Characterization of Macroglia Response during Tissue Repair in a Laser-Induced Model of Retinal Degeneration

Reactive gliosis is a hallmark of chronic degenerative diseases of the retina. As gliosis involves macroglia, we investigated their gliotic response to determine the role of S100β and intermediate filaments (IFs) GFAP, vimentin, and nestin during tissue repair in a laser-induced model of retinal degeneration. We validated the results with human retinal donor samples. Ex-periments were performed in zebrafish and mice using an argon laser (532 nm) to induce focal lesions in the outer retina. At different time points following injury induction, the kinetics of retinal degeneration and regeneration were assessed using hematoxylin and eosin staining (H&E). Immunofluorescence was performed to evaluate Müller cell (GS) and astrocyte (GFAP) injury response and to distinguish between both cell types. Additionally, staining was per-formed in human retinal sections containing drusen. Focal laser treatment elevated the expres-sion of gliotic markers in the area of the damage, which was associated with increased expres-sion of S100β, GFAP, vimentin, and nestin in mice and humans. In zebrafish, we detected S100β at the first time point, but not GFAP or nestin. Double-positive cells with the selected glia markers were detected in all models. However, in zebrafish, no double-positive GFAP/GS cells were found on days 10 and 17, nor were S100β/GS double-positive cells found on day 12. Macro-glia cells showed a different pattern in the expression of IFs in degenerative and regenerative models. In particular, S100β may prove to be a target for suppressing chronic gliosis in retinal degeneration

Caspase-3-independent photoreceptor degeneration by N-methyl-N-nitrosourea (MNU) induces morphological and functional changes in the mouse retina

Background: Retinal degeneration is followed by significant changes in the structure and function of photoreceptors in humans and several genetic animal models. However, it is not clear whether similar changes occur when the degeneration is induced pharmacologically. Therefore, our aim was to investigate the influence of retinotoxic N-methyl-N-nitrosourea (MNU) on the function, morphology and underlying molecular pathways of programmed cell death. Methods: C57/BL6 mice were injected with different doses of MNU, and function was determined by analysing optokinetic reflex measurements and cued water maze results at several time points post-injection. Morphometric measurements were also taken from H&E-stained paraffin eye sections. TUNEL-positive cells and caspase-3 and -6 were detected by immunohistochemistry. To assess the molecular changes leading to cell death, qRT-PCR from neurosensory retina mRNA was performed. Results: The application of MNU led to an instant decrease in function and a delayed decrease in the thickness of the retinal outer nuclear layer. These responses were observed in the absence of any structural changes in the retinal pigment epithelium. The degeneration of the photoreceptor cell layer was highest with 60mg/kg MNU. The assessment of TUNEL-positive cells visualised cell death after treatment, but no detectable caspase-3 activity was observed concomitant with these changes. qRT-PCR revealed the possible involvement of the inflammatory mediator caspase-1 and endoplasmic reticulum stress-mediated apoptosis by caspase-12. Conclusion: MNU leads to the dose-dependent degeneration of photoreceptor cells in mice by caspase-3-independent pathways and is, therefore, a suitable model to study retinal degeneration in an animal mode

characteristics of rod regeneration in a novel zebrafish retinal degeneration model using N-methyl-N-nitrosourea (MNU)

Primary loss of photoreceptors caused by diseases such as retinitis pigmentosa is one of the main causes of blindness worldwide. To study such diseases, rodent models of N-methyl-N-nitrosourea (MNU)-induced retinal degeneration are widely used. As zebrafish (Danio rerio) are a popular model system for visual research that offers persistent retinal neurogenesis throughout the lifetime and retinal regeneration after severe damage, we have established a novel MNU-induced model in this species. Histology with staining for apoptosis (TUNEL), proliferation (PCNA), activated Müller glial cells (GFAP), rods (rhodopsin) and cones (zpr-1) were performed. A characteristic sequence of retinal changes was found. First, apoptosis of rod photoreceptors occurred 3 days after MNU treatment and resulted in a loss of rod cells. Consequently, proliferation started in the inner nuclear layer (INL) with a maximum at day 8, whereas in the outer nuclear layer (ONL) a maximum was observed at day 15. The proliferation in the ONL persisted to the end of the follow-up (3 months), interestingly, without ongoing rod cell death. We demonstrate that rod degeneration is a sufficient trigger for the induction of Müller glial cell activation, even if only a minimal number of rod cells undergo cell death. In conclusion, the use of MNU is a simple and feasible model for rod photoreceptor degeneration in the zebrafish that offers new insights into rod regeneration

Characteristics of Müller glial cells in MNU-induced retinal degeneration.

Retinal Müller glial cells have been shown to undergo reactive gliosis in a variety of retinal diseases. Upregulation of glial fibrillary acidic protein (GFAP) is a hallmark of Müller cell activation. Reactive gliosis after retinal detachment or ischemia/reperfusion is characterized by hypertrophy and downregulation of inwardly rectifying K+ (Kir) currents. However, this kind of physiological alteration could not be detected in slowly progressing retinal degenerations. The photoreceptor toxin N-methyl-N-nitrosourea (MNU) leads to the rapid loss of cells in the outer nuclear layer and subsequent Müller cell activation. Here, we investigated whether Müller cells from MNU-treated mice exhibit reactive gliosis. We found that Müller cells showed increased GFAP expression and increased membrane capacitance, indicating hypertrophy. Membrane potential and Kir channel-mediated K+ currents were not significantly altered whereas Kir4.1 mRNA expression and Kir-mediated inward current densities were markedly decreased. This suggests that MNU-induced Müller cell gliosis is characterized by plasma membrane increase without alteration in the membrane content of Kir channels. Taken together, our findings show that Müller cells of MNU-treated mice are reactive and respond with a form of gliosis which is characterized by cellular hypertrophy but no changes in Kir current amplitudes

Long-term cellular and regional specificity of the photoreceptor toxin, iodoacetic acid (IAA), in the rabbit retina

This study investigated the anatomical consequences of a photoreceptor toxin, iodoacetic acid (IAA), in the rabbit retina. Retinae were examined 2 weeks, 1, 3, and 6 months after systemic IAA injection. The retinae were processed using standard histological methods to assess the gross morphology and topographical distribution of damage, and by immunohistochemistry to examine specific cell populations in the retina. Degeneration was restricted to the photoreceptors and was most common in the ventral retina and visual streak. In damaged regions, the outer nuclear layer was reduced in thickness or eliminated entirely, with a concomitant loss of immunoreactivity for rhodopsin. However, the magnitude of the effect varied between animals with the same IAA dose and survival time, suggesting individual differences in the bioavailability of the toxin. In all eyes, the inner retina remained intact, as judged by the thickness of the inner nuclear layer, and by the pattern of immunoreactivity for protein kinase C-α (rod bipolar cells) and calbindin D-28 (horizontal cells). Müller cell stalks became immunoreactive for glial fibrillary acidic protein (GFAP) even in IAA-treated retinae that had no signs of cell loss, indicating a response of the retina to the toxin. However, no marked hypertrophy or proliferation of Müller cells was observed with either GFAP or vimentin immunohistochemistry. Thus the selective, long lasting damage to the photoreceptors produced by this toxin did not lead to a reorganization of the surviving cells, at least with survival as long as 6 months, in contrast to the remodeling of the inner retina that is observed in inherited retinal degenerations such as retinitis pigmentosa and retinal injuries such as retinal detachmen

Characteristics of Müller glial cells in MNU-induced retinal degeneration

Retinal Müller glial cells have been shown to undergo reactive gliosis in a variety of retinal diseases. Upregulation of glial fibrillary acidic protein (GFAP) is a hallmark of Müller cell activation. Reactive gliosis after retinal detachment or ischemia/reperfusion is characterized by hypertrophy and downregulation of inwardly rectifying K+ (Kir) currents. However, this kind of physiological alteration could not be detected in slowly progressing retinal degenerations. The photoreceptor toxin N-methyl-N-nitrosourea (MNU) leads to the rapid loss of cells in the outer nuclear layer and subsequent Müller cell activation. Here, we investigated whether Müller cells from MNU-treated mice exhibit reactive gliosis. We found that Müller cells showed increased GFAP expression and increased membrane capacitance, indicating hypertrophy. Membrane potential and Kir channel-mediated K+ currents were not significantly altered whereas Kir4.1 mRNA expression and Kir-mediated inward current densities were markedly decreased. This suggests that MNU-induced Müller cell gliosis is characterized by plasma membrane increase without alteration in the membrane content of Kir channels. Taken together, our findings show that Müller cells of MNU-treated mice are reactive and respond with a form of gliosis which is characterized by cellular hypertrophy but no changes in Kir current amplitudes

Modeling MOG Antibody-Associated Disorder and Neuromyelitis Optica Spectrum Disorder in Animal Models: Visual System Manifestations.

BACKGROUND AND OBJECTIVES Mechanisms of visual impairment in aquaporin 4 antibody (AQP4-IgG) seropositive neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein antibody (MOG-IgG)-associated disorder (MOGAD) are incompletely understood. The respective impact of optic nerve demyelination and primary and secondary retinal neurodegeneration are yet to be investigated in animal models. METHODS Active MOG35-55 experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6Jrj mice, and monoclonal MOG-IgG (8-18C5, murine), recombinant AQP4-IgG (rAb-53, human), or isotype-matched control IgG (Iso-IgG, human) was administered 10 days postimmunization. Mobility impairment was scored daily. Visual acuity by optomotor reflex and ganglion cell complex thickness (GCC, 3 innermost retinal layers) by optical coherence tomography (OCT) were longitudinally assessed. Histopathology of optic nerve and retina was investigated during presymptomatic, acute, and chronic disease phases for immune cells, demyelination, complement deposition, natural killer (NK) cell, AQP4, and astrocyte involvement, retinal ganglion cells (RGCs), and Müller cell activation. Groups were compared by nonparametric tests with a p value <0.05 indicating statistical significance. RESULTS Visual acuity decreased from baseline to chronic phase in MOG-IgG (mean ± standard error of the mean: 0.54 ± 0.01 to 0.46 ± 0.02 cycles/degree, p < 0.05) and AQP4-IgG EAE (0.54 ± 0.01 to 0.43 ± 0.02, cycles/degree, p < 0.05). Immune cell infiltration of optic nerves started in presymptomatic AQP4-IgG, but not in MOG-IgG EAE (5.85 ± 2.26 vs 0.13 ± 0.10 macrophages/region of interest [ROI] and 1.88 ± 0.63 vs 0.15 ± 0.06 T cells/ROI, both p < 0.05). Few NK cells, no complement deposition, and stable glial fibrillary acid protein and AQP4 fluorescence intensity characterized all EAE optic nerves. Lower GCC thickness (Spearman correlation coefficient r = -0.44, p < 0.05) and RGC counts (r = -0.47, p < 0.05) correlated with higher mobility impairment. RGCs decreased from presymptomatic to chronic disease phase in MOG-IgG (1,705 ± 51 vs 1,412 ± 45, p < 0.05) and AQP4-IgG EAE (1,758 ± 14 vs 1,526 ± 48, p < 0.01). Müller cell activation was not observed in either model. DISCUSSION In a multimodal longitudinal characterization of visual outcome in animal models of MOGAD and NMOSD, differential retinal injury and optic nerve involvement were not conclusively clarified. Yet optic nerve inflammation was earlier in AQP4-IgG-associated pathophysiology. Retinal atrophy determined by GCC thickness (OCT) and RGC counts correlating with mobility impairment in the chronic phase of MOG-IgG and AQP4-IgG EAE may serve as a generalizable marker of neurodegeneration

Sodium Iodate-Induced Degeneration Results in Local Complement Changes and Inflammatory Processes in Murine Retina

Age-related macular degeneration (AMD), one of the leading causes of blindness worldwide, causes personal suffering and high socioeconomic costs. While there has been progress in the treatments for the neovascular form of AMD, no therapy is yet available for the more common dry form, also known as geographic atrophy. We analysed the retinal tissue in a mouse model of retinal degeneration caused by sodium iodate (NaIO3)-induced retinal pigment epithelium (RPE) atrophy to understand the underlying pathology. RNA sequencing (RNA-seq), qRT-PCR, Western blot, immunohistochemistry of the retinas and multiplex ELISA of the mouse serum were applied to find the pathways involved in the degeneration. NaIO3 caused patchy RPE loss and thinning of the photoreceptor layer. This was accompanied by the increased retinal expression of complement components c1s, c3, c4, cfb and cfh. C1s, C3, CFH and CFB were complement proteins, with enhanced deposition at day 3. C4 was upregulated in retinal degeneration at day 10. Consistently, the transcript levels of proinflammatory ccl-2, -3, -5, il-1β, il-33 and tgf-β were increased in the retinas of NaIO3 mice, but vegf-a mRNA was reduced. Macrophages, microglia and gliotic Müller cells could be a cellular source for local retinal inflammatory changes in the NaIO3 retina. Systemic complement and cytokines/chemokines remained unaltered in this model of NaIO3-dependent retinal degeneration. In conclusion, systemically administered NaIO3 promotes degenerative and inflammatory processes in the retina, which can mimic the hallmarks of geographic atrophy

Antineonatal Fc Receptor Antibody Treatment Ameliorates MOG-IgG-Associated Experimental Autoimmune Encephalomyelitis.

BACKGROUND AND OBJECTIVES Myelin oligodendrocyte glycoprotein antibody-associated disorder (MOGAD) is a rare, autoimmune demyelinating CNS disorder, distinct from multiple sclerosis and neuromyelitis optica spectrum disorder. Characterized by pathogenic immunoglobulin G (IgG) antibodies against MOG, a potential treatment strategy for MOGAD is to reduce circulating IgG levels, e.g., by interference with the IgG recycling pathway mediated by the neonatal Fc receptor (FcRn). Although the optic nerve is often detrimentally involved in MOGAD, the effect of FcRn blockade on the visual pathway has not been assessed. Our objective was to investigate effects of a monoclonal anti-FcRn antibody in murine MOG-IgG-associated experimental autoimmune encephalomyelitis (EAE). METHODS We induced active MOG35-55 EAE in C57Bl/6 mice followed by the application of a monoclonal MOG-IgG (8-18C5) 10 days postimmunization (dpi). Animals were treated with either a specific monoclonal antibody against FcRn (α-FcRn, 4470) or an isotype-matched control IgG on 7, 10, and 13 dpi. Neurologic disability was scored daily on a 10-point scale. Visual acuity was assessed by optomotor reflex. Histopathologic hallmarks of disease were assessed in the spinal cord, optic nerve, and retina. Immune cell infiltration was visualized by immunohistochemistry, demyelination by Luxol fast blue staining and complement deposition and number of retinal ganglion cells by immunofluorescence. RESULTS In MOG-IgG-augmented MOG35-55 EAE, anti-FcRn treatment significantly attenuated neurologic disability over the course of disease (mean area under the curve and 95% confidence intervals (CIs): α-FcRn [n = 27], 46.02 [37.89-54.15]; isotype IgG [n = 24], 66.75 [59.54-73.96], 3 independent experiments), correlating with reduced amounts of demyelination and macrophage infiltration into the spinal cord. T- and B-cell infiltration and complement deposition remained unchanged. Compared with isotype, anti-FcRn treatment prevented reduction of visual acuity over the course of disease (median cycles/degree and interquartile range: α-FcRn [n = 16], 0.50 [0.48-0.55] to 0.50 [0.48-0.58]; isotype IgG [n = 17], 0.50 [0.49-0.54] to 0.45 [0.39-0.51]). DISCUSSION We show preserved optomotor response and ameliorated course of disease after anti-FcRn treatment in an experimental model using a monoclonal MOG-IgG to mimic MOGAD. Selectively targeting FcRn might represent a promising therapeutic approach in MOGAD