慢性脑缺血模型大鼠学习记忆障碍与脑白质神经纤维、周细胞变化相关性分析

Correlation Between Learning and Memory Impairment and Changes in Cerebral White Matter Nerve Fibers and Pericytes in Rats with Chronic Cerebral Ischemia

目的:分析慢性脑缺血模型大鼠学习记忆行为改变与其胼胝体、海马脑区的白质神经纤维和周细胞进行性退变的相关性,探讨慢性脑缺血所致学习记忆障碍的相关机制,为血管性认知障碍提供可能的预测靶标。方法:选择24只SPF级SD雄性大鼠,按照随机数字表法分为假手术组、术后28 d组、术后56 d组,每组8只。假手术组大鼠只分离双侧颈总动脉但不结扎,术后28 d组和术后56 d组大鼠用双侧颈总动脉结扎的方法制备慢性脑缺血大鼠模型。分别于造模后28、56 d采用Morris水迷宫空间探索实验评估学习记忆能力;采用弥散张量成像(DTI)观察胼胝体和海马脑区白质神经纤维变化的各向异性分数(FA)和平均弥散率(MD);采用坚牢蓝(LFB)染色法观察大鼠胼胝体髓鞘脱失情况;采用免疫印迹法检测胼胝体和海马脑区的髓鞘碱性蛋白(MBP)和周细胞标志物血小板衍生生长因子受体β(PDGFR-β)蛋白表达情况。结果:(1)学习记忆能力:与假手术组比较,术后28 d组第2~4天逃避潜伏期均明显延长,术后56 d组第3天逃避潜伏期明显延长,差异均具有统计学意义(P<0.05)。与假手术组比较,术后28 d组和术后56 d组大鼠穿越平台次数、目标象限时间占比均明显减少,差异具有统计学意义(P<0.05)。(2)胼胝体和海马脑区白质神经纤维变化:与假手术组比较,术后28 d组、术后56 d组左、右侧胼胝体和左、右侧海马脑区白质神经纤维变化的MD值均明显降低,差异具有统计学意义(P<0.05)。(3)大鼠胼胝体、海马脑区MD值与学习记忆能力的相关性分析:大鼠胼胝体、海马脑区MD值均与穿越平台次数呈正相关关系(r=0.832,P<0.01;r=0.777,P<0.01)。(4)大鼠胼胝体神经纤维形态:与假手术组比较,术后28 d组、术后56 d组大鼠胼胝体部位发生髓鞘结构紊乱以及脱失现象,术后28 d组和术后56 d组髓鞘紊乱分级均明显升高(P<0.05)。(5)胼胝体和海马脑区MBP和PDGFR-β蛋白表达:与假手术组比较,术后28 d组、术后56 d组大鼠胼胝体、海马脑区的髓鞘碱性蛋白MBP蛋白表达水平均明显下降,差异均具有统计学意义(P<0.05);术后28 d组胼胝体PDGFR-β表达水平明显升高(P<0.05),术后28 d组、术后56 d组海马脑区PDGFR-β表达水平均明显下降,差异均具有统计学意义(P<0.05)。与术后28 d组比较,术后56 d组胼胝体PDGFR-β表达水平明显下降,差异具有统计学意义(P<0.05)。结论:慢性脑缺血所致学习记忆障碍与胼胝体和海马脑区白质神经纤维结构损伤密切相关,其中胼胝体PDGFR-β变化可能是预测慢性脑缺血所致白质神经纤维损伤的关键靶标之一。

Objective:To analyze the correlation between the changes of learning and memory and the progressive degeneration of white matter nerve fibers and pericytes in the corpus callosum and hippocampus of rats with chronic cerebral ischemia,then to explore the mechanism of learning and memory impairment caused by chronic cerebral ischemia,and provide a possible predictive target for vascular cognitive impairment.Methods:A total of 24 SPF male SD rats were divided randomly into sham operation group,28 days after operation group and 56 days after operation group,with eight rats in each group.The sham operation group were treated with bilateral common carotid arteries separation without ligation,and chronic cerebral ischemia models were established by ligation of bilateral common carotid arteries in the 28 days after operation group and the 56 days after operation group.At 28 d and 56 d after operation,morris water maze was used to detect spatial learning and memory ability;diffusion tensor imaging was used to observe the fractional anisotropy(FA) and mean diffusivity(MD) rate of white matter nerve fibers in corpus callosum and hippocampus;luxol fast blue(LFB) staining was used to observe demyelination in corpus callosum;Western blot was used to detect the expression of myelin basic protein(MBP) and pericyte marker platelet-derived growth factor receptor β(PDGFR-β) in corpus callosum and hippocampus.Results:(1) Learning and memory ability:compared with the sham operation group,the escape latency prolonged significantly from day 2 to day 4 after operation in the 28 days after operation group and on the 3rd day in the 56 days after operation group(P<0.05).Compared with the sham operation group,the times of crossings of the platform and the proportion of target quadrant time in the 28 days after operation group and the 56 days after operation group were significantly lower(P<0.05).(2) Changes of white matter nerve fibers in corpus callosum and hippocampus:compared with the sham operation group,the MD values of white matter nerve fibers in the left and right corpus callosum and the left and right hippocampus in the 28 days after operation group and the 56 days after operation group were significantly lower(P<0.05).(3) Correlation analysis between learning and memory ability and MD values of corpus callosum or hippocampus:there was a positive correlation between the times of crossings of the platform and MD values of corpus callosum or hippocampus(r=0.832,P<0.01;r=0.777,P<0.01).(4) Morphology of nerve fibers in the corpus callosum:compared with the sham operation group,the structure disorder and loss of myelin sheath occurred in the corpus callosum after chronic cerebral ischemia.The grade of myelin disorder in the 28 days after operation group and the 56days after operation group was significantly higher(P<0.05).(5) Expression of MBP and PDGFR-β protein in corpus callosum and hippocampus:compared with the sham operation group,the expression of myelin basic protein in the corpus callosum and hippocampus decreased significantly in the 28 days after operation group and the 56 days after operation group(P<0.05).The expression level of PDGFR-β in corpus callosum increased significantly in the 28 days after operation group,and the expression level of PDGFR-β in hippocampus decreased significantly in the 28 days after operation group and the 56 days after operation group.Compared with the 28 days after operation group,the expression of PDGFR-β in corpus callosum decreased significantly in the 56 days after operation group(P<0.05).Conclusion:Learning and memory impairment caused by chronic cerebral ischemia is closely related to the structural damage of corpus callosum and hippocampus,and the alteration of PDGFR-β in the corpus callosum may be one of the key targets for predicting nerve fiber damage caused by chronic cerebral ischemia.