脂多糖诱导黑质多巴胺能神经元变性中小胶质细胞的反应

目的探讨小胶质细胞活化规律与脂多糖(Lipopolysaccharide,LPS)诱导黑质多巴胺(DA)能神经元变性的关系。方法脑立体定位注射LPS入大鼠脑黑质后,采用特异性抗体0x．42标记不同时间点小胶质细胞的激活情况;酪氨酸羟化酶(tyrosine-hydroxylase,TH)免疫组织化学观察DA能神经元损害变化。结果小胶质细胞在LPS注入黑质6h后开始出现部分激活。12h大部分激活,为“灌木丛样”小胶质细胞;24h已完全激活,呈现“阿米巴样”,在随后的30d内,基本维持在此形态。而TH阳性细胞数在第3d开始出现下降,与对照组相比下降达45％,14d时下降至5～10％,至30d时几乎完全消失。结论小胶质细胞的激活先于DA能神经元变性,其激活介导的炎症反应在PD发病中具有重要的神经破坏作用。     

6-OHDA制备的帕金森病大鼠黑质神经元的超微结构改变

目的观察6-羟多巴胺(6-OHDA)单侧注射制备的帕金森病(PD)大鼠多巴胺(DA)能神经元的超微结构改变。方法单侧微量注射6-OHDA制备PD大鼠模型,用免疫荧光组织化学方法观察正常侧与6-OHDA注射侧黑质酪氨酸羟化酶(TH)阳性神经细胞及神经纤维的变化;并利用免疫电镜技术观察大鼠正常侧与注射侧黑质致密部DA能神经元的超微结构。结果免疫荧光法显示注射侧黑质致密部TH阳性细胞数和网状部TH阳性纤维面积与正常侧的百分比平均值分别为21.83%,23.19%。免疫电镜显示:TH免疫反应阳性产物表达于PD大鼠正常侧DA能神经元的高尔基复合体质膜面及胞质内,电子密度较高,注射侧很少见或几乎未见,且注射侧线粒体嵴有不同程度的溶解,呈空泡样变或髓样变,粗面内质网脱颗粒。结论6-OHDA可引起DA能神经元发生凋亡的超微结构改变。     

Lactacystin诱导帕金森病大鼠黑质胶质细胞的变化

目的观察蛋白酶体抑制剂Lactacystin诱导大鼠黑质胶质细胞的变化、炎性介质NF-κB的表达。方法采用立体定向术将蛋白酶体抑制剂Lactacystin 10μg注射至大鼠黑质部位,免疫组织化学法观察黑质区多巴胺(DA)能神经元、小胶质细胞、星形胶质细胞的变化,炎性介质核转录因子κB(NF-κB)的表达。结果注射Lactacystin 3周,阿朴吗啡腹腔注射后出现典型旋转行为;8周后实验组大鼠损毁侧黑质酪氨酸羟化酶(TH)阳性细胞数明显减少,黑质小胶质细胞及星形胶质细胞的数量均增加,NF-κB表达增强。结论蛋白酶体抑制剂Lactacystin能激活大鼠黑质小胶质细胞及星形胶质细胞,诱导炎性介质表达。     

甲基苯丙胺中毒大鼠相关脑区多巴胺能神经毒性研究

目的:探讨MA中毒多巴胺能神经毒性的损伤机制。方法:将Wistar大鼠40只,随机分成对照组10只和实验组30只(实验组分成三个亚组,分为末次给药后1天组、4天组和7天组,n=10)。实验组给予20mg/kg的MA腹腔注射,对照组给予同样剂量的生理盐水,每天注射一次,注射时间为20:00,连续注射4天。分别于末次给药后1天,7天,14天处死实验大鼠,用免疫组织化学染色法(S-P法)和荧光分光光度计法检测大鼠中脑黑质致密区(SNC)、中脑腹侧被盖区(VTA)、前额叶皮质(PFC)以及纹状体(CPu)四个脑区的多巴胺神经元细胞的形态和数量的变化,对神经纤维进行灰度值分析。结果:1、黑质致密区和腹侧被盖区TH阳性细胞图像分析结果与细胞计数分析结果一致:与对照组相比,各实验组TH免疫反应阳性降低,差异具显著性(P<0.05),d1组开始降低(P<0.05),d7组达到低谷(P<0.01),d14天组黑质致密区和腹侧被盖区TH免疫反应阳性有不同程度的恢复(P<0.05)。2、纹状体和前额叶皮质TH阳性纤维图像定量分析结果:各实验组TH免疫反应阳性均减低(P<0.05),d7组阳性反应最弱(P<0.01),d14组仍未恢复(P<0.05)。3、黑质致密区、腹侧被盖区、纹状体及前额叶皮质荧光分光光度计检测DA递质含量结果:与上述免疫组化结果基本一致。结论:1、大鼠各脑区TH阳性表达和DA含量,均出现不同程度的减低。2、MA中毒大鼠各脑区DA递质含量的变化与TH的变化结果基本一致。     

帕金森病大鼠模型中脑腹侧被盖区多巴胺能神经元改变及c-Jun蛋白表达

目的探讨帕金森病(Parkinson disease,PD)大鼠模型中脑腹侧被盖区(ventral tegmental area,VTA)多巴胺能神经元的改变及其c-Jun蛋白表达情况,探讨其可能机制。方法应用6-羟基多巴胺(6-hydroxydopamine,6-OHDA)单侧一点注射大鼠黑质致密区(substantia nigra compacta,SNc),特异性毁损DA能神经元;术后1d、7d、14d、21d腹腔注射阿朴吗啡(apomorphine,APO)诱发行为学观察、电镜、尼氏染色观察中脑VTA神经元的改变,免疫组织化学ABC观察其DA能神经元酪氨酸羟化酶(tyrosine hydroxylase,TH)及c-Jun的改变并进行图像分析,Western blot观察c-Jun蛋白表达。结果APO诱发PD大鼠模型异常旋转行为,尼氏染色及电镜见PD大鼠中脑VTA有神经细胞肿胀、坏死等变化,VTA TH阳性(TH )神经元数量减少,形态学改变。APO诱导的旋转实验≥7转/min,VTA毁损侧c-Jun表达。结论中脑VTA DA能神经参与PD模型大鼠的改变;APO能诱导6-OHDA PD模型大鼠的旋转行为,其强弱可能与TH 神经元数量直接相关;c-Jun表达与DA能神经元毁损程度有一定的关系。     

DJ-1保护大鼠中脑黑质多巴胺能神经元抵抗鱼藤酮损伤的研究

目的:探讨过表达DJ-1蛋白能否保护大鼠中脑黑质多巴胺能神经元抵抗鱼藤酮所致的急性损伤.方法:构建表达DJ-1基因的腺相关病毒载体(rAAV-DJ-1)并转染HEK-293细胞,进行腺相关假病毒颗粒包装,所得假病毒颗粒注射到大鼠脑内感染黑质神经细胞;4周后,注射鱼藤酮于大鼠黑质相同脑区;通过免疫组织化学和免疫印迹试验鉴定TH蛋白表达情况,采用动物行为轨迹分析软件计算大鼠30 min内运动距离以及强迫游泳试验鉴定大鼠精神症状.结果:与对照组相比,过表达D J-1组大鼠运动损伤症状较轻,精神症状改善明显;损伤侧黑质TH阳性神经元数目和TH蛋白表达水平显著高于对照组.结论:过表达DJ-1蛋白能保护大鼠黑质多巴胺能神经元抵抗鱼藤酮所致的急性损伤,延缓多巴胺能神经元的退行性变,提示DJ-1可能对帕金森病的治疗有较好的应用前景.     

甲基苯丙胺中毒大鼠相关脑区多巴胺能神经毒性研究

目的：探讨MA中毒多巴胺能神经毒性的损伤机制。方法：将Wistar大鼠40只,随机分成对照组10只和实验组30只（实验组分成三个亚组,分为末次给药后1天组、4天组和7天组,n=10）。实验组给予20mg/kg的MA腹腔注射,对照组给予同样剂量的生理盐水,每天注射一次,注射时间为20：00,连续注射4天。分别于末次给药后1天,7天,14天处死实验大鼠,用免疫组织化学染色法（S-P法）和荧光分光光度计法检测大鼠中脑黑质致密区（SNC）、中脑腹侧被盖区（VTA）、前额叶皮质（PFC）以及纹状体（CPu）四个脑区的多巴胺神经元细胞的形态和数量的变化,对神经纤维进行灰度值分析。结果：1、黑质致密区和腹侧被盖区TH阳性细胞图像分析结果与细胞计数分析结果一致：与对照组相比,各实验组TH免疫反应阳性降低,差异具显著性（P〈0.05）,d1组开始降低（P〈0.05）,d7组达到低谷（P〈0.01）,d14天组黑质致密区和腹侧被盖区TH免疫反应阳性有不同程度的恢复（P〈0.05）。2、纹状体和前额叶皮质TH阳性纤维图像定量分析结果：各实验组TH免疫反应阳性均减低（P〈0.05）,d7组阳性反应最弱（P〈0.01）,d14组仍未恢复（P〈0.05）。3、黑质致密区、腹侧被盖区、纹状体及前额叶皮质荧光分光光度计检测DA递质含量结果：与上述免疫组化结果基本一致。结论：1、大鼠各脑区TH阳性表达和DA含量,均出现不同程度的减低。2、MA中毒大鼠各脑区DA递质含量的变化与TH的变化结果基本一致。     

PD大鼠胼胝体和扣带胶质原纤维酸性蛋白的表达

目的观察不同病程帕金森病(PD)大鼠胼胝体和扣带胶质原纤维酸性蛋白(GFAP)的表达。方法大鼠右侧前脑内侧束注射六羟基多巴胺制备PD模型。大鼠分为正常对照组,2周、4周和6周模型组。以酪氨酸羟化酶(TH)和GFAP抗体免疫组化阳性分别显示黑质的多巴胺能神经元、胼胝体与扣带处的星形胶质细胞。结果 2、4、6周模型组右侧黑质TH阳性细胞数较正常对照组均显著降低,而2、4、6周模型组之间无显著差异。正常对照组和4周模型组胼胝体和扣带处星形胶质细胞呈未活化状态,两组之间的GFAP表达强度和细胞密度均无显著差异。2周和6周模型组两部位星形胶质细胞呈活化状态,其表达强度和细胞密度均显著高于正常对照组和4周模型组。结论急性完全损伤PD模型大鼠注射侧胼胝体和扣带处GFAP表达随病程呈增高、降低和增高趋势。     

电针足三里穴对糖尿病胃轻瘫大鼠延髓多巴胺能神经元和 星形胶质细胞活性的影响

目的:研究电针足三里穴对糖尿病胃轻瘫大鼠延髓多巴胺能神经元内酪氨酸羟化酶(tyrosine hydroxylase,TH)和星形胶质细胞内胶质原纤维酸性蛋白(Glial Fibrillary Acidic Protein,GFAP)表达的影响。方法:32只实验大鼠分为空白对照(空白)组、糖尿病胃轻瘫模型(模型)组、模型组+电针足三里穴(足三里)组和模型组+电针非经非穴(非经非穴)组(每组8只)。模型制备采用腹腔注射5%四氧嘧啶和熟地灌胃诱导的方法。实验3周后取大鼠延髓进行抗TH和抗GFAP的单一和双重免疫组化染色,观察并记数TH和GFAP在延髓内的表达。结果:与空白组比较,各实验组TH阳性多巴胺能神经元和GFAP阳性星形胶质细胞集中表达于延髓迷走孤束复合体内,有明显的定位特点;高倍镜下观察到TH阳性神经元周围有大量GFAP阳性星形胶质细胞包绕。各组TH和GFAP表达以模型组最高;而足三里组TH阳性多巴胺能神经元数量明显减少(31.3±4.4→16.8±3.2),GFAP阳性产物表达明显降低(113.8±7.6→95.4±8.4),且它们之间有统计学意义(P<0.01);非经非穴组与模型组之间差异没有统计学意义。结论:针刺调节糖尿病胃运动功能障碍大鼠与其调控延髓多巴胺能神经元及其周围的星形胶质细胞功能活动有关。     

HLRP 蛋白在大鼠脑中的定位及LPS 对其表达的影响

目的:研究HLRP分子在大鼠脑中的细胞定位和表达特点,观察LPS刺激对动物脑HLRP表达的影响。方法:对原代培养的大鼠神经元、星形胶质细胞、小胶质细胞和大鼠脑组织冰冻切片分别进行免疫荧光染色,观察HLRP的细胞定位和表达特点;给大鼠侧脑室注射LPS,提脑组织蛋白,进行Western blot检测,半定量分析LPS刺激后,大鼠脑HLRP的表达变化。结果:①HLRP选择性表达于部分神经元的细胞核中,正常的星形胶质细胞和小胶质细胞不表达HLRP。②从嗅脑到脑干各节段,HLRP在大鼠脑组织中均匀分布,未发现HLRP阳性神经元聚集的现象。③侧脑室注射LPS 1天以后,HLRP表达明显升高(P<0.05)。结论:大鼠脑中正常表达HLRP,侧脑室注射LPS能刺激HLRP表达。     

Changes in somatodendritic but not terminal dopamineregulation in aged rhesus monkeys

For these studies, young (8-9 years), middle-aged (14-17 years) and aged (23-28 years) rhesus monkeys were used as a model of normal aging in humans to investigate changes in dopamine (DA)-containing neurons in senescence. Aged monkeys exhibited significant age-related motoric declines as compared to the young animals. In vivo microdialysis studies showed that basal levels of the DA metabolites, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were diminished by 44% and 79%, respectively, in the substantia nigra (SN) of aged monkeys. In addition, d-amphetamine-evoked overflow of DA in the SN was diminished by 30% in the middle-aged animals and 67% in the aged monkeys. Post-mortem measures of DA and DA metabolites showed significant decreases in DA (20%), DOPAC (47%) and HVA (22%) levels in the putamen and a 25% decline in HVA tissue levels in the SN of the aged monkeys as compared to the young animals. Unbiased stereological cell counting of tyrosine hydroxylase (TH)-immunoreactive neurons in the SN showed a small (15-20%) but significant age-related decline in TH-positive neurons. In addition, there was a small (15-20%) but significant decline in TH-positive fiber density and TH-positive cell size. In comparison to the massive loss of DA neurons responsible for the movement dysfunctions seen in Parkinson's disease, pronounced functional changes in DA release in the SN and putamen may significantly contribute to the motoric dysfunctions characterizing normal aging in rhesus monkeys.     

Persistent MDMA-induced dopaminergic neurotoxicity in the striatum and substantia nigra of mice

Acute administration of repeated doses of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) dramatically reduces striatal dopamine (DA) content, tyrosine hydroxylase (TH), and DA transporter-immunoreactivity in mice. In this study, we show for the first time the spatiotemporal pattern of dopaminergic damage and related molecular events produced by MDMA administration in mice. Our results include the novel finding that MDMA produces a significant decrease in the number of TH-immunoreactive neurons in the substantia nigra (SN). This decrease appears 1 day after injection, remains stable for at least 30 days, and is accompanied by a dose-dependent long-lasting decrease in TH- and DA transporter-immunoreactivity in the striatum, which peaked 1 day after treatment and persisted for at least 30 days, however, some recovery was evident from day 3 onwards, evidencing sprouting of TH fibers. No change is observed in the NAc indicating that MDMA causes selective destruction of DA-containing neurons in the nigrostriatal pathway, sparing the mesolimbic pathway. The expression of Mac-1 increased 1 day after MDMA treatment and glial fibrillary acidic protein increased 3 days post-treatment in the striatum and SN but not in the NAc, in strict anatomical correlation with dopaminergic damage. These data provide the first evidence that MDMA causes persistent loss of dopaminergic cell bodies in the SN.     

鼠胚中脑多巴胺神经元的体外发育及靶细胞对其影响

１４天大鼠胚中脑腹侧神经元在体外进行分散培养,用抗酪氨酸羟化酶血清（ＴＨ）检测多巴胺（ＤＡ）神经元。培养２４小时即可见ＴＨ阳性神经元,３天后数量开始增加,高峰出现在１４天左右,此后逐渐减少。ＴＨ阳性神经元胞体随培养时间的延长渐增大,核浆比例减少,表明神经元趋向成熟。收集培养液用高效液相色谱一电化学仪（ＨＰＬＣ－ＥＣ）测定其中ＤＡ递质的含量,在培养最初的７２小时内其浓度极低,至１５天时为初７２小时的５０余倍,此后开始下降,与ＤＡ神经元的形态发育同步。将中脑腹侧多巴胺神经元与其靶细胞纹状体神经元共培养时．ＴＨ阳性神经元数明显增加．培养液中的ＤＡ浓度均为同期中脑腹侧神经元单纯培养时的一倍,ＴＨ活性也较单纯培养时为高,表明靶细胞能明显提高ＤＡ神经元的存活能力。     

Altered glutathione homeostasis in animals prenatally exposed to lipopolysaccharide

We previously reported that injection of bacterial lipopolysaccharide (LPS) into gravid female rats at embryonic day 10.5 resulted in a birth of offspring with fewer than normal dopamine (DA) neurons along with innate immunity dysfunction and many characteristics seen in Parkinson's disease (PD) patients. The LPS-exposed animals were also more susceptible to secondary toxin exposure as indicated by an accelerated DA neuron loss. Glutathione (GSH) is an important antioxidant in the brain. A disturbance in glutathione homeostasis has been proposed for the pathogenesis of PD. In this study, animals prenatally exposed to LPS were studied along with an acute intranigral LPS injection model for the status of glutathione homeostasis, lipid peroxidation, and related enzyme activities. Both prenatal LPS exposure and acute LPS injection produced a significant GSH reduction and increase in oxidized GSH (GSSG) and lipid peroxide (LPO) production. Activity of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo GSH synthesis, was up-regulated in acute supranigral LPS model but was reduced in the prenatal LPS model. The GCS light subunit protein expression was also down-regulated in prenatal LPS model. GSH redox recycling enzyme activities (glutathione peroxidase, GPx and glutathione reducdase, GR) and glutathione-S-transferase (GST), gamma-glutamyl transpeptidase (gamma-GT) activities were all increased in prenatal LPS model. Prenatal LPS exposure and aging synergized in GSH level and GSH-related enzyme activities except for those (GR, GST, and gamma-GT) with significant regional variations. Additionally, prenatal LPS exposure produced a reduction of DA neuron count in the substantia nigra (SN). These results suggest that prenatal LPS exposure may cause glutathione homeostasis disturbance in offspring brain and render DA neurons susceptible to the secondary neurotoxin insult.     

6-羟基多巴胺损毁大鼠内侧前脑束早期黑质-纹状体系统的铁水平与多巴胺能神经元退变的关系

应用快速周期伏安法(fast cyclic vohammetry,FCV)、原子吸收分光光度法及免疫组织化学方法,观察了6-羟基多巴胺(6-hydroxydopamine,6-OHDA)单侧损毁大鼠内侧前脑束(medial forebrain bundle,MFB)早期黑质(sub-stantia nigra,sN)铁水平与多巴胺(dopamine,DA)神经元损伤的变化,以及纹状体(striatum,Str)的DA释放。结果如下：6-OHDA单侧损毁大鼠MFB1d和3d后,SN的酪氨酸羟化酶(tyrosine hydroxylase,TH)阳性细胞分别下降了45％和66％;与正常鼠和未损毁侧相比,损毁侧SN的铁染色增强,铁浓度增加,而Str的DA释放量不变;6-OHDA损毁后1d与3d组相比,损毁侧的铁染色、铁浓度及DA释放量差别无显著性。上述结果表明,6-OHDA单侧损毁大鼠MFB的早期阶段,SN的DA能神经元数目中等程度减少时,铁染色及铁浓度即有增加,由于DA能神经系统有强大的代偿功能,使得Str的DA释放量仍趋于正常。     

Acidic Fibroblast Growth Factor and Catecholamines Synergistically Up-Regulate Tyrosine Hydroxylase Activity in Developing and Damaged Dopamine Neurons in Culture

Abstract: Our previous studies indicate that, in certain non-catecholamine (CA) neurons, expression of the gene for the CA biosynthetic enzyme tyrosine hydroxylase (TH) can be initiated by the obligatory interaction of acidic fibroblast growth factor (aFGF) and a CA activator. In this study, we sought to determine whether these same differentiation factors also play a role in regulating existing TH expression in CA neurons. Thus, the effects of exogenous aFGF and CAs on TH were studied in developing or toxin-damaged dopamine (DA) neurons from the embryonic day 15 rat ventral midbrain, where it was likely to be at physiologically low levels. Cultures were incubated with various concentrations of aFGF, DA, or aFGF and DA. Some cultures were first damaged with 2.5 µ M 1-methyl-4-phenylpyridinium. In developing DA neurons, an 80% increase in TH activity was found only after cotreatment with aFGF (100 ng/ml) and DA (1 µ M ) or other monoamines. Likewise, in damaged DA neurons, aFGF and DA reversed the 50% loss in TH activity caused by toxin. This was observed within 4 h of treatment and was not associated with changes in the number or appearance of DA neurons, suggesting a biochemical rather than a trophic effect. Pretreatment with protein or RNA synthesis inhibitors eliminated the increase. In PC12 cells, where TH is highly expressed, activity was unaltered by treatment. We conclude that the aFGF and CAs may be involved in not only the initiation but also the regulation of TH.     

A comprehensive study on long‐term injury to nigral dopaminergic neurons following intracerebroventricular injection of lipopolysaccharide in rats

Parkinson's disease (PD) is characterized by selective and progressive degeneration of dopaminergic neurons in the substantia nigra (SN). Lipopolysaccharide (LPS) can induce chronic inflammation and has been widely used to study the pathogenesis of PD. In this study, a single intracerebroventricular injection of LPS was used to induce neurotoxic effects on dopaminergic neurons in Sprague–Dawley rats. The long‐term neurotoxic effects of LPS were evaluated at different time points. Microglia were activated in the hippocampus and striatum at 4 weeks, and in the SN at 24 weeks. Astrocytes were activated in the hippocampus and nigrostriatal system at 2 and 24 weeks. The expression of brain‐derived neurotrophic factor in the SN increased at 4 weeks and decreased after 12 weeks, and tyrosine hydroxylase‐positive neurons in the SN were shown to have an atrophic appearance, with cell loss evident after 24 weeks. Phospho‐α‐synuclein expression, a reflection of parkinsonian pathogenesis, increased at 12 weeks, and peaked at 24 weeks. Abnormal motor behavior appeared at 16 weeks and lasted up to 48 weeks. These results indicate that microglia are activated for several months after a single, low dose injection of LPS, which eventually results in progressive and selective damage to dopaminergic neurons in the SN.     

Nigral GFRα1 Infusion in Aged Rats Increases Locomotor Activity, Nigral Tyrosine Hydroxylase, and Dopamine Content in Synchronicity

Delivery of exogenous glial cell line-derived neurotrophic factor (GDNF) increases locomotor activity in rodent models of aging and Parkinson’s disease in conjunction with increased dopamine (DA) tissue content in substantia nigra (SN). Striatal GDNF infusion also increases expression of GDNF’s cognate receptor, GFRα1, and tyrosine hydroxylase (TH) ser31 phosphorylation in the SN of aged rats long after elevated GDNF is no longer detectable. In aging, expression of soluble GFRα1 in the SN decreases in association with decreased TH expression, TH ser31 phosphorylation, DA tissue content, and locomotor activity. Thus, we hypothesized that, in aged rats, replenishing soluble GFRα1 in SN could reverse these deficits and increase locomotor activity. We determined that the quantity of soluble GFRα1 in young adult rat SN is ~3.6 ng. To replenish age-related loss, which is ~30 %, we infused 1 ng soluble GFRα1 bilaterally into SN of aged male rats and observed increased locomotor activity compared to vehicle-infused rats up to 4 days following infusion, with maximal effects on day 3. Five days after infusion, however, neither locomotor activity nor nigrostriatal neurochemical measures were significantly different between groups. In a separate cohort of male rats, nigral, but not striatal, DA, TH, and TH ser31 phosphorylation were increased 3 days following unilateral infusion of 1 ng soluble GFRα1into SN. Therefore, in aged male rats, the transient increase in locomotor activity induced by replenishing age-related loss of soluble GFRα1is temporally matched with increased nigral dopaminergic function. Thus, expression of soluble GFRα1 in SN may be a key component in locomotor activity regulation through its influence over TH regulation and DA biosynthesis.