Adult Neurogenesis and Stroke Recovery

描述(由申请人提供)： 摘要目的：本研究的总体目标是确定成人神经发生在卒中后康复过程中的重要性。成体出生的神经元是局灶性缺血损伤后潜在的修复基质，神经干细胞是公认的 (NSCs)在实验性卒中后增殖，其后代迁移到损伤部位。越来越多的证据表明，卒中后成体产生的神经元大量迁移到受损的纹状体和皮质，但很少有神经元整合并长期存活。此外，关于存活下来的神经元的表型以及它们整合到大脑电路中的程度，数据是相互矛盾的。很难确定细胞的命运或功能整合，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被用于特定标记或消融实验性卒中背景下的成人产生的神经元。此外，我们对成年神经干细胞关键程序性细胞死亡途径缺陷的Bax基因敲除小鼠的初步数据表明，在这些小鼠的兴奋毒性纹状体损伤后，更多的新生神经元存活下来。使用这些遗传学方法，我们将检验我们的中心假设，即成年产生的神经元通过神经元替代促进局灶性缺血后的修复；此外，抑制这一过程将不利于中风的恢复，反过来，刺激成年NSC后代的存活将促进恢复。特定目标1的研究将表征局灶性脑缺血损伤后成年出生神经元的长期存活、表型和整合。在特定目标2的实验将确定成年神经发生是否是修复过程的关键，因为它会消融中风后新分裂的细胞。在目标3中，我们将使用条件性Bax缺失的小鼠来检验在成体产生的神经元中选择性地丢失促凋亡蛋白Bax是否会导致这些神经元的存活和整合增加，并促进局灶性脑缺血损伤后的功能恢复。方法：采用成年小鼠短暂性大脑中动脉闭塞模型。目标1将通过使用分裂细胞的逆转录病毒标记和条件转基因NSC报告鼠来跟踪成人产生的神经元的命运来实现。在目标2中，我们将使用在神经干细胞中选择性表达药物激活的自杀基因的条件转基因小鼠模型来去除成人产生的神经元。Aim 3的方法将涉及将神经母细胞特异性cre驱动小鼠系(Doublecortin-CreerT2)与带花的bax系杂交，以删除中风后产生的神经元前体中的bax。对于AIMS 2和AIMS 3，我们将从组织学上确定实验操作对梗塞面积的影响，并将对动物进行行为学测试，以确定抑制神经发生损害或刺激神经发生分别改善局灶性脑缺血损伤后运动技能恢复。 临床意义：中风尚无再生疗法。这些目标的进展将增加我们对局灶性缺血损伤后大脑修复过程的理解，并为中风的新的再生疗法提供潜力，中风是一种在退伍军人群体中导致大量疾病负担的疾病。

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.