Retinal Astrocytes Research Articles

Oxygen-Induced Retinopathy from Recurrent Intermittent Hypoxia Is Not Dependent on Resolution with Room Air or Oxygen, in Neonatal Rats.

Preterm infants often experience intermittent hypoxia (IH) with resolution in room air (RA) or hyperoxia (Hx) between events. Hypoxia is a major inducer of vascular endothelial growth factor, which plays a key role in normal and aberrant retinal angiogenesis. This study tested the hypothesis that neonatal IH which resolved with RA is less injurious to the immature retina than IH resolved by Hx between events. Newborn rats were exposed to: (1) Hx (50% O2) with brief hypoxia (12% O2); (2) RA with 12% O2; (3) Hx with RA; (4) Hx only; or (5) RA only, from P0 to P14. Pups were examined at P14 or placed in RA until P21. Retinal vascular and astrocyte integrity; retinal layer thickness; ocular and systemic biomarkers of angiogenesis; and somatic growth were determined at P14 and P21. All IH paradigms resulted in significant retinal vascular defects, disturbances in retinal astrocyte template, retinal thickening, and photoreceptor damage concurrent with elevations in angiogenesis biomarkers. These data suggest that the susceptibility of the immature retina to changes in oxygen render no differences in the outcomes between RA or O2 resolution. Interventions and initiatives to curtail O2 variations should remain a high priority to prevent severe retinopathy.

The soluble epoxide hydrolase attenuates pathological vascularization by preventing astrocyte loss in a retinopathy of prematurity model in mice

Retinopathy of prematurity (ROP) is a leading cause of childhood blindness. Several signalling pathways have been implicated in the process of ROP but a novel drugable target is the cytochrome P450 (CYP)/soluble epoxide hydrolase (sEH) pathway. The latter enzymes play a crucial role in biological processes by generating bioactive epoxides and diols from endogenous polyunsaturated fatty acids (PUFAs). This study aimed to characterize the effects of the newly identified sEH‐derived w‐3 PUFA diol; 19,20‐dihydroxydocosapentaenoic acid (DHDP) in a mouse ROP model.ROP in mice was achieved by placing pups in 75% oxygen for 5 days from postnatal day (P) 7 until P12, before moving animals back to room air for an additional 5 days. This procedure resulted in the development of avascular areas that were significantly larger in retinas from sEH−/− mice than in their wild‐type (WT) littermates at P17. Avascularization was accompanied by angiogenesis in the form of characteristic vascular “tufts” which were more evident in retinas from sEH−/− mice. Although there was no difference in vascular obliteration, a marked decrease in the astrocyte network was observed in sEH−/− mice after exposure to high oxygen. Mechanistically, astrocyte loss in hyperoxic conditions was a consequence of elevated mitochondrial (mt) DNA damage mediated by increased interaction of the PARP‐1 DNA binding domain with mtDNA, as revealed by chromatin immunoprecipitation studies. Exposure of astrocytes to 75% O2 in vitro resulted in elevated PARP‐1 cleavage/activation; a phenomenon reversed in the presence of 19,20‐DHDP. Administration of 19,20‐DHDP during hyperoxia preserved the complex of presenilin 1 with mitochondrial carrier homolog 1 (MTCH‐1) in the outer mitochondria membrane, inhibiting the translocation of the MTCH‐1 to the inner mitochondria membrane and subsequently inhibiting PARP‐1 cleavage. The intravitreal injection of 19,20‐DHDP at day 7 significantly prevented astrocyte loss caused by high O2 levels at day 8, and reduced both the avascular area as well as vascular tuft formation at P17 in the sEH−/− mice.Our date indicate that the sEH dependent generation of 19,20‐DHDP from w‐3PUFA is required for retinal astrocyte survival and preservation of DNA integrity under hyperoxic conditions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The AMPK-PGC-1α signaling axis regulates the astrocyte glutathione system to protect against oxidative and metabolic injury

Neurons are highly sensitive to metabolic and oxidative injury, but endogenous astrocyte mechanisms have a critical capacity to provide protection from these stresses. We previously reported that the master regulator PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1α) is necessary for retinal astrocytes to mount effective injury responses, with particular regard to oxidative stress. Yet, this pathway has not been well studied in glia. PGC-1α is a transcriptional co-activator that is dysregulated in a variety of neurodegenerative diseases. It functions as a master regulator of cellular bioenergetics, with the ability to regulate tissue specific responses. A key inducer of PGC-1α signaling is adenosine monophosphate-activated kinase (AMPK). Thus, the AMPK-PGC-1α signaling axis coordinates metabolic and oxidative damage responses in the central nervous system (CNS). Here we report that AMPK selectively regulates expression of GCLM (glutamate cysteine ligase modulatory subunit) in astrocytes, but not neurons, through PGC-1α activation. Glutamate cysteine ligase (GCL) is the rate limiting enzyme in the biosynthesis of glutathione (GSH); a critical antioxidant and detoxifying peptide in the CNS. Through this mechanism we describe PGC-1α-dependent induction of GSH synthesis and antioxidant activity in astrocytes, and in the rodent retina in vivo. Furthermore, we demonstrate that therapeutic agonism of this pathway with the AMP mimetic, AICAR, rescues GSH levels in vivo, while reducing RGC death and astrocyte reactivity, following retinal ischemia/reperfusion injury. This mechanism presents a novel strategy for enhancing protective astrocyte antioxidant capacity in the CNS.

Significance of Endothelin-1 in Glaucoma - a Short Overview

This review article is focused on the various facets of possible endothelin's role in glaucoma; involvement of endothelin in other ocular, in particular vascular, diseases is not specifically discussed. Endothelin is an ubiquitous molecule that occurs in practically all ocular tissues. Its primary physiological function is regulation of the blood vessel diameter and hence regulation of the blood supply in tissues. It is secreted locally, and exerts its effect also predominantly locally. This limits the value of venous blood sampling for estimation of the endothelin function in a particular patient, or in study cohorts as well. Endothelin is involved in the regulation of intraocular pressure, in the regulation of blood flow and in activation of retinal and optic nerve head astrocytes. All these functions are of high importance when it comes to pathogenesis of glaucoma. Possible future directions for glaucoma treatment should encompass pharmacological antagonism to endothelin, an avenue which is at present hindered by potentially serious side-effects of available endothelin-antagonists.

Sox2 regulates astrocytic and vascular development in the retina.

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.

Retinal putative glial alterations: implication for glaucoma care.

PurposeGliosis‐like retinal alterations, presumed to be activated retinal astrocytes and Müller cells (ARAM), have been reported to occur frequently in patients with glaucoma but rarely in controls. We investigated the association between glaucomatous abnormality and the presence, the extent of retinal region, and the spatial distribution, of hyperreflective retinal alterations on optical coherence tomography (OCT) en‐face images, presumed to be ARAM.MethodsFindings of hyperreflective structures, presumed to be ARAM, in the central retinal ±24 degrees of OCT en‐face images (acquired with the SPECTRALIS® OCT) were compared between 35 younger controls, 42 older controls and 38 patients with glaucoma. Presumed ARAM was defined as reflective structures on the en‐face images other than retinal vasculature and retinal nerve fibre bundles. Chi‐square tests were used to compare the proportion of younger controls vs older controls with presumed ARAM to investigate the effect of ageing, and the proportion of patients vs age‐similar older controls with presumed ARAM to investigate the effect of disease. We also investigated the effect of glaucoma on the retinal area with presumed ARAM when it was present; we used an analysis of covariance (ancova) to compare the retinal area with hyperreflectivity in patients vs controls, adjusting for the effects of age and axial length.ResultsThe mean (S.D.) age of the younger controls, older controls, and patients with glaucoma was 26 (3), 62 (10) and 69 (8) years, respectively. The median (25th quartile, 75th quartile) of the retinal region with the hyperreflective structures, presumed to be ARAM, was zero (0,0), 1 (0,6), and 11 (0,43) degrees square in the younger controls, older controls and patients with glaucoma respectively. The chi‐square test investigating the effect of ageing found χ2(1, N = 77) = 24.8, p < 0.001, and that investigating the effect of disease found χ2(1, N = 80) = 2.3, p = 0.1. The ancova found F(1, 46) = 10.32, P = 0.02.ConclusionsThere was an effect of ageing on the presence of the hyperreflective structures, presumed to be ARAM, on OCT images. Compared to the presence of hyperreflective structures, the extent of retinal region with the hyperreflective structures has a greater potential of being an indicator of glaucomatous degeneration. Further study is needed to investigate the nature of the relation between glaucomatous abnormality and the extent of the retina with the hyperreflective structures, presumed to be ARAM.

Astrocyte-derived lipoxins A4 and B4 promote neuroprotection from acute and chronic injury.

Astrocytes perform critical non-cell autonomous roles following CNS injury that involve either neurotoxic or neuroprotective effects. Yet the nature of potential prosurvival cues has remained unclear. In the current study, we utilized the close interaction between astrocytes and retinal ganglion cells (RGCs) in the eye to characterize a secreted neuroprotective signal present in retinal astrocyte conditioned medium (ACM). Rather than a conventional peptide neurotrophic factor, we identified a prominent lipid component of the neuroprotective signal through metabolomics screening. The lipoxins LXA4 and LXB4 are small lipid mediators that act locally to dampen inflammation, but they have not been linked directly to neuronal actions. Here, we determined that LXA4 and LXB4 are synthesized in the inner retina, but their levels are reduced following injury. Injection of either lipoxin was sufficient for neuroprotection following acute injury, while inhibition of key lipoxin pathway components exacerbated injury-induced damage. Although LXA4 signaling has been extensively investigated, LXB4, the less studied lipoxin, emerged to be more potent in protection. Moreover, LXB4 neuroprotection was different from that of established LXA4 signaling, and therapeutic LXB4 treatment was efficacious in a chronic model of the common neurodegenerative disease glaucoma. Together, these results identify a potential paracrine mechanism that coordinates neuronal homeostasis and inflammation in the CNS.

Human retinal endothelial cells and astrocytes cultured on 3-D scaffolds for ocular drug discovery and development

Topical ocular ketorolac improves the outcomes of severe retinopathy of prematurity and when administered with systemic caffeine, decreases the severity of oxygen-induced retinopathy. We tested the hypothesis that co-cultures of human retinal endothelial cells (HRECs) and human retinal astrocytes (HRAs) on 3-dimensional (3-D) hydrogel scaffolds is a more representative biomimetic paradigm of the blood-retinal-barrier (BRB) than 2-D cultures, and should be utilized for preclinical drug discovery and development. Mono- and co-cultures of HRECs and HRAs were treated with standard doses of ketorolac, ibuprofen, and/or caffeine, and exposed to hyperoxia, intermittent hypoxia (IH), or normoxia on 2-D surfaces or 3-D biodegradable hydrogel scaffolds (AlgiMatrix or Geltrex). Media and cells were collected at 72h post treatment for arachidonic acid metabolites. Cells cultured on 3-D scaffolds exhibited less oxidative stress and variability in drug responses. HRAs enhanced the responses of HRECs to drugs and changes in oxygen environment. PGE2 and PGI2 were the predominant prostanoids produced in response to IH, reflecting COX-2 immunoreactivity. We conclude that HRECs and HRAs co-cultured on 3-D scaffolds may recapitulate drug responses of the dynamic BRB and therefore should be implemented for preclinical ocular drug discovery and development.

Retinal Astrocytes and GABAergic Wide-Field Amacrine Cells Express PDGFRα: Connection to Retinal Ganglion Cell Neuroprotection by PDGF-AA.

PurposeOur previous experiments demonstrated that intravitreal injection of platelet-derived growth factor-AA (PDGF-AA) provides retinal ganglion cell (RGC) neuroprotection in a rodent model of glaucoma. Here we used PDGFRα-enhanced green fluorescent protein (EGFP) mice to identify retinal cells that may be essential for RGC protection by PDGF-AA.MethodsPDGFRα-EGFP mice expressing nuclear-targeted EGFP under the control of the PDGFRα promoter were used. Localization of PDGFRα in the neural retina was investigated by confocal imaging of EGFP fluorescence and immunofluorescent labeling with a panel of antibodies recognizing different retinal cell types. Primary cultures of mouse RGCs were produced by immunopanning. Neurobiotin injection of amacrine cells in a flat-mounted retina was used for the identification of EGFP-positive amacrine cells in the inner nuclear layer.ResultsIn the mouse neural retina, PDGFRα was preferentially localized in the ganglion cell and inner nuclear layers. Immunostaining of the retina demonstrated that astrocytes in the ganglion cell layer and a subpopulation of amacrine cells in the inner nuclear layer express PDGFRα, whereas RGCs (in vivo or in vitro) did not. PDGFRα-positive amacrine cells are likely to be Type 45 gamma-aminobutyric acidergic (GABAergic) wide-field amacrine cells.ConclusionsThese data indicate that the neuroprotective effect of PDGF-AA in a rodent model of glaucoma could be mediated by astrocytes and/or a subpopulation of amacrine cells. We suggest that after intravitreal injection of PDGF-AA, these cells secrete factors protecting RGCs.

Ocular inflammation as a motor for axonal regeneration in the optic nerve

SummaryRetinal ganglion cells (RGCs) normally fail to regenerate injured axons and die upon axotomy. However, induction of intraocular inflammation (IS), which is associated with an activation of retinal astrocytes and microglia transforms RGCs into a regenerative state. It delays neuronal degeneration and enables axon growth into the injured nerve. CNTF, LIF and IL‐6 released from retinal astrocytes have been shown to be critical for this stimulation. However, the role of microglia has not yet been investigated in this context. Using a pharmacological approach based on CSF1R inhibition we show that almost all microglia are eliminated in murine retinae and optic nerves without affecting peripheral monocytes/macrophages or other cells of the visual system. We surprisingly found that the time course and extent of RGC degeneration after optic nerve crush remained unaffected despite the presence of apoptotic RGC remnants after microglia depletion. In addition, microglia depletion neither affected the induction of regeneration associated genes upon optic nerve injury nor the increased regenerative potential of RGCs upon IS. Thus, microglia are not essentially involved in RGC degeneration or axonal regeneration after acute optic nerve injury.

Directed migration of retinal astrocytes by PDGF signaling

PurposeProper patterning of astrocytes is crucial for retinal angiogenesis in both rodents and human. Platelet‐derived growth factor (PDGF) is one of the key regulators of cell migration, but distinctive downstream pathways have been implicated in a variety of cell types. Herein we investigated the detailed mechanism of PDGFA‐directed astrocyte migration in early postnatal mouse retina.MethodsTransgenic mice with glia‐specific deletion of PDGFRα and multiple potential downstream effectors of PDGF signaling pathway are generated. Perinatal astrocytes and retinal vasculature are evaluated by whole mount IHC.ResultsAstrocyte migration and retinal angiogenesis are severely impaired in knockout mice of PDGFRα. This is phenocopied by mutations in PI3K catalytic subunits p110αβ, as well as in mutations of PI3K binding site in PDGFRα. Rac/Rap mediated cytoskeleton rearrangement is also imperative for the patterning of astrocytic network. On the other hand, disruption of mTOR signaling by knocking out binding partner Raptor or Rictor had no effect on astrocyte motility. PLCγ pathway, which is essential for PDGF chemotaxis in mesenchymal cells, is also dispensable for astrocyte migration.ConclusionsThis study demonstrated that PDGFA‐directed astrocyte migration is mediated through PI3K and Rac/Rap signaling, but not PLCγ or Akt/mTOR pathway. These findings add mechanistic insight into cell type‐specific regulation of migration by PDGF.

Interleukin-6 Deficiency Attenuates Retinal Ganglion Cell Axonopathy and Glaucoma-Related Vision Loss.

The pleotropic cytokine interleukin-6 (IL-6) is implicated in retinal ganglion cell (RGC) survival and degeneration, including that associated with glaucoma. IL-6 protects RGCs from pressure-induced apoptosis in vitro. However, it is unknown how IL-6 impacts glaucomatous degeneration in vivo. To study how IL-6 influences glaucomatous RGC axonopathy, accompanying glial reactivity, and resultant deficits in visual function, we performed neural tracing, histological, and neurobehavioral assessments in wildtype (B6;129SF2/J; WT) and IL-6 knock-out mice (B6;129S2-IL6tm1kopf/J; IL-6-/-) after 8 weeks of unilateral or bilateral microbead-induced glaucoma (microbead occlusion model). IOP increased by 20% following microbead injection in both genotypes (p < 0.05). However, deficits in wound healing at the site of corneal injection were noted. In WT mice, elevated IOP produced degenerating axon profiles and decreased axon density in the optic nerve by 15% (p < 0.01). In IL-6-/- mice, axon density in the optic nerve did not differ between microbead- and saline-injected mice (p > 0.05) and degenerating axon profiles were minimal. Preservation of RGC axons was reflected in visual function, where visual acuity decreased significantly in a time-dependent manner with microbead-induced IOP elevation in WT (p < 0.001), but not IL-6-/- mice (p > 0.05). Despite this preservation of RGC axons and visual acuity, both microbead-injected WT and IL-6-/- mice exhibited a 50% decrease in anterograde CTB transport to the superior colliculus, as compared to saline-injected controls (p < 0.01). Assessment of glial reactivity revealed no genotype- or IOP-dependent changes in retinal astrocytes. IOP elevation decreased microglia density and percent retinal area covered in WT mice (p < 0.05), while IL-6-/- mice exhibited only a decrease in density (p < 0.05). Together, our findings indicate that two defining features of RGC axonopathy—axon transport deficits and structural degeneration of axons—likely occur via independent mechanisms. Our data suggest that IL-6 is part of a mechanism that specifically leads to structural degeneration of axons. Furthermore, its absence is sufficient to prevent both structural degeneration of the optic nerve and vision loss. Overall, our work supports the proposition that functional deficits in axon transport represent a therapeutic window for RGC axonopathy and identify IL-6 signaling as a strong target for such a therapeutic.

LB47 - Making multiple therapeutic cell products from a cGMP-compliant iPSC line

Induced pluripotent stem cell (iPSC) can differentiate into multiple phenotypes, and indeed multiple protocols for such differentiation have been established. Although the efficiency of different protocols is variable, most protocols work with most lines with some tweaking. To confirm this hypothesis and to make a clinically compliant line available for independent evaluation, we have taken the current Good Manufacture Practice (cGMP)-compliant iPSC line developed by the NIH, and used it to develop cellular products that are currently being considered for therapy. We focused on retinal cells, MSC, NSC, dopaminergic neurons and astrocytes. We obtained a WCB from the NIH and prepared a stock at passage 20. After testing quality we used it to generate multiple products using standard protocols. To confirm that these protocols could also be used for other iPSC lines we obtained a second clinically compliant line and tested the reproducibility of our methodology. Our results confirmed that the same protocols could be used with minimal modifications with multiple qualified lines. We believe that our demonstration that multiple products can be made from the same WCB bank and the same protocols can be used with multiple lines offers a path to a cost effective strategy for developing cellular products.

Cellular retinoic acid bioavailability in various pathologies and its therapeutic implication.

Retinoic acid (RA), an active metabolite of vitamin A, is a critical signaling molecule in various cell types. We found that RA depletion caused by expression of the RA-metabolizing enzyme CYP26A1 promotes carcinogenesis, implicating CYP26A1 as a candidate oncogene. Several studies of CYP26s have suggested that the biological effect of RA on target cells is primarily determined by "cellular RA bioavailability", which is defined as the RA level in an individual cell, rather than by the serum concentration of RA. Consistently, stellate cells store approximately 80% of vitamin A in the body, and the state of cellular RA bioavailability regulates their function. Based on the similarities between stellate cells and astrocytes, we demonstrated that retinal astrocytes regulate tight junction-based endothelial integrity in a paracrine manner. Since diabetic retinopathy is characterized by increased vascular permeability in its early pathogenesis, RA normalized retinal astrocytes that are compromised in diabetes, resulting in suppression of vascular leakiness. RA also attenuated the loss of the epithelial barrier in murine experimental colitis. The concept of "cellular RA bioavailability" in various diseases will be directed at understanding various pathologies caused by RA insufficiency, implying the potential feasibility of a therapeutic strategy targeting the stellate cell system.

The P2X7 receptor links mechanical strain to cytokine IL-6 up-regulation and release in neurons and astrocytes.

Mechanical strain in neural tissues can lead to the up-regulation and release of multiple cytokines including interleukin 6 (IL-6). In the retina, the mechanosensitive release of ATP can autostimulate P2X7 receptors on both retinal ganglion cell neurons and optic nerve head astrocytes. Here, we asked whether the purinergic signaling contributed to the IL-6 response to increased intraocular pressure (IOP) invivo, and stretch or swelling invitro. Rat and mouse eyes were exposed to non-ischemic elevations in IOP to 50-60mmHg for 4h. A PCR array was used to screen cytokine changes, with quantitative (q)PCR used to confirm mRNA elevations and immunoblots used for protein levels. P2X7 antagonist Brilliant Blue G (BBG) and agonist (4-benzoyl-benzoyl)-ATP (BzATP) were injected intravitreally. ELISA was used to quantify IL-6 release from optic nerve head astrocytes or retinal ganglion cells. Receptor identity was confirmed pharmacologically and in P2X7-/- mice, acute elevation of IOP altered retinal expression of multiple cytokine genes. Elevation of IL-6 was greatest, with expression of IL1rn, IL24, Tnf, Csf1, and Lif also increased more than twofold, while expression of Tnfsf11, Gdf9, and Tnfsf4 were reduced. qPCR confirmed the rise in IL-6 and extracellular ATP marker ENTPD1, but not pro-apoptotic genes. Intravitreal injection of P2X7 receptor antagonist BBG prevented the pressure-dependent rise in IL-6 mRNA and protein in the rat retina, while injection of P2X7 receptor agonist BzATP was sufficient to elevate IL-6 expression. IOP elevation increased IL-6 in wild-type but not P2X7R knockout mice. Application of mechanical strain to isolated optic nerve head astrocytes increased IL-6 levels. This response was mimicked by agonist BzATP, but blocked by antagonists BBG and A839977. Stretch or BzATP led to IL-6 release from both astrocytes and isolated retinal ganglion cells. The mechanosensitive up-regulation and release of cytokine IL-6 from the retina involves the P2X7 receptor, with both astrocytes and neurons contributing to the response.

Large is required for normal astrocyte migration and retinal vasculature development

BackgroundPersistent fetal vasculature (PFV) is a congenital developmental anomaly of the eye that accounts for about 5% of childhood blindness. The molecular mechanism of PFV remains unclear. As a glycosyltransferase of α-dystroglycan, LARGE mutations have been found in congenital muscular dystrophy patients with brain abnormalities. Spontaneous Large mutant mice displayed similar symptoms of human muscle–eye–brain disorders. However, the detailed roles of Large in ocular vasculature development still need to be uncovered.ResultsIn this paper, we report that a novel Large mutation generated by the piggyBac transposon insertion leads to PFV and abnormal retinal vasculature in mice. Glycosylation of α-DG, an essential component of the extracellular matrix, was significantly impaired in these Large mutants, leading to broken inner limiting membrane (ILM). As a guide of the retinal vasculature development, the distribution of retinal astrocytes became irregular within the retina, and many astrocytes abnormally migrated into the vitreous along with the hyaloid vessels in Large mutants.ConclusionsLarge is essential for ILM formation and retinal astrocyte migration. The novel Large mutant mouse can serve as a new PFV model to further dissect LARGE functions in ocular vasculature development.

Microglia activation is essential for BMP7-mediated retinal reactive gliosis

BackgroundOur previous studies have shown that BMP7 is able to trigger activation of retinal macroglia. However, these studies showed the responsiveness of Müller glial cells and retinal astrocytes in vitro was attenuated in comparison to those in vivo, indicating other retinal cell types may be mediating the response of the macroglial cells to BMP7. In this study, we test the hypothesis that BMP7-mediated gliosis is the result of inflammatory signaling from retinal microglia.MethodsAdult mice were injected intravitreally with BMP7 and eyes harvested 1, 3, or 7 days postinjection. Some mice were treated with PLX5622 (PLX) to ablate microglia and were subsequently injected with control or BMP7. Processed tissue was analyzed via immunofluorescence, RT-qPCR, or ELISA. In addition, cultures of retinal microglia were treated with vehicle, lipopolysaccharide, or BMP7 to determine the effects of BMP7-isolated cells.ResultsMice injected with BMP7 showed regulation of various inflammatory markers at the RNA level, as well as changes in microglial morphology. Isolated retinal microglia also showed an upregulation of BMP-signaling components following treatment. In vitro treatment of retinal astrocytes with conditioned media from activated microglia upregulated RNA levels of gliosis markers. In the absence of microglia, the mouse retina showed a subdued gliosis and inflammatory response when exposed to BMP7.ConclusionsGliosis resulting from BMP7 is mediated through an inflammatory response from retinal microglia.

Glioprotection of Retinal Astrocytes After Intravitreal Administration of Memantine in the Mouse Optic Nerve Crush Model.

BackgroundIn glaucoma, non-intraocular pressure (IOP)-related risk factors can result in increased levels of extracellular glutamate, which triggers a cascade of neurodegeneration characterized by the excessive activation of N-methyl-D-aspartate (NMDA). The purpose of our study was to evaluate the glioprotective effects of memantine as a prototypic uncompetitive NMDA blocker on retinal astrocytes in the optic nerve crush (ONC) mouse model for glaucoma.Material/MethodsOptic nerve crush was performed on all of the right eyes (n=8), whereas left eyes served as contralateral healthy controls (n=8) in Balb/c/Sca mice. Four randomly assigned mice received 2-μl intravitreal injections of memantine (1 mg/ml) after ONC in the experimental eye. One week after the experiment, optic nerves were dissected and stained with methylene blue. Retinae were detached from the sclera. The tissue was immunostained. Whole-mount retinae were investigated by fluorescent microscopy. Astrocyte counts for each image were performed manually.ResultsHistological sections of crushed optic nerves showed consistently moderate tissue damage in experimental groups. The mean number of astrocytes per image in the ONC group was significantly lower than in the healthy control group (7.13±1.5 and 10.47±1.9, respectively). Loss of astrocytes in the memantine-treated group was significantly lower (8.83±2.2) than in the ONC group. Assessment of inter-observer reliability showed excellent agreement among observations in control, ONC, and memantine groups.ConclusionsThe ONC is an effective method for investigation of astrocytic changes in mouse retina. Intravitreally administered memantine shows a promising glioprotective effect on mouse retinal astrocytes by preserving astrocyte count after ONC.

Roles of Hypoxia Response in Retinal Development and Pathophysiology.

The hypoxia response is a fundamental phenomenon mainly regulated by hypoxia-inducible factors (HIFs). For more than a decade, we have investigated and revealed the roles of the hypoxia response in the development, physiology, and pathophysiology of the retina by generating and utilizing cell-type-specific conditional knockout mice. To investigate the functions of genes related to the hypoxia response in cells composing the retina, we generated various mouse lines that lack HIFs and/or related genes specifically in retinal neurons, astrocytes, myeloid cells, or retinal pigment epithelium cells. We found that these genes in the different types of retinal cells contribute in various ways to the homeostasis of ocular vascular and visual function. We hypothesized that the activation of HIFs is likely involved in the development and progress of retinal diseases, and we subsequently confirmed the pathological roles of HIFs in animal models of neovascular and atrophic ocular diseases. Currently, anti-vascular endothelial growth factor (anti-VEGF) therapy is a first-line treatment widely used for neovascular retinal diseases. However, alternative or additional targets are now required because several recent large-scale clinical trials and animal studies, including our own research, have indicated that VEGF antagonism may induce retinal vascular and neuronal degeneration. We have identified and confirmed a microRNA as a candidate for an alternative target against neovascular retinal diseases, and we are now working to establish a novel HIF inhibitor for clinical use based on the disease mechanism that we identified.

Structure and Function Relationship of Activated Retinal Glia in Primary Open-Angle Glaucoma Patients.

Purpose To evaluate clinically activated retinal astrocytes and Müller cells (ARAM) regarding retinal sensitivity and retinal nerve fiber layer (RNFL) thickness in primary open-angle glaucoma (POAG). Methods Central visual field (VF; i.e., retinal sensitivity) was measured with a custom-made macular pattern by microperimetry and correlated with the presence (ARAM+) or absence (ARAM−) of ARAM on red-free fundus photography and with the corresponding RNFL by optical coherence tomography (OCT). Results In the eyes of POAG patients, ARAM+ had overall a significantly lower retinal sensitivity (ARAM+: 7.34 dB, ARAM−: 11.9 dB; p < 0.001) and lower RNFL thickness in the inferior peripapillary quadrants compared to ARAM− (RNFL superior: ARAM+ 74.2 μm, ARAM− 77.5 μm; RNFL temporal: ARAM+ 46.8 μm, ARAM− 53.0 μm, p < 0.001; and RNFL inferior: ARAM+ 63.2 μm, ARAM− 73.1 μm, p < 0.001). Within the same eye, ARAM+ showed a lower retinal sensitivity compared to ARAM− ([ARAM− (11.13 dB)] − [ARAM+ (9.56 dB) = 1.57 dB; p = 0.25). The proportion of ARAM+ per eye correlated strongly with reduced retinal light sensitivity (p = 0.02), corresponding lower peripapillary RNFL thickness (p = 0.02), and lower RNFL temporal quadrant thickness (p < 0.01), but not with greater age (p = 0.45). Conclusion ARAM was more frequently identified in the eyes with a lower retinal sensitivity and peripapillary RNFL thickness and may be a clinical sign in the macula for an advanced stage of POAG.