Protective Effect of Hemin Preconditioning after Intracerebral Hemorrhage

描述（由申请人提供）：脑出血（ICH）是10-15%中风的主要事件。越来越多的实验证据支持血肿释放血红素可能导致邻近组织氧化细胞损伤的假设。血红素加氧酶（HO）酶催化血红素分解的限速步骤，以复杂的方式调节中枢神经系统细胞对血红素的反应。HO-1是诱导型异构体，与实验性脑出血后病变体积和炎症增加以及行为结果恶化有关。然而，申请人实验室最近的研究表明，在星形胶质细胞中特异性表达HO-1的小鼠明显比野生型小鼠更不容易患脑出血。此外，目前临床用于治疗急性卟啉症的hemin全身给药可增加小鼠大脑中HO-1的表达。在脑出血后但在红细胞溶解前使用，这种治疗可以减轻纹状体血肿周围的血脑屏障破坏和细胞损伤。这些结果表明，临床耐受剂量的全身性血红素提供了一种预处理刺激，保护中枢神经系统细胞免受随后从血肿中释放的高浓度血红素的影响。这种安全、易于施用和廉价的化合物可能是治疗脑出血的一种新型和高效的方法。该项目的目标是在两种已建立的脑出血模型中确定全身性血凝素的治疗益处。具体目的如下：1)每天给小鼠腹腔注射血凝素，持续1 ~ 3天；末次注射后24小时，采集纹状体，定量HO-1蛋白表达及活性。通过免疫染色鉴定表达HO-1的细胞群。2)立体定向注射自体血或胶原酶到小鼠纹状体诱导脑出血。1、3、6或12小时后给予血红素或对照治疗，24小时后随访

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.