Hemorrhage Induced Brain Injury

描述(申请人提供)：脑出血(ICH)是一种毁灭性的中风。脑出血对大脑的损害是由于不断增长的血肿和凝块形成，通过水肿和细胞凋亡损害邻近的大脑。虽然这些都是脑出血后发生的不同的病理实体，但我们有数据表明它们有一些共同的机制。我们发现血肿周围脑组织的细胞凋亡部分是通过谷氨酸的兴奋性毒性来实现的。这让我们产生了非受体酪氨酸激酶pp60-Src(Src)，因为它通过直接磷酸化NR2A亚单位来增强NMDA受体的功能。我们发现，大鼠实验性脑出血后，Src激酶活性增加了4倍，Src家族激酶(SFK)成员Lyn在脑出血后增加了21倍以上，而Src抑制剂减少了细胞凋亡，改善了脑出血后的行为结局。我们假设Src在ICH介导的细胞凋亡和水肿中起中心作用：ICH导致凝血酶受体激活Src，激活介导细胞凋亡的NR2A亚单位；ICH激活凝血酶受体，通过Src激活HIF和MMPs，从而产生水肿和不良行为结局。这些假说是基于先前的研究，表明凝血酶介导脑出血后的急性脑水肿，凝血酶通过凝血酶受体激活Src。因此，这些研究将检测：(A)脑出血对Src的影响；(B)凝血酶对Src的激活；(C)脑出血引起的NMDA受体的磷酸化；(D)对HIF-1、HIF-1靶基因和MMPs的Src激活；以及(E)通过建立大鼠脑出血(ICH)模型，阻断Src对细胞存活、水肿和行为结局的影响。将解决以下目标：目标#1a-d：证明脑出血后Src mRNA、Src蛋白、Src磷酸化和Src活性增加。确定凝血酶拮抗剂是否阻断脑出血引起的血沉变化，以及凝血酶和凝血酶受体激动剂是否复制脑出血引起的血沉变化。目的#2.观察药物阻断前后脑出血大鼠脑出血后HIF-1、靶基因血管内皮生长因子、基质金属蛋白酶及NMDA受体亚基磷酸化状态的变化。目的#3.在大鼠脑出血后(A)采用脑出血的输血模型和(B)使用缝线诱导的脑出血血管破裂模型，证明药物抑制src可改善脑出血后的细胞存活、脑水肿和改善行为结局。

DESCRIPTION (provided by applicant): Intracerebral hemorrhage (ICH) is a devastating stroke. The damage that occurs in brain from ICH is due to the growing hematoma and clot formation that damages the adjacent brain through edema and apoptosis. Though these are distinct pathologic entities that occur following ICH, we have data that they share some common mechanisms. We found that apoptosis in peri-hematoma brain is mediated in part by glutamate excitotoxicity. This led us to the non-receptor tyrosine kinase, pp60-Src (Src) because it potentiates function of NMDA receptors through direct phosphorylation of the NR2A subunit. We found that Src kinase activity increases 4 fold following experimental ICH in rats, the Src family kinase (SFK) member Lyn increases over 21 fold following ICH, and Src inhibitors decrease apoptosis and improve behavioral outcome following ICH. We hypothesize that Src plays a central role in ICH mediated apoptosis and edema: ICH causes thrombin receptor activation of Src that activates NR2A subunits that mediate apoptosis; and ICH activates thrombin receptors which activates HIF and MMPs via Src to produce edema and poor behavioral outcome. These hypotheses are based upon previous studies showing that thrombin mediates the acute brain edema following ICH, and that thrombin activates Src via the thrombin receptor. Therefore, these studies will examine: (a) the effect of ICH on Src; (b) thrombin activation of Src; (c) ICH induced phosphorylation of NMDA receptors by Src; (d) Src activation of HIF-1, HIF-1 target genes and MMPs; and (e) the effect of Src blockade on cell survival, edema and behavioral outcome using rat models of intracerebral hemorrhage (ICH). The following aims will be addressed: Aims #1a-d: Demonstrate that Src mRNA, Src protein, Src phosphorylation and Src activity increase following ICH. Determine whether thrombin antagonists block these ICH induced changes of Src and whether thrombin and thrombin receptor agonists reproduce the ICH induced changes of Src. Aim #2. Demonstrate changes of HIF-1, of the HIF-1 target gene VEGF, and the Matrix Metalloproteinases and the phosphorylation state of NMDA receptor subunits after ICH in rats with and with out pharmacological blockade of Src. Aim #3. Demonstrate that pharmacological inhibition of Src improves cell survival, decreases brain edema and improves behavioral outcome following ICH in rats (a) using a blood infusion model of ICH and (b) using a suture induced vessel rupture model of ICH.