TRPV1-mediated induction of a protective heat shock response after stroke

描述（由申请人提供）：缺血性卒中是美国死亡和残疾的主要原因之一;然而，目前的治疗选择非常有限。我们最近证明了通过选择性药理学激动清醒小鼠TRPV 1通道促进治疗性低温（Tc=33 ℃）对卒中后的神经保护作用。我们在本提案中扩展了这些发现，证明TRPV 1激动剂产生的神经保护作用的重要部分实际上独立于低温效应。在寻找这种新的非低温依赖性效应的机制时，我们发现TRPV 1激动剂促进脑中的保护性热休克反应（HSR）。TRPV 1是在体温调节系统的热敏感觉纤维以及大脑中的多种其他细胞类型内发现的Ca 2+渗透性非选择性阳离子通道。HSR是一种内源性细胞反应，涉及热休克蛋白（Hsps）的快速转录激活，有助于预防或逆转细胞损伤。这种细胞保护机制有助于身体对各种侮辱和伤害（包括中风）的生存反应。在大脑中，Hsp 27和Hsp 70似乎在中风的神经恢复中起关键作用。 本项目的总体目标是证明TRPV 1通道激动作为一种有效的治疗策略，以促进HSR激活和持续的缺血性卒中后的神经保护。具体而言，我们将1）确定TRPV 1介导的HSR诱导在未受伤和中风脑中的机制和细胞特异性，2）确定TRPV 1介导的HSR诱导是否在中风后提供持续的神经保护。将使用培养的脑星形胶质细胞和神经元进行初步研究，以测试TRPV 1介导的Hsp 27和Hsp 70诱导的拟议机制，使用药理学调节剂和从机制特异性敲除（KO）小鼠建立的培养物。这些研究之后将进行整体动物研究，以确定野生型和机制特异性KO小鼠中未损伤脑和局灶性缺血性中风后脑中TRPV 1激动诱导HSR的体内剂量反应、时间过程和细胞特异性。最后，将对中风小鼠进行研究，以确定TRPV 1介导的HSR诱导是否在28天的再灌注中提供持续的神经保护。将通过组织学/免疫荧光方法评价神经保护，以确定细胞特异性存活、BBB完整性和细胞凋亡，并通过行为测试证明最终功能恢复。Hsp 27和Hsp 70在神经保护中的具体作用将用各自的KO小鼠确定。预计这些研究将证明TRPV 1激动剂在大脑中的新的神经保护机制，并为改善中风和其他神经退行性疾病的治疗策略奠定基础。

DESCRIPTION (provided by applicant): Ischemic stroke is among the leading causes of death and disability in the United States; however, current treatment options are very limited. We recently demonstrated significant neuroprotection following stroke by promoting therapeutic hypothermia (Tc=33� C) through selective pharmacological agonism of TRPV1 channels in conscious mice. We extend these findings in the present proposal by demonstrating that a significant portion of the neuroprotection derived from TRPV1 agonism is actually independent of the hypothermic effect. While seeking the mechanism of this novel hypothermia- independent effect, we discovered that TRPV1 agonists promote a protective heat shock response (HSR) in the brain. TRPV1 is a Ca2+-permeable non-selective cation channel found within heat- sensitive sensory fibers of the thermoregulatory system as well as multiple other cell types in the brain. The HSR is an endogenous cellular response that involves rapid transcriptional activation of heat shock proteins (Hsps) which contribute to the prevention or reversal of cellular damage. This cytoprotective mechanism contributes to the body's survival response to a variety of insults and injuries, including stroke. In the brain, Hsp27 and Hsp70 appear to play critical roles in neurorecovery from stroke. The overall aim of this project is to demonstrate TRPV1 channel agonism as an effective therapeutic strategy to promote HSR activation and sustained neuroprotection following ischemic stroke. Specifically, we will 1) determine the mechanism and cell specificity of TRPV1-mediated HSR induction in the uninjured and stroke brain and 2) determine if TRPV1-mediated HSR induction provides sustained neuroprotection following stroke. Initial studies will be performed with cultured brain astrocytes and neurons to test proposed mechanisms of TRPV1-mediated induction of Hsp27 and Hsp70, using pharmacological modulators and cultures established from mechanism-specific knockout (KO) mice. These studies will be followed by whole animal studies to determine the in vivo dose- response, time course, and cell specificity of HSR induction by TRPV1 agonism in the uninjured brain and the brain following focal ischemic stroke in wild type and mechanism-specific KO mice. Lastly, studies will be performed with stroke mice to determine if TRPV1-mediated HSR induction provides sustained neuroprotection through 28 days of reperfusion. Neuroprotection will be evaluated by histological/immunofluorescence methods to determine cell specific survival, BBB integrity, and apoptosis and by behavior testing to demonstrate ultimate functional recovery. The specific roles of Hsp27 and Hsp70 in neuroprotection will be determined with respective KO mice. It is expected that these studies will demonstrate novel neuroprotective mechanisms of TRPV1 agonism in the brain and lay the groundwork for improved treatment strategies for stroke and other neurodegenerative diseases.