持续惊厥后幼年大鼠海马电生理学变化及脑源性神经营养因子表达变化

Alteration of electrophysiology and expression of brain-derived neurotrophic factor in hippocampus of juvenile rats following a status convulsion

摘要:

目的：研究幼年期大鼠长时程惊厥（status convulsion，SC）后海马电生理学与海马脑源性神经营养因子（brain-derived neurotrophic factor，BDNF）表达变化，探索两者之间的可能关系。方法：选择生后 21 d Wistar 鼠160只，随机分为对照组和实验组，每组 80 只。腹腔注射氯化锂-匹罗卡品制造SC模型；以腹腔注射生理盐水为对照组。造模后 1、7、14、21 d 膜片钳技术检测海马场兴奋性突触后电位（field excitatory postsynaptic potentials，fEPSP）斜率与波幅的变化，比较长时程增强（long-term potenti－ation，LTP）现象。造模后12 h、1 d、3、7、14、21 d免疫组织化学观察海马区BDNF表达的定位情况，Western blot方法鉴定其表达情况。结果：（1）SC后7 d，实验组fEPSP斜率为（162.30±28.50）%高于对照组（124.01±26.46）%（P＜0.05）；SC后14 d实验组和对照组fEPSP斜率分别为（83.06±8.32）%和（121.64±23.12）%，波幅分别为（100.54±16.03）%和（135.65±35.85）%，实验组较对照组斜率及波幅明显降低，有统计学差异（P＜0.05）。（2）免疫组织检查发现SC后大鼠海马CA1，CA3区BDNF阳性着色均有不同程度的增强，实验组在SC后12 h、1 d、3、7 d 较正常组有增多趋势，14 d及21 d组无明显差别。（3）Western blot测定BDNF表达情况，SC后 12 h、1 d、3、7 d分别为：1.08±0.10、1.39±0.08、0.85±0.04、0.53±0.06，对照组分别为：0.39±0.16、0.37±0.03、0.39±0.02、0.37±0.04，实验组与对照组比较有统计学差异（P＜0.05）。结论：（1）幼年大鼠 SC 后 fEPSP 斜率在 SC 后 7 d 较正常组增高；在SC后14 d fEPSP斜率及波幅较对照组和其他实验组明显降低。提示 SC 后急性期 LTP 增高，潜伏期LTP降低。（2）经免疫组化和Western blot方法检测，提示BDNF表达受 SC 刺激影响，在 SC 后急性期表达明显增多。（3）BDNF在LTP 形成过程中可能起重要作用。

Abstract:

Objective：To investigate the alteration of electrophysiology and the change of expression of brain-derived neurotrophic factor（BDNF） in hippocampus of juvenile rats following a status convulsion（SC） and to explore the relationship between the two. Methods：Totally 21-day-old Wistar rats were divided into control group（n=80） and experiment group（n=80）. Rats in control group were injected with normal saline. Intraperitoneal injection of lithium chloride-pilocarpine was conducted to establish SC model. Changes in slope and amplitude of field excitatory postsynaptic potentials（fEPSP） on 1，7，14，21 d after model establishment were detected by patch clamp technique and long-term potentiation（LTP was compared. Location of BDNF in the hippocampus was observed by immunohistochemistry at 12 h and on 1，3，7，14，21 d after SC. Expression of BDNF was quantitatively analyzed by Western blot. Results：（1）Slope of fEPSP on 7 d after SC was significantly higher in experiment group（162.30±28.50）% than in control group（124.01±26.46）%（P<0.05）. Slopes of fEPSP on 14 d after SC in experiment group and control group were （83.06±8.32）% and（121.64±23.12）%，respectively；amplitudes in experiment group and control group were （100.54±16.03）% and （135.65±35.85）%，respectively；slope and amplitude were decreased in experiment group than in control group with statistical differences（P<0.05）. （2）Im－munohistochemistry showed that positive staining of BDNF in CA1 and CA3 region of hippocampus was enhanced after SC；enhance－ment of positive staining of BDNF in CA1 and CA3 region of hippocampus was higher in experiment group than in control group at 12 h and on 1，3，7 d after SC and there was no differences between two group on 14，21 d after SC. （3）According to the results of Western blot，expression of BDNF in experiment group and control group at 12 h and on 1，3，7 d after SC was 1.08±0.10，1.39±0.08，0.85±0.04，0.53±0.06 and 0.39±0.16，0.37±0.03，0.39±0.02，0.37±0.04 respectively，with statistical differences between the two groups（P<0.05）. Conclusions：（1）Slope of fEPSP of juvenile rats on 7 d after SC is significantly increased in experiment group than in con－trol group and slope and amplitude of juvenile rats on 14 d after SC are significantly decreased in experiment group and control group，which indicates that LTP can be increased at acute stage after SC and be decreased at latency stage. （2）Expression of BDNF can be influenced by SC based on results of immunohistochemistry and Western blot and expression of BDNF can be increased at a－cute stage after SC. （3）BDNF plays an important role in the formation of LTP.