Inflammation and Degeneration of Optic Nerve in EAE

描述（由申请人提供）：视神经损伤导致的视力受损是多发性硬化症（MS）患者最常见的症状之一。多发性硬化症相关的视神经损伤是由于自身反应性T细胞和其他免疫细胞的涌入。炎症过程被认为有助于脱髓鞘，轴突变性，少突胶质细胞和视网膜神经节细胞（RGCs）的损失。了解EAE视神经退行性变的病理生理对开发恢复视觉功能的药物具有潜在的治疗意义。在Lewis大鼠的MS，实验性过敏性脑脊髓炎（EAE）动物模型中，钙（Ca2+）依赖性蛋白酶calpain在视神经中的活性和表达增加，暗示了calpain在EAE视神经损伤中的作用。由于炎症的确切时间以及视神经细胞和轴突损伤的机制尚不完全清楚，因此预防炎症和导致轴突和细胞损伤的分子事件的潜力可能会改善和恢复功能。我们假设calpain介导的自身反应性T细胞激活和免疫细胞浸润到中枢神经系统将导致视觉功能丧失，这是由于炎症导致EAE视神经轴突和少突胶质细胞损伤，随后可能在疾病临床症状出现之前发生RGCs的丧失。一个必然的假设是，在挑战后的不同时间点抑制钙蛋白酶可以通过防止周围和视神经的炎症来恢复视觉功能，并改善视神经损伤开始后的神经退行性变。数据表明，在EAE视神经中，Ca2+内流、钙蛋白酶表达、轴突损伤、细胞死亡和视网膜损伤增加，并且在发病前几个参数受到影响。与未治疗的EAE动物相比，使用钙蛋白酶抑制剂治疗可减少免疫细胞浸润、钙蛋白酶表达、细胞死亡和视网膜损伤，从而改善视觉反应。体外干扰素（IFN?）诱导神经胶质细胞calpain活化，MBP特异性T细胞上清降解MBP。以下具体目标已被设计来验证这些假设：(1)确定炎症反应的时间，Ca2+内流，Ca2+依赖事件，细胞死亡，以及EAE视神经轴突/髓鞘变性，并检查RGCs的状态与EAE动物视觉功能障碍的相关性；(2)研究钙蛋白酶抑制剂治疗是否会通过改变急性EAE的免疫臂（T细胞活化，免疫细胞浸润到视神经）和/或神经退行性臂（细胞死亡，轴突损伤）来恢复视觉功能，与载药治疗的动物相比；(3)研究calpain抑制剂在促炎细胞因子作用下或与活化的MBP特异性T细胞上清孵育时，在体外保存和保护RGCs功能的作用。了解视神经损伤的过程和时间将进一步发展治疗策略，以最好地恢复MS视神经变性引起的损伤。

DESCRIPTION (provided by applicant): Impaired vision due to optic nerve damage is one of the most common presenting symptoms of patients with multiple sclerosis (MS). MS-associated optic nerve damage is due to an influx of auto-reactive T cells and other immune cells. The inflammatory process is thought to contribute to demyelination, axonal degeneration, and loss of oligodendrocytes and retinal ganglion cells (RGCs). Understanding the pathophysiology of optic nerve degeneration in EAE has potential therapeutic implications to develop agents that restore visual function. Demonstration of increased activity and expression of calpain, a calcium (Ca2+)-dependent protease, in optic nerve using an animal model of MS, experimental allergic encephalomyelitis (EAE), in Lewis rats has implied a role for calpain in optic nerve damage in EAE. Since the precise timing of inflammation and mechanisms of cell and axon damage in optic nerve are not fully understood, the potential to prevent inflammation and molecular events that lead to axonal and cell damage, may improve and restore function. We hypothesize that calpain-mediated activation of auto-reactive T cells and immune cell infiltration into the CNS will result in loss of visual function due to inflammation leading to axon and oligodendrocyte damage in EAE optic nerve with subsequent loss of RGCs which may occur prior to clinical symptoms of disease. A corollary hypothesis is that inhibiting calpain at different time-points following challenge will restore visual function by preventing inflammation in the periphery and in optic nerve, and ameliorate neurodegeneration after damage to the optic nerve have begun. Data indicate that Ca2+ influx, calpain expression, axonal damage, cell death, and retinal damage are increased in EAE optic nerve with several parameters affected before disease onset. Treatment with calpain inhibitors reduced immune cell infiltration, calpain expression, cells death, and retinal damage, resulting in improved visual responses compared to untreated EAE animals. In vitro interferon (IFN?) induced calpain activation in glial cells and MBP-specific T cell supernatant degraded MBP. The following specific aims have been designed to test these hypotheses: (1) determine the timing of inflammatory responses, Ca2+ influx, Ca2+-dependent events, cell death, and axonal/myelin degeneration in EAE optic nerve and examine the status of RGCs in correlation with visual dysfunction in EAE animals following challenge; (2) investigate whether treatment with calpain inhibitors will restore visual function by altering the immune arm (T cell activation, immune cell infiltration into optic nerve) and/or the neurodegenerative arm (cell death, axonal damage) in acute EAE, as compared to vehicle treated animals; and (3) examine the effects of calpain inhibitors in preserving and protecting function of RGCs in vitro when subjected to pro-inflammatory cytokines or incubated with supernatant from activated MBP- specific T cells. Understanding the process and timing of optic nerve damage will further the development of treatment of strategies to best restore the impairment caused by optic nerve degeneration in MS. PUBLIC HEALTH RELEVANCE: Damage to the optic nerve resulting in impaired vision is one of the most common symptoms of patients with multiple sclerosis (MS). MS-associated inflammation of the optic nerve due to an influx of immune cells is thought to contribute to deficits leading to impaired vision, including demyelination, axonal degeneration, and loss of retinal ganglion cells (RGCs) and oligodendrocytes. Understanding the mechanisms by which inflammatory and neurodegenerative events contribute to optic nerve damage may have important and potentially therapeutic implications for developing agents that protect cells, preserve axons and myelin, and thus, improve vision and ultimately retard the development of MS.