帕金森病大鼠胃肠功能障碍的机制

目的 探讨6-羟基多巴胺(6-OHDA)诱导的SD大鼠帕金森病(PD)模型胃肠功能障碍的发生机制.方法 60只SD大鼠随机分为6-OHDA组和对照组;以6-OHDA诱导制备PD大鼠模型.4周后收集大鼠1h粪便排出量,计算粪便含水量,测定餐后2 h大鼠胃内固体食物残留率.采用免疫组化法检测黑质酪氨酸羟化酶(TH)、胃肠神经丛α-突触核蛋白(α-syn)、神经元型一氧化氮合酶(nNOS)的表达;应用逆转录(RT)-PCR方法检测胃、结肠组织nNOS mRNA的表达.结果 与对照组相比,6-OHDA组大鼠1 h粪便排出量及含水量明显降低,胃内固体食物残留率明显增加(均P<0.01);损伤侧黑质TH阳性细胞明显减少(P<0.01);胃肠肌间神经丛α-syn表达明显升高,nNOS表达明显降低(均P<0.01);胃肠组织nNOS mRNA表达明显降低(均P<0.01).结论 PD大鼠胃肠功能障碍可能与胃肠神经系统nNOS水平降低有关.     

司来吉兰对帕金森病模型大鼠结肠α-突触核蛋白和神经元型一氧化氮合酶表达的影响

目的观察司来吉兰(selegiline)对帕金森病(Parkinson disease,PD)模型大鼠结肠功能障碍及结肠α-突触核蛋白(α-Synuclein,α-Syn)及神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)表达的影响,探讨司来吉兰对PD患者结肠功能障碍的治疗作用及可能机制。方法将72只健康SD大鼠随机分为正常对照组、PD模型组和司来吉兰治疗组,后两组采用颈背部皮下注射鱼藤酮方法制备PD模型。模型组每天给予生理盐水灌胃,治疗组每天给予司来吉兰0.5mg/kg灌胃。分别于治疗后4d、8d测定大鼠1h粪便排出量及含水量,并采用免疫组化法和蛋白质印迹法检测结肠α-Syn和nNOS的表达。结果 (1)与对照组比较,模型组各时间点1h粪便排出量及含水量均减少(均P0.01);与模型组相比较,治疗组各时间点1h粪便排出量及含水量均增加(均P0.05),且治疗8d亚组高于治疗4d亚组(均P0.05)。(2)与对照组比较,模型组各时间点结肠α-Syn、nNOS表达均增加(均P0.01);与模型组比较,治疗组各时间点结肠α-Syn、nNOS表达均降低(均P0.05)。与治疗4d亚组比较,治疗8d亚组的α-Syn、nNOS表达含量降低(均P0.05)。结论司来吉兰能够改善PD大鼠结肠功能障碍,其作用机制可能与其抑制α-Syn、nNOS表达有关。     

帕金森病大鼠模型神经元型一氧化氮合酶(nNOS)阳性神经元的实验研究

目的 观察研究帕金森病(PD)大鼠模型纹状体神经元型一氧化氮合酶(nNOS)阳性神经元，探讨一氧化氮(NO)在PD发病机制中所起作用。方法 应用立体定向技术建立6-OHDA毁损的大鼠PD模型，通过多巴胺受体激动剂阿扑吗啡(APO)测试大鼠旋转行为，免疫组化方法观察黑质酪氨酸羟化酶(TH)阳性神经元和纹状体nNOS阳性神经元的变化。结果 大鼠6-OHDA损毁侧黑质TH阳性神经元数目较对侧明显减少，双侧纹状体nNOS阳性神经元数目无显著差异。结论 6-OHDA对TH阳性神经元有损伤作用，而NOS阳性神经元对其具有抵抗作用。NO可能参与了PD发病机制。     

偏侧帕金森病大鼠模型的行为,免疫组化和脑微透析的实验研究

评价6-羟多巴胺(6-OHDA)损毁大鼠单侧黑质制备的偏侧帕金森病动物模型。应用6-羟多巴胺损毁SD大鼠单侧黑质制备偏侧PD鼠模型。3周后根据药物诱发试验,TH免疫组化证实模型制作成功。进一步用脑微透析技术结合HPLC-ECD在体检测PD鼠纹状体多巴胺及代谢产物含量。结果:82只大鼠中有36只阿朴吗啡(APO)诱发的旋转次数>7转/min。6-OHDA注射侧黑质DA神经末稍已绝大多数被损毁。6-OHDA损毁侧纹状体多巴胺及代谢产物明显低于健侧(P<0.05,P<0.01)。应用6-OHDA制备的偏侧PD鼠模型是PD研究的理想模型之一。     

尼莫地平对脑缺血早期神经元型一氧化氮合酶的影响

目的观察局灶性脑缺血大鼠早期缺血部位神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)的变化,以及钙拮抗剂尼莫地平对其的影响。方法以线栓法制作大鼠大脑中动脉闭塞模型为基础,选取雄性SD大鼠共75只,随机分成假手术组(n=25)、模型对照组(n=25)和尼莫地平处理组(n=25)。以NDAPH-d组织化学方法观测各组大鼠脑缺血后30min、2h和6h纹状体中nNOS阳性细胞的变化;利用生物化学法和Hoechst凋亡染色分别检测各组大鼠脑缺血30min纹状体nNOS活性和6h半暗区细胞凋亡。结果脑缺血30min,与假手术组相比,模型对照组大鼠缺血侧纹状体中nNOS活性和nNOS阳性细胞数均升高(P<0.01),而尼莫地平组无明显变化(P>0.05);随脑缺血时间延长,模型对照组和尼莫地平组nNOS阳性细胞数都下降(P<0.05);脑缺血6h,尼莫地平组半暗区的凋亡细胞少于模型对照组(P<0.01)。结论钙拮抗剂尼莫地平可抑制脑缺血早期的nNOS上升,并减轻随后的脑组织损伤。     

灵芝孢子粉对帕金森病黑质神经细胞caspase-3影响的实验研究

目的：研究灵芝孢子对帕金森病（PD）大鼠模型黑质神经细胞caspase-3的影响。方法：SD大鼠随机分为3组，PD组：经立体定向向黑质部注入6-羟多巴（6-OHDA）；灵芝孢子组：先用灵芝孢子粉灌胃3d，立体定向注入6-OHDA，继续灌胃4周；正常对照组：立体定向注入抗坏血酸生理盐水。实验大鼠4周处死后用免疫组化、原位杂交检测caspase-3及其mRNA的阳性细胞数，Western blot检测caspase-3的半定量。结果：灵芝孢子组术侧黑质caspase-3及其mRNA阳性神经元数量较PD组明显降低，Western blot显示caspase-3蛋白较PD组显著降低。结论：灵芝孢子能够降低caspase-3的表达，对PD大鼠有脑保护作用。     

还原型谷胱苷肽治疗帕金森病大鼠模型的实验研究

目的观察还原型谷胱苷肽(GSH)治疗帕金森病(PD)大鼠模型的疗效。方法35只SD大鼠采用内侧前脑束注射6-羟多巴胺(6-OHDA)制作偏侧PD模型。术后第6周根据阿朴吗啡旋转试验将模型鼠分为部分PD组和完全PD组,各组再随机分为GSH亚组和对照亚组,并分别给予GSH或生理盐水腹腔注射,每日1次,共4周。治疗后第2周、4周、6周、8周进行阿朴吗啡旋转试验。结果共有27只大鼠制模成功,13只为部分PD模型,14只为完全PD模型;部分PD GSH亚组治疗后第4周、6周、8周阿朴吗啡旋转试验每分钟旋转次数较治疗前及相应的对照亚组显著减少(P<0.05~0.01);完全PD GSH亚组较治疗前及相应的对照亚组无显著变化。结论GSH治疗6-OHDA所致的部分PD大鼠模型有效,对完全PD大鼠模型无效。     

重组人促红细胞生成素预处理对帕金森病大鼠胶质细胞源性炎症因子表达的影响

目的探讨重组人促红细胞生成素(rhEPO)预处理对帕金森病(PD)大鼠胶质细胞源性炎症因子表达的影响。方法 40只SD大鼠随机分为4组,A组:右侧纹状体内注射rhEPO 24 h后,同侧黑质内注射6-羟基多巴胺(6-OHDA);B组:右侧纹状体内立体定向注射与rhEPO等量的生理盐水,24 h后同侧黑质内立体定向注射6-OHDA;C组:右侧黑质内立体定向注射6-OHDA;D组:右侧黑质内立体定向注射与6-OHDA等量的生理盐水。4周后采用酶联免疫吸附法检测血清诱导型一氧化氮合酶(iNOS)和肿瘤坏死因子(TNF)-α含量;逆转录(RT)-PCR法检测黑质iNOS和TNF-αmRNA的表达。结果与D组比较,A、B、C组大鼠血清iNOS、TNF-α含量增多,黑质iNOS、TNF-αmRNA表达增高(均P<0.05);与B组和C组比较,A组大鼠血清iNOS、TNF-α含量显著减少,黑质iNOS、TNF-αmRNA表达显著降低(均P<0.05)。结论 rhEPO可能通过抑制黑质TNF-α、iNOS表达,减轻6-OHDA对多巴胺能神经元的毒性损害,具有神经保护作用。     

左旋多巴对帕金森病大鼠模型学习记忆能力的影响及其机制

目的 观察左旋多巴(levodopa, L-dopa)对帕金森病(Parkinson disease, PD)模型大鼠学习记忆能力的影响,并探讨其机制.方法 应用6-羟多巴胺(6-hydroxydopamine, 6-OHDA)制作PD大鼠模型.将228只造模成功的PD大鼠分为对照组和实验组.对实验组大鼠分别按体质量10、20、30 mg/(kg·d)腹腔注射L-dopa,连续28 d,然后分别于腹腔注射L-dopa后1、3、5、7、14、28 d测定其行为学及血浆同型半胱氨酸(Homocysteine,Hcy)和叶酸水平.同时测定大鼠海马区乙酰胆碱酯酶(acetylcholinesterase,AChE)活力.结果 (1)随L-dopa剂量增大、应用时间延长可明显降低大鼠学习记忆能力(P<0.01),降低大鼠海马区AChE活力(P<0.01),升高血浆Hcy水平,降低叶酸水平(P<0.01).结论 大量使用L-dopa可造成大鼠学习记忆能力明显下降,引起海马区AChE活力下降,其机制可能与升高Hcy有关.     

帕金森病大鼠黑质细胞凋亡与Bax蛋白表达的相关性

鉴于在体研究帕金森病(Parkinson disease，PD)模型成功前黑质细胞凋亡的研究罕见报道，我们采用神经损毁剂6-羟基多巴胺(6-OHDA)制作大鼠PD模型，采用免疫组织化学、电镜观察的方法，动态观察PD大鼠模型成功前促凋亡基因Bax蛋白表达与6-OHDA诱发的黑质细胞凋亡的关系。     

重组人促红细胞生成素(rhEPO)对帕金森病大鼠模型小胶质细胞活化的影响

目的探讨重组人促红细胞生成素(recombinant human erythropoietin,rhEPO)对6-羟基多巴胺(6-OHDA)诱导的SD大鼠帕金森病(PD)模型小胶质细胞活化的影响。方法 40只SD大鼠随机分为A组(rhEPO+6-OHDA)、B组(生理盐水+6-OHDA)、C组(6-OHDA)、D组(生理盐水),每组10只。(1)A组:右侧纹状体内立体定向注射重组促红细胞生成素(rhEPO),24h后同侧黒质内立体定向注射6-OHDA;(2)B组:右侧纹状体内立体定向注射与rhEPO等量的生理盐水,24h后同侧黒质内立体定向注射6-OHDA;(3)C组:右侧黒质内立体定向注射6-OHDA;(4)D组:右侧黒质内立体定向注射与6-OHDA等量的生理盐水。4w后采用免疫组化检测黒质内酪氨酸羟化酶(TH)阳性神经元和CD11b阳性细胞数量及CD11b阳性细胞形态变化。结果与D组比较,A组大鼠黒质TH阳性神经元明显减少,CD11b阳性细胞明显增多,大部分小胶质细胞胞体小,突起细长;与B组和C组比较,A组大鼠黒质TH阳性神经元显著增多,CD11b阳性细胞显著减少,仅有少量小胶质细胞胞体大,突起短粗。结论重组人促红细胞生成素(rhEPO)可能通过抑制小胶质细胞活化,减轻6-OHDA对多巴胺(DA)能神经元的毒性损害,对DA能神经元产生神经保护作用。     

Deep brain stimulation in a rat model modulates TH, CaMKIIa and Homer1 gene expression

High-frequency stimulation (HFS) of subthalamic nucleus (STN) is a therapy for late-stage Parkinson's disease. Its mechanisms of action are not yet fully understood. In the present study, gene expression analyses were performed in a rat model of Parkinson's disease, i.e. striatal 6-hydroxydopamine (6-OHDA) lesion. Using microarrays, gene expression was analysed in 1-mm-thick sagittal brain slices, including basal ganglia of five groups of male Wistar rats. These were unmanipulated rats (group A), unlesioned rats with implanted electrode but without stimulation (group B), unlesioned, stimulated rats (group C), 6-OHDA-lesioned rats with implanted electrode but without stimulation (group D), and finally 6-OHDA-lesioned and stimulated rats (group E). A statistically significant downregulation of tyrosine hydroxylase (TH) mRNA expression induced by 6-OHDA lesion and an HFS-induced TH upregulation in 6-OHDA-lesioned rats could be detected. It could be hypothesized that the HFS-induced upregulation of TH is the result of neuronal STN modulation and mediated via projections from STN to substantia nigra pars compacta. Furthermore, a downregulation of calcium/calmodulin-dependent protein kinase type IIA and Homer1 was observed. This downregulation could result in a reduced sensitivity towards glutamate in basal ganglia downstream of STN.     

Effects of pallidotomy on motor symptoms in an animal model of Parkinson's disease

The present study was designed to evaluate the motor effects of lesioning the internal globus pallidus in an animal model of Parkinson's disease. Fourty rats were divided into four groups (each of 10 rats) which received either unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle (mfb) plus sham surgery to the pallidum, sham surgery of mfb plus N-methyl-D-aspartate (NMDA) induced pallidal lesions, combined 6-OHDA mfb + NMDA pallidal lesions or sham surgery to both structures. Animals with 6-OHDA lesions developed significant ipsilateral biases in head position, body axis and circling after amphetamine challenge (all P < 0.05). Prominent contralateral deficits were present in sensorimotor response latency and contralateral circling was induced by apomorphine challenge (both P < 0.05). The addition of an NMDA pallidal lesion, improved the head position and body axis biases, as well as dopamine-agonist induced rotation and contralateral reaction time in a sensorimotor task (all P < 0.05). There was, however, a slight worsening of sensorimotor response on the ipsilateral side (P < 0.05). Pallidal lesions in the absence of 6-OHDA lesions produced contralateral head position and body axis biases (both P < 0.05). These data indicate that pallidotomy improves some, but not all aspects of parkinsonian motor dysfunction in an animal model of Parkinson's disease (PD).     

死亡相关蛋白小分子干扰RNA对帕金森病大鼠黑质酪氨酸羟化酶表达的影响

目的:利用动物模型探讨死亡相关蛋白(FADD)小分子干扰RNA(siRNA)对帕金森病(PD)大鼠黑质酪氨酸羟化酶(TH)表达的影响。方法:40只雄性Wistar大鼠随机分为4组。经立体定向定位黑质,PD组:注入6-羟基多巴(6-OHDA);siRNA组:注入FADD siRNA2次后再注入6-OHDA;SiRNA阴性对照组:注入FADD siRNA阴性对照物2次后再注入6-OHDA;正常对照组:注入抗坏血酸生理盐水。各组大鼠术后4周处死,取鼠的术侧中脑黑质,利用原位杂交检测FADD、TH mRNA表达。结果:PD组与正常对照组比较FADD mRNA表达显著增加(P<0.01);siRNA组与PD组比较FADD mRNA表达显著减少(P<0.05);siRNA阴性对照组与正常对照组比较FADD mRNA表达量无显著差异(P>0.05)。PD组与正常对照组比较TH mRNA表达显著减少(P<0.01);siRNA组与PD组比较TH mRNA表达显著增加(P<0.05);siRNA阴性对照组与正常对照组比较TH mRNA表达量差异无统计学意义(P>0.05)。结论:FADDsiRNA可能通过抑制凋亡对PD大鼠模型有一定的保护和治疗作用。     

Subthalamic Nucleus Lesion in Rats Prevents Dopaminergic Nigral Neuron Degeneration After Striatal 6-OHDA Injection: Behavioural and Immunohistochemical Studies

Several studies have shown that antagonists of N -methyl-D-aspartate receptors provide protection of the dopaminergic nigrostriatal pathway in animal models of Parkinson's disease. Since the substantia nigra compacta receives a moderate glutamatergic innervation from the subthalamic nucleus, we tried to determine whether subthalamic nucleus lesion could prevent the toxicity of the selective dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). Experiments were carried out on four groups of rats. Group 1 ( n = 10) received a unilateral injection of 6-hydroxydopamine in the striatum and group 2 ( n = 10) received kainic acid in the subthalamic nucleus. Group 3 ( n = 10) received an injection of kainic acid in the subthalamic nucleus and 1 week later an injection of 6-OHDA in the striatum. Group 4 ( n = 5) received the same treatment but kainic acid was replaced by saline. Apomorphine induced an ipsilateral rotation in rats of groups 2 and 3 and a contralateral rotation in rats of groups 1 and 4. The number of tyrosine hydroxylase-immunoreactive cells in the pars compacta of the substantia nigra was not significantly different between injected and non-injected sides in rats of groups 2 and 3, but was significantly decreased on the side ipsilateral to 6-OHDA striatal injection in rats of groups 1 and 4. These results show that subthalamic nucleus lesion provides neuroprotection of the dopaminergic nigrostriatal pathway against 6-OHDA toxicity and opens a new way for slowing or stopping the progression of Parkinson's disease.     

6-羟基多巴胺诱导SH-SY5Y细胞帕金森病模型中Peroxiredoxin6表达的研究

目的 应用蛋白质组学技术研究6-羟基多巴胺(6-OHDA)诱导SH-SY5Y细胞帕金森病(PD)模型中Peroxiredoxin6(Prx6)的表达变化.方法 在建立6-OHDA诱导SH-SY5Y细胞帕金森病模型的基础上,分别提取6-OHDA实验组和对照组孵育24h的细胞总蛋白,应用荧光差异双向凝胶电泳(DIGE)技术获得蛋白点的差异表达信息,运用基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOF MS)鉴定出差异蛋白质.结果 DIGE分析发现实验组有一个表达明显上调的差异蛋白质点(P<0.01),经质谱分析鉴定确认为Prx6.结论 6-OHDA诱导SH-SY5Y细胞的帕金森病模型中Prx6表达上调,提示PD中有氧化应激存在,Prx6上调及氧化应激可能与PD的发病机制有关.     

N-terminal tripeptide of IGF-1 (GPE) prevents the loss of TH positive neurons after 6-OHDA induced nigral lesion in rats

The effect of the N-terminal tripeptide of insulin-like growth factor (IGF)-1, glycine-proline-glutamate (GPE), as a neuroprotective agent for nigro-striatal dopaminergic neurons was examined in the present study using a rat model of Parkinson's disease. A unilateral nigro-striatal lesion was induced in rats by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle (MFB). GPE (3 microgram) or its vehicle was administered intracerebroventricularly (i.c.v.) 2 h after the 6-OHDA lesion. Tyrosine-hydroxylase (TH) immunohistochemistry in the substantia nigra compacta (SNc) and the striatum were examined 2 weeks after the lesion. Following 6-OHDA injection, the number of TH immunopositive neurons in the ipsilateral SNc was reduced. The density of TH immunostaining was also reduced in the ipsilateral SNc and the striatum. Treatment with a single dose of GPE (n=9) significantly prevented the loss of TH immunopositive neurons (p<0. 001) and restored the TH immunoreactivity in both the SNc and the striatum compared with the vehicle control group (n=9, p<0.001). The results suggest that GPE showed promise as a potential treatment for Parkinson's disease.     

Effects of electrolytic and 6-hydroxydopamine lesions of rat nigrostriatal pathway on nitric oxide synthase and nicotinamide adenine dinucleotide phosphate diaphorase

The aim of the present study was to assess degenerative changes in the nitric oxide (NO) system of basal ganglia in animals with experimentally induced Parkinson's disease. In one procedure, rats were stereotaxically injected with 6-hydroxydopamine (6-OHDA) in the right medial forebrain bundle; in a second procedure, electrodes were implanted in the right substantia nigra pars compacta (SNc). After 15 and 30 days animals were tested for rotational asymmetry induced by apomorphine. Apomorphine induced rotation in lesioned animals, towards the ipsilateral side after electrolytic lesion and towards contralateral side in 6-OHDA animals. Structural deficits in basal ganglia were quantified by nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and by nitric oxide synthase (NOS) immunoreactivity. 6-OHDA and electrolytic lesions induced a significant decrease in the number of NADPH-d/NOS positive cells in the lesion ipsilateral to SNc, in contrast with cell number increase in the ipsilateral dorsal striatum. By contrast, 6-OHDA-treated animals showed a decrease in the number of NOS immunoreactive cells in the contralateral nucleus accumbens. We conclude that populations of NO-synthesizing neurons are differentially regulated in Parkinson's disease induced by different experimental procedures.