脑室内注射GDNF改善新生大鼠术后远期认知功能与海马PKMζ和Kalirin表达的关系

Relationship between intracerebroventricular GDNF-induced improvement in long-term postoperative cognitive function and expression of PKMζ and Kalirin in hippocampus of neonatal rats

目的评价脑室内注射胶质细胞源性神经营养因子(GDNF)改善新生大鼠术后远期认知功能与海马蛋白激酶Mζ(PKMζ)和Kalirin表达的关系。方法取出生后第7天的SD大鼠60只,雄雌不拘,采用随机数字表法为4组(n=15):对照组(C组)、GDNF组(G组)、手术组(S组)、手术+GDNF组(S+G组)。C组未接受麻醉、手术或药物治疗;G组脑室内注射重组大鼠GDNF 0.3μg;S组和S+G组在3%七氟烷麻醉下行右颈动脉暴露手术,S+G组术后脑室内注射重组大鼠GDNF 0.3μg。于出生后第33天开始,进行Barnes迷宫测试及恐惧条件测试。行为学测试后处死大鼠,取大脑,左半脑用于高尔基染色,观察树突形态和测量树突棘密度;右半脑提取海马蛋白,采用Western blot法检测PKMζ和Kalirin的表达。结果与C组比较,S组Barnes迷宫中识别目标盒所需时间延长,恐惧条件测试中环境相关冻结时间缩短,海马树突分支总长度、分支数量及与同心圆交点数量和树突棘密度减少,PKMζ和Kalirin表达下调(P<0.05),G组上述指标差异无统计学意义(P>0.05)。与S组比较,S+G组识别目标盒所需时间缩短,环境相关冻结时间延长,海马树突分支总长度、分支数量及与同心圆交叉点数量和树突棘密度增加,PKMζ和Kalirin表达上调(P<0.05)。结论脑室内注射GDNF改善新生大鼠术后远期认知功能的机制可能与上调海马PKMζ和Kalirin的表达,促进树突和树突棘的发育有关。

Objective To evaluate the relationship between intracerebroventricular glial cell line-derived neurotrophic factor(GDNF)-induced improvement in long-term postoperative cognitive function and expression of PKMζand Kalirin in the hippocampus of neonatal rats.Methods Sixty 7-day old Sprague-Dawley rats,male or female,were divided into 4 groups(n=15 each)using a random number table method:control group(group C),GDNF group(group G),surgery group(group S)and surgery plus GDNF group(group S+G).Group C did not receive anesthesia,surgery or drug treatment.Group G received intracerebroventricular injection of 0.3μg recombinant rat GDNF.Group S and group S+G underwent right carotid artery exposure surgery under 3%sevoflurane anesthesia,and in addition group S+G received intracerebroventricular injection of GDNF.The Barnes maze test and the fear conditioning test were performed starting from postnatal day 33.The animals were then sacrificed under sevoflurane anesthesia after behavioral testing,the brains were obtained and divided into 2 halves sagittally along the midline.The left half of the brain was used for Golgi staining to observe dendritic morphology and measure dendritic spine density.The hippocampal protein was extracted from the right half of the brain to detect the expression of PKMζand Kalirin by Western blot.Results Compared with group C,the time to identify the target box in the Barnes maze test was significantly prolonged,the context-related freezing time in the fear conditioning test was shortened,the total dendritic length,the number of branches,the number of intersections in sholl analysis and spinal density in the hippocampus were reduced,and the expression of PKMζand Kalirin was down-regulated in group S(P<0.05),and no significant change was found in the aforementioned parameters in group G(P>0.05).Compared with group S,the time to identify the target box in the Barnes maze test was significantly shortened,the context-related freezing time in the fear conditioning test was prolonged,the total dendritic length,the number of branches,the number of intersections in sholl analysis and spinal density in the hippocampus were increased,and the expression of PKMζand Kalirin was up-regulated in group S+G(P<0.05).Conclusions The mechanism by which intracerebroventricular GDNF improves long-term postoperative cognitive function may be related to up-regulating the expression of PKMζand Kalirin and promoting the development of dendrites and dendritic spines in the hippocampus of neonatal rats.