6-羟基多巴胺诱发帕金森病大鼠模型的制作和评价

目的 向大鼠中脑黑质区注入6-羟基多巴胺(6-OHDA)建立帕金森病(PD)大鼠模型,并从行为学(ethology)及组织病理、生化角度对该模型进行评价.方法 将6-OHDA立体定向微量注入大鼠右侧中脑黑质(SN)区,观察阿朴吗啡(APO)诱发的大鼠旋转行为及黑质细胞形态学变化,测定脑组织液中儿茶酚胺类物质含量及黑质酪氨酸羟化酶的免疫活性.结果 120只大鼠中经APO诱导后有67只(占55.8%)持续转向健侧(旋转圈数＞7r/min),帕金森病大鼠模型复制成功.PD鼠注射侧黑质区多巴胺能神经元数量较对侧明显减少,体积缩小,结构欠清晰.注射侧脑组织液中多巴胺(DA)、3,4-二羟基苯酸(DOPAC)、高香草酸(HVA)、5-羟色胺(5-HT)含量均低于对侧,注射侧黑质致密部酪氨酸羟化酶(TH)免疫阳性细胞较健侧明显减少.连续观察10个月,PD模型大鼠的异常旋转行为无自发性恢复.结论 用6-OHDA选择性损毁大鼠黑质多巴胺能神经元,可造成与PD患者相似的基本病理变化,建立起可靠而稳定的PD大鼠模型.     

雌激素对PD模型大鼠黑质纹状体通路的保护作用及其机制探讨

目的研究雌激素（Estrogen）对6-羟基多巴（6-OHDA）制备的去卵巢（OVX）帕金森病（PD）模型大鼠黑质纹状体通路的保护作用及其可能机制。方法应用6-OHDA两点注射单侧损毁内侧前脑束（MFB）制备OVXPD模型大鼠,侧脑室给予17-β雌二醇（17-βestradiol,1μg/5μl）,观察大鼠旋转行为、黑质酪氨酸羟化酶（TH）基因表达、黑质铁染色阳性细胞数量和纹状体内多巴胺（DA）及其代谢产物含量的变化。结果雌激素用药组可明显减少阿朴吗啡诱导的PD模型大鼠单侧旋转行为（P〈0.01）。在损毁侧黑质,雌激素用药组TH基因的表达较PD模型组明显增加（P〈0.01）;纹状体DA及其代谢产物亦较PD模型组显著升高（P〈0.01）。黑质铁染色阳性细胞数量较PD模型组明显减少（P〈0.01）。结论雌激素对PD模型大鼠黑质DA能神经元有明显的保护作用,其作用机制可能与降低铁负载有关。     

抑颤汤治疗帕金森病的作用机制研究

目的　探讨抑颤汤治疗帕金森病 (PD)的作用机制。方法　运用 6 羟基多巴诱发法建立大鼠PD模型 ,通过随机分组 ,观察抑颤汤对PD大鼠行为特征的影响 ;采用脑组织液微透析技术 ,用高效液相色谱法测定各组大鼠脑组织细胞外液中儿茶酚胺类物质含量的变化 ;用放免法测定各组大鼠双侧脑组织M、DA受体数量及亲和力的变化 ;用光镜和电镜观察各组大鼠脑黑质细胞形态学变化。结果　抑颤汤能明显改善PD模型大鼠的旋转行为(P <0 0 1) ;抑颤汤组PD大鼠损毁侧脑组织透析液中 3,4 二羟基苯酸 (DOPAC)、高香草酸(HVA)、多巴胺 (DA)、5 羟色胺(5 HT)水平以及脑组织DA和M受体最大结合量 (Bmax)和亲和常数 (KD)均明显高于生理盐水组 (P <0 0 5 )。病理学观察见抑颤汤组大鼠受损侧脑黑质神经细胞数量明显多于生理盐水组 ,且神经元体积较饱满 ,结构较清晰 ,细胞内高尔基体、线粒体等接近正常。结论　抑颤汤能减轻PD模型大鼠脑黑质细胞的受损程度 ,促进其修复 ,改善黑质纹状体系统的分泌功能 ,提高脑组织中内源性儿茶酚胺类物质的含量 ,刺激PD模型大鼠受体系统 ,使DA、M受体的亲和力和敏感性提高 ,从而改善PD大鼠的旋转行为。     

乌鸡黑色素对6-OH-DA诱导帕金森病大鼠模型的神经保护作用

目的 探讨乌鸡黑色素对于6-羟多巴胺(6-OH-DA)单侧损毁纹状体诱导的帕金森病模型大鼠的神经保护作用及其机制.方法 将60只SD大鼠造模成功后,随机分为对照组、6-OH-DA诱导的帕金森病单纯模型组和治疗组,治疗组分别按6 mg/kg或20 mg/kg乌鸡黑色素灌胃给药,1次/d,共3w.然后比较各组中脑黑质多巴胺能神经元的数目、纹状体酪氨酸羟化酶(TH)阳性神经纤维的数量、阿朴吗啡诱导的旋转行为以及大鼠自主运动情况的差异.结果 6 mg/kg和20 mg/kg乌鸡黑色素灌胃给药能显著减少6-OH-DA所导致的中脑黑质多巴胺能神经元的死亡,减少纹状体TH阳性神经纤维的丢失,并有效改善阿朴吗啡诱导的旋转行为.结论 乌鸡黑色素能缓解6-OH-DA诱导的中脑黑质多巴胺能神经元的损伤,对于帕金森病有潜在的治疗作用.     

脑源性神经营养因子对多巴胺能神经元的影响

目的观察脑源性神经营养因子对帕金森氏病大鼠模型黑质多巴胺能神经元的影响。方法选用Wistar种系大白鼠，体重230～250g，随机分3组，通过左侧中脑黑质立体定向注射法，组1为生理盐水对照组（简称对照组）10只，注射相应量（5μl）的生理盐水；组2为注射6-OHDA制作帕金森氏病模型组（简称6-OHDA组）10只，注射6-OHDA，5μl（2μg／μl）；组3为（6-OHDA＋BDNF组），在制成帕金森氏病模型后再向同侧中脑黑质注射BDNF5μl（3μg／μl），连续6d，qd。分别观察动物的旋转行为，免疫组化染色方法观察黑质酪氨酸羟化酶（TH）阳性神经元的数量，高效液相法测定纹状体部多巴胺（DA）含量的变化。结果单侧黑质内注入6-OHDA制成帕金森氏病大鼠模型后，6-OHDA组与对照组比较，产生旋转行为，（6-OHDA＋BDNF）组在观察旋转行为时，症状明显改善；镜下见TH阳性神经元主要见于对照组的黑质致密部，数量为42．3±7．56个／μm^2，模型组黑质致密部TH阳性神经元数明显减少为2．41±1．07个／μm^2，（6-OHDA＋BDNF）组黑质致密部TH阳性神经元数为15．36±3．04个／μm^2；纹状体部多巴胺含量：生理盐水组为11．4±1．2μg／g，6-OHDA组3．6±0．5μg／g，（6-OHDA＋BDNF）组5．5±0．6μg／g。结论①6-OHDA对黑质多巴胺能神经元有损害作用，制成偏侧帕金森病大鼠模型。②BDNF能改善6-OHDA所致的帕金森氏病大鼠黑质多巴胺能神经元数目的减少；BDNF能明显抑制6-OHDA引起的纹状体部多巴胺含量降低；BDNF可抑制6-OHDA对黑质多巴胺能神经元的毒性作用。     

高场强~1H-MRS对帕金森病大鼠模型的应用价值探讨

目的:利用高场强氢质子磁共振波谱(1H-MRS)观察帕金森病大鼠模型纹状体区的神经代谢变化,探讨高场强1H-MRS对PD大鼠模型的应用价值。方法:7只正常大鼠经6-羟基多巴胺(6-OHDA)单侧(右侧)损毁制备偏侧帕金森病模型前后应用1.5T磁共振进行波谱分析。分析造模术前后双侧纹状体区N-乙酰天门冬氨酸/肌酸(NAA/Cr)、胆碱/肌酸(Cho/Cr)比值的变化。并对黑质致密部进行黑质酪氨酸羟化酶免疫组织化学染色。结果:6只大鼠造模成功。损毁侧纹状体内NAA/Cr比值明显低于对侧及造模前同侧(P<0.05),而Cho/Cr比值与对侧及造模前同侧相比无显著性差异(P>0.05)。损毁侧黑质酪氨酸羟化酶阳性神经元较对侧显著减少(P<0.05)。结论:1.5T临床专用型磁共振1H-MRS可以作为帕金森病大鼠模型纹状体区细胞代谢有价值的无创性检测方法。     

MRI对于恒河猴PD模型建立和治疗的评价

目的：应用MRI评价建立恒河猴帕金森病（PD）模型，以及在中脑黑质受损区进行细胞移植的疗效。材料与方法：选取6只恒河猴，雌性2只，雄性4只，体重6．0－8．5kg。经颈总动脉注射甲基－苯基－四氢吡啶（MPTP）建立PD模型，并行MRI模型。获取横断和冠状位T1WI和T2WI，以单侧注射MPTP的对侧脑组织作为正常对照。建立PD模型后，再对病例中脑黑质区进行细胞移植，并复查MRI。最后应用抗酪氨酸羟化酶免疫组化和Grino染色对猴脑组织切片进行病理检查。结果：MRI的T2WI显示注射MPTP侧中脑黑质缩小，内部有局灶性高信号。细胞移植前后的MPRI所见无显著性差异。免疫组化检查证实注射MPTP侧中脑多巴胺能神经元明显减少，Grino染色显示注射侧黑质内大量胶质细胞增生。经细胞移植症状缓解的猴脑切片显示神经元的减少无明显改变。结论：MRI是恒河猴PD模型建立的一种评价手段，可以用于细胞移植疗效的评价。     

CT引导脑内植入携带人类酪氨酸羟化酶基因腺相关病毒载体治疗实验性帕金森病猴

目的 观察CT引导脑内注射入类酪氨酸羟化酶基因腺相关病毒载体(AAV—hTH)对神经细胞的转染能力，以及对帕金森病(PD)猴的治疗效应。方法 经右侧颈内动脉注射1-甲基4-苯基-1，2，3，6一四氢吡啶(MPTP)制作6只偏侧帕金森病恒河猴模型，经CT引导将AAV—hTH注入5只帕金森病猴右侧尾状核内，观测猴模型的行为学改变6个月以上，以高效液相色谱法(HLPC)测定转染后尾状核内多巴胺(DA)及其代谢产物含量，并以免疫组化检测法及逆转录-聚合酶链式反应(RT—PCR)方法检测酪氨酸羟化酶(TH)基因的表达情况。结果 CT引导定位穿刺有实时操作、定位精确、微创的优点。所有注射AAV—hTH的偏侧PD猴模型术后2周即有帕金森病症状改善，持续6个月以上，其中1只动物阿朴吗啡旋转实验示完全停止了帕金森病旋转现象，其余动物旋转次数较术前减少约42％～70％。实验侧与健侧尾状核区多巴胺及其代谢产物含量比值较未治疗模型增高。免疫组化染色见穿刺针道部位有大量TH阳性细胞；RT—PCR检测到实验侧尾状核区有TH-mRNA存在；而对照侧尾状核区、其他部位脑组织与心、肝、肾组织及未注射模型实验侧尾状核区均未见阳性发现。结论 CT引导立体定向穿刺PD猴尾状核注入AAV—hTH能使TH基因精确地在特定部位有效表达，提高尾状核区DA含量，改善模型猴症状，研究结果提示这是一种有效、安全的治疗PD非人类灵长类动物模型的方法。为临床基因治疗PD的研究提供了实验研究依据。     

帕金森病模型大鼠脑D2受体与血流灌注的对比研究

目的 对偏侧帕金森病（ＰＤ）模型大鼠脑多巴胺（ＤＡ）Ｄ２受体改变与脑血流灌注进行对比研究。方法 立体定位脑黑质、腹侧被盖区、以６－羟基多巴胺定向损毁制作偏侧ＰＤ大白鼠模型，通过阿朴吗啡诱发其向健侧旋转的转数筛选模型，并用高效液相－电化学检测器（ＨＰＬＣ－ＥＣＤ）检测ＰＤ模型及对照组大鼠脑纹状体ＤＡ含量；彩用＾１２５Ｉ－左旋－３－碘－２－羟基－６－甲氧基－Ｎ（１－乙基－２－吡咯烷）甲基）苯酰胺（ＩＢ     

三维磁共振波谱成像在健康对照组及帕金森病病人黑质中的表现

目的研究帕金森病病人黑质在3T磁场高空间分辨率的三维磁共振波谱成像中的表现。评价健康对照组及帕金森病病人黑质喙尾之间及中脑被盖区波谱数据的区域性变化。方法9例帕金森病病人及与之年龄、性别相     

Manganese-enhanced MRI in a rat model of Parkinson's disease

PURPOSE: To measure intra- and inter-hemispheric connectivity within the basal ganglia (BG) nuclei in healthy and in unilateral 6-hydroxydopamine (6-OHDA) Parkinson disease rat model in order to test the BG interhemispheric connectivity hypothesis. MATERIAL AND METHODS: The manganese-enhanced MRI (MEMRI) method with direct injection of manganese chloride into the entopeduncular (EP), substantia nigra (SN), and the Habenula nuclei in unilateral 6-OHDA (N = 22) and sham-operated (N = 16) rat groups was used. MEMRI measurements were applied before, 3, 24, and 48 hours post-manganese injection. Signal enhancements in T1-weighted images were compared between groups. RESULTS: Manganese injection into the EP nucleus resulted with bihemispheric signal enhancements in the habenular complex (Hab) at both groups with stronger enhancements in the 6-OHDA group. It also exhibited lower sensorimotor cortex signal enhancement in the 6-OHDA rat group. SN manganese injection caused enhanced anteroventral thalamic and habenular nuclei signals in the 6-OHDA rat group. Manganese habenula injection revealed enhanced interpeduncular (IP) and raphe nuclei signals of the 6-OHDA rat group. CONCLUSION: Modulations in the effective intra- and interhemispheric BG connectivity in unilateral 6-OHDA Parkinson's disease (PD) rat model support the BG interhemispheric connectivity hypothesis and suggest a linkage between the dopaminergic and serotonergic systems in PD, in line with clinical symptoms.     

阿扑吗啡阻抗6-羟基多巴胺毁损纹状体的实验研究

 目的 探讨阿扑吗啡是否对6-羟基多巴胺毁损纹状体帕金森病鼠模型有神经保护作用.方法 6-羟基多巴胺毁损大鼠左侧纹状体,在毁损前15 min阿扑吗啡(10mg/kg,皮下注射),连续注射11 d.毁损2周后,分别进行行为学(苯丙胺引起的旋转数目)和神经化学(高压液相测定纹状体多巴胺及代谢物含量)的研究.结果 阿扑吗啡能降低苯丙胺引起的向损伤侧旋转的数目.而且,显著减低多巴胺的损耗使其恢复到正常,并使DOPAC/DA比率恢复到正常.结论 在6-羟基多巴胺毁损纹状体模型中,阿扑吗啡不仅改善运动功能,而且,恢复纹状体的多巴胺含量.     

天麻对帕金森病模型大鼠行为学、生物化学的影响

 目的 探讨天麻剂量对帕金森病模型大鼠行为学及生物化学的影响及防治的机制.方法 采用6-羟基多巴胺大鼠模拟帕金森病,治疗2周后,分别进行行为学(阿朴吗啡引起的旋转数目)和神经化学(高压液相测定纹状体多巴胺及代谢物含量)的研究.结果 天麻小剂量组能降低阿朴吗啡引起的向损伤对侧旋转的数目,而且显著减少多巴胺的损耗,使其它含量恢复正常.结论 在6-羟基多巴胺毁损纹状体模型中,天麻小剂量组不仅运动功能改善,而且,可恢复纹状体的多巴胺含量.     

Detection of dopaminergic neurotransmitter activity using pharmacologic MRI: Correlation with PET,microdialysis, and behavioral data

The metabolic activation resulting from direct dopaminergic stimulation can be detected using auto-radiography, positron emission tomography (PET) or, potentially, fMRI techniques. To establish the validity of the latter possibility, we have performed a number of experiments. We measured the regional selectivity of two different dopaminergic ligands: the dopamine release compound D-amphetamine and the dopamine transporter antagonist 2β-carbomethoxy-3β-(4-fluoropheny) tropane (CFT). Both compounds led to increased signal intensity in gradient echo images in regions of the brain with high dopamine receptor density (frontal cortex, striatum, cingulate cortex ? parietal cortex). Lesioning the animals with unilaterally administered 6-hydroxydopamine (6-OHDA) led to ablation of the phMRI response on the ipsilateral side; control measurements of rCBV and rCBF using bolus injections of Gd,-DTPA showed that the baseline rCBV and rCBF values weire intact on the lesioned side. The time course of the BOLD signal changes paralleled the changes observed by microdialyisis measurements of dopamine release in the striatum for both amphetamine and CFT; peaking at 20–40 min after injection and returning to baseline at about 70–90 min. Signal changes were not correlated with either heart rate, blood pressure or pCO₂. Measurement of PET binding in the same animals showed an excellent correlation with the phMRI data when compared by either measurements of the number of pixels activated or percent signal change in a given region. The time course for the behavioral measurements of rotation in the 6-OHDA lesioned animals correlated with the phMRI. These experiments demonstrate that phMRI will become a valuable, noninvasive tool for investigation of neurotransmitter activity in vivo. Key words: fMRI; dopamine; amphetamine; CFT.     

Bilateral increase in striatal dopamine D<Subscript>2</Subscript> receptor density in the 6-hydroxydopamine-lesioned rat: a serial in vivo investigation with small animal PET

Unilateral destruction of the substantia nigra by local application of 6-hydroxydopamine (6-OHDA) serves as an animal model for Parkinson's disease. In this study, the changes in neostriatal dopamine D(2) receptor density were investigated with a small animal positron emission tomograph (PET) before and after 6-OHDA lesion. PET scans were performed in 14 rats after injection of the D(2) receptor radioligand [(18)F] N-methylbenperidol. After the first scan (day 0), nigrostriatal pathways were lesioned by unilateral injections of 6-OHDA. Further PET scans were performed on days 2 and 14 post-lesion. For both striata, B(max) values were determined from saturation binding curves with non-linear regression analysis. In the striatum ipsilateral to the lesion, B(max) initially amounted to 19.3+/-1. 9 fmol/mg (mean+/-SD) and increased to 19.7+/-2.2 and 29.9+/-5.7 fmol/mg on days 2 and 14 post-lesion, respectively. Contralateral B(max) values increased from 19.2+/-2 fmol/mg prior to the lesion to 21.2+/-2.9 and 28.6+/-5.7 fmol/mg on days 2 and 14, respectively. On day 14, the ipsilateral saturation binding curve differed from the ipsilateral pre-lesion curve (P=0.04; F test). When the contralateral pre-lesion saturation binding curve was compared with the contralateral post-lesion curve on day 14, a P value of 0.08 was obtained. This first serial in vivo imaging study of 6-OHDA-lesioned rats showed a time-dependent increase in striatal D(2) receptor density on both sides, the increase being more pronounced ipsilateral to the lesion. This result implies that compensatory mechanisms in the intact hemisphere contribute to regenerative processes following nigrostriatal dopaminergic denervation. Overall, our findings show the feasibility of repetitive in vivo studies of striatal receptor density with a small animal tomograph. Moreover, the applied in vivo saturation binding technique provides a versatile method for the quantification of time-dependent changes in the concentration of receptor binding sites.     

99mTc-TRODAT-1 SPECT in healthy and 6-OHDA lesioned parkinsonian monkeys: comparison with 18F-FDOPA PET

Parkinson's disease (PD) is a degeneration of the nigrostriatal dopaminergic pathway, leading to a selective loss of dopamine in the striatum. 99mTc-TRODAT-1 is a recently developed radiotracer that selectively binds to the dopamine transporters, which are significant because loss of these transporters corresponds with a loss of dopaminergic neurons. The present investigation compared 99mTc-TRODAT-1 single photon emission computed tomography (SPECT) with 18F-FDOPA positron emission tomography (PET) in the evaluation of PD using a primate model. Three monkeys, including one 6-hydroxydopamine lesioned PD model and two controls, were examined by both 99mTc-TRODAT-1 SPECT and 18F-FDOPA PET. For the PD monkey, expression of parkinsonian behaviour and 18F-FDOPA PET were used to evaluate the severity of the lesion. 99Tc-TRODAT-1 was prepared from a lyophilized kit. After intravenous injection of the radiotracer, SPECT was acquired over 4 h using a dual-head camera equipped with ultra-high resolution fan-beam collimators. Both uptake measurement and visual assessment were performed. Data were compared with motor behaviour and PET imaging. The striatal uptake in both healthy and PD monkeys increased continuously during the study, although the gradient of increase was less prominent in the diseased monkey. The increased uptake in the controls appeared to become blunt around 4 h after injection. A profound decrease of 99Tc-TRODAT-1 uptake was found in the striatum of the PD monkey compared with the controls (0.91 vs 2.16). In the PD monkey, the decrease of striatal uptake contralateral to the more affected side of the body was more prominent compared to the ipsilateral side (0.77 vs 1.06). In addition, greater loss occurred in the contralateral side of the putamen (0.54 vs 1.04). Changes of uptake ratios in the striatum and its subnuclei of the PD monkey were significantly correlated with those measured from PET. The loss of striatal uptake appeared greater in SPECT than the corresponding PET with both visual and uptake analyses. In conclusion, our data in a limited series of cases indicate that 99Tc-TRODAT-1 with a conventional nuclear medicine camera system may provide a suitable tool in evaluating parkinsonism in a primate model.