Adult Neurogenesis and Stroke Recovery

描述（由申请人提供）： 摘要目的：本提案的总体目标是确定成人神经发生对卒中后恢复过程的重要性。成年神经元是局灶性缺血损伤后修复的潜在基质，并且已经确定神经干细胞可以促进神经元的修复。 在实验性中风后，神经干细胞（NSC）增殖并且它们的后代迁移到损伤部位。越来越多的证据表明，大量的成人产生的神经元迁移到受损的纹状体和皮层中风后，但这些神经元整合和生存的时间很长。此外，关于存活的神经元的表型以及它们整合到大脑回路中的程度，数据是相互矛盾的。确定细胞命运或功能整合是困难的，因为用于标记这些细胞的工具有限。 研究计划：在拟议的研究中，创新的转基因小鼠模型和逆转录病毒技术将用于在实验性中风的背景下特异性标记或消融成人产生的神经元。此外，我们的初步数据从Bax基因敲除小鼠线缺陷的一个关键的程序性细胞死亡途径的成年神经干细胞表明，更多的新生神经元存活后，兴奋毒性纹状体损伤在这些小鼠。使用这些遗传学方法，我们将测试我们的中心假设，即成人产生的神经元有助于修复局灶性缺血后，通过神经元替代;此外，抑制这一过程将不利于中风恢复，相反，刺激成年NSC后代的生存将改善恢复。特定目标1的研究将描述局灶性缺血损伤后成年出生的神经元的长期存活、表型和整合。具体目标2的实验将通过消融中风后新分裂的细胞来确定成人神经发生是否对修复过程至关重要。在目标3中，我们将使用条件性Bax基因敲除小鼠来检查促凋亡蛋白Bax在成人产生的神经元中的选择性缺失是否导致其存活和整合增加，以及局灶性缺血损伤后功能恢复改善。 方法：采用成年小鼠大脑中动脉短暂性闭塞模型。目的1将通过使用分裂细胞的逆转录病毒标记和条件性转基因NSC报告小鼠来追踪成年产生的神经元的命运来实现。在目标2中，我们将使用条件性转基因小鼠模型消融成人产生的神经元，该模型选择性地在NSC中表达一种激活的自杀基因。目标3的方法将涉及将成神经细胞特异性cre驱动小鼠系（doublecortin-CreERT 2）与floxed Bax系杂交，以删除中风后产生的神经元前体中的Bax。对于目标2和3，我们将确定实验操作对组织学梗死面积的影响，并对动物进行行为学测试，以确定抑制神经发生是否会损害或刺激神经发生是否会分别改善局灶性缺血损伤后的运动技能恢复。 临床相关性：卒中无再生疗法。这些目标的进展将增加我们对局灶性缺血性损伤后大脑修复过程的理解，并有可能为中风的新型再生疗法做出贡献，中风是一种导致退伍军人人群严重疾病负担的疾病。

DESCRIPTION (provided by applicant): Abstract Objectives: The overall goal of this proposal is to determine the importance of adult neurogenesis to the recovery process following stroke. Adult-born neurons are a potential substrate for repair after focal ischemic injury, and it is well established that neural stem cells (NSCs) proliferate and their progeny migrate to sites of injury after experimental stroke. Accumulating evidence indicates that large numbers of adult-generated neurons migrate to the injured striatum and cortex following stroke, but few of these neurons integrate and survive for prolonged periods. Data are conflicting, moreover, as to the phenotypes of the neurons that do survive and the extent to which they integrate into brain circuitry. Determination of cell fate or functional integration is difficult because the tools used to label these cells have been limited. Research Plan: In the proposed studies, innovative transgenic mouse models and retroviral techniques will be used to specifically label or ablate adult-generated neurons in the setting of experimental stroke. In addition, our preliminary data from a Bax knockout mouse line defective in a critical programmed cell death pathway for adult NSCs suggest that more newborn neurons survive after excitotoxic striatal injury in these mice. Using these genetic approaches, we will test our central hypothesis that adult-generated neurons contribute to repair after focal ischemia via neuronal replacement; moreover, inhibiting this process will be detrimental to stroke recovery and, conversely, stimulating the survival of adult NSC progeny will improve recovery. Studies in Specific Aim 1 will characterize the long-term survival, phenotypes and integration of adult-born neurons after focal ischemic injury. Experiments in Specific Aim 2 will determine if adult neurogenesis is critical to the repair process by ablating newly dividing cells following stroke. In Aim 3, we will use conditional Bax null mice to examine whether loss of the pro-apoptotic protein Bax selectively in adult-generated neurons leads to their increased survival and integration, and improved functional recovery after focal ischemic injury. Methods: We will use the transient middle cerebral artery occlusion model in adult mice. Aim 1 will be accomplished by using retroviral labeling of dividing cells and conditional transgenic NSC reporter mice to track the fate of adult-generated neurons. In Aim 2, we will ablate adult-generated neurons using a conditional transgenic mouse model that expresses a pharmacologically activated suicide gene selectively in NSCs. The methods for Aim 3 will involve crossing a neuroblast-specific cre driver mouse line (doublecortin-CreERT2) with a floxed Bax line to delete Bax in neuronal precursors generated after stroke. For Aims 2 and 3, we will determine the influence of experimental manipulations on infarct size histologically, and animals will be behaviorally tested to determine if suppressing neurogenesis impairs or stimulating neurogenesis improves, respectively, motor skill recovery after focal ischemic injury. Clinical Relevance: No regenerative therapies exist for stroke. Progress in these aims will increase our understanding of reparative processes in the brain following focal ischemic injury and offers the potential to contribute to novel regenerative therapies for stroke, a disorder that leads to a substantial disease burden in the Veteran population.