A fluorescent method to quantify neuronal injury 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。虽然对血肿周围组织的损伤最初归因于其质量效应，但实验证据表明，血肿本身可能对邻近细胞具有毒性。一种可能的神经毒素是血红蛋白（Hb），它在血块中以毫摩尔浓度存在，并在出血后几天从红细胞中释放出来。血红素加氧酶（HO）分解血红素部分可能导致铁依赖性氧化损伤。然而，胶原酶和血液注射脑出血模型中相互矛盾的结果表明，对血红素分解的后果进行更详细的研究是有必要的。实验性脑出血周围组织的神经元损伤通常通过细胞计数来量化。具体的组织学方法因实验室而异，但都有费力、依赖操作人员和相当昂贵的共同特点。后一种特征可能解释了许多研究范围有限的原因，这些研究通常只测试一种出血量和一种或两种药物剂量。由于任何治疗干预的效果都可能是血块大小和药物剂量的函数，因此迫切需要一种更有效的方法来量化细胞损伤。荧光蛋白表达已成功地用作体外和体内视网膜研究中细胞活力的标记物，但迄今为止尚未用于任何脑出血模型。为了评估这种方法的潜力，申请人已经建立了一个在神经元中组成表达红色荧光蛋白变体dTomato的转基因小鼠群体。初步研究表明：1)d通过行为表型和组织学分析发现，番茄表达本身无毒；2)纹状体荧光与实验性脑出血后MTT细胞活力测定有很好的相关性。本项目的目的是验证纹状体荧光作为实验性脑出血后神经元损失的快速有效标记。目前正在将该转基因纳入缺乏HO-2的小鼠中，以进一步评估该酶对出血性中枢神经系统损伤的影响。具体目的如下：1)通过立体定向注射自体血液（5、10、205l）或胶原酶（0.01、0.03、0.075单位）诱导HO-2敲除dtomato和HO-2野生型dtomato小鼠纹状体出血。在第3天和第8天，比较纹状体细胞分离后的神经元损伤，将福马嗪的产生正常化到对侧纹状体；b)纹状体细胞裂解，随后测量裂解液荧光，也归一化到对侧纹状体；c)细胞计数，由基于设计的立体程序指导。比较神经元损失与血肿周围星形胶质细胞增生和炎症细胞浸润。2)将纹状体神经元损伤与行为结果相关联，通过转弯、去除黏着剂、升高的身体摆动测试和家庭笼子视频记录的数字分析进行量化。希望所提出的研究将建立这种快速荧光方法定量实验性脑出血后神经元损伤的有效性。

DESCRIPTION (provided by applicant): Intracerebral hemorrhage (ICH) is the primary event in 10-15% of strokes. Although injury to tissue surrounding a hematoma was initially attributed to its mass effect, experimental evidence suggests that the hematoma per se may be toxic to neighboring cells. One likely neurotoxin is hemoglobin (Hb), which is present in millimolar concentrations in the clot and is released from erythrocytes in the days after hemorrhage. Breakdown of heme moieties by the heme oxygenase (HO) enzymes may then result in an iron-dependent oxidative injury. However, conflicting results in the collagenase and blood injection ICH models suggest that more detailed investigation of the consequences of heme breakdown is warranted. Neuronal injury in tissue surrounding an experimental ICH is usually quantified by cell counts. Specific histological methods vary considerably from laboratory to laboratory, but do share common features of being laborious, operator- dependent, and quite expensive. The latter characteristic may account for the limited scope of many studies, which typically test only one hemorrhage volume and one or two drug doses. Since the efficacy of any therapeutic intervention will likely be a function of clot size and drug dose, a more efficient method to quantify cell injury is urgently needed. Fluorescent protein expression has been successfully used as a marker of cell viability in vitro and in retinal studies in vivo, but not in any ICH model to date. In order to evaluate the potential of this approach, a colony of transgenic mice that constitutively express the red fluorescent protein variant dTomato in neurons has been established by the applicant. Preliminary studies indicate that: 1) dTomato expression is nontoxic per se, as detected by behavioral phenotyping and histological analysis; 2) Striatal fluorescence correlates very well with the MTT cell viability assay after experimental ICH. The goal of this proposed project is to validate striatal fluorescence as a rapid and efficient marker of neuronal loss after experimental ICH. The transgene is currently being incorporated into mice lacking HO-2 for further evaluation of the effect of this enzyme on hemorrhagic CNS injury. Specific aims are as follows: 1) Induce striatal hemorrhage in HO-2 knockout-dTomato and HO-2 wild-type-dTomato mice by stereotactic injection of autologous blood (5, 10, or 20 5l) or collagenase (0.01, 0.03 or 0.075 units). At 3 and 8 days, compare neuronal injury as quantified by: a) MTT assay after striatal cell dissociation, normalizing formazan production to that in the contralateral striatum; b) Striatal cell lysis, followed by measurement of lysate fluorescence, also normalized to that in the contralateral striatum; c) Cell counts, guided by a design-based stereology program. [Compare neuronal loss with perihematomal astrogliosis and inflammatory cell infiltration. 2) Correlate striatal neuronal injury with behavioral outcome, quantified with corner turn, adhesive removal, and elevated body swing tests, and digital analysis of home cage video recordings.] It is hoped that the proposed studies will establish the validity of this rapid fluorescent method for quantifying neuronal injury after experimental ICH. PUBLIC HEALTH RELEVANCE: The information gained in this project may lead to more efficient and accurate evaluation of new treatments for victims of hemorrhagic stroke. It is expected that the fluorescent method that is the focus of this study will facilitate preclinical testing of drug and surgical therapies at a range of hemorrhage volumes and drug doses. The appropriate use and potential toxicity of these therapies will then be more clearly defined prior to clinical trials, increasing the likelihood of a successful outcome.