外源性VEGF对脑缺血模型大鼠认知能力的保护作用及机制探究

发布时间：2018-05-11 06:51

本文选题：VEGF + 脑缺血　；参考：《南开大学》2014年博士论文

【摘要】：目的 (1)通过在体(in vivo)实验观察外源性血管内皮生长因子VEGF对慢性低灌注型脑缺血模型大鼠认知功能的保护作用及电生理学和组织学机制。 (2)利用离体(in vitro)海马脑片缺血缺氧模型探讨VEGF对海马神经元保护作用的细胞学及电生理学机制。方法 (1)In vivo:将30只雄性Wistar大鼠随机分成3组,每组10只：假手术组(Sham)、缺血组(Ischemia)、VEGF处理组(Ischemia+VEGF)。采用双侧颈总动脉结扎(2-VO)2周建立慢性低灌注型脑缺血大鼠模型。采用鼻饲法给予外源性VEGF,然后利用Morris水迷宫实验(MWM)比较各组大鼠的空间认知功能；行为学实验后,进行电生理实验,内容包括海马CA3-CA1区的长时程增强LTP以及局部场电位(LFP),并应用神经信息流算法分析各组大鼠CA3与CA1区神经网络的振荡模态的变化。实验结束后取脑,使用ELISA的方法测定各组海马内VEGF的含量并通过组织学方法(HE染色)观察海马CA1区锥体细胞的形态结构。 (2) In vitro:首先利用糖氧剥夺(OGD)的方法建立离体缺血缺氧海马脑片模型,实验分为4组,即正常组(Control)、糖氧剥夺组(OGD)、VEGF处理组(OGD+VEGF)、VEGF及其受体抑制剂处理组(OGD+VEGF+SU5416)。使用碘化丙啶(PI)染色的方法并利用共聚焦显微镜观察各组脑片海马CA1区神经元在OGD处理20min后的存活情况。然后分别记录各组脑片在OGD处理10min以及恢复正常脑脊液后的电生理信号的变化,包括神经元膜电位、自发放电频率以及sEPSCs的变化。结果 (1)水迷宫(MWM)实验结果发现,缺血组大鼠的逃避潜伏期从训练的第3天开始比假手术组显著延长(Ischemia:13.5±1.9s vs. Sham:8.1±1.2s,P0.05),而VEGF处理后的脑缺血大鼠的逃避潜伏期也从第3天开始比缺血组大鼠明显地缩短了(Ischemia+VEGF:8.5±1.1s, P0.05vs. Ischemia);但是,三组大鼠的平均游泳速度无显著性差别(P0.05)。测试日的实验发现,缺血组大鼠在目标象限停留的百分比比假手术组的大鼠明显减少(Ischemia:34.2±1.6%vs. Sham:42.9±1.5%, P0.05), VEGF处理后缺血大鼠的目标象限百分比几乎恢复到假手术组的水平(Ischemia+VEGF:40.1±2.6%). (2)记录并分析各组大鼠海马CA3-CA1通路的长时程增强LTP的结果发现,缺血组的场兴奋性突触后电位(fEPSPs)斜率百分比比假手术组显著降低(Ischemia:117.8±6.23%vs. Sham:136.2±5.96%,P0.01),而VEGF组则比缺血组明显要高(130.6±5.36%, P0.01vs. Ischemia)。 (3)记录并计算各组大鼠海马CA3与CA1区局部场电位(LFP)的结果发现,脑缺血能够引起海马CA3与CA1脑区间的相位同步性降低,CA3区theta相位调控CA1低频gamma(LG,30-60Hz)幅值的程度显著降低,而加入VEGF后,CA3、CA1脑区间的耦合强度均有上升的趋势。 (4)HE染色结果发现,假手术组大鼠海马CA1区锥体神经元形态正常。而缺血组的CA1神经元排列松散,分布不均,细胞出现水肿以及核固缩的现象。而VEGF处理组大鼠的CA1神经元可见部分形态正常,部分形态异常,分布及排列趋近于正常组。 (5)分析各组大鼠海马内VEGF的含量发现,缺血组大鼠海马内的VEGF含量明显比假手术组要低(Sham:745.33±42.34pg/g vs. Ischemia:420.09±40.82pg/g,P0.01),而VEGF处理组的海马内的VEGF含量有显著提高(592.83±38.76pg/g, P0.05vs. Ischemia),但仍然低于假手术组(P0.05)。 (6)在离体实验中检测各组脑片海马CA1神经元存活情况的结果发现,OGD处理20min后,神经元的死亡率显著上升(Control vs. OGD, P0.05),而VEGF孵育组存活细胞的数量增多,接近正常组。然而与VEGFR-2的抑制剂SU5416共孵育之后,神经元的死亡率升高,接近OGD组。 (7)膜片钳实验发现,OGD处理后CA1神经元的电生理特性能够发生可逆性变化的时间窗口为10min,在恢复正常aCSF处理后绝大部分神经元的膜电位能够恢复正常,因此将其确定为本实验的时间窗口。 (8)在10min OGD处理期间分析各组神经元的兴奋性发现,OGD组神经元的膜电位在OGD结束时去极化了14.7±1.23mV(n=5),而VEGF处理组神经元则去极化了6.1±1.57mV (n=8, OGD vs. OGD+VEGF, P0.01)。同时共孵育VEGFR-2抑制剂SU5416组(OGD+VEGF+SU5416)则去极化了12.1±1.92mV(n=6)。同样的在自发放电频率的统计结果中发现,OGD+VEGF组的神经元的在OGD期间的自发放电频率比base时增加了5.1±0.6倍,远远低于OGD组(9.2±0.56倍,P0.01,n=5),而OGD+VEGF+SU5416放电频率增加了8.1±0.47倍,高于VEGF处理组且具有显著差异(P0.01,n=5),但是与OGD组相比却不具有统计学差异(P0.05)。 (9)在记录并分析了各组大鼠海马CA1神经元的sEPSCs事件结果发现,在10min OGD处理期间,各组sEPSCs的幅值和频率均时间依赖性的增加,而在VEGF处理组,这两个指标增加的程度被缓解,但仍不能恢复到正常水平。而加入VEGF受体抑制剂SU5416之后,VEGF的缓解效果被解除,说明VEGF的作用是通过VEGFR-2受体发挥作用的。结论 (1)外源性给予VEGF可显著改善VD大鼠的空间认知障碍,其机制包括改善缺血大鼠海马锥体神经元水肿和核固缩情况,维持海马神经元正常功能。 (2)通过鼻饲法给予外源性VEGF是一种可行的方法,可以使缺血大鼠海马内VEGF的含量有效升高,对缺血产生了保护作用。 (3) VEGF能够改善VD大鼠海马内突触可塑性的降低,增强CA3-CA1的长时程增强LTP,这一现象可能与VEGF改善了它们之间theta节律与gamma节律的交叉耦合模式有关。 (4)在离体缺血缺氧脑片模型中,外源性VEGF能够通过其VEGFR-2受体来发挥对海马神经元的保护作用,其机制是对抗缺氧导致的神经元凋亡,从而促进其存活。 (5)VEGF能够对抗由缺氧引起的海马CA1神经元兴奋性的升高,维持了神经元的正常电生理特性,这可能是VEGF在缺血早期发挥保护作用的电生理学机制。 (6)VEGF能够降低由缺氧引起的海马神经元突触传递的异常升高,维持了神经元正常的突触传递功能,这可能是VEGF能够改善突触可塑性并进一步改善认知功能的机制之一。
[Abstract]:objective
(1) the protective effect of exogenous vascular endothelial growth factor VEGF on cognitive function of chronic hypoplastic cerebral ischemia model rats and the electrophysiological and histological mechanism were observed by the in vivo experiment.
(2) the cellular and electrophysiological mechanisms of VEGF on hippocampal neurons were studied by using in vitro hippocampal slices.
Method
(1) In vivo: randomly divided 30 male Wistar rats into 3 groups, with 10 rats in each group: the sham operation group (Sham), the ischemic group (Ischemia) and the VEGF treatment group (Ischemia+VEGF). The rat model of chronic low perfusion cerebral ischemia was established by bilateral common carotid artery ligation (2-VO) for 2 weeks. The exogenous VEGF was given by the nasal feeding method, and the Morris water maze test (MWM) ratio was used. The spatial cognitive function of the rats in each group was compared. After the behavior experiment, the electrophysiological experiments were carried out, including the long time enhancement LTP and the local field potential (LFP) in the hippocampal CA3-CA1 region, and the changes of the oscillatory modes of the CA3 and CA1 neural networks in each group were analyzed by the neural information flow algorithm. The brain was taken after the experiment and the method of ELISA was used. The content of VEGF in hippocampus of each group was determined, and the morphological structure of pyramidal cells in hippocampus CA1 was observed by histological method (HE staining).
(2) In vitro: first used the method of glucose oxygen deprivation (OGD) to establish an isolated ischemic hypoxic hippocampal slice model. The experiment was divided into 4 groups, namely, normal group (Control), glucose oxygen deprivation group (OGD), VEGF treatment group (OGD+VEGF), VEGF and its receptor inhibitor treatment group (OGD+VEGF+ SU5416). The method of staining with propidium iodide (PI) was used and confocal microscopy was used. The survival of hippocampal CA1 neurons in each group was observed after OGD treatment of 20min. The changes of electrophysiological signals in each group of brain slices after OGD treatment and recovery of normal cerebrospinal fluid were recorded, including the neuronal membrane potential, spontaneous discharge frequency and the change of sEPSCs.
Result
(1) the results of water maze (MWM) experiment found that the escape latency of rats in the ischemic group was significantly longer than that of the sham group (Ischemia:13.5 + 1.9s vs. Sham:8.1 + 1.2s, P0.05), and the escape latency of ischemic rats after VEGF treatment was also significantly shorter than that of the ischemic group (Ischemia+VEGF:8.5 + 1.1s). P0.05vs. Ischemia); however, the average swimming speed of the three groups was not significantly different (P0.05). The test day showed that the percentage of the rats in the ischemic group was significantly lower than that of the sham group (Ischemia:34.2 + 1.6%vs. Sham:42.9 + 1.5%, P0.05). The percentage of the target quadrants of the ischemic rats after VEGF treatment was few. It returned to the level of the sham operation group (Ischemia+VEGF:40.1 + 2.6%).
(2) the results of recording and analyzing the long time enhanced LTP of the CA3-CA1 pathway in the hippocampus of rats showed that the percentage of the field excitatory postsynaptic potential (fEPSPs) slope in the ischemic group was significantly lower than that of the sham group (Ischemia:117.8 + 6.23%vs. Sham:136.2 + 5.96%, P0.01), while VEGF group was significantly higher than that of the ischemic group (130.6 + 5.36%, P0.01vs. Ischemia).
(3) the results of recording and calculating the local field potential (LFP) of CA3 and CA1 regions in the hippocampus of rats were recorded and calculated. It was found that cerebral ischemia could cause the phase synchronization of CA3 and CA1 in the hippocampus to be reduced, and the degree of low frequency gamma (LG, 30-60Hz) amplitude of CA1 in CA3 region theta phase regulated significantly decreased. Potential.
(4) the results of HE staining showed that the hippocampal pyramidal neurons in the hippocampal CA1 area of the sham operation group were normal, while the CA1 neurons in the ischemic group were loosely arranged and distributed unevenly. The cells appeared edema and nuclear condensation. The CA1 neurons in the VEGF treatment group showed normal part of the morphology, and the partial morphology was abnormal. The distribution and arrangement of the CA1 neurons were close to the normal group.
(5) the content of VEGF in hippocampus of rats in each group was analyzed. The content of VEGF in hippocampus of ischemia group was significantly lower than that of sham group (Sham:745.33 + 42.34pg/g vs. Ischemia:420.09 + 40.82pg/g, P0.01), while the VEGF content in the hippocampus of the VEGF treatment group was significantly increased (592.83 + 38.76pg/g, P0.05vs.), but still lower than the sham operation group. P0.05).
(6) the results of the survival of hippocampal CA1 neurons in each group were detected in isolated experiments. It was found that after OGD treatment 20min, the mortality of neurons increased significantly (Control vs. OGD, P0.05), while the number of surviving cells in the VEGF incubating group increased, close to that of the normal group. However, the mortality of neurons increased after reincubation with the inhibitor SU5416 of VEGFR-2. Close to group OGD.
(7) the patch clamp experiment found that the time window for the electrophysiological characteristics of CA1 neurons after OGD treatment was 10min. The membrane potential of most of the neurons was restored to normal after the recovery of normal aCSF, so it was determined as the time window of this experiment.
(8) the excitability of neurons in each group was analyzed during the 10min OGD treatment. The membrane potential of the neurons in group OGD was 14.7 + 1.23mV (n=5) at the end of OGD, while the neurons in the VEGF treatment group depolarized 6.1 + 1.57mV (n=8, OGD vs. OGD+VEGF). 1.92mV (n=6). In the same statistical results of spontaneous discharge frequency, the spontaneous discharge frequency of neurons in group OGD+VEGF increased by 5.1 + 0.6 times than that of base, far lower than that of OGD group (9.2 + 0.56 times, P0.01, n=5), and OGD+VEGF+SU5416 discharge frequency increased by 8.1 + 0.47 times, higher than that of VEGF treatment group with significant difference (P0.0) 1, n=5), but there was no statistical difference compared with group OGD (P0.05).
(9) the results of sEPSCs event of hippocampal CA1 neurons in each group were recorded and analyzed. The amplitude and frequency of sEPSCs in each group increased during 10min OGD treatment, while in VEGF treatment group, the increase of these two indexes was relieved, but still could not be restored to normal level. After SU5416, the VEGF receptor inhibitor SU5416 was added. The relieving effect of VEGF was relieved, indicating that VEGF plays a role through VEGFR-2 receptors.
conclusion
(1) exogenous VEGF can significantly improve the spatial cognitive impairment of VD rats. The mechanism includes improving hippocampal pyramidal neuron edema and nuclear pyknosis in ischemic rats and maintaining normal function of hippocampal neurons.
(2) giving exogenous VEGF through nasal feeding is a feasible method, which can effectively increase the content of VEGF in the hippocampus of ischemic rats and protect against ischemia.
(3) VEGF can improve the decrease of synaptic plasticity in the hippocampus of VD rats and enhance the long term enhanced LTP of CA3-CA1. This phenomenon may be related to the improvement of the cross coupling mode between theta rhythm and gamma rhythm with VEGF.
(4) in the isolated ischemic hypoxic brain model, exogenous VEGF can play a protective role in hippocampal neurons through its VEGFR-2 receptor, and its mechanism is to antagonize the apoptosis of neurons induced by hypoxia and thus promote its survival.
(5) VEGF can antagonize the increase of excitatory activity of hippocampal CA1 neurons caused by hypoxia and maintain the normal electrophysiological characteristics of neurons, which may be the electrophysiological mechanism of VEGF's protective effect in the early stage of ischemia.
(6) VEGF can reduce the abnormal increase of synaptic transmission in hippocampal neurons caused by hypoxia and maintain the normal synaptic transmission function of neurons, which may be one of the mechanisms that VEGF can improve synaptic plasticity and further improve cognitive function.

【学位授予单位】：南开大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R-332;R318.04

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