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，全世界有数百万人致残。 在病理生理学上，它的两个主要成分是与急性组织损伤直接相关的急性组织损伤 煽动伤害，以及在接下来的几天里发生的延迟伤害的后期阶段。后一个阶段是 主要是炎症和炎症性水肿的结果，这会减少实质灌注量， 从而导致最初损伤的缺血性延伸。值得注意的是，这种延迟性炎症损伤可能 导致比最初的损伤更多的结构性损伤，通常占神经损伤的大部分 脊髓损伤患者的发病率。大多数当代研究都集中在这种炎性反应的后期效应上。 答复；很少有人试图查明这一进程的上游发起者。因此，这项提案将考验 新的假设是创伤性脊髓损伤的炎症反应是由星形胶质细胞释放的ATP启动的， 它以依赖于嘌呤受体的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中被释放。该提案的基础是 根据我们的初步观察，创伤性脊髓损伤与ATP的病理性释放有关，并且 嘌呤能受体拮抗剂能有效减轻炎症反应，促进脊髓损伤后的运动功能恢复。 目标1将检验这一假设，即创伤后失调的ATP释放既是必要的，也是必要的 足以激活小胶质细胞。这些实验将使用新的转基因小鼠，这些小鼠已经 被设计为释放异常高水平或低水平的星形胶质细胞ATP以响应脊髓损伤：ATP释放是 在缺乏星形细胞连接蛋白半通道(Cx43/Cx30 KO)的小鼠中减弱，而在患有AFP的小鼠中增强 星形细胞半突细胞数量增加(Cx43 G138R突变)。目标2将尝试定义 在创伤后炎症反应中，通路是嘌呤能激活下游的中介。这个 嘌呤能信号的遗传和药物调控对转录激活的影响 将对脊髓损伤的趋化因子和细胞因子效应因子进行评估，以确定脊髓损伤的作用途径。 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 通过对FACS分选的小胶质细胞进行微阵列评估来分析表达情况。然后，目标3将测试这个想法 小胶质细胞炎症介质引发星形胶质细胞增生症，从而形成星形胶质细胞疤痕，进而 为局部ATP释放的持续增加提供了病灶。因此，这一目标将检验以下可能性 脊髓损伤后，星形胶质细胞的三磷酸腺苷释放增加，持续数周至数月。我们的假设是，ATP 从反应性星形胶质细胞释放的物质可能推动促炎介质的进一步释放，从而 增强星形胶质细胞增生症。总之，这些实验有望填补我们在理解这一角色方面的关键空白 研究了脊髓损伤中的嘌呤能信号转导，并使我们能够确定关键的嘌呤调节基因和基因产物。 这可能允许对延迟性脊髓损伤进行治疗性抑制。

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.