Protective effect of astrocyte heme oxygenase-1 after intracerebral hemorrhage

项目摘要/摘要 脑出血(ICH)是约10%的卒中的主要事件，其发病率较高。 发病率和死亡率。除了它的质量效应，实验证据表明 从血肿中释放出的毒素，如血红蛋白，会导致邻近区域的细胞丢失。 组织。细胞对血红蛋白的易感性在很大程度上是血红素活性的函数。 加氧酶(HO)酶，由诱导型HO-1和结构性表达的HO-2组成。 HO活性具有抗氧化和抗炎作用，这是由于去除了血红素/氯化血红素和 产生两种分解产物，胆绿素(由胆绿素还原酶转化为胆红素) 和一氧化碳。以往的研究表明，通过培养HO-1过表达 星形胶质细胞对血红素介导的损伤提供强大的保护，而HO-1基因敲除是 有害的。在体内，初步结果表明，转基因小鼠在体内过表达HO-1 星形胶质细胞术后死亡率和血脑屏障破坏显著减少 实验性脑出血比野生型脑出血更严重。此外，HO-1的全身治疗 诱导剂增加血管周围星形胶质细胞中的HO-1，也具有保护作用。这样做的总体目标是 该项目旨在进一步确定脑出血后星形胶质细胞HO-1过度表达的治疗潜力， 使用特定的遗传学方法进行概念验证，然后进行随机、盲法试验 翻译相关的药物疗法。我们的实验目标如下：1)生产 野生型纹状体血肿(WT)、GFAP-CRE-HMOX1fl/fl(星形胶质细胞HO-1 KO)和 GFAP.HMOX1小鼠(星形胶质细胞HO-1过表达)立体定向注射血或 胶原酶。比较死亡率、血脑屏障破坏、纹状体细胞丢失、炎症 反应和行为/认知结果。2)量化血肿周围血流量、血氧 WT、GFAP-Cre-Hmox1fl/fl和GFAP.HMOX1小鼠脑出血后的饱和度和血肿大小 采用显微超声与光声成像相结合的方法。用来量化血管 以设计为基础的体视学指导下的无偏组织学分析。3)随机化GFAP-CRE- Hmox1fl/fl小鼠和Hmox1fl/fl对照用HO-1诱导剂(萝卜硫素、氯化高铁血红素)或 车辆，接受ip。在注射纹状体血或胶原酶后3小时开始。量化 如上所述对结果的影响。希望这些目标的实现将 确定自发性脑出血后星形胶质细胞HO-1过度表达的益处，以及 论证通过选定的药物疗法实现这一目标的可行性。这 然后，信息将为中风新药的临床试验提供合理的基础 目前治疗选择很少且预后不佳的亚型。

Project Summary/Abstract Intracerebral hemorrhage (ICH) is the primary event in about 10% of strokes, and has high morbidity and mortality rates. In addition to its mass effect, experimental evidence indicates that release of toxins such as hemoglobin from the hematoma contributes to cell loss in adjacent tissue. Cellular vulnerability to hemoglobin is largely a function of the activity of the heme oxygenase (HO) enzymes, which consist of inducible HO-1 and constitutively-expressed HO-2. HO activity has antioxidant and anti-inflammatory effects due to heme/hemin removal and generation of two breakdown products, biliverdin (converted to bilirubin by biliverdin reductase) and carbon monoxide. Prior studies have demonstrated that HO-1 overexpression by cultured astrocytes provides robust protection against heme-mediated injury, while HO-1 knockout is deleterious. In vivo, initial results indicate that transgenic mice overexpressing HO-1 in astrocytes sustain significantly less mortality and blood-brain barrier disruption after experimental ICH than their wild-type counterparts. Furthermore, systemic treatment with HO-1 inducers increases HO-1 in perivascular astrocytes and is also protective. The broad goal of this project is to further define the therapeutic potential of astrocyte HO-1 overexpression after ICH, using specific genetic methods for proof of concept followed by randomized blinded trials of translationally-relevant pharmacotherapies. Our experimental aims are as follows: 1) Produce striatal hematomas in wild-type (WT), GFAP-Cre-HMOX1fl/fl (astrocyte HO-1 KO), and GFAP.HMOX1 mice (astrocyte HO-1 overexpression) by stereotactic injection of blood or collagenase. Compare mortality, blood-brain barrier breakdown, striatal cell loss, inflammatory response, and behavioral/cognitive outcome. 2) Quantify perihematomal blood flow, oxygen saturation, and hematoma size in WT, GFAP-Cre-Hmox1fl/fl and GFAP.HMOX1 mice after ICH using micro ultrasound combined with photoacoustic imaging. Quantify blood vessels with unbiased histological analysis guided by design-based stereology. 3) Randomize GFAP-Cre- Hmox1fl/fl mice and Hmox1fl/fl controls to treatment with HO-1 inducers (sulforaphane, hemin) or vehicle, administered i.p. beginning 3 hours after striatal blood or collagenase injection. Quantify the effect on outcome as described above. It is hoped that completion of these aims will establish the benefit of astrocyte HO-1 overexpression after spontaneous ICH, and also demonstrate the feasibility of accomplishing this end with selected pharmacotherapies. This information will then provide the rational basis for clinical trials of novel agents for a stroke subtype that currently has few therapeutic options and a grim prognosis.