雌激素对去卵巢大鼠学习记忆功能及海马神经元的保护作用

目的 研究雌激素对去卵巢(OVX)大鼠学习记忆功能及海马神经元的保护作用.方法 切除雌性SD大鼠双侧卵巢制作OVX大鼠模型,给予雌二醇(E2)200 μg/kg(OVX+E2组)皮下注射,每周2次,共5周.用Morris水迷宫试验检测大鼠学习记忆功能,HE染色及Bielschowski染色法观察海马神经元形态,免疫组化法观察海马tau蛋白磷酸化变化;并与OVX组和正常对照组比较.结果 与OVX组大鼠相比,OVX+E2组大鼠Morris水迷宫测试成绩明显改善(均P<0.05);海马CA1区神经元及纤维形态较规则,tau蛋白磷酸化阳性细胞数明显减少(均P<0.05).结论 OVX雌性大鼠应用雌激素可改善学习记忆功能障碍、保护海马神经元及降低tau蛋白磷酸化水平.     

长期口服雌激素对去卵巢大鼠海马结构内胆碱能神经元的影响

目的观察长期雌激素缺乏大鼠海马结构内胆碱能神经元的变化,同时比较长期口服复方尼尔雌醇和小剂量17β-雌二醇对去卵巢大鼠海马结构内胆碱能神经元的作用及效果。方法7月龄SD大鼠随机分成5组:正常对照组、假手术组(SHAM)、去卵巢组(OVX)、17β-雌二醇预防干预组(OVX/ERT)和复方尼尔雌醇预防干预组(OVX/NL),5组均在去卵巢后35周处死。用免疫组织化学的方法(SABC法)显示老年大鼠海马结构内的胆碱能神经元,细胞计数及图象分析法观察其数量、形态结构和胆碱乙酰基转移酶(ChAT)表达的变化。结果OVX组海马结构各亚区的胆碱能神经元较少。OVX组海马结构各亚区胆碱能神经元的数量和平均光密度均低于正常组、SHAM组、OVX/ERT组和OVX/NL组(P<005),在其他各组之间无显著性差异。结论长期雌激素缺乏导致了大鼠海马结构内胆碱能神经元数量及ChAT表达的下降,推测是导致去卵巢老年大鼠记忆力下降的原因之一。长期口服小剂量17β-雌二醇和复方尼尔雌醇补充治疗可防止上述变化的出现,有助于胆碱能神经元的生长和存活。复方尼尔雌醇获得了与小剂量17β-雌二醇相同的效果。     

雌激素对去卵巢大鼠中脑黑质多巴胺能神经元的保护作用

目的观察雌激素对去卵巢大鼠中脑黑质多巴胺能神经元的作用,探讨雌激素防治帕金森病的可能性。方法成年雌性大鼠行卵巢切除术、随机分为雌激素组(Ⅰ组)和对照组(Ⅱ组),应用立体定向技术注射六羟基多巴胺(6OHDA)于大鼠中脑黑质,采用免疫组织化学的方法对黑质多巴胺能神经元酪氨酸羟化酶(TH)进行标记并记数阳性细胞。同时,对大鼠进行行为学观测。结果在没有给予雌激素的去卵巢大鼠组,阿朴吗啡(APO)诱导的旋转次数与对照组相比有显著增加(P<0.05)、大鼠中脑黑质TH阳性神经元数目与对照组比较也有显著减少(P<0.01)。结论雌激素对雌性大鼠中脑黑质多巴胺能神经元具有保护作用。     

去卵巢及穹窿海马伞切断复合模型大鼠不同脑区雌激素α受体mRNA的表达及倍美力的干预作用

目的观察倍美力对去卵巢及穹窿海马伞切断后大鼠脑皮质、基底前脑及海马CA_1区雌激素α受体(estrogen receptor alpha,ERa)mRNA表达的干预作用,探讨其脑保护作用的机制。方法健康雌性Wister大鼠30只,随机分成对照组、模型组、倍美力组;制备去卵巢及穹窿海马伞切断复合模型大鼠,采用Morris水迷宫观察其学习、记忆能力,利用RT-PCR及Western blotting法观察每组大鼠不问脑区雌激素α受体mRNA及蛋白的表达。结果模型组大鼠脑皮质、基底前脑、海马CA_1区雌激素α受体mRNA及蛋白的表达明显降低,学习、记忆能力下降,与对照组、倍美力组比较有显著差异(P<0.01);对照组与倍美力组比较无明显差异(P>0.05)。结论倍美力可上调大鼠脑皮质、基底前脑及海马CA_1区雌激素α受体mRNA及蛋白的表达,可能参与了学习、记忆能力的改善。     

苯甲酸雌二醇对去卵巢大鼠慢性前脑缺血脑保护作用的研究

目的观察苯甲酸雌二醇对去卵巢大鼠慢性前脑缺血脑组织病理形态、学习记忆以及死亡率的影响,探讨雌激素对慢性缺血性脑损害的保护作用。方法50只健康雌性Wistar大鼠,随机分为4组。A组:正常对照组,n=5;B组:假去卵巢缺血组,n=15;C组:去卵巢缺血组n=15;D组:去卵巢缺血苯甲酸雌二醇治疗组。各组按要求制备模型,应用Morris水迷宫筛选并检测记忆功能,坚劳蓝 焦油紫染色、CD31免疫组化染色观察额叶皮质和海马CA1区神经元毛细血管变化。结果A组大鼠额叶皮质和海马CA1区神经元、毛细血管形态正常,学习记忆功能良好,B、C、D组与A组相比上述指标改变明显,差异具有显著性(P<0.05),而且C组改变明显重于B、D组(P<0.05);C组额叶皮质神经元、毛细血管和海马CA1区神经元数量减少,与A、B、D组相比差异具有显著性(P<0.05),B、D组相比上述改变无差异(P>0.05);缺血后各组大鼠急性期死亡率比较,差异无显著性(P>0.05)。结论雌激素对去卵巢慢性前脑缺血大鼠额叶皮质及海马CA1区病理变化以及学习记忆功能均产生了有益的影响,但未能降低急性期大鼠的死亡率。     

去松果树对大鼠学习记忆及大脑皮质胆碱能纤维的影响

目的　探讨松果体功能减退致褪黑素 (MT)分泌减少对大鼠学习记忆及大脑皮质胆碱能纤维分布的影响。方法　将大鼠进行Y型迷宫测试 ,淘汰学习障碍的大鼠 ,将学习正常的大鼠随机分 2组 ,实验组手术摘除松果体 ,对照组给予假手术 ,饲养 40d后再行Y型迷宫测试 ,乙酰胆碱酯酶 (AChE)组织化学法测AChE。结果　实验组大鼠学习成绩由术前 (14.7± 5 .0 )次增加到术后(2 8.7± 2 .4)次 ,学习记忆能力明显低于术前自身及对照组 (13.8± 8.3)次 (P <0 .0 1) ,运动皮质、体感皮质第Ⅱ～Ⅳ层 ,海马CA1、CA2、CA3区辐状层、腔隙层、分子层和齿状回多形细胞层AChE阳性纤维密度明显低于对照组 (P <0 .0 1)。结论　大鼠松果体摘除可以引起大鼠学习记忆障碍及大脑皮质胆碱能纤维密度降低     

长期口服雌激素或复方雌激素对去卵巢大鼠海马结构内β-淀粉样蛋白沉积的影响

目的观察长期口服雌激素或复方雌激素对去卵巢大鼠大脑海马结构内β-淀粉样蛋白(Aβ)沉积的影响。方法7月龄SD大鼠随机分成5组:正常对照组、假手术组、去卵巢组、17β-雌二醇预防干预组(雌二醇干预组)和复方尼尔雌醇预防干预组(尼尔雌醇干预组)。去卵巢组、雌二醇干预组及尼尔雌醇干预组手术切除卵巢,并分别对雌二醇干预组及尼尔雌醇干预组饲喂相应的药物35周,处死各组大鼠,用免疫组化法、细胞计数及图像分析观察各组大鼠海马结构内Aβ沉积的变化。结果去卵巢组海马结构内各亚区Aβ阳性神经元数量和平均光密度明显高于其他4组(均P<0·05)。结论长期雌激素缺乏导致大鼠海马结构内Aβ沉积增加,减少Aβ的沉积可能是雌激素神经保护的重要机制之一,雌激素与复方雌激素效果相同。     

切除卵巢对大鼠学习记忆和海马雌激素受体α的影响及电针的干预作用

目的 研究电针对去卵巢大鼠行为学及海马区雌激素受体α(estrogen receptor alpha,ERα)蛋白和mRNA表达的影响.方法 将40只雌性SD大鼠随机分为4组:假手术组、模型组、假电针组、电针组,各10只.采用卵巢切除大鼠模型,造成低雌激素记忆障碍,去势2周后进行电针刺激,连续治疗3个月.以Morris水迷宫测试空间学习记忆能力,酶联免疫吸附分析(enzyme-linked immunosorbent assay,ELISA)检测血清雌二醇(estradiol,E2)浓度,免疫印迹法(Western blot,WB)和实时荧光定量PCR分别检测海马区ERα蛋白和mRNA的相对表达量.结果 模型组、假手术组、电针组和假电针组大鼠逃避潜伏期分别为(49.65±3.82)s、(15.26±3.37)s、(14.33±3.62)s、(25.46±2.87)s,游泳距离分别为(941.88±37.77) cm、(291.66±23.42) cm、(261.52±28.76)cm、(471.01±33.31)cm,跨越平台数分别为(2.35±0.44)、(8.94±0.91)、(8.53±0.83)、(3.64±0.88)次,血清E2浓度分别为(38.16±5.44) pg/mL、(65.13±7.72) pg/mL、(56.47±6.70) pg/mL、(49.29±5.90)pg/mL,海马ERa蛋白表达分别为(0.406±0.040)、(0.985±0.086)、(0.796±0.072)、(0.498±0.063),海马ERamRNA表达分别为(1.037±1.021)、(2.303±0.065)、(1.410±0.062)、(1.153±0.035),模型组较假手术组其逃避潜伏期和游泳距离延长,跨越平台数增加,血清E2浓度及海马ERa蛋白和mRNA表达均降低,电针组和假电针组均较模型组有所改善,但电针组改善更明显,上述差异均有统计学意义(均P＜ 0.01).结论 电针能够提高去卵巢大鼠学习记忆能力,其机制可能通过升高体内雌激素浓度上调海马ERα蛋白和mRNA的表达有关.     

去卵巢、去松果体对大鼠杏仁核细胞凋亡的影响

阿尔茨海默病(Alzheimer’s disease，AD)临床主要表现为记忆力减退，其中受情绪影响的长时记忆减退主要与杏仁核的神经元减少有关。AD的病因至今不清，近来研究结果显示，体内雌激素(estrogen，E)、褪黑素(melatonin，MT)缺乏与AD发病有关，而细胞凋亡可能参与了发病过程心。     

雌激素对去卵巢大鼠缺血再灌注脑损伤的保护作用

目的探讨雌激素对去卵巢大鼠缺血再灌注脑损伤的保护作用。方法将大鼠切除双侧卵巢后30d给予肌肉注射苯甲酸雌二醇100μg/(kg·d)连续14d,然后制作大鼠局灶性脑缺血再灌注模型;制模12h后取脑组织行免疫组化染色,检测细胞间黏附分子(CD54)、肿瘤坏死因子α(TNF-α)的表达;TUNEL法检测脑组织凋亡细胞数;电镜观察脑细胞膜超微结构的变化。结果与缺血再灌注组及去卵巢组比较,雌激素组脑组织CD54、TNF-α表达明显降低,凋亡细胞数明显减少(均P<0.05);脑细胞膜结构非特异性损伤减轻。结论雌激素通过减少缺血再灌注大鼠脑组织炎性细胞因子表达、减轻炎症反应、降低脑组织细胞凋亡而发挥脑保护作用。     

Relationship of calbindin D-28k and cholinergic neurons in the nucleus basalis of Meynert of the monkey and the rat

Double-labeling immunocytochemistry reactions were carried out in the monkey and the rat nucleus basalis of Meynert (NBM) to determine the extent of overlap between cholinergic neurons and neurons immunoreactive for calbindin-D-28k (CaBP), a Vitamin D-dependent calcium binding protein. The results indicate that most, but not all, NBM cholinergic neurons in the monkey are immunoreactive for CaBP. On the other hand, none of the rat NBM cholinergic neurons are immunoreactive for CaBP.     

The cerebrovascular role of the cholinergic neural system in Alzheimer's disease

The intrinsic cholinergic innervation of the cortical microvessels contains both subcortical pathways and local cortical interneurons mediated by muscarinic and nicotinic acetylcholine receptors. Stimulation of this system leads to vasodilatation. In the extrinsic innervation, choline acts as a selective agonist for the α7-nicoticinic acetylcholine receptor on the sympathetic nerves to cause vasodilatation, and through this mechanism, cholinergic modulation may affect this sympathetic vasodilatation. Alzheimer's disease is characterized by a cerebral cholinergic deficit and cerebral blood flow is diminished. Cholinesterase inhibitors, important drugs in the treatment of Alzheimer's disease, could influence the cerebral blood flow through stimulation of the intrinsic cholinergic cerebrovascular innervation. Indeed, cholinesterase inhibitors improve cerebral blood flow in Alzheimer patients who respond to treatment. Further, cerebrovascular reactivity and neurovascular coupling are impaired in Alzheimer's disease and both can be improved by cholinesterase inhibitors. Conversely, cholinesterase inhibitors inhibit the α7-nicoticinic acetylcholine receptor on extrinsic sympathetic nerves and thus may impair vasodilatation. The net outcome of these opposing effects in clinical practice remains unknown. Moreover, it is uncertain whether the regulation of cerebral blood flow during blood pressure changes (cerebral autoregulation) is impaired in patients with Alzheimer's disease. Technological developments now allow us to dynamically measure blood pressure, cerebral blood flow, and cerebral cortical oxygenation. Using simple maneuvers like single sit-stand and repeated sit-stand maneuvers, the regulation of cerebral perfusion in patients with Alzheimer's disease can easily be measured. Sit-stand maneuvers can be considered as a provocation test for cerebral autoregulation, and provide excellent opportunities to study the cerebrovascular effects of cholinesterase inhibitors.     

Receptor-mediated transport of human recombinant nerve growth factor from olfactory bulb to forebrain cholinergic nuclei

Receptors for nerve growth factor are present in the olfactory bulb and in cholinergic nuclei that send projections to the olfactory bulb. The retrograde transport of¹²⁵I-labeled recombinant human nerve growth factor (rhNGF) was demonstrated in the rat 18 h following an injection of [¹²⁵I]rhNGF into the left olfactory bulb. In each of six animals, [¹²⁵I]rhNGF label was observed in the ipsilateral horizontal limb of the diagonal band and, in four of the 6 animals, in the vertical limb of the diagonal band. Label was not observed in any other brain region except within the injected olfactory bulb. The transport of label to the diagonal band was blocked by the injection of 170-fold greater concentration of unlabeled rhNGF. Emulsion autoradiography of hematoxylin/eosin counterstained sections revealed silver grains clustered over numerous cell profiles that resembled neurons. In contrast, cerebellar injections of [¹²⁵I]rhNGF, with or without unlabeled rhNGF, did not label diagonal band neurons, nor the lateral vestibular or red nuclei, from which originate the primary cholinergic afferents to cerebellum. The receptor-dependent transport of NGF from olfactory bulb to forebrain cholinergic nuclei suggests that this projection, unlike pontomesencephalic cholinergic pathways, may be responsive to endogenous NGF or exogenously administered rhNGF.     

The history of the cholinergic hypothesis

The cholinergic hypothesis of cognitive impairment and Alzheimer's disease has been for decades a “polar star” for studies on dementia and neurodegenerative diseases. Aim of the present article is to briefly summarize its birth and its evolution throughout years and discoveries. Putting the cholinergic hypothesis in an historical perspective, allows to appreciate the enormous amount of experimental and clinical research that it has stimulated over years and the impressive extent of knowledge generated by this research. While some of the assumptions at the basis of its original formulation are disputable in the light of recent developments, the cholinergic hypothesis has, however, constituted an invaluable stimulus to better understand not only the anatomy and the biochemistry of the cholinergic systems of brain connections but also its developmental biology, its complex relationships with trophic factors, its role in cognitive functions. Thus, rather than being consigned to history, the cholinergic hypothesis will likely contribute to further understanding dementia and neurodegenerative diseases and will hopefully be integrated in novel therapies and treatments.     

Chronic estrogen treatment replaces striatal dopaminergic function in ovariectomized rats

Eight-week-old female Sprague–Dawley rats were divided into three groups: ovariectomized rats (OVX); ovariectomized rats treated with estradiol valerate (E2), 20 μg subcutaneously (s.c.) twice weekly for 12 weeks (OVX+E2 group); and sham-operated control rats treated with vehicle alone (controls). Spontaneous locomotor activity was measured for 24 h, and then again after the administration of methamphetamine (1 mg/kg, i.p.). In addition, striatal contents of dopamine (DA) and its metabolites were measured. Using an in vivo microdialysis technique, changes in extracellular striatal dopamine concentration were studied in a separate set of similarly treated rats after the administration of methamphetamine (0.2 mg/kg, i.p.). Spontaneous locomotor activity decreased in the OVX group, and estradiol replacement reversed this decreased activity. No significant differences were observed in the contents of DA and its metabolites at the striatum among the three groups. The basal output of DA at the striatum was lower in the OVX group than in those of the other two groups. Extracellular DA concentration following methamphetamine administration was also lower in the rats of OVX group. These results indicate that ovariectomy decreases spontaneous locomotor activity, response to methamphetamine, and striatal DA release in the female rats. Chronic replacement of estrogen reversed spontaneous locomotor activity and DA release by the striatum. These results suggest that chronic administration of estrogen may be beneficial in the treatment of female menopausal patients with Parkinson’s disease.     

Evidence for the presence of antibodies to cholinergic neurons in the serum of patients with Alzheimer's disease

Summary A blind study showing that serum from patients with Alzheimer's disease causes immunolysis of mammalian brain synaptosomes is reported. Control, aged-matched, sera were largely without effect. The immunolysis was directed mainly against cholinergic synaptosomes. The data presented support the hypothesis that autoimmune mechanisms may operate in the pathogenesis of Alzheimer's disease.     

Colocalization of androgen, estrogen and cholinergic receptors on cultured astrocytes of rat central nervous system

By means of immunohistochemical and electrophysiological methods, we have investigated the presence of androgen receptors on astrocytes in explant and primary cultures from various regions of rat central nervous system. Our studies have shown that a great number of astrocytes and neurones express androgen receptors as recognized by a specific monoclonal antibody. Immunoreactivity was mainly distributed over the soma of the astrocytes, the nuclei being intensely stained. In contrast, glial processes were only faintly stained or not stained. Double-immunostaining studies have provided evidence for a colocalization of androgen and estrogen alpha- and beta-receptors on many astrocytes. Furthermore, there was also a coexistence of glial androgen receptors with cholinergic muscarinic and nicotinic sites. Our immunohistochemical findings are supported by electrophysiological investigations demonstrating that 5alpha-androstan, 17beta-estradiol as well as the cholinergic agonists muscarine and nicotine caused hyperpolarizations on the same astrocytes. Our studies suggest that there is a coexistence of functional receptors for androgen, estrogen as well as for the cholinergic agonists on glial cells. Further investigations are needed to elucidate the physiological role of glial androgen, estrogen and cholinergic receptors and to define their function in neurodegenerative diseases.     

Deficits of the cholinergic system in early AD

Impairment of cholinergic neurotransmission is a well-established fact in Alzheimer's disease (AD) but there is controversy about its relevance at the early stages of the disease. In the recent years new techniques for in vivo imaging of key components of the cholinergic system in humans have developed. They are beginning to be applied to the very early stages of AD. Preliminary results suggest that there is early impairment of presynaptic receptors and acetylcholine esterase, the main degrading enzyme for acetylcholine, in cerebral cortex. The relation of these findings to neuronal function and post-mortem findings is being discussed.