本文選題：VEGF + 腦缺血��； 參考：《南開大學》2014年博士論文

【摘要】：目的 (1)通過在體(in vivo)實驗觀察外源性血管內皮生長因子VEGF對慢性低灌注型腦缺血模型大鼠認知功能的保護作用及電生理學和組織學機制。 (2)利用離體(in vitro)海馬腦片缺血缺氧模型探討VEGF對海馬神經元保護作用的細胞學及電生理學機制。 方法 (1)In vivo:將30只雄性Wistar大鼠隨機分成3組,每組10只：假手術組(Sham)、缺血組(Ischemia)、VEGF處理組(Ischemia+VEGF)。采用雙側頸總動脈結扎(2-VO)2周建立慢性低灌注型腦缺血大鼠模型。采用鼻飼法給予外源性VEGF,然后利用Morris水迷宮實驗(MWM)比較各組大鼠的空間認知功能；行為學實驗后,進行電生理實驗,內容包括海馬CA3-CA1區(qū)的長時程增強LTP以及局部場電位(LFP),并應用神經信息流算法分析各組大鼠CA3與CA1區(qū)神經網絡的振蕩模態(tài)的變化。實驗結束后取腦,使用ELISA的方法測定各組海馬內VEGF的含量并通過組織學方法(HE染色)觀察海馬CA1區(qū)錐體細胞的形態(tài)結構。 (2) In vitro:首先利用糖氧剝奪(OGD)的方法建立離體缺血缺氧海馬腦片模型,實驗分為4組,即正常組(Control)、糖氧剝奪組(OGD)、VEGF處理組(OGD+VEGF)、VEGF及其受體抑制劑處理組(OGD+VEGF+SU5416)。使用碘化丙啶(PI)染色的方法并利用共聚焦顯微鏡觀察各組腦片海馬CA1區(qū)神經元在OGD處理20min后的存活情況。然后分別記錄各組腦片在OGD處理10min以及恢復正常腦脊液后的電生理信號的變化,包括神經元膜電位、自發(fā)放電頻率以及sEPSCs的變化。 結果 (1)水迷宮(MWM)實驗結果發(fā)現(xiàn),缺血組大鼠的逃避潛伏期從訓練的第3天開始比假手術組顯著延長(Ischemia:13.5±1.9s vs. Sham:8.1±1.2s,P0.05),而VEGF處理后的腦缺血大鼠的逃避潛伏期也從第3天開始比缺血組大鼠明顯地縮短了(Ischemia+VEGF:8.5±1.1s, P0.05vs. Ischemia);但是,三組大鼠的平均游泳速度無顯著性差別(P0.05)。測試日的實驗發(fā)現(xiàn),缺血組大鼠在目標象限停留的百分比比假手術組的大鼠明顯減少(Ischemia:34.2±1.6%vs. Sham:42.9±1.5%, P0.05), VEGF處理后缺血大鼠的目標象限百分比幾乎恢復到假手術組的水平(Ischemia+VEGF:40.1±2.6%). (2)記錄并分析各組大鼠海馬CA3-CA1通路的長時程增強LTP的結果發(fā)現(xiàn),缺血組的場興奮性突觸后電位(fEPSPs)斜率百分比比假手術組顯著降低(Ischemia:117.8±6.23%vs. Sham:136.2±5.96%,P0.01),而VEGF組則比缺血組明顯要高(130.6±5.36%, P0.01vs. Ischemia)。 (3)記錄并計算各組大鼠海馬CA3與CA1區(qū)局部場電位(LFP)的結果發(fā)現(xiàn),腦缺血能夠引起海馬CA3與CA1腦區(qū)間的相位同步性降低,CA3區(qū)theta相位調控CA1低頻gamma(LG,30-60Hz)幅值的程度顯著降低,而加入VEGF后,CA3、CA1腦區(qū)間的耦合強度均有上升的趨勢。 (4)HE染色結果發(fā)現(xiàn),假手術組大鼠海馬CA1區(qū)錐體神經元形態(tài)正常。而缺血組的CA1神經元排列松散,分布不均,細胞出現(xiàn)水腫以及核固縮的現(xiàn)象。而VEGF處理組大鼠的CA1神經元可見部分形態(tài)正常,部分形態(tài)異常,分布及排列趨近于正常組。 (5)分析各組大鼠海馬內VEGF的含量發(fā)現(xiàn),缺血組大鼠海馬內的VEGF含量明顯比假手術組要低(Sham:745.33±42.34pg/g vs. Ischemia:420.09±40.82pg/g,P0.01),而VEGF處理組的海馬內的VEGF含量有顯著提高(592.83±38.76pg/g, P0.05vs. Ischemia),但仍然低于假手術組(P0.05)。 (6)在離體實驗中檢測各組腦片海馬CA1神經元存活情況的結果發(fā)現(xiàn),OGD處理20min后,神經元的死亡率顯著上升(Control vs. OGD, P0.05),而VEGF孵育組存活細胞的數(shù)量增多,接近正常組。然而與VEGFR-2的抑制劑SU5416共孵育之后,神經元的死亡率升高,接近OGD組。 (7)膜片鉗實驗發(fā)現(xiàn),OGD處理后CA1神經元的電生理特性能夠發(fā)生可逆性變化的時間窗口為10min,在恢復正常aCSF處理后絕大部分神經元的膜電位能夠恢復正常,因此將其確定為本實驗的時間窗口。 (8)在10min OGD處理期間分析各組神經元的興奮性發(fā)現(xiàn),OGD組神經元的膜電位在OGD結束時去極化了14.7±1.23mV(n=5),而VEGF處理組神經元則去極化了6.1±1.57mV (n=8, OGD vs. OGD+VEGF, P0.01)。同時共孵育VEGFR-2抑制劑SU5416組(OGD+VEGF+SU5416)則去極化了12.1±1.92mV(n=6)。同樣的在自發(fā)放電頻率的統(tǒng)計結果中發(fā)現(xiàn),OGD+VEGF組的神經元的在OGD期間的自發(fā)放電頻率比base時增加了5.1±0.6倍,遠遠低于OGD組(9.2±0.56倍,P0.01,n=5),而OGD+VEGF+SU5416放電頻率增加了8.1±0.47倍,高于VEGF處理組且具有顯著差異(P0.01,n=5),但是與OGD組相比卻不具有統(tǒng)計學差異(P0.05)。 (9)在記錄并分析了各組大鼠海馬CA1神經元的sEPSCs事件結果發(fā)現(xiàn),在10min OGD處理期間,各組sEPSCs的幅值和頻率均時間依賴性的增加,而在VEGF處理組,這兩個指標增加的程度被緩解,但仍不能恢復到正常水平。而加入VEGF受體抑制劑SU5416之后,VEGF的緩解效果被解除,說明VEGF的作用是通過VEGFR-2受體發(fā)揮作用的。 結論 (1)外源性給予VEGF可顯著改善VD大鼠的空間認知障礙,其機制包括改善缺血大鼠海馬錐體神經元水腫和核固縮情況,維持海馬神經元正常功能。 (2)通過鼻飼法給予外源性VEGF是一種可行的方法,可以使缺血大鼠海馬內VEGF的含量有效升高,對缺血產生了保護作用。 (3) VEGF能夠改善VD大鼠海馬內突觸可塑性的降低,增強CA3-CA1的長時程增強LTP,這一現(xiàn)象可能與VEGF改善了它們之間theta節(jié)律與gamma節(jié)律的交叉耦合模式有關。 (4)在離體缺血缺氧腦片模型中,外源性VEGF能夠通過其VEGFR-2受體來發(fā)揮對海馬神經元的保護作用,其機制是對抗缺氧導致的神經元凋亡,從而促進其存活。 (5)VEGF能夠對抗由缺氧引起的海馬CA1神經元興奮性的升高,維持了神經元的正常電生理特性,這可能是VEGF在缺血早期發(fā)揮保護作用的電生理學機制。 (6)VEGF能夠降低由缺氧引起的海馬神經元突觸傳遞的異常升高,維持了神經元正常的突觸傳遞功能,這可能是VEGF能夠改善突觸可塑性并進一步改善認知功能的機制之一。
[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.

【學位授予單位】：南開大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R-332;R318.04

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