Effect of Inducible Antioxidants on Hemoglobin Toxicity

描述(由申请人提供)：出血是大约15%的中风的主要事件，并伴随着最严重的脑损伤。越来越多的实验证据表明，溶解的红细胞释放的血红蛋白可能会对血肿周围的组织造成氧化损伤。以前的研究表明，血红蛋白的毒性是通过其血红素部分转移到附近的细胞而介导的，这是通过其自发氧化成高铁血红蛋白来促进的。亚铁血红素是亚铁血红素的氧化形式，然后被亚铁血红素加氧酶分解为铁、一氧化碳和胆绿素。如果铁隔离不足，后两种产品的细胞保护作用可能会被压倒。星形胶质细胞对血红蛋白暴露的反应是通过快速诱导铁蛋白，铁蛋白是一种24聚体的杂聚体，其矿物质核心可容纳4000多个铁原子。然而，经血红蛋白处理后，细胞培养和活体中的皮质神经元表达的铁蛋白很少。细胞铁蛋白水平主要由铁调节蛋白(Irp1和IRP2)调节，它们与其mRNA 5‘-非翻译区的铁反应元件(IRE)结合并抑制翻译。使用细胞系的体外研究表明，IRP结合的抑制剂增加了铁蛋白的表达，并保护其免受氧化损伤。为了评估这种方法在减轻血红蛋白神经毒性方面的治疗潜力，我们建立了IRP1和IRP2基因敲除小鼠的克隆。在已完成的研究中，我们观察到：1)IRP2基因敲除的神经元在基线和氧化暴露后均可过表达铁蛋白，而IRP1基因敲除的作用要弱得多；2)IRP2基因敲除显著降低了神经元对血红蛋白和过氧化氢的易感性；3)在IRP2基因敲除的小鼠中，纹状体血肿周围的氧化损伤得到缓解。这一竞争性修订应用的目标是比较野生型和IRP2基因敲除小鼠在实验性脑出血后的功能恢复，使用直接血液注射和胶原酶注射模型。除了标准的行为测试，我们建议通过出血性中风研究中的一种新方法来量化功能缺陷：自动分析家庭笼子运动行为，测试由两家美国公司销售的专有软件。具体地说，将对鼠标活动进行24小时的视频记录。活动水平、转身/转圈和刻板行为将通过任意迷宫和HomeCageScan软件进行量化。 公共卫生相关性：从该项目中获得的信息可能为出血性中风和头部创伤的受害者提供新的治疗方法。最终的目标是通过解毒铁来减少血液凝块周围组织的脑损伤，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): Hemorrhage is the primary event in approximately 15% of strokes, and accompanies most significant brain trauma. A growing body of experimental evidence suggests that hemoglobin release from lysing erythrocytes may contribute to oxidative injury to tissue surrounding a hematoma. Prior studies have demonstrated that hemoglobin toxicity is mediated by transfer of its heme moieties to nearby cells, which is facilitated by its spontaneous oxidation to methemoglobin. Hemin, the oxidized form of heme, is then catabolized by the heme oxygenase enzymes to iron, carbon monoxide, and biliverdin. The cytoprotective effects of the latter two products may be overwhelmed if iron sequestration is inadequate. Astrocytes respond to hemoglobin exposure by rapidly inducing ferritin, a 24-mer heteropolymer with a capacity for over 4000 iron atoms in its mineral core. However, cortical neurons in cell culture and in vivo express very little ferritin after hemoglobin treatment. Cell ferritin levels are primarily regulated by iron regulatory proteins (IRP1 and IRP2), which bind to an iron responsive element (IRE) in the 5'-untranslated region of its mRNA and repress translation. In vitro studies using cell lines have demonstrated that inhibitors of IRP binding increase ferritin expression and protect against oxidative injury. In order to evaluate the therapeutic potential of this approach in attenuating hemoglobin neurotoxicity, we have established colonies of IRP1 and IRP2 knockout mice. In completed studies, we have observed that: 1) Ferritin is overexpressed by IRP2 knockout neurons at baseline and after oxidant exposure, while IRP1 knockout has a much weaker and variable effect; 2) IRP2 gene knockout markedly reduces neuronal vulnerability to hemoglobin and hydrogen peroxide; 3) Oxidative injury surrounding a striatal hematoma is attenuated in IRP2 knockout mice. The goal of this competitive revision application is to compare functional recovery in wild-type and IRP2 knockout mice after experimental intracerebral hemorrhage, using both direct blood injection and collagenase injection models. In addition to standard behavioral tests, we propose to quantify functional deficits via an approach that is novel in hemorrhagic stroke research: automated analysis of home cage locomotor behavior, testing proprietary software marketed by two U.S.-based companies. Specifically, mouse activity will be video recorded for 24 hour intervals. Activity levels, turning/circling, and stereotypic behaviors will be quantified with ANY-maze and HomeCageScan software. PUBLIC HEALTH RELEVANCE: 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 by detoxifying iron, and to thereby improve the likelihood of survival and return to an independent, productive life.