Astrocyte-specific TLR4 signaling and Blood Brain Barrier permeability following acute focal cerebral ischemia

项目总结/摘要 目前批准的卒中治疗包括未充分利用的、有时限的、全身性溶栓和机械性溶栓。 再通选择。因此，需要有更负担得起和更灵活的治疗方法， 可以广泛传播。一种方法包括靶向稳健表达的先天性途径，如 局灶性脑缺血后Toll样受体（TLR）信号通路激活。都缺乏 关于下游途径激活的时间和细胞特异性的知识， TLR 4在星形胶质细胞和神经血管单位的其他组分中。在这个建议中，我们将确定关键 星形胶质细胞TLR 4信号传导的下游靶点和星形胶质细胞特异性TLR 4信号传导对血液的影响 急性局灶性脑缺血后的脑屏障通透性（BBB）和神经行为结果。我们 将使用临床相关的内源性危险相关分子模式（DAMP），HMGB 1，一种已知的 TLR 4配体来确定星形胶质细胞TLR 4信号传导中的关键下游靶标。我们还将在体外使用 缺血模型，如氧葡萄糖脱氢酶（OGD），以确定TLR 4依赖性 由星形胶质细胞中的其他DAMP激活的下游通路。下游效应物的表征 星形胶质细胞TLR 4信号通路的改变对降低血脑屏障通透性和继发性脑损伤具有重要意义。 损害和改善中风后的结果。这个提议的总体假设是， 星形胶质细胞特异性TLR 4信号传导在中风急性期诱导BBB破坏， 星形胶质细胞中缺血相关的DAMP-TLR 4信号传导将降低急性局灶性脑缺血后BBB通透性 脑缺血和改善行为结果。这一核心假设将在以下几个方面得到检验： 目的：目的1：我们将确定在TLR 4反应性和TLR 4非反应性中激活的信号通路。 使用大脑中动脉闭塞（MCAO）模型和转录组学， 急性脑缺血目的2）使用HMGB 1刺激培养的星形胶质细胞和体外模型， 脑缺血，OGD，我们将通过Western blot鉴定星形胶质细胞中TLR 4信号传导的下游靶点 和磷酸化蛋白质组学。目的3：使用具有可诱导的星形胶质细胞特异性TLR 4缺失的小鼠，我们将 确定星形胶质细胞特异性TLR 4缺失对BBB通透性和神经行为结果的影响 在MCAO之后。在这些研究的最后，我们将对分子机制有更好的了解 这是星形胶质细胞中TLR 4信号的基础。这些研究的结果将为 开发新的治疗方法，可以减少中风后的脑损伤。

PROJECT SUMMARY/ABSTRACT Currently approved stroke therapies include underutilized, time-limited, systemic thrombolysis and mechanical recanalization options. Therefore, there is a need for more affordable and flexible approaches to treatment that can be disseminated widely. One approach includes targeting robustly expressed innate pathways such as the Toll-like Receptor (TLR) signaling pathways activated after focal cerebral ischemia. There is a lack of knowledge regarding the timing and cellular specificity of downstream pathways activated by pathways such as TLR4 in astrocytes and other components of the neurovascular unit. In this proposal, we will determine the key downstream targets in astrocyte TLR4 signaling and the effect of astrocyte-specific TLR4 signaling on blood brain barrier permeability (BBB) and neurobehavioral outcomes following acute focal cerebral ischemia. We will use a clinically relevant, endogenous danger associated molecular pattern (DAMP), HMGB1, a known TLR4 ligand to determine the key downstream targets in astrocyte TLR4 signaling. We will also use in vitro models of ischemia, such as Oxygen Glucose Deprivation (OGD), to determine the TLR4-dependent downstream pathways activated by other DAMPs in astrocytes. Characterization of the downstream effectors of astrocyte TLR4 signaling has important implications for decreasing BBB permeability and secondary brain damage and improving outcomes after stroke. The overall hypothesis of this proposal is that stroke-induced, astrocyte-specific TLR4 signaling induces BBB disruption in the acute phase of stroke, and that inhibiting ischemia-relevant DAMP-TLR4 signaling in astrocytes will decrease BBB permeability following acute focal cerebral ischemia and improve behavioral outcomes. This central hypothesis will be tested in the following aims: Aim 1: We will determine the signaling pathways active in TLR4-reactive and TLR4 non-reactive penumbral astrocytes using a model of middle cerebral artery occlusion (MCAO) and transcriptomics following acute cerebral ischemia Aim 2) Using HMGB1 stimulation of cultured astrocytes and an in vitro model of cerebral ischemia, OGD, we will identify downstream targets of TLR4 signaling in astrocytes via Western blot and phosphoproteomics. Aim 3: Using mice with inducible, astrocyte-specific deletion of TLR4, we will determine the effect of astrocyte-specific TLR4 deletion on BBB permeability and neurobehavioral outcomes following MCAO. At the end of these studies, we will have a better understanding of the molecular mechanisms that underlie TLR4 signaling in astrocytes. Results from these studies will lay the foundation for the development of novel therapeutics that can decrease brain damage after stroke.