血管活性肠肽对帕金森病大鼠黑质胶质细胞活化及相关炎性因子释放的影响

目的探讨血管活性肠肽(VIP)对帕金森病(PD)大鼠模型中脑黑质胶质细胞活化及相关炎性因子表达的影响。方法将6-羟多巴胺(6-OHDA)定向注入大鼠右侧纹状体制备PD模型。32只制备成功的PD大鼠随机分为VIP组和模型组,VIP组大鼠腹腔注射VIP 1ml(20μg/L),另10只正常大鼠为对照组。分别采用免疫组织化学、Western blotting、RT-PCR方法观察大鼠中脑黑质多巴胺(DA)能神经元、小胶质细胞、星形胶质细胞的数量和形态变化以及肿瘤坏死因子α(TNF-α)和环氧化酶2(COX-2)的表达变化。结果模型组大鼠黑质损毁侧小胶质细胞即白细胞分化抗原11b(CD11b)阳性细胞,数量较对照组明显增加(P0.05)并呈阿米巴样改变,星形胶质细胞(GFAP阳性细胞)数量明显增加(P0.05),炎性因子表达水平也明显上升(P0.05),DA能神经元数量较对照组明显下降(P0.05);与模型组相比,VIP组大鼠损毁侧黑质小胶质细胞和星形胶质细胞数量明显下降(P0.05),炎性因子表达水平显著降低(P0.05),DA能神经元数量较模型组增加(P0.05)。结论 VIP对帕金森病大鼠黑质小胶质细胞和星形胶质细胞的活化具有抑制作用,并可减少相关炎症因子的表达,从而保护黑质DA能神经元。     

激活小胶质细胞对帕金森病模型大鼠黑质多巴胺能神经元的影响

目的 探讨脂多糖(LPS)激活小胶质细胞(MG)对黑质损伤多巴胺(DA)能神经元细胞存活的影响.方法 采用立体定向技术向大鼠单侧黑质(SNpc)内注入LPS激活MG后,隔48h于原位注射高、低剂量鱼藤酮(BT)分别建立重度及轻度损伤模型;采用免疫组化法观察黑质酪氨酸羟化酶(TH)阳性细胞和离子钙结合转接分子1(Ib α...     

帕金森病大鼠黑质小胶质细胞及星形胶质细胞的变化

目的探讨帕金森病(PD)发病中黑质小胶质细胞和星形胶质细胞的变化。方法采用立体定向术将神经毒素6-羟基多巴胺(6-OHDA)注入大鼠右侧黑质和内侧前脑束内,制备大鼠PD模型。将制模成功的16只PD大鼠随机分为2周和8周模型组,另6只正常大鼠作为对照组。观察各组大鼠黑质致密带内多巴胺(DA)能神经元、OX-42(小胶质细胞的特异性标志物)及神经胶质纤维酸性蛋白(GFAP,星形胶质细胞的特异性标志物)阳性细胞的分布和形态变化。结果 2周和8周模型组损毁侧黑质致密部DA能神经元较健侧显著减少(P0.01),损毁侧OX-42阳性细胞的数量较健侧明显增加(P0.01),形态呈"阿米巴状"。损毁侧GFAP阳性细胞数量较健侧明显增加(P0.01),突起变短,染色加深。2周模型组和8周模型组DA能神经元及两种胶质细胞的变化情况相似。结论 PD大鼠模型中存在着小胶质细胞和星形胶质细胞的激活,且两种胶质细胞的活化程度在PD发病过程中的不同时间无明显差别。     

硫辛酸对帕金森模型大鼠黑质内神经胶质细胞及多巴胺能神经元的影响

目的:探讨硫辛酸(LA)对帕金森病(PD)模型大鼠黑质内星形胶质细胞胶质纤维酸性蛋白(GFAP)、小胶质细胞离子钙接头蛋白(Iba-l)、酪氨酸羟化酶(TH)表达的影响。方法:130只雄性SD大鼠随机分为对照组(颅内注射生理盐水)30只、模型组(颅内注射6-OHDA)100只。30只对照组大鼠术后4周再随机取20只作为假手术组;100只模型组大鼠4周后随机取80只成功模型再随机分为PD模型组和硫辛酸干预低、中、高剂量组,每组20只。模型组采用立体定位仪定向注射6-OHDA。对照组定向注射等体积的生理盐水。硫辛酸低、中、高剂量组于PD模型大鼠成功术后4周分别每日腹腔注射15、30、60 mg/kg,连续注射14 d。干预结束后取各组大鼠右侧中脑黑质采用Western Blot方法检测GFAP和Iba-l的表达,采用免疫组化方法检测黑质内TH的表达情况。结果:(1)与假手术组比较,PD模型组及硫辛酸干预低、中、高剂量组大鼠黑质内GFAP及Iba-1的表达均明显有所增加(P<0.05),但TH阳性细胞数则明显有所减少(P<0.01);(2)与PD模型组比较,硫辛酸干预低、中、高剂量组大鼠黑质内GFAP及Iba-1表达均明显减少(P<0.05),而TH阳性细胞数则明显增加(P<0.05);(3)与硫辛酸干预低剂量组比较,硫辛酸干预中、高剂量组大鼠黑质内GFAP及Iba-1表达均明显减少(P<0.05),而TH阳性细胞数则明显有所增加(P<0.01);(4)硫辛酸干预中、高剂量组间大鼠黑质内GFAP及Iba-1的表达虽无显著差异(P>0.05),但TH阳性细胞数则明显有所增加(P<0.05)。结论:硫辛酸通过抑制帕金森病模型大鼠黑质内星形胶质细胞和小胶质细胞的过度表达,保护多巴胺能神经元,该结果可为帕金森病的治疗提供新的思路。     

尼古丁对Parkinson小鼠黑质多巴胺能神经元及小胶质细胞的影响

为探讨尼古丁对多巴胺能神经元的保护作用,本研究采用尼古丁(0.25mg/kg)预处理C57BL/6J小鼠,再给予1-甲基-2-乙基-1,2,3,6-四氢吡啶(MPTP;20mg/kg)诱导Parkinson病(PD)小鼠模型。通过行为学检测和中脑黑质致密部(SNc)的酪氨酸羟化酶(TH)以及OX-42的免疫组织化学染色,观察了尼古丁对PD小鼠的行为以及黑质多巴胺能神经元和小胶质细胞的影响。结果显示:经尼古丁预处理可以明显减轻PD小鼠的行为障碍,增加TH免疫阳性的多巴胺能神经元的数量,并且可以抑制SNc内小胶质细胞的增生。本研究结果提示,尼古丁可保护多巴胺能神经元,而抑制小胶质细胞的增生可能是其作用机制。     

硫辛酸对LPS诱导的帕金森病小鼠黑质多巴胺能神经元损伤的影响

目的:探讨硫辛酸(LA)对脂多糖(LPS)诱导的帕金森病(PD)模型小鼠黑质多巴胺(DA)能神经元损伤的作用及机制。方法:10月龄雌性C57BL/6小鼠,随机分为生理盐水对照组、PD模型组和LA干预组。采用鼻腔滴入LPS制作PD小鼠模型,通过爬竿实验及酪氨酸羟化酶和p-NF-κB/p65的免疫组化技术观察LA对DA能神经元的保护作用。结果:LPS经鼻可以成功诱导小鼠PD模型,给予LA可明显恢复小鼠运动,减少黑质DA能神经元的丢失、抑制小胶质细胞及其NF-κB信号通路激活。结论:LA干预可缓解LPS经鼻诱导的小鼠PD行为学和病理学的改变,其作用机制可能与抑制脑内的炎症反应有关。     

血管活性肠肽对树突状细胞免疫调节作用研究进展

神经系统与免疫系统通过神经肽、神经递质和细胞因子相互作用。血管活性肠肽(VIP)作为一种重要的神经肽参与炎症反应和免疫应答的调控。近年研究表明VIP能通过与树突状细胞(DC)表面的受体结合调节DC的表型和功能成熟,在DC体内迁移和功能成熟中发挥重要作用。VIP主要通过调节DC膜表面黏附分子和共刺激分子的表达、趋化因子及其受体的表达和趋化活性,诱导Th细胞分化以及抗原提呈功能发挥作用。     

血管活性肠肽在MPTP帕金森病模型小鼠的抗氧化应激作用

目的:研究血管活性肠肽(VIP)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导的帕金森病(PD)模型小鼠发挥抗氧化应激和神经保护作用。方法:雄性C57BL/6J小鼠随机分为生理盐水(NS)组、MPTP组和MPTP+VIP组。Elisa法检测纹状体丙二醛(MDA)以及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的变化;免疫组织化学法观察中脑黑质纹状体系统酪氨酸羟化酶(TH)、星形胶质细胞特异性标记物胶质细胞纤维酸性蛋白(GFAP)和小胶质细胞标志物离子钙结合蛋白(Iba-1)的表达变化;透射电子显微镜观察中脑黑质多巴胺能神经元的超微结构变化。结果:MPTP组与对照组相比,MDA水平显著增高,SOD和CAT的表达显著降低;给予VIP可显著抑制MDA的水平(P0.01),增强SOD和CAT的表达(P0.05)。与对照组相比,MPTP组小鼠GFAP和Iba-1的表达明显上升,TH表达明显下降;给予VIP可显著降低GFAP和Iba-1的表达(P0.05),而TH表达明显增强。透射电镜观察显示:NS组神经细胞和细胞器结构清晰完整;MPTP组神经细胞核膜内陷,线粒体空泡样变;MPTP+VIP组神经细胞和细胞器结构基本正常。结论:VIP能够抑制MPTP诱导PD小鼠中脑黑质星形胶质细胞和小胶质细胞的活化,对抗氧化应激,发挥神经保护作用。     

尼古丁对PD大鼠黑质多巴胺能神经元变性的影响

目的 探讨尼古丁对帕金森病(PD)大鼠黑质多巴胺能神经元变性的影响及其机制. 方法 45只大鼠随机分为PBS对照组(CON)、生理盐水+ 脂多糖(NS)组、尼古丁+脂多糖(NIC)组,每组15只.黑质内立体定向注射脂多糖(LPS)或PBS后24h,免疫印迹法检测黑质诱导性一氧化氮合酶(iNOS)蛋白表达变化;黑质注射药物后14d,采用免疫组织化学法观察大鼠黑质酪氨酸羟化酶(TH)阳性神经元数量及OX-42阳性细胞形态学变化,RT-PCR及免疫印迹检测黑质TH mRNA及TH蛋白的表达水平. 结果 与CON组相比,NS组大鼠黑质iNOS表达明显增多,TH阳性神经元、TH mRNA及TH蛋白明显减少,小胶质细胞大多呈胞体大突起短粗的形态;NIC组黑质iNOS表达明显少于NS组,黑质TH阳性神经元、TH mRNA及TH蛋白表达较NS组明显增多,大部分小胶质细胞呈胞体小,突起细长的形态. 结论 尼古丁可以减轻LPS介导的多巴胺能神经元变性,对多巴胺能神经元有保护作用,其保护机制与抑制小胶质细胞激活、减少iNOS的表达有关.     

6-OHDA所致PD大鼠模型黑质多巴胺能神经元与胶质细胞的变化

目的 应用6-羟多巴(6.OHDA)建立帕金森病(PD)大鼠模型。方法将6-OHDA立体注入大鼠前脑内侧束,应用免疫组织化学法检测6-OHDA注射后第3、7及14天后多巴胺能神经元、小胶质细胞与星形胶质细胞数量及形态的变化。用显微镜专业摄像头采集图像,计算机图像分析软件测量平均灰度值。结果 损伤侧与对侧相比,TH阳性多巴胺能神经元数量减少,损伤侧平均灰度值增加;小胶质细胞与星形胶质细胞显著增生,二者损伤侧的平均灰度值下降,经配对t检验,各组内比较差异均具有显著性意义(P<0.01);增生活化的小胶质细胞与星形胶质细胞形态发生明显的改变。结论 6-OHDA能成功建立PD大鼠模型。     

GABAergic control of rat substantia nigra dopaminergic neurons: role of globus pallidus and substantia nigra pars reticulata

Dopaminergic neurons in vivo fire spontaneously in three distinct patterns or modes. It has previously been shown that the firing pattern of substantia nigra dopaminergic neurons can be differentially modulated by local application of GABA(A) and GABA(B) receptor antagonists. The GABA(A) antagonists, bicuculline or picrotoxin, greatly increase burst firing in dopaminergic neurons whereas GABA(B) antagonists cause a modest shift away from burst firing towards pacemaker-like firing. The three principal GABAergic inputs to nigral dopaminergic neurons arise from striatum, globus pallidus and from the axon collaterals of nigral pars reticulata projection neurons, each of which appear to act in vivo primarily on GABA(A) receptors (see preceding paper). In this study we attempted to determine on which afferent pathway(s) GABA(A) antagonists were acting to cause burst firing. Substantia nigra dopaminergic neurons were studied by single unit extracellular recordings in urethane anesthetized rats during pharmacologically induced inhibition and excitation of globus pallidus. Muscimol-induced inhibition of pallidal neurons produced an increase in the regularity of firing of nigral dopaminergic neurons together with a slight decrease in firing rate. Bicuculline-induced excitation of globus pallidus neurons produced a marked increase in burst firing together with a modest increase in firing rate. These changes in firing rate were in the opposite direction to what would be expected for a monosynaptic GABAergic pallidonigral input. Examination of the response of pars reticulata GABAergic neurons to similar manipulations of globus pallidus revealed that the firing rates of these neurons were much more sensitive to changes in globus pallidus neuron firing rate than dopaminergic neurons and that they responded in the opposite direction. Pallidal inhibition produced a dramatic increase in the firing rate of pars reticulata GABAergic neurons while pallidal excitation suppressed the spontaneous activity of pars reticulata GABAergic neurons. These data suggest that globus pallidus exerts significant control over the firing rate and pattern of substantia nigra dopaminergic neurons through a disynaptic pathway involving nigral pars reticulata GABAergic neurons and that at least one important way in which local application of bicuculline induces burst firing of dopaminergic neurons is by disinhibition of this tonic inhibitory input.     

色钉菇乙酸乙酯提取物对帕金森病模型小鼠黑质多巴胺神经元的保护作用

目的:探讨色钉菇乙酸乙酯提取物能否改善帕金森病(PD)小鼠的行为症状以及对黑质多巴胺(DA)能神经元保护作用。方法:以成年C57BL/6小鼠为对象,设置空白对照组、MPTP模型组和100 mg/kg、200 mg/kg、400 mg/kg三个剂量的色钉菇乙酸乙酯提取物的干预组。通过站立次数和爬杆得分等指标评价行为症状,以TH的免疫荧光方法检测存活的黑质DA能神经元,FJC染色法显示黑质致密部变性的DA能神经元。结果:在行为上,实验处理之前各组之间无显著性差异(P0.05);而实验处理后,于站立次数指标上,400 mg/kg剂量干预组显著地高于实验对照组(P0.05),与空白对照组无显著性差异(P0.05);在爬杆得分指标上,200 mg/kg组和400 mg/kg组均显著地高于实验对照组(P0.05),但与空白对照组无显著性差异(P0.05)。在TH标记的黑质多巴胺能神经元数量上,只有400 mg/kg组显著地高于实验对照组(P0.05),与空白对照组无显著性差异(P0.05)。而FJC标记的变性细胞只在MPTP组、100 mg/kg和200 mg/kg组表达,空白对照组和400 mg/kg组未见FJC阳性细胞。结论:色钉菇乙酸乙酯提取物能显著地改善PD的行为症状和保护黑质DA能神经元,且呈剂量依赖性。     

Meloxicam reduces lipopolysaccharide-induced degeneration of dopaminergic neurons in the rat substantia nigra pars compacta

Inflammation is believed to play an important role in the etiology and pathogenesis of Parkinson's disease (PD). However, experimental and epidemiological evidences from various non-steroidal anti-inflammatory drugs, including cyclooxygenase-2 (COX-2) inhibitors, seem contradictive. Using the intranigral lipopolysaccharide (LPS) rat model, we show that meloxicam, a preferential COX-2 inhibitor, diminishes the activation of OX-42-immunoreactive (ir) microglia and reduces the loss of tyrosine hydroxylase (TH)-ir dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) that is normally induced by exposure to LPS. Double-labelling immunohistochemistry identified that activated microglia rather than intact resting microglia are the main intracellular venues for COX-2 expression. These findings suggest that inhibition of COX-2 activity in activated microglial cells may be potentially neuroprotective for DA neurons in the SNpc.     

血管活性肠肽对MPTP亚急性帕金森病模型小鼠突触的影响

目的 探讨血管活性肠肽(VIP) 对1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）诱导的帕金森病(PD)小鼠模型突触及其行为学的影响. 方法 雄性C57BL/6 J小鼠30只,随机分为生理盐水(NS)组、MPTP组、MPTP+VIP组。利用Tru Scan系统测定小鼠的行为学变化;免疫组织化学染色法检测黑质和纹状体区酪氨酸羟化酶(TH)的表达及纹状体区突触小泡膜蛋白 2（VAMP2）和突触素1 (SYN1)的表达变化;运用免疫印迹法检测VAMP2和SYN1的表达变化;并通过透射电子显微镜观察黑质区突触的结构改变。 结果 行为学结果统计学分析,MPTP组小鼠的垂直运动距离比对照组明显减少,而给予VIP的小鼠明显高于MPTP组小鼠。 MPTP组TH、VAMP2和SYN1表达均明显少于NS组,而MPTP+VIP组显著高于MPTP组。透射电子显微镜结果显示,NS组神经突触结构完整; 模型组神经突触数量少,突触间隙不清,突触前成分的线粒体出现髓样变,并且突触小泡数量减少;MPTP+VIP组神经突触结构基本正常。 结论 VIP可使MPTP模型小鼠VAMP2和SYN1的表达上调,减少突触结构的损伤,保护黑质纹状体系统TH的表达,从而改善其运动状态。     

6-Hydroxydopamine induces the loss of the dopaminergic phenotype in substantia nigra neurons of the rat

Intraparenchymal injections of the neurotoxin 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle in rats destroys the dopaminergic neurons in the pars compacta of the substantia nigra. In other transmitter systems it has been found that axotomy or neurotoxin exposure produces an initial loss of neurotransmitter phenotype, with cell death occurring over a much slower time course. To determine whether this also occurs in dopamine neurons after 6-OHDA, two approaches were utilized. First, the effect of injections of 6-OHDA into the medial forebrain bundle on nigral dopaminergic neurons was studied using combined fluorogold and immunocytochemical labeling. Four weeks after the 6-OHDA injection, there was an 85% reduction in the number of tyrosine hydroxylase (TH)-immunoreactive cells on the lesioned side. In contrast, there was only a 50% reduction in the number of fluorogold-labeled cells on the lesioned side. Second, the time course of the rescue of dopaminergic neurons after 6-OHDA by glial cell line-derived neurotrophic factor (GDNF) was determined using TH immunocytochemistry. Greater numbers of dopamine neurons were rescued 9 weeks after GDNF, compared with counts made 5 weeks after GDNF. Taken together, these results suggest loss of dopaminergic phenotype is greater than cell loss following 6-OHDA injections, and that GDNF restores the phenotype of affected cells.     

黑质内炎症反应对恒河猴多巴胺能神经元的毒性作用

目的 后存活24周、48周和生理盐水注射后存活48周.上述5只动物于存活前0周、8周、16周,向右侧黑质分3次注射脂多糖或生理盐水.术后用免疫组织化学染色及高效液相色谱法观察黑质TH阳性神经元的变化和纹状体神经递质含量的改变. 结果 急性实验组注射侧与对照侧TH表达量无明显差异,但注射侧可见小胶质细胞活化,人类白细胞DR抗原表达量上调并有大量肿瘤坏死因子α、白介素1β、环氧化酶2(COX-2)生成.慢性实验组单侧黑质脂多糖注射24周和48周后,各个动物注射侧黑质阳性神经元数量以及纹状体神经递质含量有不同程度的减少,而生理盐水注射动物无变化. 结论 脂多糖注射后在1周内可引起黑质剧烈的炎症反应,但这种病理过程并不伴随大量多巴胺能神经元死亡.随病程的发展,炎症反应能引起恒河猴黑质多巴胺能神经元慢性进行性的损伤.该病理过程较好模拟了帕金森病的发病特点.     

Expression and functional properties of TRPM2 channels in dopaminergic neurons of the substantia nigra of the rat

Transient receptor potential melastatin 2 (TRPM2) channels are sensitive to oxidative stress, and their activation can lead to cell death. Although these channels have been extensively studied in expression systems, their role in the brain, particularly in the substantia nigra pars compacta (SNc), remains unknown. In this study, we assessed the expression and functional properties of TRPM2 channels in rat dopaminergic SNc neurons, using acute brain slices. RT-PCR analysis revealed TRPM2 mRNA expression in the SNc region. Immunohistochemistry demonstrated expression of TRPM2 protein in tyrosine hydroxylase-positive neurons. Channel function was tested with whole cell patch-clamp recordings and calcium (fura-2) imaging. Intracellular application of ADP-ribose (50-400 μM) evoked a dose-dependent, desensitizing inward current and intracellular free calcium concentration ([Ca(2+)](i)) rise. These responses were strongly inhibited by the nonselective TRPM2 channel blockers clotrimazole and flufenamic acid. Exogenous application of H(2)O(2) (1-5 mM) evoked a rise in [Ca(2+)](i) and an outward current mainly due to activation of ATP-sensitive potassium (K(ATP)) channels. Inhibition of K(+) conductance with Cs(+) and tetraethylammonium unmasked an inward current. The inward current and/or [Ca(2+)](i) rise were partially blocked by clotrimazole and N-(p-amylcinnamoyl)anthranilic acid (ACA). The H(2)O(2)-induced [Ca(2+)](i) rise was abolished in "zero" extracellular Ca(2+) concentration and was enhanced at higher baseline [Ca(2+)](i), consistent with activation of TRPM2 channels in the cell membrane. These results provide evidence for the functional expression of TRPM2 channels in dopaminergic SNc neurons. Given the involvement of oxidative stress in degeneration of SNc neurons in Parkinson's disease, further studies are needed to determine the pathophysiological role of these channels in the disease process.