大鼠脑片损伤模型和新型定量评价方法的建立

The establishment of the models of brain injury in rat brain slices and the nov el method of quantitative analysis

目的 建立不同性质脑损伤实验模型和一种方便、快速、灵敏的定量评价方法。方法 制备大鼠皮层和海马脑片 ,检测活性后 ,分别接受缺氧缺糖 (OGD)、谷氨酸、过氧化氢 (H2 O2 )损伤 ,氯胺酮、D AP5、异丙酚预处理 ,随后与TTC溶液共同孵育 ,有机溶剂抽提 ,酶标仪测定OD4 90 值 ,同时测定孵育上清液乳酸脱氢酶 (LDH)释放率。结果 室温下恢复孵育 90min后 ,海马脑片CA1区锥体细胞层可记录到群体峰电位。随着OGD时间延长 ,皮层和海马脑片TTC染色明显降低 ,孵育上清液LDH释放率逐渐增加 ,组织损伤百分率与LDH释放率明显正相关 :皮层r =0 960 9,P <0 0 1 ;海马r =0 892 1 ,P <0 0 5。和对照组相比 ,谷氨酸 1mmol·L- 1 和H2 O2 2mmol·L- 1 明显降低脑片TTC染色。氯胺酮5μmol·L- 1 、D AP550 μmol·L- 1 、异丙酚 5μmol·L- 1 分别抑制OGD 1 0min、谷氨酸、H2 O2 损伤所致脑片TTC染色降低。结论 利用大鼠脑片建立的缺氧缺糖、谷氨酸和过氧化氢损伤实验模型 ,以及新鲜脑片与TTC溶液共同孵育 ,有机溶剂抽提、比色的定量评价方法具有方便、快速、灵敏的特点 。

AIM To establish the models of d ifferent kinds of brain injuries and a simple, sensitive, rapid method of quanti tative analysis. METHODS The rat cortical and hippocampal slices were prepared and the tissue activi ties were evaluated by the population spikes in the CA1 region. Brain slices wer e damaged by oxygen and glucose deprivation (OGD), glutamate, hydrogen peroxide (H 2O 2) injury, and pretreated with ketamine, D-AP 5, propofol when subject ed to these three damages respectively, then incubated in 2,3,5-triphenyltetrazolium chloride (TTC). The red formazan was extracted by solvent extraction and measured by ELIS A reader. Lactic dehydrogenase (LDH) effluent rate in the incubated supernate we re also measured. RESULTS The population spikes of the CA1 regi on in hippocampal slices could be recorded. Progressive prolongation of OGD inju ry resulted in decreased TTC staining in the cortical and hippocampal slices. Th ere was a parallel increase in LDH effluent rate. The brain injury percentage ha d a positive correlation with the LDH effluent rate:cortical slices r=0 960 9, P<0 01; hippocampal slices r=0 8921, P<0 05. Compared with the control groups, the TTC staining was decreased when slices were incubated in 1 mmol·L -1 glutamate and 2 mmol·L -1 H 2O 2. The decreases of TTC s taining induced by 10 min of OGD injury, glutamate and H 2O 2 injury were sign ificantly attenuated when pretreated with ketamine 5μmol·L -1 , D-AP 5 5 0 μmol·L -1 or propofol 5 μmol·L -1 respectively. CONCLUSI ON The models OGD, glutamate, H 2O 2 injuries in rat brain slices and the method of quantitative analysis as TTC staining, solvent extraction, and co lorimetry with ELISA reader can be objectively, rapidly, and sensitively used in the studies of brain ischemia and drug evaluation.