Inflammation and Degeneration of Optic Nerve in EAE

描述(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、轴突变性以及少突胶质细胞和视网膜神经节细胞(RGC)的丢失。了解EAE视神经变性的病理生理学机制对开发恢复视功能的药物具有潜在的治疗意义。利用实验性变态反应性脑脊髓炎(EAE)的多发性硬化动物模型，在Lewis大鼠的视神经中证明了Calain的活性和表达增加，这是一种钙(Ca2+)依赖的蛋白酶，暗示了Calain在EAE的视神经损伤中所起的作用。由于视神经炎症的确切时间和细胞和轴突损伤的机制尚不完全清楚，因此预防导致轴突和细胞损伤的炎症和分子事件的潜力可能会改善和恢复功能。我们推测，Calain介导的自身反应性T细胞的激活和免疫细胞向中枢神经系统的渗透将导致炎症导致的视觉功能丧失，导致EAE视神经中的轴突和少突胶质细胞损伤，继而RGC丢失，这可能发生在疾病临床症状之前。一个必然的假设是，在挑战后的不同时间点抑制Calain将通过预防外周和视神经的炎症来恢复视觉功能，并在视神经损伤开始后改善神经退化。数据表明，EAE视神经中钙离子内流、钙蛋白表达、轴突损伤、细胞死亡和视网膜损伤在发病前增加，并有几个参数受到影响。与未经治疗的EAE动物相比，使用Calain抑制剂治疗减少了免疫细胞渗透、Calain表达、细胞死亡和视网膜损伤，从而改善了视觉反应。体外干扰素(干扰素？)诱导神经胶质细胞中钙蛋白酶的激活，MBP特异性T细胞上清液降解MBP。为了验证这些假说，设计了以下特定目标：(1)确定EAE视神经炎症反应、钙离子内流、钙依赖事件、细胞死亡和轴突/髓鞘变性的时间，并检测RGCs的状态与EAE动物视觉功能障碍的相关性；(2)与赋形剂治疗动物相比，观察Calain抑制剂治疗是否通过改变急性EAE动物的免疫臂(T细胞激活、免疫细胞进入视神经)和/或神经变性臂(细胞死亡、轴突损伤)来恢复视功能；(3)观察钙蛋白酶抑制剂在体外作用于促炎症细胞因子或与活化的MBP特异性T细胞培养上清液共同培养时，对RGCs功能的保护作用。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视神经变性造成的损害。 公共卫生相关性：视神经损伤导致视力受损是多发性硬化症(MS)患者最常见的症状之一。由于免疫细胞的涌入，与MS相关的视神经炎症被认为是导致视力受损的原因之一，包括脱髓鞘、轴突变性以及视网膜神经节细胞(RGC)和少突胶质细胞的丢失。了解炎症和神经退行性变导致视神经损伤的机制可能对开发保护细胞、保存轴突和髓鞘的药物具有重要和潜在的治疗意义，从而改善视力并最终延缓MS的发展。

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.