Astrocytic TGFB Signaling Pathways and Neuroinflammation Following Stroke

描述(申请人提供)：中风是世界上导致死亡和残疾的主要原因之一。FDA批准的唯一治疗中风的药物是组织纤溶酶原激活剂，它只在中风后大约4.5小时内有效。一个有希望的治疗窗口更长的策略是限制中风后过度的神经炎症。过度的炎症会加重梗塞面积，使炎症反应持久。在允许其有益作用发生后促进神经炎症的消退可能会改善中风的预后。转化生长因子β(TGFR？‘S)是一种主要的调节性细胞因子，以非活性的形式分泌，并滞留在细胞外基质中。它们主要是抗炎的，而且它们的信号在中风后几天会升高。星形胶质细胞是对转化生长因子水平升高作出反应的细胞类型之一。在中风后的这段亚急性期。由于星形胶质细胞是与神经元、血管和免疫细胞相互作用的胶质细胞，它们处于协调神经炎性反应的良好位置。然而，转化生长因子？S在星形胶质细胞中的调节和功能还不完全清楚。我们的初步数据表明，卒中边缘星形胶质细胞产生的血栓反应蛋白-1激活了转化生长因子？在细胞外基质中，并且这种增加上调了转化生长因子？和星形胶质细胞中的凝血酶敏感蛋白-1。此外，抑制星形细胞转化生长因子？信号大约使炎症反应增加一倍，减少梗塞附近存活神经元中神经保护性Akt信号，并恶化中风后的预后。因此，我们的中心假设是星形胶质细胞利用转化生长因子？在中风后的亚急性时间段，触发正反馈循环，导致更高水平的活性转化生长因子？，从而减少神经炎症，改善预后。我们的目标是确定星形细胞转化生长因子？信号转导是必要且充分的，以触发增加活性转化生长因子的正反馈循环。中风后的亚急性。我们的目标也是确定是全球性的还是星形细胞型的转化生长因子增加？超过基线的信号足以减少神经炎症并改善中风后的功能结果。以确定星形细胞转化生长因子？信号对于这个正反馈循环是必要的，我们将使用双转基因模型来降低转化生长因子？特别是在星形胶质细胞中，并通过激活全球转化生长因子？来补充这一缺陷。我们还将确定这种星形细胞转化生长因子？通过另一种不同的双转基因模型，上调星形胶质细胞转化生长因子？发信号。我们将使用ELISA法、免疫印迹法、免疫组织化学法和定量聚合酶链式反应(QPCR)来检测全球范围内的转化生长因子？和血栓反应蛋白-1，以及转化生长因子的信号转导途径？在这些模型中的每一个中。此外，我们将使用这些模型来确定哪些条件下的转化生长因子？信号传递减少了中风后的神经炎症，改善了功能结果。不管我们的假设是正确的，拟议的实验将提供更好的理解，星形胶质细胞用来影响中风后神经炎症的机制，也适用于其他类型的中枢神经系统炎症。

DESCRIPTION (provided by applicant): Stroke is one of the leading causes of death and disability in the world. The only FDA approved therapy for stroke is tissue plasminogen activator and it is only effective within approximately 4.5 hours following stroke. One promising strategy with a longer treatment window is limiting excessive neuroinflammation following stroke. Excessive inflammation worsens infarct sizes and perpetuates the inflammatory response. Promoting resolution of neuroinflammation after allowing its beneficial effects to occur may improve outcome in stroke. Transforming growth factor betas (TGF?'s) are master regulatory cytokines that are secreted in an inactive form and sequestered in the extracelluar matrix. They are primarily anti-inflammatory and their signaling is elevated days after stroke. Astrocytes are one of the cell types responding to increased levels of TGF? during this subacute period after stroke. Because astrocytes are glial cells that interact with neurons, blood vessels, and immune cells, they are well situated to coordinate neuroinflammatory responses. However TGF?'s regulation and function in astrocytes is not completely understood. Our preliminary data suggests that thrombospondin-1 production by astrocytes in the stroke border activates TGF? in the extracellular matrix, and the increase upregulates TGF? and thrombospondin-1 in astrocytes. Additionally, inhibiting astrocytic TGF? signaling approximately doubles the inflammatory response, decreases neuroprotective Akt signaling in surviving neurons adjacent to the infarct, and worsens outcomes after stroke. Our central hypothesis is therefore that astrocytes utilize TGF? in the subacute time period after stroke to trigger a positive feedback loop that leads to higher levels of active TGF?, which then reduces neuroinflammation and improves outcomes. We aim to determine whether astrocytic TGF? signaling is necessary and sufficient to trigger a positive feedback loop that increases active TGF? subacutely after stroke. We also aim to determine whether increasing global or astrocytic TGF? signaling over baseline is sufficient to decrease neuroinflammation and improve functional outcomes after stroke. To determine whether astrocytic TGF? signaling is necessary for this positive feedback loop we will use a double transgenic model with reduced TGF? specifically in astrocytes and supplement this deficit by activating global TGF?. We will also determine whether this astrocytic TGF? signaling is sufficient for this positive feedback loop with a different double transgenic model, upregulating astrocytic TGF? signaling. We will use ELISA, Western blot, immunohistochemistry, and qPCR to measure global levels of TGF? and thrombospondin-1, and signaling pathways of TGF? in each of these models. In addition, we will use these models to determine which conditions of TGF? signaling provide reduced neuroinflammation and improved functional outcomes following stroke. Regardless of our hypothesis being correct, the proposed experiments will provide a better understanding of the mechanisms that astrocytes use to influence neuroinflammation following stroke, which be applicable to other types of central nervous system inflammation as well.