Optimizing Heme Oxygenase Activity after CNS Hemorrhage

中枢神经系统出血后优化血红素加氧酶活性

• 批准号: 7046350
• 负责人: RAYMOND F REGAN
• 金额: $ 24.24万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7046350
• 关键词: astrocytes; culture; genes; heme; heme oxygenase; hemorrhage; injury; neurons; stroke; tissues

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.

描述（由申请人提供）：大多数中枢神经系统外伤和约 15% 的中风都会伴有脑出血。越来越多的 9 个证据表明，溶解红细胞释放的血红蛋白 (Hb) 可能会导致周围组织的氧化应激。由于其病程较长，血红蛋白毒性可能是治疗干预的理想目标。先前的研究表明，Hb 通过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，主要存在于神经胶质细胞中，具有保护作用； HO-2 主要在神经元中表达，它会加剧损伤。不幸的是，HO 抑制剂会阻断这两种异构体，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 H2O2 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 激活调节的差异； 2) 转移有义和反义 HO 基因以及将转录靶向特定细胞类型的启动子。我们的实验目的如下：1) 使用特定的苯并三唑抑制剂，通过抑制 CK2 的激活来降低皮质培养物中神经元 HO-2 的活性。确定这种处理对培养物 H2O 活性、活性氧 (ROS) 形成以及 Hb 或氯高铁血红素暴露后细胞活力的影响。 2)构建由星形胶质细胞特异性GFAP启动子驱动的编码HO-1基因的腺病毒。量化其在星形胶质细胞和神经元中的表达，并确定其对血红素介导的 ROS 形成和细胞死亡的影响。 3) 构建含有反义方向 HO-2 基因的腺病毒，由神经元特异性突触蛋白-1 启动子驱动。量化其对培养的星形胶质细胞和神经元中 HO-2 表达以及血红素介导的氧化损伤的影响。 4) 将血液单独或与有义 HO-1、反义 HO-2 或两种载体一起立体定向注射到小鼠纹状体中。或者，用腹膜内 CK2 抑制剂治疗注射血液的小鼠。在 72 小时和 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。该项目中获得的信息可能会为出血性中风和头部外伤患者带来新的治疗方法。最终目标是减少血栓周围组织的脑损伤，从而提高生存和恢复独立、富有成效的生活的可能性。