Protective Effect of Hemin Preconditioning after Intracerebral Hemorrhage

描述(申请人提供)：脑出血(ICH)是10%-15%的中风的主要事件。越来越多的实验证据支持这一假设，即血肿中释放的氯化血红素可能有助于邻近组织的氧化细胞损伤。血红素加氧酶(HO)催化氯化血红素分解的限速步骤，以一种复杂的方式调节中枢神经系统细胞对氯化血红素的反应。HO-1是一种可诱导的亚型，与实验性脑出血后损伤体积的增加和炎症反应有关，并使行为结局恶化。然而，申请人实验室最近的研究表明，在星形胶质细胞中过度表达HO-1的小鼠明显比野生型小鼠更不容易患脑出血。此外，全身注射氯化血红素，目前正用于治疗急性卟啉病，增加了小鼠大脑中HO-1的表达。当在脑出血后但在红细胞溶解之前给药时，这种治疗方法减轻了纹状体血肿周围血脑屏障的破坏和细胞损伤。这些结果表明，临床耐受剂量的全身氯化血红素提供了一种预适应刺激，保护中枢神经系统细胞免受随后从血肿中释放的高浓度氯化血红素的影响。这种安全、易于使用和廉价的化合物可能是一种新的、高效的脑出血治疗方法。该项目的目标是确定系统性氯化血红素在两种已建立的脑出血模型中的治疗益处。具体目的如下：1)小鼠每日ip氯化高铁血红素。注射1-3天；最后一次注射后24小时，采集纹状体并量化HO-1蛋白的表达和活性。通过免疫染色鉴定表达HO-1的细胞群。2)向小鼠纹状体立体定向注射自体血或胶原酶诱导脑出血。在1、3、6或12小时后用氯化血红素或车辆对照组治疗，24小时后 几个小时后再加一剂。量化3天后血脑屏障的破坏和纹状体的水肿。3)量化氯化血红素对脑出血后3天和8天的纹状体细胞活性和血肿周围炎性细胞浸润的影响。使用拐角、粘合剂去除和抬高身体摆动试验评估这些时间点的焦点缺陷，并通过对家庭笼子视频记录的数字分析来评估活动缺陷。4)比较氯化血红素治疗对野生型小鼠血脑屏障破坏、水肿、神经元活性、炎症和行为缺陷的影响。 在HO-1基因敲除小鼠中。通过使用血红素结合蛋白基因敲除小鼠进行额外的实验，确定血红素结合蛋白对氯化血红素治疗的影响。希望该项目的结果将确定脑出血后全身使用氯化血红素的益处，并迅速导致对一种目前几乎没有治疗选择的疾病过程进行临床试验。

DESCRIPTION (provided by applicant): Intracerebral hemorrhage (ICH) is the primary event in 10-15% of strokes. A growing body of experimental evidence supports the hypothesis that release of hemin from the hematoma may contribute to oxidative cell injury in adjacent tissue. The heme oxygenase (HO) enzymes, which catalyze the rate-limiting step in hemin breakdown, regulate the response of CNS cells to hemin in a complex fashion. HO-1, the inducible isoform, has been associated with increased lesion volume and inflammation after experimental ICH, and worsened behavioral outcome. However, recent studies in the applicant's laboratory suggest that mice overexpressing HO-1 specifically in astrocytes are markedly less vulnerable to ICH than wild-type mice. Moreover, systemic administration of hemin, which is currently in clinical use to treat acute porphyrias, increased HO-1 expression in the mouse brain. When administered after ICH but before erythrocyte lysis, this treatment attenuated blood- brain barrier breakdown and cell injury surrounding a striatal hematoma. These results suggest that systemic hemin at clinically-tolerated doses provides a preconditioning stimulus that protects CNS cells from the hemin subsequently released in high concentrations from the hematoma. This safe, easily administered, and inexpensive compound may be a novel and highly effective treatment for ICH. The goal of this project is to define the therapeutic benefit of systemic hemin in two established ICH models. The specific aims are as follows: 1) Administer hemin to mice via daily i.p. injections for 1-3 days; 24 hours after the last injection, harvest striata and quanify HO-1 protein expression and activity. Identify cell populations that express HO-1 via immunostaining. 2) Induce ICH by stereotactic injection of autologous blood or collagenase into the striata of mice. Treat with hemin or vehicle control 1, 3, 6, or 12 hours later, followed in 24 hours by an additional dose. Quantify blood-brain barrier disruption and striatal edema at 3 days. 3) Quantify the effect of hemin on striatal cell viability and perihematomal inflammatory infiltrates 3 and 8 days after ICH. Assess focal deficits at these time points using corner, adhesive removal and elevated body swing tests, and activity deficits by digital analysis of home cage video recordings. 4) Compare the effects of hemin treatment on blood-brain barrier disruption, edema, neuronal viability, inflammation, and behavioral deficits in wild-type mice with those in HO-1 knockout mice. Determine the effect of the heme binding protein hemopexin on hemin therapy by performing additional experiments using hemopexin knockout mice. It is hoped that the results of this project will define the benefits of systemic hemin after ICH, and rapidly lead to clinical trials for a disease process that currently has few therapeutic options.