| Glaucoma is a complex neurodegenerative disease leading to blindness. Glaucomatous neurodegeneration involves specific combinations of genetic predispositions, epigenetic risk factors, and environmental stressors that increase the aging-related stress in retinal ganglion cells (RGCs) and optic nerve axons. Intraocular pressure, however, has been one of the risk factors for the development and progression of the disease that has been identified to date and the only one that is modifiable. IOP reduction forms the basis for our current therapy of glaucoma. Present evidence indicates highly interconnected downstream pathways of cellular processes in RGCs exposed to glaucomatous stress, which include mitochondrial dysfunction, proteolytic caspase cascade, endoplasmic reticulum stress, and oxidative stress. The precise molecular mechanism of initiating the neuronal injury and the glaucomatous neurodegeneration are under intensive investigation and great debate.There are many factors of glaucomatous ganglion cell damage, and some theories attempt to explain the pathogenesis of glaucoma, such as the mechanical theory, oxidative stress theory, recently scentists pay more attention on two aspects:autoimmunity and abnormal protein precipitation:Studies highlight the invovement of the immune system in glaucomatous neurodegeneration. Different components, including both innate and adaptive immunity, exhibit prominent activity in glaucoma. Recently, growing evidence points to the involvement of complement activation in the pathogenesis of glaucoma. Complement activation products, such as Clq, C3and membrane attack complex (MAC) have been found both in animal glaucoma models and in the retinas of glaucoma patients. Part Ⅰ and part Ⅱ of the research attempted to investigate the role of complement system activation in the pathogenesis of glaucoma. Part Ⅰ was in vivo work, continuous observing the alteration of complement associated proteins in the chronic hypertention rat model of glaucoma, while part Ⅱ was in vitro work, discussing the effect of exposure to the complement C5b-9complex sensitizes retinal ganglion cells to both apoptosis and necroptosis. Neurodegeneration is a collective term for the progressive loss of the structure, function, or even death of neurons. The pathogenesis of neurodegeneration involves the aberrant processing, misfolding, and subsequent aggregation of normal proteins that are then deposited both intracellularly and extracellularly. The retina is part of the central nervous system (CNS), therefore, the retina and optic nerve could be affected by similar degenerative processes during neurodegeneration Abnormal protein deposition in neurons, including A(3and abnormal Tau protein deposition is the other factor of the injure of ganglion cells. Part Ⅲ of the research is observation of the variation of Tau protein in glaucomatous retina and discussion the effect and mechanism of treatment of LiCl on chronic glaucomatous neurodegeneration.Part I Alteration of complement associated proteins in the chronic hypertention rat model of glaucoma.PURPOSE:This study tempted to investigate the role of complement system in retinal ganglion cells (RGCs) in chronic ocular hypertension model of glaucoma.METHODS:Construct of a chronic ocular hypertension model on DA rats which were divided into4groups,5of each. Intraocular pressure (IOP) was elevated in the left eye of DA rats by injection of hypertonic saline into episcleral veins, while right eyes did not receive treatment and served as control. Animals were sacrificed at lweek,3weeks,8weeks and16weeks post-injection. Levels of membrane attack complex (MAC, C5b-9), C3and Decay accelerating facor (DAF) were elevated by immunefluorescence in the retina of eyes with increased IOP and the control.RESULTS:C5b-9was found to be upregulated in the retina of glaucomatowus rat from8weeks after IOP elevated. The express of C3on RGC layer of IOP elevated eyes was also upregulated. And complement activation inhibitor DAF was found decreased in both8weeks and16weeks high IOP groups.CONCLUSIONS:Complement cascade produts were upregulated and the complement regulatory protein DAF was deceased on the GCL of in chronic glaucoma models suggests that complement activation and the imbalance of complement regulation plays a potential role in the pathogenesis of glaucoma.Part II Exposure to the complement C5b-9complex sensitizes retinal ganglion cells to both apoptosis and necroptosisPURPOSE:Retinal ganglion cell (RGC) loss is the defining characteristic of glaucoma, but the mechanisms that regulate RGC death are not fully understood. The aim of this work was to determine whether exposure to the terminal complement complex C5b-9induces RGC death and/or modulates the sensitivity of RGC cells to other cellular stressors.METHODS:RGC-5cells were exposed to normal human serum following antibody blockade of CD59. Apoptosis induction was assessed morphologically, by flow cytometry, and on western blotting by probing for cleaved PARP and activated caspase-3. Necroptosis was assessed by flow cytometry and inhibition using AGK2. The sensitivity of RGC-5cells to ionomycin, staurosporine, peroxide and chelerythrine was also investigated, with or without prior formation of C5b-9.RESULTS:RGC-5cells underwent apoptotic cell death following exposure to C5b-9, as judged by PARP cleavage and activation of caspase-3. We also observed apoptotic cell death in response to staurosporine, but RGC-5cells were resistant to both ionomycin and peroxide. Interestingly, C5b-9significantly increased RGC-5sensitivity to staurosporine-but not chelerythrine-induced apoptosis and necroptosis.CONCLUSIONS:We have shown that low levels of C5b-9on RGC-5cells can induce apoptosis, and by using a range of different agonists we have further demonstrated that C5b-9specifically sensitizes RGC-5cells to certain apoptotic and necroptotic pathways. Our observations provide new insight into the potential role of the complement system in RGC loss, with implications for the cellular aetiology of glaucoma. Part Ⅲ Alteration of Tau protein in the chronic hypertention rat model of glaucoma.Objective This study attempted to investigate the variation of tau phosphorylation in chronic glaucomatous retina, with the treatment of lithium chloride (LiCl), inhibited the activity of the up stream kinase Glycogen synthase kinase-3beta (GSK-3β), and provided experimental evidence for the clinical treatment of LiCl on chronic glaucomatous neurodegeneration.Methods Construct of a chronic ocular hypertension model on SD rats which were divided into2groups,10of each. Group1were high intra ocular pressure (IOP) ones with no treatment. The right eyes were high IOP ones, and the left eyes were normal control. Group2were high IOP ones treated with lithium chloride (LiCl). On the first day of constructing the model, the SD rat of group2were intraperitoneal injected with0.6M LiCl, which was the inhibitor of GSK3beta.5SD rats of each group were executed at the week2and week4, and their retina was taken for immunofluorescence stain and Western Blot, and the variation of tau phosphorylation in retina was investigated.Results1) Variations of Tau and phosphorylation of Tau on chronic hyperocular model::the amount of total tau was decreased to77.3%and60.4%of the control at week2and4respectively. The ratio of p-tau/total tau was unchanged at week2and increased to135.4%of the control at week4, and the phosphorylation of tau was increased significantly.2) The effect of LiCl on high IOP models:The total tau in retina was increased compared to the no treated group, reached to99%of the normal control. The ratio of p-tau/total tau declined as well, almost to the level of normal control.Conclusion In ocular hypertension model, total-Tau was decreased in retina compared with controls. Meanwhile expression of phospho-Tau was increased. The Tau phosphorylation could be inhibited by blocking the activation of GSK3beta with LiCl. SUMMARY and CONCLUSIONSMain findings:1. C5b-9was found to be increased in the ganglion cell layer of retina in chronic ocular hypertention rat model from8weeks after IOP elevated. The express of C3which is a important protein in three pathway of complement activation was also upgrade in the chronic ocular hypertention model. And complement regulating protein Decay Accelerating Facor (DAF) was found decreased obviously from8weeks and. the decrease was relatively with the increase of C5b-9.2. In vitro, the membrane attack complex C5b-9could be formed on RGC-5. C5b-9formation promotes apoptosis of RGC-5cells. C5b-9significantly increased RGC-5sensitivity to staurosporine-induced apoptosis and necroptosis.3. In retina of chronic ocular hypertension rat model, the amount of total tau began decreased from2weeks time point and was decreased dramatically at4weeks time point. The phosphorylation of tau was increased significantly at week4.2) The total tau in retina was increased with the treatment of LiCl compared to the no treated group. The phosphorylation of tau declined as well.Conclusions:1. Complement cascade produts were upregulated and the complement regulatory protein DAF was deceased on the GCL of in chronic glaucoma models suggests that complement activation and the imbalance of complement regulation plays a potential role in the pathogenesis of glaucoma. 2. Low levels of C5b-9on RGC-5cells can induce apoptosis, and by using a range of different agonists we have further demonstrated that C5b-9specifically sensitizes RGC-5cells to certain apoptotic and necroptotic pathways.3. In ocular hypertension model, total-Tau was decreased in retina compared with control. Meanwhile expression of phospho-Tau was increased. The Tau phosphorylation caused by sustained ocular hypertension could be inhibited by LiCl. |
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