Neuronal Regeneration of Stroked Brain with Perlecan Domain V

描述（由申请人提供）：卒中是死亡和严重发病的主要原因。不幸的是，仍然迫切需要新的缺血性中风疗法来减轻这种重大的社会负担，因为组织纤溶酶原激活剂（t-PA）（唯一FDA批准的缺血性中风药物疗法）具有有限的治疗窗口并产生显著的风险。此外，仅基于神经保护的实验性疗法在很大程度上未能转化为人类中风。相反，更好的治疗方法可能会出现一个创新的方法，基于对大脑自身中风后修复机制的更好理解-识别和利用既有神经保护作用又能促进大脑修复的内源性药物。为此，我们最近已经确定，C-末端片段的细胞外基质成分串珠素，称为域V（DV），是持续产生缺血性中风后。当作为重组蛋白外源给药时，DV既具有神经保护作用又具有血管生成作用（一种修复过程），最终可恢复大鼠和小鼠受中风影响的运动功能。初步数据表明，DV增加了体内梗死和缺血区域的神经元再增殖，并增强了体外神经突延伸和连接。此外，我们最近已经证明，DV治疗抑制神经胶质瘢痕形成的缺血核心，从而减轻了潜在的显着障碍神经元再生。因此，我们假设中风后给予DV将提供持久、稳定的恢复，部分是通过增加到达并建立在损伤部位的新神经元的量。为此，我们现在提出以下具体目标：1）确定长期DV卒中治疗对梗死和缺血区域神经元再增殖和连接的影响，2）确定DV影响体外神经突延伸的机制细节。对于特定目的1，将在3月龄雄性C57/B16小鼠中使用串联同侧颈总动脉和大脑中动脉闭塞模型通过短暂局灶性皮质缺血诱导中风。在中风后4-8周进行BrdU注射，随后进行运动试验和梗塞和缺血区域的BrdU和神经元标记物的免疫组织化学，以确定DV是否提供稳定的恢复和神经元再增殖，DV最初在中风后7天施用以使其神经保护作用的影响最小化。将监测动物疾病体征，以评估长期DV治疗的潜在不良反应。对于具体目标2，DV对神经突延伸的影响和先前鉴定的神经元DV受体α 2 β 1整联蛋白的潜在作用将通过用野生型或α 2 β 1缺失的成年小鼠神经球的神经突延伸测定来确定。这些研究的成功完成将通过提供对脑对缺血性损伤的反应的新见解以及进一步支持我们将DV建立为人类新型中风治疗的长期目标而具有重要意义。

DESCRIPTION (provided by applicant): Stroke is a leading cause of death and significant morbidity. Unfortunately, there is still a critical need for new ischemic stroke therapies to reduc this significant societal burden as tissue plasminogen activator (t-PA), the only FDA approved ischemic stroke pharmaceutical therapy, has a limited therapeutic window and engenders significant risk. Furthermore, experimental therapies based solely on neuroprotection have largely failed to translate to human stroke. Instead, better therapies may emerge from an innovative approach based on improved understanding of the brain's own post-stroke repair mechanisms - identifying and exploiting endogenous agents that are both neuroprotective and promote brain repair. To this end, we have recently determined that the c-terminal fragment of the extracellular matrix component perlecan, termed domain V (DV), is persistently generated after ischemic stroke. When exogenously administered as a recombinant protein, DV is both neuroprotective and angiogenic (a reparative process), culminating in the restoration of stroke affected motor function in rats and mice. Preliminary data now suggest that DV increases neuronal repopulation of the infarcted and ischemic areas in vivo, and enhances neurite extension and connectivity in vitro. Furthermore, we have recently demonstrated that DV treatment suppresses glial scar formation in the ischemic core, thereby alleviating a potentially significant barrier to neuronal regeneration. Therefore, we hypothesize that post-stroke administered DV will provide a lasting, stable recovery, in part, by increasing the amount of new neurons that reach and establish themselves at the injury site. To that end, we now propose the following specific aims: 1) Determine the effect of long-term DV stroke therapy on neuronal repopulation and connectivity in the infarcted and ischemic areas, 2) Determine in mechanistic detail the potential of DV to influence neurite extension in vitro. For specific aim 1, stroke willbe induced by transient focal cortical ischemia using the tandem ipsilateral common carotid and middle cerebral artery occlusion model in 3-month-old male C57/Bl6 mice. BrdU injection followed by motor tests and immunohistochemistry of the infarcted and ischemic areas for BrdU and neuronal markers 4-8 weeks post-stroke will be performed to determine if DV, initially administered 7 days after stroke to minimize the influence of its neuroprotective effect, provides stable recovery and neuronal repopulation. Signs of animal illness will be monitored to assess potential adverse effects of long-term DV treatment. For specific aim 2, DV's effects on neurite extension and the potential role of the previously identified neuronal DV receptor, alpha2beta1 integrin, will be determined via neurite extension assays with wild-type or alpha2beta1 null adult mouse neurospheres. The successful completion of these studies will be significant by providing new insight into the brain's response to ischemic injury, and by further supporting our long-term goal of establishing DV as a novel stroke therapy for humans.