神经细胞黏附分子在GDNF保护大鼠多巴胺能神经元中的作用

目的研究神经细胞黏附分子(neural cell adhesion molecule,NCAM)在胶质细胞系源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)保护帕金森(Parkinson's disease,PD)模型大鼠受损多巴胺(dopamine,DA)能神经元中的作用。方法右侧纹状体内立体定位注射6-羟多巴胺(6-OHDA)制备早期PD模型,而后分为4组:对照组(同侧黑质内注射PBS)、NCAM组(同侧黑质内仅注射anti-NCAM抗体)、GDNF组(同侧黑质内注射GDNF)、NCAM阻断组(同侧黑质内注射anti-NCAM抗体30min后注射GDNF),采用免疫组织化学染色技术和免疫印迹技术,观察各组酪氨酸羟化酶(tyrosine hydroxylase,TH)的表达变化。结果GDNF组黑质致密部TH阳性神经元数目及表达的量明显多于PBS组,差别有统计学意义;NCAM阻断组与GDNF组相比,该处TH阳性神经元数目及表达的量明显减少,差别有统计学意义。结论NCAM参与了GDNF保护DA能神经元的作用。     

NCAM介导GDNF保护多巴胺能神经元作用的研究

目的研究神经细胞黏附分子(neural cell adhesion molecule,NCAM)在胶质细胞系源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)保护帕金森(Parkinson's disease,PD)模型大鼠受损多巴胺(dopamine,DA)能神经元中的作用。方法SD大鼠右侧纹状体内立体定位注射6-羟多巴胺(6-OHDA)制备早期PD模型,而后分为4组:对照组(同侧黑质内注射PBS),NCAM组(同侧黑质内仅注射anti-NCAM抗体),GDNF组(同侧黑质内注射GDNF),NCAM阻断组(同侧黑质内注射anti-NCAM抗体30min后注射GDNF),采用免疫组织化学染色技术和免疫印迹技术,观察各组酪氨酸羟化酶(tyrosine hydroxylase,TH)的表达变化。结果GDNF组黑质致密部TH阳性神经元数目(92.44±16.96)及表达的量(44731.50±9765.30)明显多于对照组(56.83±14.27;22218.75±5925.39),差别有统计学意义(P<0.05);NCAM阻断组与GDNF组相比,该处TH阳性神经元数目及表达的量明显减少(NCAM阻断组:67.57±12.71,26891.00±6848.87;GDNF组:92.44±16.96,44731.50±9765.30),差别有统计学意义(P<0.05)。结论NCAM参与了GDNF保护DA能神经元的作用。     

尼古丁对帕金森病大鼠纹状体GDNF和多巴胺含量的影响

目的 研究尼古丁对帕金森病（PD）大鼠纹状体脑胶质细胞源性神经营养因子(GDNF)和多巴胺（DA）含量的影响。方法 将6－羟多巴胺（6－OHDA）立体定向注射到大鼠右侧中脑腹侧背盖部（VTA）和黑质致密部（SNpc)，建立PD大鼠模型。采用生化、免疫组织化学方法观察不同剂量尼古丁对PD大鼠的作用，检测纹状体GDNF表达及DA含量的变化。结果 造模前及造模后皮下注射尼古丁的PD大鼠，纹状体GDNF表达及DA含量较PD组有明显改善（P＜0．05）。结论 尼古丁可减轻6－OHDA对黑质DA能神经元的损伤，对PD大鼠具有保护作用。     

左旋多巴诱发异动症大鼠黑质胶质细胞变化的实验研究

目的研究异动症(LID)大鼠黑质致密部(SNpc)胶质细胞的变化。方法6-羟多巴胺(6-OHDA)立体定位注射制备偏侧帕金森病(PD)大鼠模型,复方左旋多巴(L-dopa)甲酯腹腔内注射治疗4周(10mg.kg-1.d-1,每日2次)诱发异动症大鼠模型。采用免疫组化方法观察大鼠黑质致密部酪氨酸羟化酶(TH)、胶质纤维酸性蛋白(GFAP)、增殖细胞核抗原(PCNA)和S-100蛋白免疫阳性细胞的面密度进行定量分析。结果免疫组化方法证实,PD组、LID组和非LID组大鼠毁损侧SNpc部位的TH免疫阳性面密度较正常组大鼠明显减少(P<0.01),而GFAP、PCNA、S-100免疫阳性面密度则明显增多(P<0.01);LID组和PD组、非LID组相比较,SNpc部位TH的表达进一步减少(P<0.05),GFAP、PCNA、S-100的表达则进一步增加(P<0.05)。结论PD大鼠毁损侧SNpc部位存在明显的胶质细胞增生。LID大鼠毁损侧SNpc部位的多巴胺(DA)能神经元变性及胶质细胞增生较非LID大鼠及PD大鼠更为明显。我们推测SNpc部位的胶质细胞通过其对DA能神经元的神经保护和神经破坏的双重作用,间接影响着LID的发生及其严重程度。     

神经胶质细胞在帕金森病发病机制中的作用

目的　探讨胶质细胞在帕金森病 ( Parkinson's disease,PD)发病机制中的作用。方法　采用立体定向术将神经毒素 6 -羟基多巴 ( 6 - hydroxydopamine,6 - OHDA)注入大鼠右侧纹状体内 ,制备经典的帕金森病动物模型。观察黑质致密带内多巴胺 ( dopamine,DA)能神经元缺失、胶质细胞的增生和肿瘤坏死因子 ( tumor necrosis factor-alpha,TNF- α)表达水平。结果　模型组右侧黑质 DA能神经元的数量明显减少 ,同时伴有星形胶质细胞和小胶质细胞的数量明显增高 ( P<0 .0 5 ) ,TNF- α在模型组右侧黑质和纹状体内有阳性表达 ,且主要分布在激活的小胶质细胞上。结论　神经胶质细胞可能是通过 TNF- α等细胞因子 ,导致或参与 DA能神经元的大量的变性、死亡。以抑制小胶质细胞的激活作为靶点的药物研究有可能为 PD的治疗提供新的思路。     

被动吸烟对大鼠纹状体GDNF和多巴胺含量的影响

目的 :研究被动吸烟对帕金森病 (PD)大鼠的影响 ,探讨其作用机制。方法 :通过 6—羟多巴胺 (6 OH DA)脑立体定向注射术建立大鼠帕金森病模型。采用生化、免疫组织化学的方法观察PD大鼠纹状体脑胶质细胞源性神经营养因子表达 (GDNF)及多巴胺 (DA)含量的变化以及术前 4周开始给予被动吸烟 (持续 6周 )和术后 3周给予的被动吸烟持续 2周对上述指标的影响。结果 :术前及术后吸烟的PD大鼠纹状体DA含量、脑胶质源性神经营养因子表达较PD组有明显改善 (P <0 0 5 )。结论 :被动吸烟能减轻黑质纹状体DA能神经元的损伤     

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。     

被动吸烟对大鼠纹状体GDNF和多巴胺含量的影响

目的：研究被动吸烟对帕金森病（PD）大鼠的影响，探讨其作用机制。方法：通过6－羟多巴胺（6－OH－DA）脑立体定向注射术建立大鼠帕金森病模型。采用生化、免疫组织化学的方法观察PD大鼠纹状体脑胶质细胞源性神经营养因子表达（GDNF）及多巴胺（DA）含量的变化以及术前4周开始给予被动吸烟（持续6周）和术后3周给予的被动吸烟持续2周对上述指标的影响。结果：术前及术后吸烟的PD大鼠纹状体DA含量、脑胶质源性神经营养因子表达较PD组有明显改善（P＜0．05）。结论：被动吸烟能减轻黑质纹状体DA能神经元的损伤。     

黑质内注射脂多糖对不同年龄大鼠DA能神经元和小胶质细胞活性的影响

目的 探讨青年和老年大鼠黑质DA能(DA)神经元对脂多糖(LPS)所诱导的损伤作用的敏感性差异和对小胶质细胞活性的影响.方法 采用立体定向技术向大鼠单侧黑质内注入LPS建立PD大鼠模型;采用免疫组化法观察黑质酪氨酸羟化酶(TH)阳性细胞和OX6阳性小胶质细胞变化;采用Fluoro-Jade B染色法检测黑质的变性神经元.结果 黑质内注射LPS后,老年组大鼠黑质区TH阳性神经元数量较青年组明显减少(P<0.01);老年组大鼠黑质区Fluoro-Jade B阳性神经元明显多于青年组(P<0.01);青年组大鼠黑质部位的OX6阳性小胶质细胞主要是处在激活期的;而老年组大鼠黑质部位的OX6阳性小胶质细胞主要是活化期的(阿米巴样或巨噬细胞样).结论 老年大鼠的黑质DA能神经元对于LPS所诱导的损伤作用较青年大鼠更为敏感.     

经侧脑室注射脂多糖诱导大鼠黑质部位小胶质细胞激活及多巴胺能神经元损伤的研究

目的观察经脑室注射脂多糖(LPS)后大鼠的黑质部小胶质细胞激活及多巴胺(DA)能神经元的变化,探讨脑内炎性反应在黑质DA能神经元慢性变性过程中的作用。方法健康雄性SD大鼠30只,随机分为生理盐水(NS)对照组和LPS组,分别向大鼠右侧脑室注射20μL NS或50μg LPS,40周后用免疫组织化学方法检测大鼠黑质小胶质细胞是否激活、激活的程度(OX-42及OX-6抗体水平),以及酪氨酸羟化酶(TH)阳性神经元的形态和数量。以Fluoro-Jade B(FJB)染色法检测黑质部位神经元变性情况。结果 (1)NS对照组大鼠黑质部位OX-42阳性小胶质细胞呈静息状态,染色浅。LPS组大鼠黑质部OX-42阳性小胶质细胞呈部分激活状态,染色深。两组大鼠黑质部位均未发现OX-6阳性小胶质细胞。(2)NS对照组大鼠黑质部位有大量深染的TH阳性神经元。LPS组大鼠黑质部位TH阳性染色神经元数目(99.11±20.31)比NS对照组(189.52±12.12)减少47.7%(P<0.01)。(3)两组大鼠黑质部位均未见FJB阳性染色神经元。结论经侧脑室单次注射LPS可能造成大鼠黑质部位小胶质细胞长期慢性激活及DA能神经元慢性迟发性功能性损伤。     

Delayed gene therapy of glial cell line-derived neurotrophic factor is efficacious in a rat model of Parkinson's disease

Gene transfer of glial cell line-derived neurotrophic factor (GDNF) in rodent models of Parkinson's disease (PD) has been shown to protect against neurodegeneration either prior to or immediately after neurotoxin-induced lesions; however, the nigrostriatal pathway was largely intact when gene delivery was completed in these models, which may not accurately reflect the clinical situation encountered with Parkinson's patients. In this study, replication-incompetent adenoviral vectors encoding the rat GDNF gene were administered into the striatum 4 weeks following 6-hydroxydopamine (6-OHDA) injection in the unilateral striatum, more closely resembling fully developed PD. Apomorphine-induced rotational behavior testing was performed every week following 6-OHDA injection. At the 10th week after gene transfer, the striatal dopamine concentrations were measured by HPLC with an electrochemical detector and the number of tyrosine hydroxylase (TH)-positive dopamine neurons in the substantia nigra (SN) was determined by immunohistochemistry. Injection of 6-OHDA into the striatum produced stable increases in rotation, which reached a plateau between 4 and 5 weeks post-injection. The number of TH-positive neuron in the SN and dopamine levels in the striatum was significantly lower in the 6-OHDA group compared to the normal group. Gene transfer of GDNF, but not beta-galactosidase, significantly increased the number of TH-positive neurons and dopamine levels, with a subsequent behavioral recovery between 5 and 10 weeks following GDNF transduction. These findings demonstrate that adenovirus-mediated gene transfer of GDNF is efficacious even in the late stages of 6-OHDA-induced PD rats. They also provide further evidence on the effectiveness of GDNF-based gene therapy for experimental Parkinson's disease.     

Behavioral and Cellular Protection of Rat Dopaminergic Neurons by an Adenoviral Vector Encoding Glial Cell Line-Derived Neurotrophic Factor

Previously, we observed that an adenoviral (Ad) vector encoding human glial cell line-derived neurotrophic factor (GDNF), injected near the rat substantia nigra (SN), protects SN dopaminergic (DA) neuronal soma from 6-hydroxydopamine (6-OHDA)-induced degeneration. In the present study, the effects of Ad GDNF injected into the striatum, the site of DA nerve terminals, were assessed in the same lesion model. So that effects on cell survival could be assessed without relying on DA phenotypic markers, fluorogold (FG) was infused bilaterally into striatae to retrogradely label DA neurons. Ad GDNF or control treatment (Ad mGDNF, encoding a deletion mutant GDNF, Ad lacZ, vehicle, or no injection) was injected unilaterally into the striatum near one FG site. Progressive degeneration of DA neurons was initiated 7 days later by unilateral injection of 6-OHDA at this FG site. At 42 days after 6-OHDA, Ad GDNF prevented the death of 40% of susceptible DA neurons that projected to the lesion site. Ad GDNF prevented the development of behavioral asymmetries which depend on striatal dopamine, including limb use asymmetries during spontaneous movements along vertical surfaces and amphetamine-induced rotation. Both behavioral asymmetries were exhibited by control-treated, lesioned rats. Interestingly, these behavioral protections occurred in the absence of an increase in the density of DA nerve fibers in the striatum of Ad GDNF-treated rats. ELISA measurements of transgene proteins showed that nanogram quantities of GDNF and lacZ transgene were present in the striatum for 7 weeks, and picogram quantities of GDNF in the SN due to retrograde transport of vector and/or transgene protein. These studies demonstrate that Ad GDNF can sustain increased levels of biosynthesized GDNF in the terminal region of DA neurons for at least 7 weeks and that this GDNF slows the degeneration of DA neurons and prevents the appearance of dopamine dependent motor asymmetries in a rat model of Parkinson's disease (PD). GDNF gene therapy targeted to the striatum, a more surgically accessible site than the SN, may be clinically applicable to humans with PD.     

Neuroprotection in the rat Parkinson model by intrastriatal GDNF gene transfer using a lentiviral vector

We used a recombinant lentiviral vector (rLV) for gene delivery of GDNF to the striatum, and assessed its neuroprotective effects in the intrastriatal 6-hydroxydopamine (6-OHDA) lesion model.The level of GDNF expression obtained with the rLV-GDNF vector was dose-related and ranged between 0.9-9.3 ng/mg tissue in the transduced striatum, as determined by ELISA, and 0.2-3.0 ng/mg tissue were detected in the ipsilateral substantia nigra (SN), due to anterograde transport of the GDNF protein. GDNF expression was apparent at 4 days and maintained for > 8 months after injection. Striatal delivery of rLV-GDNF efficiently protected the nigral dopamine (DA) neurons and their projection, against the 6-OHDA lesion (65-77% of intact side). Sprouting of the lesioned axons was observed along the nigrostriatal pathway, precisely corresponding to the areas containing anterogradely transported GDNF.     

被动吸烟对大鼠纹状体多巴胺及黑质自由基含量的影响

目的研究被动吸烟对帕金森病(PD)大鼠的影响,探讨其作用机制。方法通过6-羟多巴胺(6-O-HDA)脑立体定向注射术建立大鼠PD模型。术前4周开始被动吸烟及术后持续2周为预防组;术后3周给予被动吸烟持续2周为治疗组。采用生化的方法观察PD大鼠纹状体黑质自由基、抗氧化剂及多巴胺含量的变化。结果吸烟治疗组和吸烟预防组大鼠黑质自由基及抗自由基酶较对照组有明显改善(P<0.05);吸烟预防组PD大鼠纹状体DA含量较对照组有明显改善(P<0.05);吸烟治疗组PD大鼠纹状体DA含量较对照组无变化(P>0.05)。结论被动吸烟能减轻黑质纹状体DA能神经元的损伤。     

帕金森病模型大鼠黑质多巴胺能神经元的氧化应激研究

目的　探索帕金森病模型大鼠黑质多巴胺能神经元的氧化应激发病机制。方法　通过立体定位仪 ,将 6-OHDA注入大鼠一侧纹状体内制备 PD模型 ,2周后观察动物的行为学改变 ,2个月后观察黑质纹状体等的病理形态学变化 ,检测模型组、假手术组和正常对照组的超氧化物歧化酶 (SOD)的活性 ,丙二醛 (MDA)、一氧化氮(NO)代谢产物 (NO x)的含量的变化。结果　成功 PD模型鼠有 2 2只。光镜下 HE染色示模型组右侧黑质的多巴胺神经元受损 ,数目减少。模型组右侧黑质的 SOD的含量下降 ,MDA及 NO x含量明显升高 ,与左侧、假手术组及正常对照组相比有显著差异 (P>0 .0 5)。结论　 6-OHDA纹状体内双靶点注射法是一种有效的制备 PD模型的方法。帕金森病大鼠模型黑质内 SOD活性下降 ,MDA、NO x含量升高 ,氧化应激在 PD的发病中起重要的作用     

提高帕金森病动物模型制作成功率的方法学探讨及模型综合评价

目的探讨提高6-OHDA帕金森病(PD)大鼠模型制作成功率的方法,并对模型进行综合评价。方法取SD大鼠90只,将6-OHDA立体定向注射于左侧黑质区及中脑腹侧被盖,观察大鼠的行为、黑质抗氧化指标、线粒体呼吸链及黑质细胞形态学的变化。结果①经阿朴吗啡诱导后共筛选出成功模型64只,成功率为71%;②模型大鼠黑质区ROS、MDA水平均明显升高,而GSH-Px活性则明显降低;③模型大鼠黑质区存在线粒体呼吸链功能障碍;④免疫组化发现注射侧黑质区多巴胺能神经元较对侧明显减少(P<0.001);⑤电镜观察发现模型大鼠黑质细胞同时存在凋亡、变性和坏死样改变。结论应用本方法可较快建立稳定的成功率较高的PD大鼠模型,该方法是通过增强氧化应激、干扰线粒体呼吸链功能、诱导凋亡甚至引发坏死等不同机制损毁多巴胺能神经元的,同PD的发病机制基本一致。     

Delivery of a GDNF gene into the substantia nigra after a progressive 6-OHDA lesion maintains functional nigrostriatal connections

The effects of delivering GDNF via an adenoviral vector (AdGDNF) 1 week after lesioning dopaminergic neurons in the rat substantia nigra (SN) with 6-hydroxydopamine (6-OHDA) were examined. Rats were unilaterally lesioned by injection of 6-OHDA into the striatum, resulting in progressive degeneration of dopaminergic neurons in the SN. One week later, when substantial damage had already occurred, AdGDNF or a control vector harboring beta-galactosidase (AdLacZ) was injected into either the striatum or SN (3.2 x 10(7) PFU/microl in 2 microl). Rats were examined behaviorally with the amphetamine-induced rotation test and for forelimb use for weight-bearing movements. On day 30 postlesion, the extent of nigrostriatal tract degeneration was determined by injecting a retrograde tracer (FluoroGold) bilaterally into the lesioned striatum. Five days later, rats were sacrificed within 2 h of amphetamine injection to examine amphetamine-induced Fos expression in the striatum, a measure of dopaminergic-dependent function in target neurons. AdGDNF injection in the SN rescued dopaminergic neurons in the SN and increased the number of dopaminergic neurons that maintained a connection to the striatum, compared to rats injected with AdLacZ. Further support that these spared SN cells maintained functional connections to the striatum was evidenced by increased Fos expression in striatal target neurons and a decrease in amphetamine-induced rotation. In contrast to the effects observed in rats injected with AdGDNF in the SN, rats injected with AdGDNF in the striatum did not exhibit significant ameliorative effects. This study demonstrates that experimentally increasing levels of GDNF biosynthesis near the dopaminergic neuronal soma is effective in protecting the survival of these neurons and their function even when therapy is begun after 6-OHDA-induced degeneration has commenced. Thus, GDNF gene therapy may ameliorate the consequences of Parkinson's disease through rescuing compromised dopaminergic neurons.     

Recombinant adeno-associated viral vector-mediated glial cell line-derived neurotrophic factor gene transfer protects nigral dopamine neurons after onset of progressive degeneration in a rat model of Parkinson's disease

Previous work has demonstrated that viral vector mediated gene transfer of glial cell line-derived neurotrophic factor (GDNF), when administered prior to a striatal injection of the specific neurotoxin, 6-hydroxydopamine (6-OHDA), can protect nigral dopamine (DA) neurons from cell death. When considering gene therapy for Parkinson's disease (PD), vector delivery prior to the onset of neuropathology is not possible and chronic delivery will likely be necessary in a GDNF-based PD therapy. The present study was undertaken to determine if GDNF delivered via a recombinant adeno-associated viral vector (rAAV) could affect nigral DA cell survival when initiated just after the administration of striatal 6-OHDA. The onset of rAAV-mediated GDNF transgene expression near the substantia nigra was determined to begin somewhere between 1 and 7 days after the 6-OHDA injection and subsequent vector administration. The cell survival data indicate that rAAV-GDNF delivery results in a highly significant sparing of nigral DA neurons. These data indicate that a single delivery of rAAV encoding GDNF is efficacious when delivered after the onset of progressive degeneration in a rat model of PD.     

KDI tripeptide of gamma1 laminin protects rat dopaminergic neurons from 6-OHDA induced toxicity

Our previous studies indicate that the KDI (Lys-Asp-Ile) tripeptide of gamma1 laminin protects central neurons from mechanical trauma and excitotoxicity. At least part of the neuroprotective effect of the KDI tripeptide may be mediated by its inhibitory function on ionotropic glutamate receptors. We studied the protective effect of the KDI tripeptide against 6-hydroxy-dopamine (6-OHDA) induced neurotoxicity in a rat experimental model of Parkinson's disease (PD). We found that a single unilateral injection of the KDI tripeptide into the substantia nigra before an injection of 6-OHDA protected the dopaminergic neurons from the neurotoxicity of 6-OHDA. Compared to rats treated with 6-OHDA alone, the KDI + 6-OHDA-treated substantia nigra was relatively intact with large numbers of dopaminergic neurons present at the injection side. In the rats treated with 6-OHDA alone, no dopaminergic neurons were detected, and the substantia nigra-area at the injection side was filled with blood-containing cavities. Quantification of the rescue effect of the KDI tripeptide indicated that, in animals receiving KDI before 6-OHDA, 33% of tyrosine hydroxylase-positive dopaminergic neurons of the substantia nigra were present as compared to the contralateral non-injected side. In animals receiving 6-OHDA alone, only 1.4% of the tyrosine hydroxylase expressing dopaminergic neurons could be verified. If this much protection were achieved in humans, it would be sufficient to diminish or greatly alleviate the clinical symptoms of PD. We propose that the KDI tripeptide or its derivatives might offer a neuroprotective biological alternative for treatment of PD.