Optimizing Heme Oxygenase Activity after CNS Hemorrhage

描述(申请人提供)：脑出血伴发大多数中枢神经系统损伤和约15%的中风。越来越多的证据表明，溶解的红细胞释放的血红蛋白可能参与了周围组织的氧化应激。由于其时间较长，Hb毒性可能是治疗干预的理想靶点。先前的研究表明，Hb通过在很大程度上依赖于血红素加氧酶(HO)的途径影响神经元和星形胶质细胞的存活。HO-1是可诱导的，主要在神经胶质细胞中发现，在那里它是保护性的；HO-2主要在神经元中表达，在那里它会加重损伤。不幸的是，HO抑制剂阻断了这两种异构体，并在保护神经元的同时增加了对星形胶质细胞的损伤。该项目的目标是开发一种策略，在出血后优化两种细胞类型的HO活性。我们假设这可能通过两种方法完成：1)利用HO-1和HO-2激活调控的差异；2)通过启动子将正义和反义HO基因转移到特定类型的细胞。我们的实验目标如下：1)使用特定的苯并三唑抑制剂，通过抑制CK2的激活来降低皮层培养的神经元HO-2的活性。确定在Hb或氯化血红素暴露后，这种处理对培养HO活性、活性氧物种(ROS)的形成和细胞活力的影响。2)构建由星形胶质细胞特异性GFAP启动子驱动的HO-1基因重组腺病毒。量化其在星形胶质细胞和神经元中的表达，并确定其在血红素介导的ROS形成和细胞死亡中的作用。3)构建神经元特异性突触素-1启动子驱动的反义HO-2基因重组腺病毒。量化其对培养的星形胶质细胞和神经元中HO-2表达的影响，以及对血红素介导的氧化损伤的影响。4)单独或与正义HO-1、反义HO-2或两者同时向小鼠纹状体内立体定向注射血液。或者，用CK2抑制剂治疗注射血液的小鼠。在72小时和144小时，用TTC染色确定对病变体积的影响。量化周围组织中的细胞蛋白质和脂肪氧化。从这个项目中获得的信息可能会为出血性中风和头部创伤的受害者带来新的治疗方法。最终目标是减少血液凝块周围组织中的脑损伤，从而提高生存的可能性，并恢复独立的、富有成效的生活。

DESCRIPTION (provided by applicant): Intracerebral hemorrhage accompanies most CNS traumatic injuries and about 15% of strokes. Increasing 9vidence suggests that hemoglobin (Hb) release from lysing erythrocytes may contribute to oxidative stress n surrounding tissue. Because of its prolonged time course, Hb toxicity may be an ideal target for therapeutic intervention. Prior studies have demonstrated that Hb affects the survival of neurons and astrocytes by pathways that are largely dependent on the heme oxygenase enzymes (HO). HO-1 is inducible, and is found primarily in glia, where it is protective; HO-2 is expressed mainly in neurons, where it worsens injury. Unfortunately, HO inhibitors block both isoforms, and increase injury to astrocytes while protecting neurons. The goal of this project is to develop a strategy that will optimize HO activity in both cell types after hemorrhage. We hypothesize that this may be accomplished by two approaches: 1) exploiting differences in the regulation of HO-1 and HO-2 activation; 2) transferring sense and antisense HO genes with promoters that target transcription to specific cell types. Our experimental aims are as follows: 1) Decrease neuronal HO-2 activity in cortical cultures by inhibiting its activation by CK2, using specific benzotriazole inhibitors. Determine the effect of this treatment on culture HO activity, reactive oxygen species (ROS) formation, and cell viability after Hb or hemin exposure. 2) Construct an adenovirus encoding the HO-1 gene driven by the astrocyte-specific GFAP promoter. Quantify its expression in astrocytes and neurons, and determine its effect on heme-mediated ROS formation and cell death. 3) Construct an adenovirus containing the HO-2 gene in antisense orientation, driven by the neuron-specific synapsin-1 promoter. Quantify its effect on HO-2 expression in cultured astrocytes and neurons, and on heme-mediated oxidative injury. 4) Stereotactically inject blood into the striata of mice, alone or with sense HO-1, antisense HO-2, or both vectors. Alternatively, treat blood-injected mice with intraperitoneal CK2 inhibitors. At 72 and 144 hours, determine the effect on lesion volume using TTC staining. Quantify cellular protein and lipid oxidation in surrounding tissue. The information gained in this project may lead to new treatments for victims of hemorrhagic stroke and head trauma. The ultimate goal is to reduce brain injury in tissue surrounding a blood clot, and to thereby improve the likelihood of survival and return to an independent, productive life.