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%卒中的主要事件。虽然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有毒。一种可能的神经毒素是血红蛋白（Hb），它以毫摩尔浓度存在于凝块中，并在出血后的几天内从红细胞中释放出来。血红素部分被血红素加氧酶（HO）分解然后可导致铁依赖性氧化损伤。然而，胶原酶和血液注射ICH模型中相互矛盾的结果表明，有必要对血红素分解的后果进行更详细的研究。实验性脑出血周围组织中的神经元损伤通常通过细胞计数来定量。具体的组织学方法在实验室之间有很大的不同，但都具有费力、依赖操作者和相当昂贵的共同特征。后一个特征可能是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功效都可能是凝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志物，但迄今为止尚未用于任何ICH模型。为了评估这种方法的潜力，申请人建立了在神经元中组成型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）dTomato表达本身是无毒的，如通过行为表型分析和组织学分析所检测的; 2）纹状体荧光与实验性ICH后的MTT细胞活力测定非常相关。本项目的目标是验证纹状体荧光作为实验性ICH后神经元丢失的快速有效标记物。转基因目前正在纳入缺乏HO-2的小鼠，以进一步评估这种酶对出血性CNS损伤的影响。具体目标如下：1）通过立体定向注射自体血（5、10或20 μ l）或胶原酶（0.01、0.03或0.075单位）在HO-2敲除-dTomato和HO-2野生型-dTomato小鼠中诱导纹状体出血。在第3天和第8天，比较神经元损伤，如通过以下定量：a）纹状体细胞解离后的MTT测定，将甲瓒产生标准化为对侧纹状体中的甲瓒产生; B）纹状体细胞裂解，随后测量裂解物荧光，也标准化为对侧纹状体中的荧光; c）细胞计数，由基于设计的体视学程序指导。[比较神经元丢失与血肿周围星形胶质细胞增生和炎性细胞浸润。2)将纹状体神经元损伤与行为结果相关联，通过转角、粘合剂去除和升高的身体摆动试验以及对家庭笼舍视频记录的数字分析进行量化。我们希望，拟议的研究将建立这种快速荧光定量方法的有效性实验性脑出血后神经元损伤。

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.