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（NSC）代表了寻求脊髓损伤（SCI）治愈的潜在机会，但NSC在损伤部位存活的机制仍不清楚。先前的记录显示，大多数脊髓损伤后立即植入的神经干细胞在免疫排斥发生前一周内死亡。因此，了解触发神经干细胞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，被认为是神经干细胞移植物急性死亡的原因。与神经元不同，NSC不表达受体（例如，NMDA受体等）介导的致命信号所必需的，这表明一个替代机制，不涉及膜受体。在可能的候选者中，一氧化氮（NO）是高度保守的信号分子。虽然NO是神经发生和NSC分化的主要调节剂，但已显示过量的NO产生导致SCI后的继发性损伤过程，包括半胱天冬酶激活和凋亡。因此，我们推测NO及其相关的自由基物种，如过氧亚硝酸盐，可能会引发SCI供体神经干细胞的死亡。利用我们建立的体外和体内系统，我们的目标是确定NO对损伤脊髓中人NSC（hNSC）移植物的死亡，存活和分化的作用。将进行体外研究，例如与NO供体孵育的hNSC的凋亡测定，以及NO介导的细胞色素c释放和半胱天冬酶活化的Western印迹分析，以提供用于构建可回收的可生物降解的PLGA聚合物支架的指导方针，该支架将用作hNSC植入和宿主微环境报告载体（特定目的I和II）。嵌入这些支架中的活性氮自由基的供体或清除剂将使我们能够研究大鼠半切SCI模型（特定目标III）中hNSC移植物的细胞反应，以及未来的其他神经系统疾病。如果确定NO诱导的hNSC死亡的信号通路，则可以用作开发新治疗策略的靶点，以增强基于NSC的SCI疗法的功效。(For公开声明：我们将研究影响注射神经干细胞在受损脊髓中存活的因素。由于大多数干细胞在注射后死亡，因此科学家和医生必须了解注射后干细胞死亡的机制，然后才能将干细胞技术用于治疗脊髓损伤。

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).