异亚丙基莽草酸对慢性低灌注大鼠突触可塑性的影响

【作者】 王晶；

【导师】 孙建宁；

【作者基本信息】 北京中医药大学 ， 中药药理学， 2008， 博士

【摘要】 血管性痴呆(Vascular Dementia, VD)是由一系列脑血管因素导致脑组织损害而引起的获得性智能损害综合征,是继阿尔茨海默氏症(Alzheimer’s Disease, AD)之后第二位最常见的痴呆类型,临床可表现出包括记忆障碍在内的一系列症状。慢性脑灌注不足是血管性痴呆,特别是低灌注性痴呆的主要病理基础。脑灌注减少影响了糖和氧的正常传递,导致大脑突触通路的能量代谢衰竭,最终引发包括记忆衰退在内的大脑整体功能的下降。记忆是中枢神经元的整合,其细胞和分子基础定位于突触。记忆形成的基础是突触效应的增强。在一定条件下,机体可调整突触功能,改变其形态以及数目,这被称之为突触可塑性。随着我国老龄人口比例加大,预防及治疗慢性低灌注引发的VD具有重大社会意义。异亚丙基莽草酸(3,4-oxo-isopropylidene shikimic acid,ISA)是从中药八角属植物八角茴香中提取的有效成分莽草酸(shikimic acid,SA)的合成衍生物,本组前期研究结果显示,ISA能够明显抑制多种诱导剂所致的血小板聚集以及实验性血栓的形成,对培养的海马以及神经细胞具有直接的神经保护作用,可对抗脑缺血—再灌注诱导的急性炎症反应。本研究进一步从行为学、神经细胞突触结构、胆碱能神经递质合成水平、突触前标志物以及核内转录因子表达水平等环节探讨了ISA对VD模型大鼠海马突触可塑性的干预作用及其可能的作用机理。实验分为三部分。第一部分建立光化学损伤以及慢性低灌注性VD大鼠模型,探讨慢性低灌注大鼠海马CA1区神经元以及突触结构的变化特点目的:探讨建立光化学损伤以及慢性低灌注性VD大鼠模型的条件以及两种模型的特点,并探讨慢性低灌注大鼠海马CA1区神经元以及突触结构的变化特征。方法:采用舌下静脉注射4%玫瑰红与绿光照射顶叶皮层相结合的方法诱导大鼠局灶性脑缺血,建立光化学损伤性VD大鼠模型,术后30天通过Morris水迷宫对大鼠进行空间记忆测试;采用永久性结扎加切断大鼠双侧颈总动脉的方法诱导前脑慢性低灌注性VD大鼠模型,60天后通过Morris水迷宫对其进行空间记忆测试。将Morris水迷宫测试结束后的大鼠进行经心灌注固定,制成大脑石蜡切片后,进行HE染色以及Nissl染色,光镜下观察顶叶皮层神经元或海马CA1区锥体神经元的形态学改变。并采用透射电子显微镜观察慢性低灌注性VD模型大鼠海马CA1区突触结构的改变。结果:①采用舌下静脉注射4%玫瑰红复合5mm直径绿光光纤照射顶叶皮层诱导的VD模型大鼠出现明显的学习记忆障碍,表现为在第2—4天定位航行测试中的逃避潜伏期明显长于假手术组(p<0.05;p<0.01);在空间探索测试中,大鼠游泳初始角度显著大于假手术组(p<0.05)。②光化学损伤造成大鼠顶叶皮质出现明显可见的圆形梗死灶。梗死灶内神经细胞消失,灶内无明显出血。外围的过渡区内从相对正常状态过渡到梗死灶,病变逐渐加重。③双侧颈总动脉永久性结扎加切断后8周,模型大鼠出现明显的学习记忆障碍,表现为在第3天定位航行测试中的路径长度显著长于假手术大鼠(p<0.05);在第2天以及第3天定位航行测试中的游泳错误初始角度显著大于假手术大鼠(p<0.05;p<0.01)。在空间探索测试中,模型大鼠的游泳错误初始角度显著大于假手术大鼠(p<0.05),但二者的原平台象限游泳距离百分比无显著差异(p>0.05)。④慢性低灌注大鼠海马CA1区的锥体细胞排列紊乱,部分脱失,胶质细胞增生显著。⑤进一步采用双侧颈总动脉结扎模型大鼠进行的透射电镜研究显示,8周后该模型大鼠海马CA1区突触间隙模糊,部分突触间隙增宽,突触前后膜肿胀、空化,囊泡数量减少。⑥光化学法VD模型大鼠死亡率较高,为25%;而双侧颈总动脉永久性结扎造成的VD模型大鼠死亡率较低,为10%。结论:①成功建立了光化学损伤和慢性低灌注两种VD大鼠模型。光化学法与双侧颈总动脉永久性结扎造成的VD模型大鼠在学习记忆以及神经元形态学改变方面均较为显著。②慢性低灌注大鼠模型由于死亡率较低,且手术操作相对简便而更适用于进一步的药效学研究。第二部分异亚丙基莽草酸对慢性低灌注大鼠的药效作用研究目的:探讨ISA对双侧颈总动脉结扎造成的慢性低灌注大鼠空间学习记忆能力、海马CA1区神经元形态学以及突触超微结构的影响。方法:采用永久性结扎大鼠双侧颈总动脉的方法诱导前脑慢性低灌注大鼠模型。实验动物分为假手术组、模型组、阳性对照药喜得镇组、ISA100mg/kg组、ISA50mg/kg组以及ISA25mg/kg组。造模后连续灌胃给予大鼠相应药物60天,通过Morris水迷宫对其进行4d定位航行及1d空间探索测试。水迷宫测试结束后,将部分大鼠进行经心灌注固定,制成大脑石蜡切片,进行HE染色。行为学测试结束后,另取部分大鼠经心灌注固定,取海马CA1区,制成超薄切片。采用透射电镜观察大鼠突触前及突触后结构参数的改变,包括突触界面曲率、活性区长度(length of active zone, L)、突触后致密物质(postsynaptic density,PSD)厚度、突触数密度(numerical density per unit volume of synapses, Nv)、面密度(surface density per unit volume, Sv)、入坞囊泡(docked vesicle, DV)的数量以及单位长度活性区中入坞囊泡的数量(DV/L)。结果:①双侧颈总动脉结扎诱导的慢性低灌注模型大鼠出现明显的学习记忆障碍。与假手术组相比,模型大鼠在第2、3天定位航行实验中的游泳初始角度增加显著(p<0.05;p<0.01);在第3天定位航行实验中,游泳路径长度延长显著(p<0.05)。在空间探索实验中游泳初始角度明显大于假手术组(p<0.05)。其游泳策略在中早期一般以边缘式为主,后期大多表现为趋向式。造模后连续给与ISA100mg/kg可明显缩短第2天定位航行的游泳路径长度(p<0.05),而对定位航行中游泳初始角度以及其他时间点的游泳路径长度以及空间探索实验中游泳初始角度无显著影响(p>0.05)。ISA50mg/kg可明显减小空间探索实验中的游泳初始角度(p<0.05),而对定位航行中游泳路径长度以及初始角度无显著影响(p>0.05)。ISA25mg/kg可明显减小在第3天定位航行实验及空间探索实验中的游泳初始角度(p<0.01; p<0.05),而对定位航行中的游泳路径长度无显著影响(p>0.05)。ISA各组的游泳策略在中早期一般以趋向式为主,后期大多表现为直线式。喜得镇可明显减小在第3天定位航行实验中的游泳初始角度(p<0.05),而对定位航行中游泳路径长度、其他时间点的初始角度以及空间探索实验中游泳初始角度无显著影响(p>0.05)。其游泳策略在中早期一般以趋向式为主,后期大多表现为直线式。②慢性低灌注大鼠海马CA1区的锥体细胞排列紊乱,部分脱失,神经细胞数明显较少,部分神经细胞水肿,细胞核固缩,有的形成碎片,胶质细胞显著增加。ISA及喜得镇组大鼠的神经元损伤明显减轻,尤以ISA100mg/kg剂量组减轻更明显,表现为海马各区神经细胞胞浆丰富、排列紧密、结构完整,核仁、核膜清晰可见,胞浆染色正常。③慢性低灌注模型大鼠海马CA1区神经毡内突触间隙模糊,突触前后膜肿胀、空化。模型大鼠海马CA1区Nv、Sv、DV、L、DV/L以及突触界面曲率降低非常显著(p<0.01),但PSD厚度未见显著变化(p>0.05)。连续灌胃给与ISA及喜得镇65d后上述损伤明显减轻。与模型组相比,各剂量ISA以及喜得镇可使Nv、Sv、DV、L以及DV/L显著增加(p<0.05;p<0.01)。ISA100mg/kg以及ISA50mg/kg还可明显增加突触界面曲率(p<0.05;p<0.01)。ISA各剂量以及喜得镇组间无显著差异(p>0.05)。结论:ISA连续灌胃给药8周后各剂量组均可提高慢性低灌注大鼠对空间记忆的获取以及保持能力;改善慢性低灌注大鼠海马CA1区神经细胞形态;显著改善慢性低灌注大鼠海马CA1区突触超微结构,促进突触的结构可塑性。第三部分异亚丙基莽草酸增强慢性低灌注大鼠突触可塑性的可能作用机制目的:探讨慢性低灌注大鼠海马CA1区突触可塑性相关蛋白突触素(synaptophysin, SYP)、胆碱乙酰转移酶(choline acetyltransferase, ChAT)、磷酸化cAMP反应元件结合蛋白(phosphorylated- cAMP responsive element binding protein, p-CREB)、脑源性神经营养因子(brain derived neurotrophic factor, BDNF)以及酪氨酸激酶B(tyrosine kinase B, TrkB)的蛋白表达以及基因转录水平的改变,并观察ISA对其影响。方法:实验动物分为假手术组、模型组、阳性对照药喜得镇组、ISA100mg/kg组、ISA50mg/kg组以及ISA25mg/kg组。将Morris水迷宫测试结束后的慢性低灌注大鼠经心灌注固定,制备大脑石蜡切片后,进行免疫组织化学染色,测定海马CA1区SYP、ChAT、p-CREB、BDNF以及TrkB的定位以及相对定量表达。提取大鼠海马CA1区组织蛋白,利用Western Blot对SYP、p-CREB、BDNF以及TrkB的蛋白表达水平进行半定量测定。抽提大鼠海马CA1区组织总RNA,通过荧光定量实时PCR技术,对BDNF、ChAT、TrkB、SYP以及CREB的mRNA表达水平进行测定。结果:①大鼠海马CA1区SYP免疫反应阳性产物主要分布于神经元周围,胞浆内颗粒状免疫反应产物少见,白质、血管及胶质细胞不着色。模型大鼠海马CA1区SYP蛋白表达水平以及SYP mRNA表达水平非常显著低于假手术组(p<0.01)。与模型组相比,ISA100mg/kg以及50mg/kg组大鼠的SYP蛋白表达水平显著增加(p<0.05);ISA100mg/kg连续灌胃给药65d可显著增加SYP mRNA表达水平(p<0.05);除ISA100mg/kg外,ISA其余各剂量以及喜得镇对SYP mRNA表达水平无显著影响(p>0.05)。②大鼠海马CA1区ChAT免疫反应阳性产物主要分布于锥体细胞的胞浆及轴突。模型大鼠的ChAT蛋白表达水平以及ChAT mRNA表达水平非常显著低于假手术组(p<0.01)。与模型组相比,除ISA25mg/kg组外,各给药组的ChAT蛋白表达水平均升高显著(p<0.05;p<0.01);ISA100mg/kg组的ChAT mRNA表达水平增加非常显著(p<0.01),而ISA其余各剂量以及喜得镇对ChAT mRNA表达水平无显著影响(p>0.05)。③p-CREB免疫反应阳性颗粒主要分布于锥体细胞的胞核中。模型组大鼠海马CA1区p-CREB蛋白表达水平以及CREB mRNA表达水平显著高于假手术组(p<0.05)。与模型组相比,ISA各剂量以及喜得镇对p-CREB蛋白表达水平无显著影响(p>0.05);ISA100mg/kg连续灌胃给药65d后,可显著增加大鼠海马CA1区CREB mRNA表达水平(p<0.05);ISA其余各剂量以及喜得镇对CREB mRNA表达水平无显著影响(p>0.05)。④B DNF免疫反应阳性产物主要分布于神经元的胞浆、胞核以及长的轴突中。模型大鼠海马CA1区BDNF蛋白表达水平以及BDNF mRNA表达水平均显著高于假手术组(p<0.05)。与模型组相比,100mg/kg的ISA可显著增加BDNF蛋白表达水平以及BDNF mRNA表达水平(p<0.05);喜得镇、ISA50mg/kg与25mg/kg组的BDNF蛋白表达水平以及BDNF mRNA表达水平无显著改变(p>0.05)。⑤TrkB的免疫反应阳性产物主要分布于神经元的胞浆及轴突内。模型大鼠海马CA1区TrkB mRNA表达水平显著高于假手术组(p<0.05),但是蛋白表达水平未发生显著改变(p>0.05)。各ISA组与喜得镇组的TrkB蛋白以及mRNA表达水平无显著变化(p>0.05)。结论:①长期慢性低灌注导致大鼠海马CA1区ChAT以及SYP表达水平降低。胆碱能神经功能降低以及突触丢失造成了海马突触结构可塑性的降低,而突触结构可塑性的降低可能是导致慢性低灌注大鼠学习记忆下降的结构基础。②BDNF参与了慢性低灌注损伤后的补偿机制。ISA可通过上调内源性保护因子BDNF的表达,进而提高ChAT的活性以及SYP的含量,提高突触传递效能。③ISA可通过增强胆碱能神经功能以及保护突触结构而促进突触可塑性,进而提高慢性低灌注性痴呆动物的学习记忆能力。全文结论:1.本研究成功建立了两种VD大鼠模型,即光化学损伤以及慢性低灌注性VD大鼠模型。两种模型大鼠均表现出明显的空间学习记忆障碍,同时顶叶皮质或海马CA1区神经元严重脱失。并且慢性低灌注大鼠海马CA1区突触超微结构出现明显损伤。2.相对于光化学损伤诱导的VD模型而言,慢性低灌注大鼠模型更稳定,动物成活率高,实验成本低,与人类VD的病理生理过程更加接近。本研究采用慢性低灌注大鼠模型进行ISA的药效学以及作用机制研究。3.本研究提示ISA对抗慢性低灌注性VD具有良好的药效作用,表现为连续灌胃给药8周后可明显改善慢性低灌注大鼠的学习记忆功能;减少海马CA1区锥体神经元的脱失,保护神经元形态的完整;促进海马突触的结构可塑性。并以ISA100mg/kg剂量组的药效作用为更好。4.本研究还提示突触丢失以及胆碱能神经功能障碍可能是导致慢性低灌注性痴呆动物学习记忆下降的主要原因。5.ISA的作用可能是通过提高BDNF在CA1区的蛋白以及mRNA表达水平,从而进一步加强慢性低灌注本身引发的内源性神经保护机制。即ISA通过加强BDNF-TrkB对其下游靶蛋白SYP以及ChAT的调节,提高突触传递效能。(1)连续给与ISA 8周后,大鼠海马CA1区单位面积中ChAT免疫反应阳性细胞数、免疫反应阳性产物面积百分率以及ChA mRNA表达水平均显著高于模型组。说明给与外源性药物ISA后,胆碱能水平的增加为突触可塑性的增强提供了物质基础。(2)ISA连续用药8周后,大鼠海马CA1区SYP蛋白以及mRNA表达水平均显著提高。这提示ISA可能是通过提高SYP蛋白以及mRNA表达水平,进而提高突触传递效能而改善实验动物的学习记忆能力。6.综上所述,ISA可通过增强胆碱能神经功能以及保护突触结构而促进突触可塑性,进而提高慢性低灌注性痴呆动物的学习记忆能力。本研究的创新点:1.首先研究ISA对慢性低灌注大鼠模型的药效学作用;2.首先采用慢性低灌注大鼠模型研究海马p-CREB表达水平,以及海马CA1区突触中入坞囊泡数量的改变。更多还原

【Abstract】 Vascular dementia (VD), which is characterized by progressive intellectual decline induced by ischemia hypoxia or hemorrhage brain lesion, represents the second most common dementia accounting for about a quarter to a half of all cases of dementia in developed countries. Chronic cerebral hypoperfusion (CCH) is one of common pathophysiologic basis of VD and Alzheimer’s disease. CCH impacts the delivery of glucose and oxygen, leading to the handicap of energy metabolism in the brain synaptic pathway, followed by the decline of the whole brain function including memory decline. The structural basis of memory is synapse. The enhancement of synaptic function contributes to the formation of memory. The rats with memory decline showed the changes in synaptic structural parameters, such as the decrease of numerical density of synapses (Nv) and docked vesicles (Dv).With increasing number of the elderly in world populations, dementia, characterized by progressive descending of memory and cognition, has given rise to enormous socioeconomic burden. In the past few decades, a great deal of investigation has been conducted. However, the efficiency of currently available strategies for the prevention of VD remains poor. 3,4-oxo-isopropylidene-shikimic acid(ISA)was extracted from Illicium verum Hook. Fil, a traditional Chinese herb. Our preliminary work showed that ISA could suppress variously experimental thrombosis and platelet aggregation significantly, improve the morphology and decrease the apoptosis rate of cultured cortical and hippocampal neurons after hypoxia-reoxygenation injury, exert neuroprotective effects on rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion by an intraluminal thread, as evidenced by decreasing the neurological severity score, infarct volume, brain water content and brain edema. It was indicated that the anti-free radical effects of ISA might account for the protection against cerebral ischemia. Thus, ISA appeared to be a promising drug to ameliorate cognitive deficits induced by cerebral ischemia. But to our knowledge, the effects and the mechanism of ISA on CCH in rats remain to be elucidated. In the present study, we made use of the permanent occlusion of bilateral common carotid arteries (two-vessel occlusion, 2VO) model of CCH to evaluate the effects of ISA on synaptic plasticity in dementia rats. The possible mechanism underlying its effect was also explored. To address this issue, a variety of multidisciplinary approaches including Morris water maze, transmission electron microscopy, immunohistochemical staining, Western blotting, fluorescent quantitative real-time polymerase chain reaction were used.There are three stories in this thesis.PartⅠCreation of two kinds of VD rat models and the investigation on the pathological changes in neurons and synaptic ultrastructure in hippocampal CA1 subfield Objective: To explore the characteristics of the photochemical model and 2VO model, and to investigate the pathological changes of neurons and synaptic ultrastructure in hippocampal CA1 subfield.Methods: A focal lesion in the left parietal cortex was induced photochemically by using Rose Bengal as a photosensitive dye and cold light beam, then the rats were treated with ISA orally once a day for 30 days. The cognitive effects of ISA were assessed in rats using the Morris water maze test for spatial learning and memory. HE staining and Nissl staining were used to study the neuronal alteration in left parietal cortex. For the reproduction of CCH as it occurs in VD, permanent occlusion of the bilateral common carotid arteries of rats (2-vessel occlusion, 2VO) was introduced. 60 days after 2VO surgery, the rats underwent spatial cognition test in Morris water maze. Subsequently the brain sections were subjected to HE staining and transmission electron microscopy. Results:①Photochemical lesion induced spatial cognition deficits, which was proven by prolonged escape latency on the 2-4 training days(p<0.05, p<0.01)and the larger initial angle in probe test, compared to the sham-operated group(p<0.05).②Photochemical lesion could induce round infarction in the left parietal cortex of rats. The neurons in the infarction almost disappeared. No obvious hemorrhage and edema existed. The pathological changes were gradiently severe from relatively normal brain region to the infarction.③8 weeks after 2VO surgery, the model rats showed significant cognition deficits, as evidenced by longer swimming path length on the third training day(p<0.05), the larger initial angles on the second and third training day(p<0.05, p<0.01), compared to the sham-operated group rats. In the probe test, the model rats exhibited larger initial angles(p<0.05), compared to the sham-operated group rats. No difference in the quadrant percentage was observed between the two groups(p>0.05).④Macroscopic evaluation of rat brains demonstrated that 2VO induced pyramidal neuron damage in hippocampus CA1 subfield 65 days after operation. The rats showed severe neuronal loss and the gliocyte proliferation in the hippocampal CA1 region.⑤The synaptic ultrastructures of neuropil field within CA1 stratum radiatum of the sham-operated group rats were intact. The synapses of GrayⅠtype showed clear synaptic clefts, typical asymmetric interfaces, intact structures of the pre- and postsynaptic membranes, round clear vesicles and thick postsynaptic densities (PSD). The synaptic ultrastructures of 2VO group presented the blurry synaptic clefts, swollen, vacuolated pre- and postsynaptic membranes and less synaptic vesicles in the synaptic terminals.⑥The mortality rate in the model rats with photochemical lesion was 25%, while 10% in the model rats with CCH. Conclusion:①Two VD models induced by the photochemical lesion and 2VO respectively in rats were created. The changes in the cognition and neuronal pathology in the 2VO rats were observed.②2VO model is more suitable for the investigating in the further pharmacodynamics research due to its lower mortality rate and easier surgical process.PartⅡPharmacodynamics research of 3, 4-oxo-isopropylidene-shikimic acid in rats with vascular dementia induced by chronic cerebral hypoperfusionObjective: To probe the effects of ISA on spatial cognition, neuronal pathology and synaptic ultrastructure within hippocampal CA1 subfield in rats with CCH.Methods: We used 2VO to induce CCH in rats. Male Sprague Dawley rats were randomly divided into six groups including vehicle-treated sham-operated group, vehicle-treated operated group, ISA 100mg/kg-treated operated group, ISA 50mg/kg-treated operated group, ISA 25mg/kg-treated operated group and Hydergine 0.6mg/kg-treated operated group. Vehicle group rats’bilateral carotid arteries were occluded. The sham-operated rats received the same operation with the exception of ligation. Other rats were intragastricly administered relevant drugs once per day. Eight weeks after 2VO, Morris water maze was used to measure spatial learning and memory. Each rat received two trials everyday for 4 consecutive days. Length of swimming path to escape onto the hidden platform and initial angle were recorded. On the fifth day, each rat was subjected to a 20-second probe trial in which the platform was removed and the initial angle and typical swim-tracking path were recorded. Subsequently, the morphological changes in neurons were evaluated with HE staining in hippocampal CA1 subfield. Transmission electron microscopy was used to evaluate the following synaptic structural parameters in hippocampal CA1 subfield. Nv, surface density (Sv), DV and DV per unit length of active zone (L) were measured.Results:①The sham-operated rats showed smaller initial angles on the second (p<0.05) and third (p< 0.01) training days, and the shorter path length on the third training day (p<0.05), compared to 2VO group. In the probe test, the initial angle of sham-operated group was smaller than 2VO rats (p<0.05). The swimming strategy was tendency at the early stage and straight at the late stage in sham-operated rats, and marginal at the early stage and random pattern at the late stage in 2VO rats. Administration of ISA at the dose of 100mg/kg could significantly shorten the path length on the second training day (p<0.05), but didn’t change the path length on other training days, and initial angle in navigation trails and probe trail, compared to 2VO group (all p>0.05). Administration of ISA at the dose of 50mg/kg could significantly reduce initial angle in probe trail (p<0.05), but didn’t change the path length and initial angle in navigation trails, compared to 2VO group (all p>0.05). Administration of ISA at the dose of 25mg/kg could significantly reduce initial angle on the third training day in navigation trail(p<0.01)and probe trail (p<0.05), but couldn’t change the path length in navigation trail, compared to 2VO group (all p>0.05). When treated with dihydroergotoxine at the dose of 0.6mg/kg, the rats showed smaller initial angle on the third training day, compared to 2VO group (p<0.05). No difference in swimming path length on other training days, and initial angle in probe trail were observed, compared to 2VO group (all p>0.05). The main swimming strategies in all drug-treated groups were tendency at the early stage and straight at the late stage.②Microscopic evaluation of rat brains demonstrated that 2VO induced pyramidal neuron damage in hippocampus CA1 subfield 65 days after operation. 2VO rats showed severe neuronal loss and the gliocyte proliferation in the hippocampal CA1 region. The structures of pyramidal neurons in all drug-treated groups were relatively intact.③The synaptic ultrastructures of neuropil field within CA1 stratum radiatum of the sham-operated group rats were intact. The synapses of GrayⅠtype showed clear synaptic clefts, typically asymmetric interfaces, intact structures of the pre- and postsynaptic membranes, round clear vesicles and thick PSD. The synaptic ultrastructures of 2VO rats presented the blurry synaptic clefts, swollen and vacuolated pre- and postsynaptic membranes. The synaptic ultrastructures were relatively intact in all drug-treated groups. There were more synaptic vesicles in the presynaptic terminals in ISA and dihydroergotoxine-treated groups, compared to 2VO group. Compared to the sham-operated group, the 2VO rats showed significantly reduced Nv, Sv, DV, DV/L and synaptic curvature (all p<0.01), but the thickness of PSD didin’t change markedly (p>0.05). All the ISA-treated and dihydroergotoxine-treated rats showed markedly increased Nv, Sv, DV and DV/L (p<0.05, p<0.01), compared to 2VO group. Administration of ISA at the dose of 50mg/kg (p<0.01) and 100mg/kg (p<0.05) could significantly increase the synaptic curvature.Conclusions: Administration of ISA could improve the decline of learning and memory, and protect the neuronal morphology of the brain in rats with CCH. It improved the structural plasticity of CA1 synapses.PartⅢProbe into the potential molecular mechanism of 3, 4-oxo-isopropylidene-shikimic acid underlying its synaptic plasticity effects on vacular dementia induced by chronic cerebral hypoperfusionObjective: To probe effects of ISA on the expressions of brain derived neurotrophic factor (BDNF), its receptor tyrosine kinase B(TrkB), Choline acetyltransferase (ChAT), synaptophysin (SYP), phosphorylated cAMP response element binding protein (p-CREB) in the mRNA and protein levels within hippocampal CA1 region of CCH rats. Methods: Male Sprague Dawley rats were randomly divided into six groups including vehicle-treated sham-operated group, vehicle-treated operated group, ISA 100mg/kg-treated operated group, ISA 50mg/kg-treated operated group, ISA 25mg/kg-treated operated group and Hydergine 0.6mg/kg-treated operated group. After the behavioral test, rats were perfused intracardially. The paraffin sections were used for immunohistochemistry staining to probe the expression of p-CREB, BDNF, TrkB, ChAT and SYP in protein level within hippocampal CA1 subfield. The proteins were extracted and used for evaluation of the semi-quantitative expression of p-CREB, BDNF, TrkB, ChAT and SYP in protein level. Fluorescent quantitative real-time polymerase chain reaction was employed to measure the mRNA expression level of BDNF, TrkB, ChAT, SYP and CREB.Results:①At week 9 after 2VO surgery, the immunoreactive products of p-CREB, some brown granules, distributed in the neucli of pyramidal neurons within hippocampal CA1 region. The expressions of p-CREB in protein level and CREB in mRNA level were higher in 2VO group than in sham-operated group (both p <0.05). Administration of ISA at the dose of 100mg/kg increased the CREB expression in mRNA level, compared to the 2VO group (p<0.05). Dihydroergotoxine and ISA at three doses didn’t significantly change the expressions of p-CREB in protein level and CREB in mRNA level (all p>0.05).②The immunoreactive products of BDNF distributed in the cytoplasm, neucli and axons of pyramidal neurons within hippocampal CA1 subfield. The BDNF expressions in protein and mRNA levels were higher in 2VO group than in sham-operated group (p<0.05). Administration of ISA at the dose of 100mg/kg could markedly increase BDNF expression in protein and mRNA levels, compared to the 2VO group (both p<0.05). Dihydroergotoxine and ISA at the doses of 50mg/kg and 25mg/kg didn’t significantly change BDNF expression in protein and mRNA levels (all p>0.05).③The immunoreactive products of TrkB distributed in the cytoplasm, neucli and axons of pyramidal neurons within hippocampal CA1 subfield. The TrkB mRNA expression was higher in 2VO group than in sham-operated group (p <0.05). But the protein expression level didn’t change significantly in 2VO rats. Administration of ISA at three dosages and Dihydroergotoxine didn’t significantly change TrkB expression in protein levels and mRNA levels (all p>0.05)④The immunoreactive products of ChAT distributed in the cytoplasm and axons of pyramidal neurons within hippocampal CA1 subfield. The ChAT expression in protein and mRNA levels were lower in 2VO group than in sham-operated group (p<0.01). Administration of Dihydroergotoxine and ISA at the dose of 50 mg/kg and 100mg/kg increased the ChAT expressions in protein level (all p<0.01). ISA significantly increased ChAT mRNA expression at the dose of 100mg/kg, compared to 2VO group (p<0.05, p<0.01). Dihydroergotoxine and ISA at the dose of 50 mg/kg and 25mg/kg didn’t change ChAT mRNA expression markedly (all p>0.05).⑤The immunoreactive products of SYP surrounded the pyramidal neurons, showing the profile of the cell body and axon in the hippocampal CA1 region. Few immunoreactive products were observed in the cytoplasm and nucleu. The SYP expression in protein and mRNA levels were lower in 2VO group than in sham-operated group (p<0.05). Administration of Dihydroergotoxine and ISA at all doses increased the SYP expressions in protein level (all p<0.01). ISA increased SYP mRNA expression at the dose of ISA100mg/kg (p<0.01). Administration of Dihydroergotoxine and ISA at other doses didn’t markedly change the SYP expressions in mRNA level (all p >0.05).Conclusions:①The declines of cholinergic function and synaptic loss accounted for the cognition deficits in rats subjected to CCH.②ISA enhanced the activity of ChAT and expression of SYP via increasing BDNF expression, subsequently improved the synaptic transmission efficiency.③ISA improved the learning and memory through enhancing the cholinergic function and synaptic plasticity in hippocampus of CCH rats.Conclusions for the whole thesis:1. The present research exhibited two kinds of VD rat model. The rats with photochemical lesion or CCH both showed significant declines in spatial learning and memory. The neuronal loss existed in the parietal cortex and hippocampal CA1 subfield respectively in the above-mentioned two sorts of model rats. The further transmission electron microscopy indicated the pathological changes of synaptic ultrastructure in hippocampal CA1 subfield of 2VO rats.2. Compared to the photochemical lesion model, the 2VO rat model showed better stability, higher living rate and lower experimental costs. Most of all, it better mimiced the pathophysiologic process of VD in patients. So in the present research, we took use of the 2VO model to investigate the effects and mechanisms of ISA on rats with CCH.3. The research showed that the treatment with ISA was benefit for rats with CCH, as evidenced by administration of ISA could ameliorate the spatial learning and memory, reduce the neuronal loss and enhance the structural plasticity of hippocampal synapse in rats. ISA showed better effects at the dose of 100mg/kg.4. The research also indicated that the decline of cholinergic function and synaptic loss contributed to the cognition deficits in rats with CCH.5. BDNF contributed to the compensation mechanism. ISA could enhance the endogenous neuroprotection induced by CCH through increasing the expression of BDNF in mRNA and protein levels in hippocampal CA1 subfield. Up-regulating the expressions of SYP and ChAT, two downstream proteins of BDNF-TrkB might be the main mechanism underlying the effects of ISA. (1) Administration of ISA for 8 weeks could significantly increase the number of ChAT immunoreaction-positive cell per unit area, the percentage of immunoreaction-positive area and ChAT mRNA expression in hippocampal CA1 subfield. This result indicated that the enhancement of cholinergic function induced by ISA could potentially supply the substance basis to the synaptic plasticity. (2) Administration of ISA could significantly increase the SYP expression in mRNA and protein levels, subsequently improved the learning and memory via enhancing synaptic transmission efficiency.6. To conclude, ISA improved the learning and memory through enhancing the cholinergic function and protecting the synaptic structure in hippocampus of CCH rats.更多还原