A fluorescent method to quantify neuronal injury after intracerebral hemorrhage
定量脑出血后神经元损伤的荧光方法
基本信息
- 批准号:8290451
- 负责人:
- 金额:$ 19.38万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2011
- 资助国家:美国
- 起止时间:2011-07-01 至 2013-12-30
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
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.
描述(由申请人提供):脑内出血(ICH)是10-15%的中风的主要事件。尽管最初对周围血肿的组织损伤归因于其质量作用,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),它以毫米浓度存在于凝块中,并在出血后的几天从红细胞释放。血红素加氧酶(HO)酶对血红素部分的分解可能会导致铁依赖性氧化损伤。但是,胶原酶和血液注射ICH模型的相互矛盾的结果表明,有必要对血红素分解后果进行更详细的研究。通常通过细胞计数来定量实验ICH周围组织中的神经元损伤。特定的组织学方法因实验室而异,但确实具有艰苦,依赖性和相当昂贵的共同特征。后一种特征可能会说明许多研究的范围有限,通常仅测试一种出血体积和一两个药剂量。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量化细胞损伤的方法。荧光蛋白表达已成功用作体外和视网膜研究中细胞活力的标记,但迄今为止的任何ICH模型中都没有。为了评估这种方法的潜力,申请人已经建立了一种在神经元中构成表达红色荧光蛋白变异dtomato的转基因小鼠的菌落。初步研究表明:1)DTOMATO表达本身是无毒的,如行为表型和组织学分析所检测到的; 2)纹状体荧光与实验性ICH后的MTT细胞活力测定法非常相关。该提出的项目的目的是验证纹状体荧光,作为实验性ICH后神经元丧失的快速有效标记。当前,该转基因被纳入缺乏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(5、10或20 5L)或胶原酶(0.01、0.01、0.03或0.075单元),通过立体次旋转自体血(5、10或20 5L)诱导纹状体出血和HO-2敲除DTOMATO和HO-2野生型-DTOMATO小鼠。在3和8天时,比较以下量化的神经元损伤:a)纹状体细胞解离后MTT分析,将甲半氮的产生标准化与对侧纹状体中的产生; b)纹状体细胞裂解,然后测量裂解物荧光,在对侧纹状体中也归一化。 C)细胞计数,在基于设计的立体学计划的指导下。 [比较神经元丧失与围日旁胶质细胞和炎症细胞浸润。 2)将纹状体神经元损伤与行为结果相关联,通过角转弯,粘合剂去除和身体挥杆测试进行量化,以及对家用笼式视频记录的数字分析。
项目成果
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数据更新时间:2024-06-01
RAYMOND F REGAN的其他基金
Protective effect of astrocyte heme oxygenase-1 after intracerebral hemorrhage
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Effect of Hemopexin Therapy after Intracerebral Hemorrhage
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Protective Effect of Hemin Preconditioning after Intracerebral Hemorrhage
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- 批准号:83463108346310
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Protective Effect of Hemin Preconditioning after Intracerebral Hemorrhage
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A fluorescent method to quantify neuronal injury after intracerebral hemorrhage
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