Host and stem cell interactions in spinal cord injury: roles of nitric oxide

描述(申请人提供)：神经干细胞(NSCs)代表着一个潜在的治疗脊髓损伤(SCI)的机会，但NSC在损伤部位存活的机制仍不清楚。以前的记录显示，大多数在脊髓损伤后立即植入的神经干细胞在免疫排斥开始前一周内死亡。因此，了解跳跳虎导致NSC早期死亡的事件至关重要。继发性损伤因素，如兴奋性毒性、细胞因子、蛋白质过氧化和自由基损伤等，被认为是移植神经干细胞急性死亡的原因。与神经元不同，神经干细胞不表达受体(如NMDA受体等)。对于介导致死信号是必要的，这表明了一种不涉及膜受体的替代机制。在可能的候选分子中，一氧化氮(NO)是一种高度保守的信号分子。虽然NO是神经发生和NSC分化的主要调节因子，但过量的NO产生会导致脊髓损伤后的继发性损伤过程，包括caspase激活和细胞凋亡。因此，我们推测，NO及其相关的自由基物种，如过氧亚硝酸盐，可能触发了脊髓损伤供体神经干细胞的死亡。利用我们成熟的体外和体内系统，我们的目标是确定NO在损伤脊髓内移植的人神经干细胞(HNSC)死亡、存活和分化中的作用。体外研究包括NO供体培养的hNSCs的细胞凋亡分析，以及NO介导的细胞色素c释放和caspase激活的Western Blot分析，为构建可回收的可生物降解的PLGA聚合物支架提供指导，用作hNSC植入和宿主微环境报告载体(特异性AIMS I和II)。这些支架中活性氮物种自由基的供体或清除者将使我们能够在大鼠半横断脊髓损伤模型(特定目标III)中研究hNSC移植物的细胞反应，以及未来的其他神经疾病。如果确定了NO诱导的hNSC死亡的信号通路，则可以作为开发新的治疗策略的靶点，以提高基于NSC的脊髓损伤治疗的疗效。(公开声明：我们将调查影响损伤脊髓内注射的神经干细胞存活的因素。由于大多数干细胞在注射后死亡，科学家和医生必须了解注射后干细胞死亡的机制，然后干细胞技术才能用于治疗脊髓损伤)。

DESCRIPTION (provided by applicant): Neural stem cells (NSCs) represent a potential opportunity in the search for a cure of spinal cord injury (SCI), but mechanisms underlying NSC survival at injury sites remain unclear. Previous records showed that majority of NSCs implanted immediately after SCI die within one week before the onset of immunorejection. It is consequently crucial to understand the events that tiggers the early demise of NSCs. Secondary injury factors, such as excitotoxicity, cytokines, protein peroxidization and radical damage, have been speculated as the cause of acute death of NSC grafts. Unlike neurons, NSCs do not express the receptors (e.g., NMDA receptors, etc.) necessary for mediating lethal signals, suggesting an alternate mechanisms that do not involve membrane receptors. Among the possible candidates, nitric oxide (NO) is a highly conserved signal molecule. While NO is a major modulator of neurogenesis and NSC differentiation, excessive NO production has been shown to result in secondary injury processes after SCI including caspase activation and apoptosis. Therefore, we hypothesize that NO and its associated radical species, such as peroxynitrite, may trigger death of donor NSCs in SCI. Using our well established in vitro and in vivo systems, we aim to define the role of NO on the death, survival and differentiation of human NSC (hNSC) grafts in the injured spinal cord. In vitro studies such as apoptosis assays for hNSCs incubated with NO donors, and Western Blot analysis of NO mediated cytochrome c release and caspase activation will be performed to provide guidelines for constructing a retrievable biodegradable PLGA polymer scaffold to be used as hNSC implanting and host microenvironment reporting vehicles (Specific Aims I & II). Donors or scavengers of the reactive nitrogen species radicals embedded in these scaffolds will allow us to study the cellular response of hNSC grafts in a rat hemisection SCI model (Specific Aim III), as well as other neurological disorders in the future. If identified signaling pathways of NO-induced hNSC death may be used as targets for the development of new therapeutic strategies to enhance the efficacy of NSC-based SCI therapies. (For public statement: We will investigate the factors that hurt the survival of injected neural stem cells in the injured spinal cord. Since most stem cells die after injection, scientists and doctors must understand the mechanism of post-injection stem cell death before the stem cell technology can be used to treat spinal cord injury).