Genistein对去卵巢大鼠基底前脑胆碱能神经元影响的体内研究

本文旨在研究染料木素(genistein)对去卵巢大鼠基底前脑胆碱能神经元的影响。雌性大鼠双侧卵巢切除2周后用genistein和雌激素替代治疗1周。称子宫重量以确定手术是否成功及雌二醇(E2)的治疗是否有效。用免疫组化染色、RT-PCR和Westernblot等方法对胆碱能神经元数量、ChAT基因和蛋白的表达量进行检测。结果显示:去卵巢3周后子宫变轻,雌激素替代治疗能增加去卵巢子宫的重量,而genistein替代治疗对去卵巢子宫的重量影响不明显;去卵巢3周后,内侧隔核(MS)和斜角带垂直臂核(VDB)内的胆碱能神经元数量、ChAT基因和蛋白的表达量均明显减少,雌激素和genistein替代治疗后能显著增加去卵巢大鼠MS和VDB内的胆碱能神经元数量、ChAT基因和蛋白的表达量。本研究结果提示:genistein对去卵巢大鼠基底前脑胆碱能神经元具有类似雌激素样神经保护作用,而对子宫影响不明显。     

植物雌激素对去卵巢大鼠基底前脑胆碱能神经元的影响

目的：观察植物雌激素对去卵巢大鼠基底前脑胆碱能神经元表达的影响，探讨植物雌激素在中枢神经系统的保护作用及机制。方法：采用乙酰胆碱转移酶(ChAT)免疫组织化学ABC法，观察去卵巢大鼠5w后各组基底前脑内侧隔核(MS)，斜角带垂直支(VDB)胆碱能神经元的数目。结果：与去卵巢对照组相比，植物雌激素用药组、雌激素用药组的内侧隔核，斜角带垂直支胆碱能神经元数目明显升高(P＜0．05)，与假手术组差别不明显。结论：本研究提示植物雌激素能明显增加去卵巢大鼠基底前脑胆碱能神经元的表达，从而对中枢神经系统退行性病变起保护作用，并有望预防和治疗老年性痴呆。     

银杏内酯对大鼠基底前脑神经元生长发育的影响

目的：观察银杏内酯对培养的胚胎大鼠基底前脑胆碱能神经元和NADPH-d阳性神经元生长发育的作用。方法：取E17Wistar大鼠胚胎基底前脑进行体外细胞培养，并给予银杏内酯。进行乙酰胆碱酯酶(acetyl cholinesterase，AChE)和NADPH-d酶组织化学染色，并在光镜下进行阳性细胞计数和显微测量胞体及突起发育状况。结果：给予银杏内酯处理后，培养物中乙酰胆碱酯酶阳性神经元和NADPH-d阳性神经元数量均较对照组显著增加，且神经元发育状况好于对照组，胞体大，突起多且长。结论：银杏内酯可以促进体外培养的基底前脑神经元中乙酰胆碱酯酶和NADPH-d的表达，并能促进表达这两种酶的神经元生长发育。     

BDNF、NGF对体外长期培养的胚基底前脑胆碱能神经元的影响

本文探讨了脑源性神经营养因子、神经生长因子对体外长期培养的胚基底前脑胆碱能神经元是否具有延缓退变的作用。实验分为脑源性神经营养因子组、神经生长因子组、脑源性神经营养因子加神经生长因子组及单纯对照组。取孕 17d SD大鼠胚基底前脑原基制成细胞悬液接种于 2 4孔培养板中 ,按分组加入含相应神经营养因子和不含神经营养因子的 DMEM培养液 ,分别于体外培养 12、18、2 4、3 0 d后进行乙酰胆碱酯酶组织化学反应。显微镜下计数各孔中乙酰胆碱酯酶阳性神经元数 ,每孔随机测量和计数 2 5个乙酰胆碱酯酶阳性神经元的平均胞体直径、发出的突起数和最长突起长度。数据用方差分析和 SNK检验进行统计学处理。结果显示 ,在培养的 4个时期 ,含脑源性神经营养因子组、神经生长因子组和脑源性神经营养因子加神经生长因子组的各项数据均明显地优于单纯对照组 ;脑源性神经营养因子加神经生长因子组的各项数据 ,特别是最长突起长度优于单独使用脑源性营养因子或神经生长因子组。提示 :脑源性神经营养因子和神经生长因子不仅对体外培养的胚胆碱能神经元发育生长具有促进作用 ,而且还可延缓体外长期培养的大鼠胚基底前脑胆碱能神经元的退变 ;两者的联合使用还可对延缓其退变具有协同作用     

胰岛素与尼莫地平对大鼠基底前脑胆碱能神经元及空间学习记忆的影响

目的探讨胰岛素与尼莫地平对基底前脑胆碱能神经元与学习记忆在1型糖尿病脑病发生中的影响。方法将成年雄性Wistar大鼠随机分成糖尿病组、干预组、载体组及正常对照组。用链脲佐菌素成功建立成1型糖尿病模型12周后,对干预组大鼠每日皮下注射长效胰岛素(3 IU)、腹腔注射尼莫地平(20 mg/kg),连续用药6周,在相同条件下对载体组大鼠注射等体积的无药物液体,但对糖尿病组或正常对照组大鼠未进行任何处理。应用Morris水迷宫及胆碱乙酰转移酶(ChAT)免疫组化方法,分别测定各组大鼠的空间学习记忆能力及基底前脑胆碱能神经元的变化。结果糖尿病组大鼠内侧隔核、斜角带核垂直支、斜角带核水平支的ChAT阳性神经元数均明显减少(P<0.05),空间学习记忆能力也明显下降(P<0.05);干预组大鼠以上三个核团的ChAT阳性神经元数与学习记忆能力均明显大于糖尿病组大鼠(P<0.05),但干预组的各项指标仍然明显低于载体组大鼠或正常对照组大鼠(P<0.05)。结论在糖尿病的长期自然发展过程中,若不进行治疗则可累及到基底前脑胆碱能神经元,导致学习记忆障碍,这可能是引发1型糖尿病脑病的一个负性因素;此时联合应用胰岛素与尼莫地平仍可有效遏制该脑病向纵深发展的恶性势头。     

秋水仙碱对基底前脑巢蛋白阳性和阴性胆碱能神经元的影响

目的:通过研究侧脑室注射秋水仙碱前后基底前脑巢蛋白阳性和阴性胆碱能神经元变化,探讨巢蛋白表达对基底前脑神经元机能的影响及其可能机制。方法:成年健康雌性SD大鼠随机分为正常对照组和侧脑室注射秋水仙碱组,术后分别于24 h、48 h、3 d、7 d、14 d和28 d取脑行冷冻切片与免疫组织化学显色,比较秋水仙碱注射后不同时间点基底前脑巢蛋白~+和巢蛋白~-胆碱能神经元的数目变化。结果:大鼠侧脑室秋水仙碱注射后24 h,基底前脑的内侧隔核(MS)、斜角带核垂直支(vDB)和水平支(hDB)的巢蛋白~+和巢蛋白的胆碱能神经元数目都急骤下降。随着时间的推移巢蛋白~+神经元数目逐渐恢复,术后14 d,巢蛋白~+神经元数目基本恢复至正常水平,但巢蛋白~-胆碱能神经元数目一直维持较低水平。结论:侧脑室注射秋水仙碱后,基底前脑巢蛋白~+和巢蛋白~-的胆碱能神经元都急骤减少,但巢蛋白~+神经元在减少后可逐渐恢复,而巢蛋白~-神经元则不能恢复。     

康复新对去卵巢大鼠学习记忆能力和基底前脑胆碱能神经元的影响

目的 探讨康复新对去卵巢大鼠的学习记忆能力的影响及其可能机制.方法 3月龄健康雌性SD大鼠36只,随机分成4组,即假手术组、去卵巢对照组、康复新低剂量组和康复新高剂量组,分别进行干预.8周后用Y迷宫检测大鼠的空间学习记忆能力,用免疫组化染色检测大鼠基底前脑不同区域胆碱能神经元的数目.结果 康复新高剂量组大鼠"学会"所需总训练次数、训练错误次数及记忆检测时错误反应次数与去卵巢对照组比较明显减少(P＜0.05),与假手术组相比差异无统计学意义(P＞0.05);康复新液高、低剂量组大鼠基底前脑内侧隔核(MS)、斜角带核垂直支(vDB)和水平支(hDB)的ChAT阳性神经元数量增多;高剂量组基底前脑各区的ChAT阳性神经元数明显高于去卵巢对照组(P＜0.05),与假手术组相比差异无统计学意义(P＞0.05);而且康复新高剂量组大鼠vDB和hDB的ChAT阳性神经元数明显高于康复新低剂量组(P＜0.05).结论 康复新液可显著改善去卵巢大鼠学习记忆能力,其作用可能是通过增加基底前脑胆碱能神经元的功能实现的;康复新液有望成为防治雌激素撤退引起的神经功能障碍的药物.     

基底前脑巢蛋白免疫阳性神经元研究现状及展望

目的阐述基底前脑nestin免疫阳性神经元的研究现状,为后续研究提供依据。方法对近年发表的有关基底前脑nestin免疫阳性神经元的文献进行分析和综述。结果在人和大鼠的基底前脑存在着一群能表达nestin的成熟神经元,这群神经元是区别于胆碱能神经元和γ-氨基丁酸能神经元的独立的神经元。其形态和分布与胆碱能神经元相似,向海马、大脑皮质及丘脑投射,与学习记忆及神经元的可塑性有关。结论基底前脑nestin免疫阳性神经元的化学属性、纤维联系及电生理特征等有待于进一步研究。这些研究将有助于我们了解成熟神经元表达nestin的功能意义,并为相关疾病的诊治提供新的视角。     

雌激素对去卵巢大鼠基底前脑胆碱能神经元及一氧化氮合酶阳性神经元的影响

目的　探讨雌激素补充治疗对去卵巢大鼠胆碱能神经元及一氧化氮合酶 (NOS)阳性神经元表达的影响及剂量效应关系。方法　 2 0只去卵巢SD大鼠分成 4个不同剂量组 :0 μg(对照组 )、2 0 μg(0 0 8mg/kg)、5 0 μg(0 2 0mg/kg)、10 0 μg(0 4 0mg/kg)组 ;1周后 ,用乙酰胆碱转移酶 (ChAT)免疫化学方法及尼克酰胺腺嘌呤二核苷酸黄递酶 (NADPH d)组织化学方法研究。结果　NOS阳性神经元在内侧隔核 (MS) ,其数目在各组间无明显差异 (P >0 0 5 )。在斜角带垂直支 (VDB) ,5 0 μg剂量组与对照组相比有明显差异 (P <0 0 5 ) ;ChAT阳性神经元在内侧隔核 (MS) ,其 2 0 μg、5 0 μg剂量组数目出现剂量递增效应 ,与对照组比较有明显差异 (P <0 0 5 ) ,但 10 0 μg剂量组与对照组比较没有明显差异 (P >0 0 5 ) ,同时 5 0 μg与 10 0 μg剂量组比较有明显差异 (P <0 0 5 )。ChAT阳性神经元在斜角带水平支 (HDB) ,各组间均无明显差异 (P >0 0 5 )。结论　雌激素补充治疗能选择性影响基底前脑各亚区NOS和胆碱能神经元 ,并有可能影响学习和记忆能力。     

剥夺卵巢雌激素对成年大鼠基底前脑神经元表达Nestin的影响

目的 探讨剥夺卵巢雌激素对成年大鼠基底前脑神经元表达Ｎｅｓｔｉｎ的影响。方法 将健康成年雌性大鼠随机分为正常对照、卵巢切除２周及４周三组,用免疫组化法观察卵巢切除后基底前脑的内侧隔核（ＭＳ）、斜角带垂直支（ｖＤＢ）及水平支（ｂＤＢ）Ｎｅｓｔｉｎ免疫反应阳性（Ｎｅｓｔｉｎ－ＩＲ） 神经元的形态和数目变化。结果 卵巢切除２周、４周组ＭＳ、ｖＤＢ及ｈＤＢ的Ｎｅｓｈｎ－ＩＲ神经元形态与正常对照组相比无明显变化;但卵巢切除２周、４周组ＭＳ、ｖＤＢ及ｈＤＢ的 Ｎｅｓｔｉｎ－ＩＲ神经元的数目与正常对照组相比均有不同程度的减少,尤以ｈＤＢ的减少最明显（Ｐ＜０．０１,Ｐ＜０．０５）。结论 提示基底前脑Ｎｅｓｔｉｎ－ＩＲ神经元的Ｎｅｓｈｎ表达受卵巢雌激素的影响,ｈＤＢ的Ｎｅｓｈｎ－ＩＲ神经元的表达受此影响更大。     

Human cholinergic basal forebrain: chemoanatomy and neurologic dysfunction

The human cholinergic basal forebrain (CBF) is comprised of magnocellular hyperchromic neurons within the septal/diagonal band complex and nucleus basalis (NB) of Meynert. CBF neurons provide the major cholinergic innervation to the hippocampus, amygdala and neocortex. They play a role in cognition and attentional behaviors, and are dysfunctional in Alzheimer's disease (AD). The human CBF displays a continuum of large cells that contain various cholinergic markers, nerve growth factor (NGF) and its cognate receptors, calbindin, glutamate receptors, and the estrogen receptors, ER and ERβ. Admixed with these cholinergic neuronal phenotypes are smaller interneurons containing the m2 muscarinic acetylcholine receptor (mAChRs), NADPH-diaphorase, GABA, calcium binding proteins and several inhibitory neuropeptides including galanin (GAL), which is over expressed in AD. Studies using human autopsy material indicate an age-related dissociation of calbindin and the glutamate receptor GluR2 within CBF neurons, suggesting that these molecules act synergistically to induce excitotoxic cell death during aging, and possibly during AD. Choline acetyltrasnferease (ChAT) activity and CBF neuron number is preserved in the cholinergic basocortical system and up regulated in the septohippocampal system during prodromal as compared with end stage AD. In contrast, the number of CBF neurons containing NGF receptors is reduced early in the disease process suggesting a phenotypic silence and not a frank loss of neurons. In end stage AD, there is a selective reduction in trkA mRNA but not p75^NTR in single CBF cells suggesting a neurotrophic defect throughout the progression of AD. These observations indicate the complexity of the chemoanatomy of the human CBF and suggest that multiple factors play different roles in its dysfunction in aging and AD.     

七氟醚处理对老龄小鼠基底前脑胆碱能系统和认知功能的影响

目的:探讨七氟醚对基底前脑胆碱能系统和认知功能的影响。方法:老年C57小鼠吸入七氟醚建立全身麻醉动物模型,利用Morris水迷宫检测认知功能,免疫荧光检测Meynert基底核中胆碱能神经元计数,Western Blot检测额叶皮层胆碱乙酰转移酶及高亲和力胆碱转运体的蛋白水平,ELISA测定额叶皮层胆碱乙酰转移酶的活性。结果:与对照组相比,七氟醚组小鼠平台潜伏期延长,平台穿越次数减少(2.1±0.4 vs 5.6±0.5,P0.05),原平台所在象限停留时间缩短[(21.5±2.4)%vs(48.6±2.8)%,P0.05]。此外,七氟醚组小鼠Meynert基底核胆碱能神经元计数减少(3748±248 vs 5942±315,P0.05),额叶皮层胆碱乙酰转移酶及高亲和力胆碱转运体表达下降,伴有胆碱乙酰转移酶活性降低[(31.4±1.1)μmol/(g.h)vs(55.8±1.3)μmol/(g.h),P0.05]。结论:七氟醚可损害老龄鼠基底前脑胆碱能系统并诱导认知功能损害。     

Pontine and basal forebrain cholinergic interaction: implications for sleep and breathing

Pontine and forebrain cholinergic nuclei contribute to the regulation of breathing and arousal. This report summarizes experiments in rat (n = 20) concerning the cholinergic interaction between pons and basal forebrain. In vitro [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) autoradiography quantified carbachol-stimulated guanine nucleotide binding (G) protein activation in seven basal forebrain nuclei. Carbachol significantly increased [(35)S]GTPgammaS binding in the vertical and horizontal limbs of the diagonal band of Broca, medial and lateral septum, and nucleus basalis (B)/substantia innominata (SI). In vitro receptor autoradiography demonstrated muscarinic receptors in the same nuclei where carbachol caused G protein activation. In vivo experiments showed that carbachol administered to the pontine reticular formation (PnO) significantly decreased the number of 7-14Hz spindles in the electroencephalogram (EEG), decreased acetylcholine release in SI, and decreased respiratory rate. Carbachol microinjection into SI did not alter the number of EEG spindles or respiratory rate. The results help clarify that EEG and rate of breathing are more effectively modulated by cholinergic neurotransmission in PnO than in SI.     

Adenosine inhibits basal forebrain cholinergic and noncholinergic neurons in vitro

Adenosine has been proposed as a homeostatic "sleep factor" that promotes the transition from waking to sleep by affecting several sleep-wake regulatory systems. In the basal forebrain, adenosine accumulates during wakefulness and, when locally applied, suppresses neuronal activity and promotes sleep. However, the neuronal phenotype mediating these effects is unknown. We used whole-cell patch-clamp recordings in in vitro rat brain slices to investigate the effect of adenosine on identified cholinergic and noncholinergic neurons of the magnocellular preoptic nucleus and substantia innominata. Adenosine (0.5-100 microM) reduced the magnocellular preoptic nucleus and substantia innominata cholinergic neuronal firing rate by activating an inwardly rectifying potassium current that reversed at -82 mV and was blocked by barium (100 microM). Application of the A1 receptor antagonist 8-cyclo-pentyl-theophylline (200 nM) blocked the effects of adenosine. Adenosine was also tested on two groups of electrophysiologically distinct noncholinergic magnocellular preoptic nucleus and substantia innominata neurons. In the first group adenosine, via activation of postsynaptic A1 receptors, reduced spontaneous firing via inhibition of the hyperpolarization-activated cation current. Blocking the H-current with ZD7288 (20 microM) abolished adenosine effects on these neurons. The second group was not affected by adenosine. These results demonstrate that, in the magnocellular preoptic nucleus and substantia innominata region of the basal forebrain, adenosine inhibits both cholinergic neurons and a subset of noncholinergic neurons. Both of these effects occur via postsynaptic A1 receptors, but are mediated downstream by two separate mechanisms.     

Evidence for a distinct group of nestin-immunoreactive neurons within the basal forebrain of adult rats

Nestin is an intermediate filament protein serving as a marker for neuroprogenitor and stem cells. Here we report that a cluster of previously unrecognized nestin immunoreactive (nestin-ir) neurons was located in the medial septum-diagonal band of Broca (MS-DBB) of the basal forebrain in adult rats. Nestin-ir neurons were exclusively located in the MS-DBB and intermingled with choline acetyltransferase-ir (ChAT-ir), parvalbumin-ir (PV-ir), or nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase reactive (NADPHd-reactive) neurons. However, there was no colocalization between nestin-ir and PV-ir in single neurons in MS-DBB; only about 35% of nestin-ir neurons were ChAT-ir, and 8%-12% of nestin-ir neurons were NADPHd-reactive. Morphologically, nestin-ir neurons showed a larger size of somata than that of ChAT-ir or PV-ir neurons and the distribution of nestin-ir neurons spread across the rostro-caudal extent of the MS-DBB. Moreover, retrograde tracing revealed that a significant portion of these nestin-ir neurons projected to the thalamus and hippocampus. These results, for the first time, provide strong evidence that there exists a cluster of previously unrecognized nestin-ir neurons in MS-DBB of the basal forebrain in adult rats and that these nestin-ir neurons are distinguishable from ChAT-ir, PV-ir, and NADPHd-reactive neurons.