ATP as the instigator of inflammatory responses to spinal cord injury

脊髓损伤（SCI）是一种毁灭性的疾病，在世界范围内使数百万人致残。 在病理生理学上，其两个主要组成部分是与急性组织损伤直接相关的， 引发损伤，以及随后几天发生的延迟损伤的后期阶段。后一阶段是 主要是炎症和炎性水肿的结果，其减少实质灌注， 从而导致初始损伤的缺血性扩展。值得注意的是，这种延迟性炎症损伤可能 导致比最初损伤更多的结构损伤，并且通常占神经系统损伤的大部分。 SCI患者的发病率。大多数当代研究都集中在这种炎症的晚期效应物上， 反应;很少有人试图确定这一进程的上游发起者。因此，这项建议将考验 一个新的假设，即创伤性SCI的炎症反应是由星形胶质细胞ATP释放启动的， 其用于以嘌呤受体依赖性方式激活局部小胶质细胞;随后 炎症效应物在这种继发性小胶质细胞反应的情况下释放。所涉提议主要 根据我们的初步观察，创伤性SCI与ATP的病理性释放有关， 嘌呤能受体拮抗剂有效地减轻炎症并改善SCI后的运动恢复。 目的1将测试创伤后ATP释放失调是必要的， 足以激活小胶质细胞。这些实验将使用新的转基因小鼠， 被设计为响应SCI释放异常高或低水平的星形胶质细胞ATP： 在缺乏星形胶质细胞连接蛋白半通道（Cx43/Cx 30 KO）的小鼠中减弱，但在具有星形胶质细胞连接蛋白半通道的小鼠中增强。 星形胶质细胞半通道数量增加（Cx43 G138 R突变）。目标2将试图定义 在创伤后炎症反应中嘌呤能活化的下游途径中间体。的 嘌呤能信号的遗传和药理学操作对转录激活的影响 将评估SCI的趋化因子和细胞因子效应物，以确定SCI的趋化因子和细胞因子效应物的途径。 嘌呤能激活的炎性后遗症是协调的。小胶质细胞基因的转录变化 通过FACS分选的小胶质细胞的微阵列评估来分析表达。目标3将测试这个想法 小胶质细胞炎症介质触发星形胶质细胞增生，从而形成星形胶质细胞瘢痕， 为局部ATP释放的持续增加提供病灶。因此，这一目标将检验以下可能性： SCI后星形胶质细胞ATP释放增加数周至数月。我们假设ATP 从反应性星形胶质细胞释放的蛋白质可以驱动促炎介质的进一步释放， 增强星形胶质细胞增生总之，这些实验有望填补我们对这种作用的理解中的关键空白。 这将使我们能够确定嘌呤调节基因和基因产物的关键 这可能允许治疗性抑制迟发性脊髓损伤。

Spinal cord injury (SCI) is a devastating condition that has disabled millions of individuals world-wide. Pathophysiologically, its two principal components are the acute tissue damage directly associated with the inciting injury, and a later phase of delayed injury that occurs over the ensuing days. This latter phase is primarily a consequence of inflammation and inflammatory edema, which reduces parenchymal perfusion, thereby resulting in ischemic extension of the initial injury. Remarkably, this delayed inflammatory injury may lead to more structural damage than the initial injury, and typically accounts for the bulk of neurological morbidity in SCI patients. Most contemporary studies have focused on the late effectors of this inflammatory response; few have sought to identify the upstream initiators of this process. This proposal will thus test the novel postulate that the inflammatory response to traumatic SCI is initiated by astrocytic ATP release, which serves to activate local microglia in a purine receptor-dependent fashion; subsequent inflammatory effectors are released in the setting of this secondary microglial response. The proposal is based on our preliminary observations that traumatic SCI is associated with the pathological release of ATP, and that purinergic receptor antagonists effectively reduced inflammation and improved locomotor recovery after SCI. Aim 1 will test the postulate that the dysregulated post-traumatic release of ATP is both necessary and sufficient for microglial activation. These experiments will employ novel transgenic mice that have been engineered to release either abnormally high or low levels of astrocytic ATP in response to SCI: ATP release is attenuated in mice lacking astrocytic connexin hemichannels (Cx43/Cx30 KO), but potentiated in mice with an increased number of astrocytic hemichannels (Cx43 G138R mutation). Aim 2 will attempt to define the pathway intermediates downstream of purinergic activation in the post-traumatic inflammatory response. The effects of genetic and pharmacological manipulations of purinergic signaling on the transcriptional activation of both chemokine and cytokine effectors of SCI will be assessed, so as to define the pathways by which the inflammatory sequelae of purinergic activation are coordinated. Transcriptional changes in microglial gene expression will be analyzed by microarray assessment of FACS-sorted microglia. Aim 3 will then test the idea that microglial inflammatory mediators trigger astrogliosis and hence astroglial scar formation, which in turn provides the nidus for a sustained increase in local ATP release. As such, this Aim will test the possibility that astrocytic ATP release is increased for weeks to months following SCI. Our hypothesis is that the ATP released from reactive astrocytes may drive the further release of pro-inflammatory mediators, thereby enhancing astrogliosis. Together, these experiments promise to fill critical gaps in our understanding of the role of purinergic signaling in SCI, and should permit us to define purine-regulated genes and gene products critical that might permit the therapeutic suppression of delayed spinal cord injury.