < 0.0001) in the early-stage OHT mouse model, prior to the onset of hypertrophy, which was accompanied by an increase in GFAP mRNA expression (p < 0.0001) and a decrease in S100B mRNA expression (p < 0.001). In vitro-stretched astrocytes tended to contract and had fewer cellular processes and more elongated cell bodies. Downregulation of S100B expression occurred in in both the in vivo (p = 0.0001) and in vitro (p = 0.0023) models. S100B-silenced ONH astrocytes were similarly characterized by a slender morphology. In the RNA-seq analysis, genes downregulated by more than fivefold were predominantly enriched in terms related to nutrient metabolism, motor proteins and morphogenesis. Meanwhile, genes upregulated by more than fivefold were primarily associated with terms related to histone modification and visual perception. As an early response to mechanical stress, S100B expression is downregulated in astrocytes, which assume a slender morphology, reminiscent of cell "weakening." Silencing intracellular S100B expression induced similar morphology changes and altered the transcriptome. Stress-induced changes were reversible, with evidence of enhanced late-stage reactivation that is likely related to S100B.

Pharmacological blocking of microfibrillar-associated protein 4 reduces retinal neoangiogenesis and vascular leakage.

Neovascular age-related macular degeneration and diabetic macular edema are leading causes of vision-loss evoked by retinal neovascularization and vascular leakage. The glycoprotein microfibrillar-associated protein 4 (MFAP4) is an integrin αVβ3/5/6 ligand present in the extracellular matrix. Single-cell transcriptomics reveal MFAP4 expression in cell-types in close proximity to vascular endothelial cells including choroidal vascular mural cells and retinal astrocytes and Müller cells. Binding of the anti-MFAP4 antibody, hAS0326, makes MFAP4 inaccessible for integrin receptor interaction and thereby hAS0326 blocked endothelial cell motility in vitro. Intravitreal hAS0326 inhibited retinal vascular lesion area and neovessel volume in a laser-induced choroidal neovascularization mouse model, vascular permeability in streptozotocin-induced retinopathy and vascular leakage area in a chronic non-human primate model of DL-2-aminoadipic acid-induced retinopathy. One dose of hAS0326 showed duration of efficacy of at least 12 weeks in the latter model. Moreover, hAS0326-treatment significantly enriched gene ontology terms involving reduction of integrin binding. Our data suggest that hAS0326 constitutes a promising treatment of neovascularization and vascular leakage in retinal diseases.

Mesenchymal stem cell-derived exosomes mitigate amyloid β-induced retinal toxicity: Insights from rat model and cellular studies.

Amyloid β (Aβ) has emerged as a pathophysiological driver in age-related macular degeneration (AMD), emphasizing its significance in the aetiology of this prevalent sight-threatening condition. The multifaceted nature of AMD pathophysiology, presumably involving diverse retinal cascades, corresponds with the complexity of Aβ-induced retinopathy. Therefore, targeting a broad array of pathogenic processes holds promise for therapeutic intervention in AMD-associated retinal pathology. This study investigates the potential of exosomes derived from adipose tissue mesenchymal stem cells (AT-MSC-Exosomes) in alleviating Aβ-induced retinotoxicity. Through intravitreal injections in wild-type rats and RPE-like cell culture experiments, we examined the protective effects of AT-MSC-Exosomes against Aβ42 retinotoxicity. Our findings reveal that pre-treatment with AT-MSC-Exosomes enabled nearly-intact retinal function in vivo and maintained retinal cell viability in vitro, evidenced by longitudinal electroretinography (ERG) and XTT proliferation assays, respectively. Fluorescent labelling demonstrated increased migration of AT-MSC-Exosomes towards retinal cells under conditions of amyloid-related toxicity. Proteomic analysis indicated a decrease in the retinal levels of heat-shock proteins activated by pathogenic Aβ fibrils following AT-MSC-Exosome treatment. Similarly, immunostaining highlighted the modulation of α-crystallin expression in retinal astrocytes by AT-MSC-Exosomes. These results suggest the potential therapeutic relevance of AT-MSC-Exosomes in Aβ-related retinal pathology, offering a promising avenue for future AMD treatment strategies.

Glucocorticoid-Dependent Retinal Degeneration and Vision Impairment in Mice Susceptible to Prenatal Stress-Induced Behavioral Abnormalities.

Chronic exposure to prenatal stress can impair neurogenesis and lead to irreversible cognitive and neuropsychiatric abnormalities in offspring. The retina is part of the nervous system; however, the impacts of prenatal stress on retinal neurogenesis and visual function remain unclear. This study examined how elevated prenatal glucocorticoid levels differentially affect retinal development in the offspring of pregnant mice exposed to chronic unpredictable mild stress (CUMS). Offspring were classified into control, stress-resilient, and stress-susceptible groups based on behavioral tests assessing spatial memory and depression-like behaviors. The stress-susceptible group exhibited significantly altered synaptogenesis, reduced ganglion cell development, decreased retinal thickness, and visiual impairment. These mice also showed a pervasive transformation of retinal astrocytes into a proinflammatory A1-like reactive state, evidenced by increased GFAP and decreased STAT3 expression levels. This astrocyte phenotype shift coincided with disruptions in neurogenesis and synaptic formation. Furthermore, prenatal exposure to exogenous corticosterone confirmed that the effects of prenatal stress are mediated by glucocorticoid-induced retinal neurodegeneration. Our findings suggest that elevated prenatal glucocorticoid levels trigger a series of neurodevelopmental disturbances leading to retinal neurodegeneration and vision impairment. This research highlights the impact of prenatal stress on retinal development and visual health, suggesting new avenues for understanding and potentially mitigating the negative effects of early-life stress on neurodevelopment.

Adverse effects of CXCR2 deficiency in mice reared under non-gnotobiotic conditions

The family of pro-inflammatory and pro-angiogenic chemokines including Interleukin-8 (IL-8, aka CXCL8) and its homologues (CXCL1,2,3,5,6, and 7) exhibit promiscuous binding and activation of several G-protein-coupled receptors (i.e., CXCR2, CXCR1, and the atypical chemokine receptor (ACKR1)). A high proportion of their biological activity is attributed to CXCR2 activation, thus many CXCR2 inhibitors are in clinical trials for several chronic diseases. However, CXCR2 inhibition is often only investigated acutely in these trials or in Cxcr2−/− mice grown in gnotobiotic conditions. Since humans do not live in germ-free environments, our first goal is to highlight novel retinal and systemic observations in Cxcr2−/− mice grown in non-gnotobiotic conditions that suggest potential harmful consequences of long-term CXCR2 deficiency or blockade. Beyond confirmation of circulating blood/immune cell-related phenotypes, we report novel findings in Cxcr2−/− mice including: (1) delayed dye transit to the retinal vasculature, (2) alterations in the density and distribution of retinal vessels, astrocytes and microglia, (3) decreased electroretinogram a- and b-wave amplitudes, (4) reduced visual acuity, and (5) increased polymorphonuclear cell accumulation in vascular lumina abutting venular walls in the retina and in vital non-ocular tissues (lung and liver). Furthermore, PheWAS of CXCR2 CXCR1, and ACKR1 gene variants using data from UK Biobank participants suggest clinical associations with both retinal and vascular disease phenotypes. We conclude that chronic CXCR2 deficiency in mice contributes to functional damage to the retina and that the long-term safety of CXCR1/2 inhibitors designed for chronic use in humans should be explored before clinical adoption to safeguard sight and overall vascular health.

Chronic hyperglycemia alters retinal astrocyte microstructure and uptake of cholera toxin B in a murine model of diabetes.

Astrocytes are the principle glial cells of the central nervous system and play an active role in maintaining proper metabolism in surrounding neurons. Because of their involvement in metabolic control, it is likely that their physiology changes in response to metabolic diseases such as diabetes and associated diabetic retinopathy. Here, we investigated whether microstructural changes in astrocyte morphology occur during the early stages of chronic hyperglycemia that may be indicative of early pathogenic programs. We used MORF3 mice in conjunction with streptozotocin-induced hyperglycemia to investigate the morphology of single retinal astrocytes at an early timepoint in diabetic disease. We report that astrocytes initiate a morphological remodeling program, which depends on both the glycemic background and the presence of intravitreal injury, to alter the amount of the neuronal-associated pad and bristle microstructural motifs. Additionally, hyperglycemia increases astrocyte uptake of cholera toxin B, possibly reflecting changes in glycolipid and glycoprotein biosynthesis. Chronic hyperglycemia coupled with intravitreal injection of cholera toxin B also causes extensive leukocyte infiltration into the retina. Our results have important clinical relevance as current therapies for diabetic retinopathy involve intravitreal injection of pharmaceuticals in individuals with often poorly controlled blood glucose levels.

Human umbilical cord mesenchymal stem cells derived-exosomes on VEGF-A in hypoxic-induced mice retinal astrocytes and mice model of retinopathy of prematurity.

To observe the effect of human umbilical cord mesenchymal stem cells (hUCMSCs) secretions on the relevant factors in mouse retinal astrocytes, and to investigate the effect of hUCMSCs on the expression of vascular endothelial growth factor-A (VEGF-A) and to observe the therapeutic effect on the mouse model of retinopathy of prematurity (ROP). Cultured hUCMSCs and extracted exosomes from them and then retinal astrocytes were divided into control group and hypoxia group. MTT assay, flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect related indicators. Possible mechanisms by which hUCMSCs exosomes affect VEGF-A expression in hypoxia-induced mouse retinal astrocytes were explored. At last, the efficacy of exosomes of UCMSCs in a mouse ROP model was explored. Graphpad6 was used to comprehensively process data information. The secretion was successfully extracted from the culture supernatant of hUCMSCs by gradient ultracentrifugation. Reactive oxygen species (ROS) and hypoxia inducible factor-1α (HIF-1α) of mice retinal astrocytes under different hypoxia time and the expression level of VEGF-A protein and VEGF-A mRNA increased, and the ROP cell model was established after 6h of hypoxia. The secretions of medium and high concentrations of hUCMSCs can reduce ROS and HIF-1α, the expression levels of VEGF-A protein and VEGF-A mRNA are statistically significant and concentration dependent. Compared with the ROP cell model group, the expression of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signal pathway related factors in the hUCMSCs exocrine group is significantly decreased. The intravitreal injection of the secretions of medium and high concentrations of hUCMSCs can reduce VEGF-A and HIF-1α in ROP model tissues. HE staining shows that the number of retinal neovascularization in ROP mice decreases with the increase of the dose of hUCMSCs secretion. In a hypoxia induced mouse retinal astrocyte model, hUCMSCs exosomes are found to effectively reduce the expression of HIF-1α and VEGF-A, which are positively correlated with the concentration of hUCMSCs exosomes. HUCMSCs exosomes can effectively reduce the number of retinal neovascularization and the expression of HIF-1α and VEGF-A proteins in ROP mice, and are positively correlated with drug dosage. Besides, they can reduce the related factors on the PI3K/AKT/mTOR signaling pathway.

Histomorphology and Histomorphometrics of the retina in Juvenile and Adult African Giant Rats (Cricetomys gambianus)

Aging is accompanied with various forms of functional ultrastructural and morphological changes in the eye, including the retina, leading to vision deterioration. However, age related retinal degenerative changes requires further elucidation especially as all previous studies on age-related change of the retina were done in mutant mouse strain models of hereditary retinal degenerations. The potential of using African giant rats (AGRs) as models for ageing in the eye, especially retina, was explored in this study. A total of 14 AGRs divided into two age groups (juvenile and adult) were used to study the histomorphology and histomorphometrics of the retina as well as retinal astrocyte morphology and heterogeneity. Histological findings included retinal atrophy and hypoplasia, with cellular swellings of neuronal cell populations and astrocytes soma and ramifications in the retina of adult compared to juvenile AGR. We suggest that AGR be used as animal model for translational research into normal aging process of the retina, as well as to elucidate the process of age-related neuronal cells loss.

Molecular hydrogen promotes retinal vascular regeneration and attenuates neovascularization and neuroglial dysfunction in oxygen-induced retinopathy mice

BackgroundRetinopathy of Prematurity (ROP) is a proliferative retinal vascular disease occurring in the retina of premature infants and is the main cause of childhood blindness. Nowadays anti-VEGF and retinal photocoagulation are mainstream treatments for ROP, but they develop a variety of complications. Hydrogen (H2) is widely considered as a useful neuroprotective and antioxidative therapeutic method for hypoxic-ischemic disease without toxic effects. However, whether H2 provides physiological angiogenesis promotion, neovascularization suppression and glial protection in the progression of ROP is largely unknown.This study aims to investigate the effects of H2 on retinal angiogenesis, neovascularization and neuroglial dysfunction in the retinas of oxygen-induced retinopathy (OIR) mice.MethodsIn this study, mice that were seven days old and either wild-type (WT) or Nrf2-deficient (Nrf2−/−) were exposed to 75% oxygen for 5 days and then returned to normal air conditions. Different stages of hydrogen gas (H2) inhalation were administered. Vascular obliteration, neovascularization, and blood vessel leakage were analyzed and compared. To count the number of neovascularization endothelial nuclei, routine HE staining of retinal sections was conducted. Immunohistochemistry was performed using DyLight 594 labeled GSL I-isolectin B4 (IB4), as well as primary antibodies against proliferating cell nuclear antigen (PCNA), glial fibrillary acidic protein (GFAP), and Iba-1. Western blots were used to measure the expression of NF-E2-related factor 2 (Nrf2), vascular endothelial growth factor (VEGF), Notch1, Dll4, and HIF-1α. Additionally, the expression of target genes such as NQO1, HO-1, Notch1, Hey1, Hey2, and Dll4 was measured. Human umbilical vein endothelial cells (HUVECs) treated with H2 under hypoxia were used as an in vitro model. RT-PCR was used to evaluate the mRNA expression of Nrf2, Notch/Dll4, and the target genes. The expression of reactive oxygen species (ROS) was observed using immunofluorescence staining.ResultsOur results indicate that 3–4% H2 does not disturb retinal physiological angiogenesis, but ameliorates vaso-obliteration and neovascularization in OIR mice. Moreover, H2 prevents the decreased density and reverses the morphologic and functional changes in retinal astrocytes caused by oxygen-induced injury. In addition, H2 inhalation reduces microglial activation, especially in the area of neovascularization in OIR mice. H2 plays a protective role in vascular regeneration by promoting Nrf2 activation and suppressing the Dll4-induced Notch signaling pathway in vivo. Also, H2 promotes the proliferation of HUVECs under hypoxia by negatively regulating the Dll4/Notch pathway and reducing ROS levels through Nrf2 pathway aligning with our findings in vivo.Moreover, the retinal oxygen-sensing mechanisms (HIF-1α/VEGF) are also involved in hydrogen-mediated retinal revascularization and neovascularization suppression.ConclusionsCollectively, our results indicate that H2 could be a promising therapeutic agent for POR treatment and that its beneficial effect in human ROP might involve the activation of the Nrf2-Notch axis as well as HIF-1α/VEGF pathways.Graphical

Transcriptional profiling of retinal astrocytes identifies a specific marker and points to functional specialization.

Astrocyte heterogeneity is an increasingly prominent research topic, and studies in the brain have demonstrated substantial variation in astrocyte form and function, both between and within regions. In contrast, retinal astrocytes are not well understood and remain incompletely characterized. Along with optic nerve astrocytes, they are responsible for supporting retinal ganglion cell axons and an improved understanding of their role is required. We have used a combination of microdissection and Ribotag immunoprecipitation to isolate ribosome-associated mRNA from retinal astrocytes and investigate their transcriptome, which we also compared to astrocyte populations in the optic nerve. Astrocytes from these regions are transcriptionally distinct, and we identified retina-specific astrocyte genes and pathways. Moreover, although they share much of the "classical" gene expression patterns of astrocytes, we uncovered unexpected variation, including in genes related to core astrocyte functions. We additionally identified the transcription factor Pax8 as a highly specific marker of retinal astrocytes and demonstrated that these astrocytes populate not only the retinal surface, but also the prelaminar region at the optic nerve head. These findings are likely to contribute to a revised understanding of the role of astrocytes in the retina.

Dysregulation of neuroprotective lipoxin pathway in astrocytes in response to cytokines and ocular hypertension​

Glaucoma leads to vision loss due to retinal ganglion cell death. Astrocyte reactivity contributes to neurodegeneration. Our recent study found that lipoxin B4 (LXB4), produced by retinal astrocytes, has direct neuroprotective actions on retinal ganglion cells. In this study, we aimed to investigate how the autacoid LXB4 influences astrocyte reactivity in the retina under inflammatory cytokine-induced activation and during ocular hypertension. The protective activity of LXB4 was investigated in vivo using the mouse silicone-oil model of chronic ocular hypertension. By employing a range of analytical techniques, including bulk RNA-seq, RNAscope in-situ hybridization, qPCR, and lipidomic analyses, we discovered the formation of lipoxins and expression of the lipoxin pathway in rodents (including the retina and optic nerve), primates (optic nerve), and human brain astrocytes, indicating the presence of this neuroprotective pathway across various species. Findings in the mouse retina identified significant dysregulation of the lipoxin pathway in response to chronic ocular hypertension, leading to an increase in 5-lipoxygenase (5-LOX) activity and a decrease in 15-LOX activity. This dysregulation was coincident with a marked upregulation of astrocyte reactivity. Reactive human brain astrocytes also showed a significant increase in 5-LOX. Treatment with LXB4 amplified the lipoxin biosynthetic pathway by restoring and amplifying the generation of another member of the lipoxin family, LXA4, and mitigated astrocyte reactivity in mouse retinas and human brain astrocytes. In conclusion, the lipoxin pathway is functionally expressed in rodents, primates, and human astrocytes, and is a resident neuroprotective pathway that is downregulated in reactive astrocytes. Novel cellular targets for LXB4’s neuroprotective action are inhibition of astrocyte reactivity and restoration of lipoxin generation. Amplifying the lipoxin pathway is a potential target to disrupt or prevent astrocyte reactivity in neurodegenerative diseases, including retinal ganglion cell death in glaucoma.

Morphological and electrophysiological characterization of a novel displaced astrocyte in the mouse retina.

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.

Dose-Related Side Effects of Intravitreal Injections of Humanized Anti-Vascular Endothelial Growth Factor in Rats: Glial Cell Reactivity and Retinal Ganglion Cell Loss.

In a previous study, we documented that the Intravitreal injections (IVIs) of bevacizumab in rats caused a retinal inflammatory response. We now study whether the IVI of other humanized anti-VEGF: ranibizumab and aflibercept also cause an inflammatory reaction in the rat retina and if it depends on the dose administered. Finally, we study whether this reaction affects retinal ganglion cell (RGC) survival. Albino Sprague-Dawley rats received a single IVI of 5 µL of PBS or ranibizumab or aflibercept at the concentration used in clinical practice (10 µg/µL or 40 µg/µL) or at a lower concentration (0.38 µg/µL and 1.5 µg/µL) calculated to obtain within the rat eye the same concentration as in the human eye in clinical practice. Others received a single 5 µL IVI of a polyclonal goat anti-rat VEGF (0.015 µg/µL) or of vehicle (PBS). Animals were processed 7days or 1month later. Retinal whole mounts were immunolabeled for the detection of microglial, macroglial, RGCs, and intrinsically photosensitive RGCs (ipRGCs). Fluorescence and confocal microscopy were used to examine retinal changes, and RGCs and ipRGCs were quantified automatically or semiautomatically, respectively. All the injected substances including the PBS induced detectable side effects, namely, retinal microglial cell activation and retinal astrocyte hypertrophy. However, there was a greater microglial and macroglial response when the higher concentrations of ranibizumab and aflibercept were injected than when PBS, the antibody anti-rat VEGF and the lower concentrations of ranibizumab or aflibercept were injected. The higher concentration of ranibizumab and aflibercept resulted also in significant RGC death, but did not cause appreciable ipRGC death. The IVI of all the substances had some retinal inflammatory effects. The IVI of humanized anti-VEGF to rats at high doses cause important side effects: severe inflammation and RGC death, but not ipRGC death.

Sustained Retinal Defocus Increases the Effect of Induced Myopia on the Retinal Astrocyte Template.

The aim of this article is to describe sustained myopic eye growth's effect on astrocyte cellular distribution and its association with inner retinal layer thicknesses. Astrocyte density and distribution, retinal nerve fiber layer (RNFL), ganglion cell layer, and inner plexiform layer (IPL) thicknesses were assessed using immunochemistry and spectral-domain optical coherence tomography on seventeen common marmoset retinas (Callithrix jacchus): six induced with myopia from 2 to 6 months of age (6-month-old myopes), three induced with myopia from 2 to 12 months of age (12-month-old myopes), five age-matched 6-month-old controls, and three age-matched 12-month-old controls. Untreated marmoset eyes grew normally, and both RNFL and IPL thicknesses did not change with age, with astrocyte numbers correlating to RNFL and IPL thicknesses in both control age groups. Myopic marmosets did not follow this trend and, instead, exhibited decreased astrocyte density, increased GFAP+ spatial coverage, and thinner RNFL and IPL, all of which worsened over time. Myopic changes in astrocyte density, GFAP+ spatial coverage and inner retinal layer thicknesses suggest astrocyte template reorganization during myopia development and progression which increased over time. Whether or not these changes are constructive or destructive to the retina still remains to be assessed.

Lipoxins A4 and B4 inhibit glial cell activation via CXCR3 signaling in acute retinal neuroinflammation

Lipoxins are small lipids that are potent endogenous mediators of systemic inflammation resolution in a variety of diseases. We previously reported that Lipoxins A4 and B4 (LXA4 and LXB4) have protective activities against neurodegenerative injury. Yet, lipoxin activities and downstream signaling in neuroinflammatory processes are not well understood. Here, we utilized a model of posterior uveitis induced by lipopolysaccharide endotoxin (LPS), which results in rapid retinal neuroinflammation primarily characterized by activation of resident macroglia (astrocytes and Müller glia), and microglia. Using this model, we observed that each lipoxin reduces acute inner retinal inflammation by affecting endogenous glial responses in a cascading sequence beginning with astrocytes and then microglia, depending on the timing of exposure; prophylactic or therapeutic. Subsequent analyses of retinal cytokines and chemokines revealed inhibition of both CXCL9 (MIG) and CXCL10 (IP10) by each lipoxin, compared to controls, following LPS injection. CXCL9 and CXCL10 are common ligands for the CXCR3 chemokine receptor, which is prominently expressed in inner retinal astrocytes and ganglion cells. We found that CXCR3 inhibition reduces LPS-induced neuroinflammation, while CXCR3 agonism alone induces astrocyte reactivity. Together, these data uncover a novel lipoxin–CXCR3 pathway to promote distinct anti-inflammatory and proresolution cascades in endogenous retinal glia.

Evaluation of the LDN-0060609 PERK Inhibitor as a Selective Treatment for Primary Open-Angle Glaucoma: An In Vitro Study on Human Retinal Astrocytes.

The term glaucoma encompasses various neurodegenerative eye disorders, among which the most common is primary open-angle glaucoma (POAG). Recently, the essential role of human retinal astrocytes (HRA) in glaucoma progression has been placed in the spotlight. It has been found that placing the endoplasmic reticulum (ER) under stress and activating PERK leads to apoptosis of HRA cells, which inhibits their neuroprotective effect in the course of glaucoma. Therefore, the aim of the present study was to evaluate the effectiveness of the small-molecule PERK inhibitor LDN-0060609 in countering ER stress conditions induced in HRA cells in vitro. The activity of LDN-0060609 was studied in terms of protein and mRNA expression, cytotoxicity, genotoxicity, caspase-3 level and cell cycle progression. LDN-0060609 at 25 μM proved to be a potent inhibitor of the major PERK substrate, p-eIF2α (49% inhibition). The compound markedly decreased the expression of pro-apoptotic ER stress-related genes (ATF4, DDIT3, BAX and Bcl-2). Treatment with LDN-0060609 significantly increased cell viability, decreased genotoxicity and caspase-3 levels, and restored cell cycle distribution in HRA cells with activated ER stress conditions. These findings indicate that the small-molecule PERK inhibitor LDN-0060609 can potentially be developed into a novel anti-glaucoma agent.

Protective effect of the natural flavonoid naringenin in mouse models of retinal injury

Glaucoma is an eye disease with a high rate of blindness and a complex pathogenesis. Ocular hypertension (OHT) is a critical risk factor, and retinal ischemia/reperfusion (I/R) is an important pathophysiological basis. This study was designed to investigate the retinal neuroprotective effect of oral naringenin in an acute retinal I/R model and a chronic OHT model and the possible mechanism involved. After the I/R and OHT models were established, mice were given vehicle or naringenin (100 mg/kg or 300 mg/kg). Hematoxylin-eosin (HE) staining and immunostaining of RBPMS and glial fibrillary acidic protein (GFAP) were used to evaluate retinal injury. GFAP, CD38, Sirtuin1 (SIRT1), and NOD-like receptor protein 3 (NLRP3) expression levels were measured by Western blotting. In the OHT model, intraocular pressure (IOP) was dynamically maintained at approximately 20–25 mmHg after injury. The retinal structure was damaged, and retinal ganglion cells (RGCs) were lost in both models. Naringenin ameliorated the abovementioned indications but also demonstrated that high concentrations of naringenin significantly inhibited retinal astrocyte activation and inhibited damage-induced increases in the expression of GFAP, NLRP3, and CD38 proteins, while SIRT1 protein expression was upregulated. This study showed for the first time that naringenin can reduce microbead-induced IOP elevation in the OHT model, providing new evidence for the application of naringenin in glaucoma. Naringenin may mediate the CD38/SIRT1 signaling pathway, inhibit astrocyte activation, and ultimately exert an anti-inflammatory effect to achieve retinal neuroprotection.

Diabetes Modulates Iodothyronine Deiodinase 2 Expression in the Mouse Retina: A Role for Thyroid Hormone in the Pathogenesis of Diabetic Retinopathy.

Clinical investigations associate hypothyroidism with an increased risk for microvascular complications, yet the mechanism by which thyroid hormone regulates the development of diabetic retinopathy is not clearly understood. We investigated the role of iodothyronine deiodinase 2 (DIO2) in the pathogenesis of diabetic retinopathy. Retinas from streptozotocin-induced diabetic and nondiabetic mice were evaluated by RNA sequencing, RT-PCR, and immunostaining. Media and cell lysates from mouse retinal microvascular endothelial cells and retinal astrocytes exposed to physiologic (5mM) and high glucose (25 mM) containing media were assessed by liquid chromatography-tandem mass spectrometry to measure tetraiodothyronine (T4) and tri-iodothyronine (T3) concentrations and by Western blot analysis to determine the relationship of T4/T3 to oxidative stress and inflammatory mediators. Cell death was determined by Trypan Blue exclusion assay. At 12 weeks of diabetes duration, retinas from diabetic mice compared with nondiabetic mice demonstrated a significant decrease in Dio2 transcripts and Dio2 gene and protein (P < 0.05) expression. When cultured in the presence of high glucose, both mouse retinal astrocytes and microvascular endothelial cells demonstrated a significant reduction of DIO2 protein compared with cells cultured in physiologic glucose. High glucose inhibited generation of T3, leading to a significantly increased T4/T3 (P < 0.0079). Supplementation of cells with T3, but not T4, prevented the high glucose-induced rise in endothelial nitric oxide synthase, intercellular cell adhesion molecule 1, and endothelial cell death (P < 0.0079). Decreased intraretinal T3 owing to diabetes-induced loss of DIO2 may lead to dysfunction and death of cells in the retina, thereby contributing to the pathogenesis of early diabetic retinopathy.

Astrocytic expression of Yes-associated protein (YAP) regulates retinal neovascularization in a mouse model of oxygen-induced retinopathy

Pathological neovascularization is the hallmark of many vascular oculopathies. There is still a great deal of uncertainty surrounding retinal neovascularization research. A working hypothesis that astrocytic Yes-associated protein (YAP) act as a key factor in retinal neovascularization was proposed. And our study was conducted to verified this hypothesis. In vivo, we successfully generated mice deficient in YAP in astrocytes (YAPf/f GFAP-Cre mice) and set up oxygen-induced retinopathy (OIR) model. Pathological neovascularization was evaluated by immunofluorescence staining and western blotting. In vitro, cultured retinal astrocytes were transfected with YAP siRNA. Enzyme-linked immunosorbent assay (ELISA) and western blot were used to determine the proteins in the supernatants and cells. The results showed that YAP was upregulated and activated in the OIR mice retinas. Conditional ablation of YAP aggravated pathological neovascularization, along with the upregulation of vascular endothelial growth factor A (VEGF-A) and monocyte chemoattractant protein-1 (MCP-1). Studies in vitro confirmed that the knockdown of YAP in astrocytes lead to increases in VEGF-A and MCP-1 levels, thus enhancing pro-angiogenic capability of YAP-deficit astrocytes. In conclusion, astrocytic YAP alleviates retinal pathological angiogenesis by inhibiting the over-activation of astrocytes, which suppresses excessive VEGF-A production and neuroinflammation.

Rapid isolation of intact retinal astrocytes: a novel approach

Astrocytes are a major category of glial support cell in the central nervous system and play a variety of essential roles in both health and disease. As our understanding of the diverse functions of these cells improves, the extent of heterogeneity between astrocyte populations has emerged as a key area of research. Retinal astrocytes, which form the direct cellular environment of retinal ganglion cells somas and axons, undergo a reactive response in both human glaucoma and animal models of the disease, yet their contributions to its pathology and progression remain relatively unknown. This gap in knowledge is largely a function of inadequate isolation techniques, driven in part by the sparseness of these cells and their similarities with the more abundant retinal Müller cells. Here, we present a novel method of isolating retinal astrocytes and enriching their RNA, tested in both normal and ocular hypertensive mice, a common model of experimental glaucoma. Our approach combines a novel enzyme assisted microdissection of retinal astrocytes with selective ribosome immunoprecipitation using the Ribotag method. Our microdissection method is rapid and preserves astrocyte morphology, resulting in a brief post-mortem interval and minimizing loss of RNA from distal regions of these cells. Both microdissection and Ribotag immunoprecipitation require a minimum of specialized equipment or reagents, and by using them in conjunction we are able to achieve > 100-fold enrichment of astrocyte RNA.