A fluorescent method to quantify neuronal injury after intracerebral hemorrhage

描述(申请人提供)：脑出血(ICH)是10%-15%的中风的主要事件。尽管血肿周围组织的损伤最初归因于血肿的质量效应，但实验证据表明，血肿本身可能会对邻近细胞产生毒性。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血栓中，并在出血后几天从红细胞中释放出来。血红素加氧酶(HO)酶对血红素部分的分解可能导致铁依赖的氧化损伤。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，有必要对血红素分解的后果进行更详细的调查。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量化。具体的组织学方法在不同的实验室有很大的不同，但都有共同的特点，即费力、依赖操作员和相当昂贵。后一种特征可能解释了许多研究的有限范围，这些研究通常只测试一个出血量和一到两个药物剂量。由于任何治疗干预的效果都可能是血栓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。荧光蛋白的表达已被成功地用作体外和体内视网膜研究中细胞活性的标志，但迄今为止还没有在任何脑出血模型中使用。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结构性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)行为表型和组织学分析表明，dTomato的表达本身是无毒的；2)纹状体荧光与实验性脑出血后的四甲基偶氮唑盐细胞活力测定有很好的相关性。该项目的目标是验证纹状体荧光作为实验性脑出血后神经元丢失的快速有效的标志物。转基因目前正在被整合到缺乏HO-2的小鼠体内，以进一步评估这种酶在出血性中枢神经系统损伤中的作用。1)立体定向注射自体血(5、10或205L)或胶原酶(0.01U、0.03U或0.075 U)诱导HO-2基因敲除番茄和HO-2野生型番茄小鼠纹状体出血。在第3天和第8天，比较神经元损伤的量化方法：a)纹状体细胞分离后的四甲基偶氮唑蓝比色法，使Formazan产生正常化，与对侧纹状体相同；b)纹状体细胞裂解，随后测量裂解产物的荧光，也与对侧纹状体的定量结果一致；c)细胞计数，由基于设计的体视学程序指导。[将神经元丢失与血肿周围星形胶质细胞增生症和炎性细胞浸润进行比较。2)纹状体神经元损伤与行为结局相关，通过转弯、粘合剂去除和抬高身体摆动试验以及对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的有效性，以量化实验性脑出血后的神经元损伤。

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.