Optimizing Heme Oxygenase Activity after CNS Hemorrhage

描述（由申请人提供）：脑出血伴随着大多数中枢神经系统创伤性损伤和约15%的中风。越来越多的证据表明，红细胞溶解释放的血红蛋白（Hb）可能导致周围组织的氧化应激。由于其延长的时间过程，Hb毒性可能是治疗干预的理想目标。先前的研究表明，血红蛋白影响神经元和星形胶质细胞的存活的途径，在很大程度上依赖于血红素加氧酶（HO）。HO-1是诱导型的，主要存在于神经胶质细胞中，具有保护作用; HO-2主要表达于神经元中，保护损伤。不幸的是，HO抑制剂阻断这两种亚型，并在保护神经元的同时增加对星形胶质细胞的损伤。该项目的目标是开发一种策略，优化出血后两种细胞类型中的HO活性。我们假设这可以通过两种方法来实现：1）利用HO-1和HO-2活化调节的差异; 2）将正义和反义HO基因与靶向转录至特定细胞类型的启动子一起转移。我们的实验目的如下：1）使用特异性苯并三唑抑制剂，通过抑制CK 2对皮层培养神经元HO-2的激活，降低其活性。确定这种处理对培养HO活性、活性氧（ROS）形成和Hb或氯化血红素暴露后细胞活力的影响。2)构建含星形胶质细胞特异性GFAP启动子的HO-1基因的腺病毒载体。定量其在星形胶质细胞和神经元中的表达，并确定其对血红素介导的ROS形成和细胞死亡的影响。3)构建一个含有HO-2基因的腺病毒，该基因在神经元特异性突触蛋白1启动子的驱动下处于反义方向。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)将血液单独或与正义HO-1、反义HO-2或两者一起立体定向注射到小鼠纹状体中。或者，用腹膜内CK 2抑制剂治疗血液注射小鼠。在72和144小时，使用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质氧化。在这个项目中获得的信息可能会导致出血性中风和头部创伤受害者的新治疗方法。最终目标是减少血块周围组织的脑损伤，从而提高生存和恢复独立，富有成效的生活的可能性。

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.