Protective Effect of Hemin Preconditioning after Intracerebral Hemorrhage

描述（由申请人提供）：脑出血（ICH）是10-15%卒中的主要事件。越来越多的实验证据支持这一假设，即从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，以复杂的方式调节CNS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ICH后病变体积和炎症增加以及行为结局恶化相关。然而，申请人的实验室中的最近研究表明，特异性地在星形胶质细胞中过表达HO-1的小鼠比野生型小鼠明显更不易于ICH。此外，全身施用氯化血红素（目前临床上用于治疗急性卟啉症）增加了小鼠脑中HO-1的表达。当在ICH之后但在红细胞溶解之前给药时，这种治疗减轻了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，全身氯化血红素在临床耐受剂量提供了一个预处理刺激，保护中枢神经系统细胞从随后释放的氯化血红素在高浓度的血肿。这种安全，易于管理，廉价的化合物可能是一种新的和高效的治疗ICH。本项目的目的是确定在两个已建立的ICH模型中全身性氯化血红素的治疗益处。具体目的如下：1）每天腹腔注射氯化血红素给小鼠，持续1-3天;最后一次注射后24小时，收获纹状体，定量HO-1蛋白表达和活性。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至小鼠纹状体诱发脑出血。在1、3、6或12小时后用氯化血红素或溶剂对照处理，随后在24小时内 小时的额外剂量。在第3天定量血脑屏障破坏和纹状体水肿。3)量化氯化血红素对脑出血后3天和8天纹状体细胞活力和血肿周围炎性浸润的影响。在这些时间点使用角落，粘合剂去除和升高的身体摆动测试评估局灶性缺陷，并通过对家庭笼视频记录的数字分析评估活动缺陷。4)比较氯化血红素治疗对野生型小鼠的血脑屏障破坏、水肿、神经元活力、炎症和行为缺陷的影响， HO-1基因敲除小鼠中的那些。通过使用血红素结合蛋白基因敲除小鼠进行额外的实验来确定血红素结合蛋白对血红素治疗的影响。希望该项目的结果将确定ICH后全身性氯化血红素的益处，并迅速导致目前几乎没有治疗选择的疾病过程的临床试验。

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.