Effect of Inducible Antioxidants on Hemoglobin Toxicity

描述（由申请人提供）：出血是约15%中风的主要事件，并伴随着最严重的脑外伤。越来越多的实验证据表明，溶解红细胞释放的血红蛋白可能导致血肿周围组织的氧化损伤。先前的研究已经证明，血红蛋白毒性是通过其血红素部分转移到附近的细胞介导的，这是通过其自发氧化为高铁血红蛋白而促进的。血红素的氧化形式氯化血红素然后被血红素加氧酶分解代谢成铁、一氧化碳和胆绿素。如果铁螯合不足，后两种产品的细胞保护作用可能会被淹没。星形胶质细胞通过快速诱导铁蛋白来响应血红蛋白暴露，铁蛋白是一种24聚体的杂聚物，在其矿物核心中具有超过4000个铁原子的能力。然而，在细胞培养和体内皮质神经元表达非常少的铁蛋白血红蛋白处理后。细胞铁蛋白水平主要由铁调节蛋白（IRP 1和IRP 2）调节，其结合其mRNA的5 '-非翻译区的铁响应元件（IRE）并抑制翻译。使用细胞系的体外研究已经证明IRP结合的抑制剂增加铁蛋白表达并保护免受氧化损伤。为了评估这种方法在减弱血红蛋白神经毒性方面的治疗潜力，我们建立了IRP 1和IRP 2敲除小鼠的集落。在已完成的研究中，我们观察到：1）铁蛋白在基线和氧化剂暴露后被IRP 2敲除神经元过表达，而IRP 1敲除具有弱得多且可变的作用; 2）IRP 2基因敲除显著降低神经元对血红蛋白和过氧化氢的脆弱性; 3）IRP 2敲除小鼠中纹状体血肿周围的氧化损伤减弱。这项竞争性修订申请的目的是比较野生型和IRP 2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，我们建议通过一种在出血性中风研究中新颖的方法来量化功能缺陷：自动分析家庭笼运动行为，测试由两家美国公司销售的专有软件。基于公司。具体而言，将以24小时间隔对小鼠活动进行视频记录。活动水平、转身/转圈和刻板行为将使用ANY迷宫和HomeCageScan软件进行量化。 公共卫生相关性：本项目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。最终目标是通过解毒铁来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): 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.