11-cis-retinal and Ischemia

Ligand-driven modulation of the mitochondrial translocator protein 18 kDa (TSPO) was recently described to dampen the neuroinflammatory response of microglia in a retinal light damage model resulting in protective effects on photoreceptors. We characterized the effects of the TSPO ligand XBD173 in the postischemic retina focusing on changes in the response pattern of the major glial cell types of the retina-microglia and Müller cells.. Retinal ischemia was induced by increasing the intraocular pressure for 60 min followed by reperfusion of the tissue in mice. On retinal cell types enriched via immunomagnetic separation expression analysis of TSPO, its ligand diazepam-binding inhibitor (DBI) and markers of glial activation were performed at transcript and protein level using RNA sequencing, qRT-PCR, lipid chromatography-mass spectrometry, and immunofluorescent labeling. Data on cell morphology and numbers were assessed in retinal slice and flatmount preparations. The retinal functional integrity was determined by electroretinogram recordings.. We demonstrate that TSPO is expressed by Müller cells, microglia, vascular cells, retinal pigment epithelium (RPE) of the healthy and postischemic retina, but only at low levels in retinal neurons. While an alleviated neurodegeneration upon XBD173 treatment was found in postischemic retinae as compared to vehicle controls, this neuroprotective effect of XBD173 is mediated putatively by its action on retinal glia. After transient ischemia, TSPO as a marker of activation was upregulated to similar levels in microglia as compared to their counterparts in healthy retinae irrespective of the treatment regimen. However, less microglia were found in XBD173-treated postischemic retinae at 3 days post-surgery (dps) which displayed a more ramified morphology than in retinae of vehicle-treated mice indicating a dampened microglia activation. Müller cells, the major retinal macroglia, show upregulation of the typical gliosis marker GFAP. Importantly, glutamine synthetase was more stably expressed in Müller glia of XBD173-treated postischemic retinae and homeostatic functions such as cellular volume regulation typically diminished in gliotic Müller cells remained functional.. In sum, our data imply that beneficial effects of XBD173 treatment on the postischemic survival of inner retinal neurons were primarily mediated by stabilizing neurosupportive functions of glial cells.

Topics: Animals; Antigens, Differentiation; Arginase; Carrier Proteins; Disease Models, Animal; Electroretinography; Gene Expression Regulation; Glutamate-Ammonia Ligase; Ischemia; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Neuroglia; Purines; Receptors, GABA; Retina; Retinal Diseases; Retinal Neurons; Rhodopsin; RNA, Messenger

Retinal ischemia is common in eye disorders, like diabetic retinopathy or retinal vascular occlusion. The goal of this study was to evaluate the potential protective effects of an intravitreally injected vascular endothelial growth factor (VEGF) inhibitor (ranibizumab) on retinal cells in an ischemia animal model via immunohistochemistry (IF) and quantitative real-time PCR (PCR). A positive binding of ranibizumab to rat VEGF-A was confirmed via dot blot. One eye underwent ischemia and a subgroup received ranibizumab. A significant VEGF increase was detected in aqueous humor of ischemic eyes (p = 0.032), whereas VEGF levels were low in ranibizumab eyes (p = 0.99). Ischemic retinas showed a significantly lower retinal ganglion cell number (RGC; IF Brn-3a: p<0.001, IF RBPMS: p<0.001; PCR: p = 0.002). The ranibizumab group displayed fewer RGCs (IF Brn-3a: 0.3, IF RBPMS: p<0.001; PCR: p = 0.007), but more than the ischemia group (IF Brn-3a: p = 0.04, IF RBPMS: p = 0.03). Photoreceptor area was decreased after ischemia (IF: p = 0.049; PCR: p = 0.511), while the ranibizumab group (IF: p = 0.947; PCR: p = 0.122) was comparable to controls. In the ischemia (p<0.001) and ranibizumab group (p<0.001) a decrease of ChAT+ amacrine cells was found, which was less prominent in the ranibizumab group. VEGF-receptor 2 (VEGF-R2; IF: p<0.001; PCR: p = 0.021) and macroglia (GFAP; IF: p<0.001; PCR: p<0.001) activation was present in ischemic retinas. The activation was weaker in ranibizumab retinas (VEGF-R2: IF: p = 0.1; PCR: p = 0.03; GFAP: IF: p = 0.1; PCR: p = 0.015). An increase in the number of total (IF: p = 0.003; PCR: p = 0.023) and activated microglia (IF: p<0.001; PCR: p = 0.009) was detected after ischemia. These levels were higher in the ranibizumab group (Iba1: IF: p<0.001; PCR: p = 0.018; CD68: IF: p<0.001; PCR: p = 0.004). Our findings demonstrate that photoreceptors and RGCs are protected by ranibizumab treatment. Only amacrine cells cannot be rescued. They seem to be particularly sensitive to ischemic damage and need maybe an earlier intervention.

Topics: Amacrine Cells; Animals; Aqueous Humor; Calcium-Binding Proteins; Cell Count; Cholinergic Neurons; Disease Models, Animal; Glial Fibrillary Acidic Protein; Humans; Ischemia; Mice; Microfilament Proteins; Microglia; Photoreceptor Cells, Vertebrate; Protective Agents; Protein Binding; Ranibizumab; Rats; Reperfusion Injury; Retina; Retinal Ganglion Cells; Rhodopsin; RNA, Messenger; Synapses; Vascular Endothelial Growth Factor A

Delivery of antibodies to monitor key biomarkers of retinopathy in vivo represents a significant challenge because living cells do not take up immunoglobulins to cellular antigens. We met this challenge by developing novel contrast agents for retinopathy, which we used with magnetic resonance imaging (MRI). Biotinylated rabbit polyclonal to chick IgY (rIgPxcIgY) and phosphorylthioate-modified oligoDNA (sODN) with random sequence (bio-sODN-Ran) were conjugated with NeutrAvidin-activated superparamagnetic iron oxide nanoparticles (SPION). The resulting Ran-SPION-rIgPxcIgY carries chick polyclonal to microtubule-associated protein 2 (MAP2) as Ran-SPION-rIgP/cIgY-MAP2, or to rhodopsin (Rho) as anti-Rho-SPION-Ran. We examined the uptake of Ran-SPION-rIgP/cIgY-MAP2 or SPION-rIgP/cIgY-MAP2 in normal C57black6 mice (n = 3 each, 40 μg/kg, i.c.v.); we found retention of Ran-SPION-rIgP/cIgY-MAP2 using molecular contrast-enhanced MRI in vivo and validated neuronal uptake using Cy5-goat IgPxcIgY ex vivo. Applying this novel method to monitor retinopathy in a bilateral carotid artery occlusion-induced ocular ischemia, we observed pericytes (at d 2, using Gd-nestin, by eyedrop solution), significant photoreceptor degeneration (at d 20, using anti-Rho-SPION-Ran, eyedrops, P = 0.03, Student's t test), and gliosis in Müller cells (at 6 mo, using SPION-glial fibrillary acidic protein administered by intraperitoneal injection) in surviving mice (n ≥ 5). Molecular contrast-enhanced MRI results were confirmed by optical and electron microscopy. We conclude that chimera and molecular contrast-enhanced MRI provide sufficient sensitivity for monitoring retinopathy and for theranostic applications.

Topics: Animals; Brain Ischemia; Carotid Arteries; Contrast Media; Eye Injuries; Ischemia; Male; Mice; Mice, Inbred C57BL; Neurons; Retinal Diseases; Rhodopsin

In this study, we used double transgenic mice with inducible expression of angiopoietin-2 (Ang2) to investigate the role of Ang2 in the retinal and choroidal circulations and in three models of ocular neovascularization (NV). Mice with induced expression of Ang2 ubiquitously, or specifically in the retina, survived and appeared grossly normal. They also had normal-appearing retinal and choroidal circulations, demonstrating that high levels of Ang2 did not induce regression of mature retinal or choroidal vessels. When Ang2 expression was induced soon after birth, there was increased density of the deep capillary bed on postnatal day (P) 11 that returned to normal by P18, the time that retinal vascular development is usually completed. In mice with ischemic retinopathy, induction of Ang2 during the ischemic period resulted in a significant increase in retinal NV, but induction of Ang2 at a later time point when ischemia (and vascular endothelial growth factor [VEGF]) was less, hastened regression of NV. In triple transgenic mice that coexpressed VEGF and Ang2, the increased expression of Ang2 inhibited VEGF-induced NV in the retina. Increased expression of Ang2 also resulted in regression of choroidal neovascularization. These data suggest that ocular neovascularization, but not mature retinal or choroidal vessels, is sensitive to Ang2; a high Ang2/VEGF ratio promotes regression, while high Ang2 in the setting of hypoxia and/or concomitantly high Ang2 and VEGF stimulate neovascularization.

Topics: Angiopoietin-2; Animals; Animals, Newborn; Blood Vessels; Capillaries; Choroid; Doxycycline; Gene Expression; Ischemia; Mice; Mice, Transgenic; Neovascularization, Physiologic; Photoreceptor Cells; Retina; Retinal Vessels; Reverse Transcriptase Polymerase Chain Reaction; Rhodopsin; RNA, Messenger; Vascular Endothelial Growth Factor A

Retinal astrocytes are located in the nerve fiber layer and along retinal blood vessels and have been hypothesized to participate in the induction and maintenance of the blood-retinal barrier. Platelet-derived growth factor-A (PDGF-A) is normally produced by retinal ganglion cells and is involved in astrocyte recruitment and proliferation. We used gain-of-function transgenic mice that express PDGF-A in photoreceptors to explore the roles of PDGF-A and astrocytes in the retina. Transgene-positive mice developed glial infiltration of the inner retina and had significantly less oxygen-induced retinal vascular closure and no neovascularization compared with littermate controls, which had prominent vascular closure and neovascularization. The increased survival of endothelial cells in transgenic mice in the face of oxygen-induced down-regulation of vascular endothelial growth factor was accompanied by an increase in astrocyte-derived fibroblast growth factor-2. Therefore, PDGF-A increases retinal astrocytes, which promote the survival of endothelial cells as well as their expression of barrier characteristics.

Topics: Animals; Capillaries; Cell Survival; Endothelial Growth Factors; Endothelium, Vascular; Fibroblast Growth Factor 2; Gliosis; Hyperoxia; Ischemia; Lymphokines; Mice; Mice, Transgenic; Neovascularization, Pathologic; Oxygen; Phenotype; Platelet-Derived Growth Factor; Retina; Retinal Vessels; Rhodopsin; RNA, Messenger; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Retinal vasculogenesis and ischemic retinopathies provide good model systems for study of vascular development and neovascularization (NV), respectively. Vascular endothelial cell growth factor (VEGF) has been implicated in the pathogenesis of retinal vasculogenesis and in the development of retinal NV in ischemic retinopathies. However, insulin-like growth factor-I and possibly other growth factors also participate in the development of retinal NV and intraocular injections of VEGF antagonists only partially inhibit retinal NV. One possible conclusion from these studies is that it is necessary to block other growth factors in addition to VEGF to achieve complete inhibition of retinal NV. We recently demonstrated that a partially selective kinase inhibitor, PKC412, that blocks phosphorylation by VEGF and platelet-derived growth factor (PDGF) receptors and several isoforms of protein kinase C (PKC), completely inhibits retinal NV. In this study, we have used three additional selective kinase inhibitors with different selectivity profiles to explore the signaling pathways involved in retinal NV. PTK787, a drug that blocks phosphorylation by VEGF and PDGF receptors, but not PKC, completely inhibited retinal NV in murine oxygen-induced ischemic retinopathy and partially inhibited retinal vascularization during development. CGP 57148 and CGP 53716, two drugs that block phosphorylation by PDGF receptors, but not VEGF receptors, had no significant effect on retinal NV. These data and our previously published study suggest that regardless of contributions by other growth factors, VEGF signaling plays a critical role in the pathogenesis of retinal NV. Inhibition of VEGF receptor kinase activity completely blocks retinal NV and is an excellent target for treatment of proliferative diabetic retinopathy and other ischemic retinopathies.

Topics: Aging; Angiogenesis Inhibitors; Animals; Animals, Newborn; Endothelial Growth Factors; Enzyme Inhibitors; Ischemia; Lymphokines; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neovascularization, Pathologic; Phosphotransferases; Phthalazines; Pyridines; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; Retinal Vessels; Rhodopsin; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors