Retinal ischemia is a common pathomechanism in many ocular disorders such as age-related macular degeneration (AMD), diabetic retinopathy, glaucoma or retinal vascular occlusion. Several studies demonstrated that ischemia/reperfusion (I/R) leads to morphological and functional changes of different retinal cell types. However, little is known about the ischemic effects on the optic nerve. The goal of this study was to evaluate these effects. Ischemia was induced by raising the intraocular pressure (IOP) in one eye of rats to 140 mmHg for 1 h followed by natural reperfusion. After 21 days, histological as well as quantitative real-time PCR (qRT-PCR) analyses of optic nerves were performed. Ischemic optic nerves showed an infiltration of cells and also degeneration with signs of demyelination. Furthermore, a migration and an activation of microglia could be observed histologically as well as on mRNA level. In regard to macroglia, a trend toward gliosis could be noted after ischemia induction by vimentin staining. Additionally, an up-regulation of glial fibrillary acidic protein (GFAP) mRNA was found in ischemic optic nerves. Counting of oligodendrocyte transcription factor 2 positive (Olig2+) cells revealed a decrease of oligodendrocytes in the ischemic group. Also, myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) mRNA expression was down-regulated after induction of I/R. On immunohistological level, a decrease of MOG was detectable in ischemic optic nerves as well. In addition, SMI-32 stained neurofilaments of longitudinal optic nerve sections showed a strong structural damage of the ischemic optic nerves in comparison to controls. Consequently, retinal ischemia impacts optic nerve degeneration. These findings could help to better understand the course of destruction in the optic nerve after an ischemic insult. Especially for therapeutic studies, the optic nerve is important because of its susceptibility to be damaged as a result to retinal ischemic injury and also its connecting function between the eye and the brain. So, future drug screenings should target not only the retina, but also the functionality and structure of the optic nerve. In the future, these results could lead to the development of new therapeutic strategies for treatment of ischemic injury.
Retinal ischemia occurs in a variety of eye diseases. Restrained blood flow induces retinal damage, which leads to progressive optic nerve degeneration and vision loss. Previous studies indicate that extracellular matrix (ECM) constituents play an important role in complex tissues, such as retina and optic nerve. They have great impact on de- and regeneration processes and represent major candidates of central nervous system glial scar formation. Nevertheless, the importance of the ECM during ischemic retina and optic nerve neurodegeneration is not fully understood yet. In this study, we analyzed remodeling of the extracellular glycoproteins fibronectin, laminin, tenascin-C and tenascin-R and the chondroitin sulfate proteoglycans (CSPGs) aggrecan, brevican and phosphacan/RPTPβ/ζ in retinae and optic nerves of an ischemia/reperfusion rat model via quantitative real-time PCR, immunohistochemistry and Western blot. A variety of ECM constituents were dysregulated in the retina and optic nerve after ischemia. Regarding fibronectin, significantly elevated mRNA and protein levels were observed in the retina following ischemia, while laminin and tenascin-C showed enhanced immunoreactivity in the optic nerve after ischemia. Interestingly, CSPGs displayed significantly increased expression levels in the optic nerve. Our study demonstrates a dynamic expression of ECM molecules following retinal ischemia, which strengthens their regulatory role during neurodegeneration.
Glaucoma is characterized by the loss of retinal ganglion cells (RGCs) and optic nerve fibres. Previous studies noted fewer RGCs after immunization with ocular antigens at 28 days. It is known that changes in extracellular matrix (ECM) components conduct retina and optic nerve degeneration. Here, we focused on the remodelling of tenascin‐C and phosphacan/receptor protein tyrosine phosphatase β/ζ in an autoimmune glaucoma model. Rats were immunized with optic nerve homogenate (ONA) or S100B protein (S100). Controls received sodium chloride (Co). After 14 days, no changes in RGC number were noted in all groups. An increase in GFAP mRNA expression was observed in the S100 group, whereas no alterations were noted via immunohistochemistry in both groups. Extracellular matrix remodelling was analyzed after 3, 7, 14 and 28 days. Tenascin‐C and 473HD immunoreactivity in retinae and optic nerves was unaltered in both immunized groups at 3 days. At 7 days, tenascin‐C staining increased in both tissues in the ONA group. Also, in the optic nerves of the S100 group, an intense tenascin‐C staining could be shown. In the retina, an increased tenascin‐C expression was also observed in ONA animals via Western blot. 473HD immunoreactivity was elevated in the ONA group in both tissues and in the S100 optic nerves at 7 days. At 14 days, tenascin‐C and 473HD immunoreactivity was up‐regulated in the ONA retinae, whereas phosphacan expression was up‐regulated in both groups. We conclude that remodelling of tenascin‐C and phosphacan occurred shortly after immunization, already before RGC loss. We assume that both ECM molecules represent early indicators of neurodegeneration.
Previous studies demonstrated that retinal damage correlates with a massive remodeling of extracellular matrix (ECM) molecules and reactive gliosis. However, the functional significance of the ECM in retinal neurodegeneration is still unknown. In the present study, we used an intraocular pressure (IOP) independent experimental autoimmune glaucoma (EAG) mouse model to examine the role of the ECM glycoprotein tenascin-C (Tnc). Wild type (WT ONA) and Tnc knockout (KO ONA) mice were immunized with an optic nerve antigen (ONA) homogenate and control groups (CO) obtained sodium chloride (WT CO, KO CO). IOP was measured weekly and electroretinographies were recorded at the end of the study. Ten weeks after immunization, we analyzed retinal ganglion cells (RGCs), glial cells, and the expression of different cytokines in retina and optic nerve tissue in all four groups. IOP and retinal function were comparable in all groups. Although RGC loss was less severe in KO ONA, WT as well as KO mice displayed a significant cell loss after immunization. Compared to KO ONA, less βIII-tubulin + axons, and downregulated oligodendrocyte markers were noted in WT ONA optic nerves. In retina and optic nerve, we found an enhanced GFAP + staining area of astrocytes in immunized WT. A significantly higher number of retinal Iba1 + microglia was found in WT ONA, while a lower number of Iba1 + cells was observed in KO ONA. Furthermore, an increased expression of the glial markers Gfap, Iba1, Nos2 , and Cd68 was detected in retinal and optic nerve tissue of WT ONA, whereas comparable levels were observed in KO ONA. In addition, pro-inflammatory Tnfa expression was upregulated in WT ONA, but downregulated in KO ONA. Vice versa, a significantly increased anti-inflammatory Tgfb1 expression was measured in KO ONA animals. We conclude that Tnc plays an important role in glial and inflammatory response during retinal neurodegeneration. Our results provide evidence that Tnc is involved in glaucomatous damage by regulating retinal glial activation and cytokine release. Thus, this transgenic EAG mouse model for the first time offers the possibility to investigate IOP-independent glaucomatous damage in direct relation to ECM remodeling.
The extracellular matrix (ECM) consists of a dynamic network of various macromolecules that are synthesized and released by surrounding cells into the intercellular space. Glycoproteins, proteoglycans and fibrillar proteins are main components of the ECM. In addition to general functions such as structure and stability, the ECM controls several cellular signaling pathways. In this context, ECM molecules have a profound influence on intracellular signaling as receptor-, adhesion- and adaptor-proteins. Due to its various functions, the ECM is essential in the healthy organism, but also under pathological conditions. ECM constituents are part of the glial scar, which is formed in several neurodegenerative diseases that are accompanied by the activation and infiltration of glia as well as immune cells. Remodeling of the ECM modulates the release of pro- and anti-inflammatory cytokines affecting the fate of immune, glial and neuronal cells. Tenascin-C is an ECM glycoprotein that is expressed during embryonic central nervous system (CNS) development. In adults it is present at lower levels but reappears under pathological conditions such as in brain tumors, following injury and in neurodegenerative disorders and is highly associated with glial reactivity as well as scar formation. As a key modulator of the immune response during neurodegeneration in the CNS, tenascin-C is highlighted in this mini-review.
Studies have suggested an involvement of the immune system in glaucoma. Hence, a rat experimental autoimmune glaucoma model (EAG) was developed to investigate the role of the immune response. Here, we transferred this model into mice. Either 0.8 mg/mL of the optic nerve antigen homogenate (ONA; ONA 0.8) or 1.0 mg/mL ONA (ONA 1.0) were injected in 129/Sv mice. Controls received sodium chloride. Before and 6 weeks after immunization, the intraocular pressure (IOP) was measured. At 6 weeks, retinal neurons, glia cells, and synapses were analyzed via immunohistology and quantitative real-time PCR (RT-qPCR). Additionally, optic nerves were examined. The IOP stayed in the normal physiological range throughout the study (p > 0.05). A significant reduction of retinal ganglion cells (RGCs) was noted in both immunized groups (p < 0.001). Remodeling of glutamatergic and GABAergic synapses was seen in ONA 1.0 retinas. Furthermore, both ONA groups revealed optic nerve degeneration and macrogliosis (all: p < 0.001). An increase of activated microglia was noted in ONA retinas and optic nerves (p < 0.05). Both ONA concentrations led to RGC loss and optic nerve degeneration. Therefore, the EAG model was successfully transferred from rats to mice. In further studies, transgenic knockout mice can be used to investigate the pathomechanisms of glaucoma more precisely.
Glaucoma is a neurodegenerative disease that is characterized by the loss of retinal ganglion cells (RGC) and optic nerve fibers. Increased age and intraocular pressure (IOP) elevation are the main risk factors for developing glaucoma. Mice that are heterozygous (HET) for the mega-karyocyte protein tyrosine phosphatase 2 (PTP-Meg2) show chronic and progressive IOP elevation, severe RGCs loss, and optic nerve damage, and represent a valuable model for IOP-dependent primary open-angle glaucoma (POAG). Previously, evidence accumulated suggesting that glaucomatous neurodegeneration is associated with the extensive remodeling of extracellular matrix (ECM) molecules. Unfortunately, little is known about the exact ECM changes in the glaucomatous retina and optic nerve. Hence, the goal of the present study was to comparatively explore ECM alterations in glaucomatous PTP-Meg2 HET and control wild type (WT) mice. Due to their potential relevance in glaucomatous neurodegeneration, we specifically analyzed the expression pattern of the ECM glycoproteins fibronectin, laminin, tenascin-C, and tenascin-R as well as the proteoglycans aggrecan, brevican, and members of the receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) family. The analyses were carried out in the retina and optic nerve of glaucomatous PTP-Meg2 HET and WT mice using quantitative real-time PCR (RT-qPCR), immunohistochemistry, and Western blot. Interestingly, we observed increased fibronectin and laminin levels in the glaucomatous HET retina and optic nerve compared to the WT group. RT-qPCR analyses of the laminins α4, β2 and γ3 showed an altered isoform-specific regulation in the HET retina and optic nerve. In addition, an upregulation of tenascin-C and its interaction partner RPTPβ/ζ/phosphacan was found in glaucomatous tissue. However, comparable protein and mRNA levels for tenascin-R as well as aggrecan and brevican were observed in both groups. Overall, our study showed a remodeling of various ECM components in the glaucomatous retina and optic nerve of PTP-Meg2 HET mice. This dysregulation could be responsible for pathological processes such as neovascularization, inflammation, and reactive gliosis in glaucomatous neurodegeneration.
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