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:AccountingAdhesivesAutologousBehavioralBiological AssayBloodBrain hemorrhageBrain regionBreedingCatabolismCell CountCell Culture TechniquesCell SurvivalCellsCerebral hemisphere hemorrhageCharacteristicsClinical TrialsCoagulation ProcessComputer softwareConflict (Psychology)ContralateralCorpus striatum structureCytolysisDevelopmentDissociationDoseEnzymesEquipmentErythrocytesEvaluationEventExcisionExclusionFluorescenceFormazansGoalsHematomaHematoxylin and Eosin Staining MethodHemeHemoglobinHemorrhageHome environmentHuman ResourcesImpaired cognitionIn VitroInfiltrationInflammatoryInjection of therapeutic agentInjuryInvestigationIronIschemic StrokeKnock-outKnockout MiceLaboratoriesLeadMeasurementMeasuresMediatingMethodsModelingMusNeuronal InjuryNeuronsNeurotoxinsOperative Surgical ProceduresOutcomeOxygenasesPharmaceutical PreparationsPhenotypePopulationPreclinical TestingProductionPropidium DiiodideProtein IsoformsProteinsReportingResearch PersonnelResidual stateResolutionRetinalSourceStaining methodStainsStrokeSurvivorsTestingTherapeutic InterventionTimeTissue StainsTissuesToxic effectToxinTrainingTransgenesTransgenic MiceVariantVideo Recordingastrogliosisbasecell injurycentral nervous system injurycollagenasecostcost effectivedesigndigitaleffective therapyfluoro jadeheme oxygenase-2in vivointerestmortalityneuron lossneuronal survivalneurotoxicityprogramsprotein expressionred fluorescent proteinresearch studyselective expressionskillsthree dimensional structuretissue processingtwo-dimensional
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):脑出血 (ICH) 是 10-15% 中风的主要事件。尽管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会导致铁依赖性氧化损伤。然而,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量化。具体的组织学方法因实验室而异,但具有共同的特点:费力、依赖操作人员且相当昂贵。后一个特征可能是许多研究范围有限的原因,这些研究通常仅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用作体外细胞活力的标记和体内视网膜研究,但迄今为止尚未在任何 ICH 模型中使用。为了评估该方法的潜力,申请人已经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) 通过行为表型分析和组织学分析检测,dTomato 表达本身是无毒的; 2) 纹状体荧光与实验性 ICH 后的 MTT 细胞活力测定密切相关。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损失的快速有效标记。该转基因目前正在被整合到缺乏 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。具体目标如下: 1) 通过立体定向注射自体血(5、10或20 5l)或胶原酶(0.01、0.03或0.075 单位)。在第 3 天和第 8 天,通过以下方式比较神经元损伤:a) 纹状体细胞解离后进行 MTT 测定,将甲臜的产生标准化为对侧纹状体的产生; 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,以基于设计的体视学程序为指导。 [将神经元丢失与血肿周围星形胶质细胞增生和炎症细胞浸润进行比较。 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测试以及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确立这种快速荧光方法的有效性用于量化实验性 ICH 后的神经元损伤。
项目成果
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{{ truncateString('RAYMOND F REGAN', 18)}}的其他基金
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- 批准号:
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- 资助金额:
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7162512 - 财政年份:2006
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