左旋多巴对帕金森病大鼠黑质多巴胺能神经元及纹状体多巴胺递质的影响

目的　研究长期应用左旋多巴对帕金森病 (PD)大鼠黑质多巴胺 (DA)能神经元和DA递质的影响。方法　采用 6 羟基多巴胺 (6 OHDA)制备部分损毁和严重损毁的PD大鼠模型 ,给两种模型口服不同剂量左旋多巴 /苄丝肼 3个月 ,通过观察大鼠旋转行为、酪氨酸羟化酶 (TH)免疫组化染色和高效液相色谱 电化学检测仪 (HPLC ECD)检测纹状体单胺类递质 ,研究左旋多巴对PD大鼠残存的黑质DA能神经元的影响。结果　 (1)左旋多巴对PD大鼠的旋转行为无明显影响 ;(2 )TH阳性细胞数损毁侧 /非损毁侧比值在左旋多巴喂药组和不喂药对照组的差异无显著意义 (P >0 0 5 ) ;(3)在严重损毁组 ,大剂量左旋多巴使PD大鼠损毁侧DA和 3,4二羟基苯乙酸 (DOPAC)水平明显升高(P <0 0 1)。结论　长期使用左旋多巴对 6 OHDA单侧损毁的PD大鼠残存的黑质DA能神经元无毒性作用。     

左旋多巴治疗对实验性帕金森病大鼠黑质纹状体TNF—a表达的影响

目的　研究左旋多巴 (L - dopa)治疗对实验性帕金森病 (PD)大鼠黑质纹状体肿瘤坏死因子(TNF-α)表达的影响。方法　黑质定位注射 6 -羟多巴胺 (6 - OHDA )制备偏侧 PD大鼠模型 ,经 5 0～ 10 0 mg/ kg体重 L - dopa灌胃治疗 4周后 ,检测双侧额叶皮质、黑质和纹状体区域 TNF-α的表达。结果　 6 - OHDA损毁侧黑质和纹状体 TNF-α含量和阳性细胞面密度分别显著高于健侧 (均 P<0 .0 5 )。损毁侧与健侧 TNF-α含量的比率随 L - dopa治疗用量的增加而增高 ,且均显著高于对照组 (P<0 .0 5 )。结论　 L - dopa治疗进一步加剧了PD大鼠黑质纹状体区域 TNF-α的高表达。     

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。     

帕金森病样大鼠模型的稳定性观察

6-OHDA损毁大鼠一侧中脑黑质细胞造成偏侧帕金森病(PD)样大鼠模型,用小动物旋转行为记录仪记录阿朴吗啡诱发大鼠的旋转行为;TH免疫细胞化学染色观察中脑黑质细胞的损毁状况.连续观察10个月其旋转行为无自发性恢复;PD样模型大鼠损毁侧中脑无TH阳性细胞存在.结果表明6-OHDA损毁大鼠一侧中脑黑质细胞可造成稳定、可靠的类似PD病人病理变化的偏侧动物模型.     

帕金病样大鼠模型的稳定性观察

6-OHDA损毁大鼠一侧中脑黑质细胞造成偏侧帕金森病(PD)样大鼠模型,用小动物旋转行为记录仪记录阿朴吗啡诱发大鼠的旋转行为;TH免疫细胞化学染色观察中脑黑质细胞的损毁状况。连续观察10个月其旋转行为无自发性恢复;PD样模型大鼠损毁侧中脑无TH阳性细胞存在。结果表明6-OHDA损毁大鼠一侧中脑黑质细胞可造成稳定、可靠的类似PD病人病理变化的偏侧动物模型。     

左旋多巴治疗对实验性帕金森病大鼠黑质纹状体TNF-α表达的影响

目的 研究左旋多巴(L-dopa)治疗对实验性帕金森病(PD)大鼠黑质纹状体肿瘤坏死因子(TNF-α)表达的影响.方法 黑质定位注射6-羟多巴胺(6-OHDA)制备偏侧PD大鼠模型,经50～100 mg/kg体重L-dopa灌胃治疗4周后,检测双侧额叶皮质、黑质和纹状体区域TNF-α的表达.结果 6-OHDA损毁侧黑质和纹状体TNF-α含量和阳性细胞面密度分别显著高于健侧(均P<0.05).损毁侧与健侧TNF-α含量的比率随L-dopa治疗用量的增加而增高,且均显著高于对照组(P<0.05).结论 L-dopa治疗进一步加剧了PD大鼠黑质纹状体区域TNF-α的高表达.     

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。     

立体定向下恒河猴偏侧部分损伤性帕金森病模型的制作

目的　制作一种能保留内侧前脑束 (MFB)内的多巴胺 (DA)神经纤维的偏侧部分损伤性帕金森病(PD)猴模型。方法　以 6 羟多巴 (6 OHDA)溶液对 12只恒河猴右侧黑质致密部行多靶点毁损。术后进行行为学观察、MR、PET、SPECT检查及酪氨酸羟化酶 (TH)的免疫组织化学染色。结果　猴术后符合帕金森病表现。MR示靶点准确位于黑质 ,PET示毁损侧黑质纹状体区代谢减低。TH染色示黑质致密部DA能神经元毁损达 80 %以上 ,而中脑腹侧被盖区和MFB区的DA能神经纤维保留较好。结论　用立体定向注射 6 OHDA毁损一侧黑质致密部的方法可制作出偏侧部分损伤性帕金森病恒河猴模型     

尼古丁对帕金森病大鼠纹状体多巴胺及黑质自由基含量的影响

目的 :研究尼古丁对帕金森病大鼠的影响 ,探讨其对 PD的作用机制。方法 :通过 6 - OHDA脑立体定向注射术建立大鼠帕金森病模型。采用生化方法观察不同剂量尼古丁对帕金森病大鼠的作用 ,检测黑质自由基、抗氧化剂及多巴胺含量的变化。结果 :造模前及造模后皮下注射尼古丁的 PD大鼠 ,黑质自由基及抗自由基酶及多巴胺含量较PD组有明显改善 (P<0 .0 5 )。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     

应用蛋白酶体抑制剂Lactacystin建立有Lewy体的帕金森病大鼠模型

目的建立符合帕金森病(PD)病理特征———黑质细胞有Lewy体的PD大鼠模型。方法分别在大鼠一侧黑质致密部注射蛋白酶体抑制剂Lactacystin8mg(Lactacystin组)、等体积生理盐水(NS组)和6-羟基多巴胺(6-OHDA组)12mg;观察大鼠自主行为和阿朴吗啡诱导的旋转行为;光镜下观察中脑组织学改变;应用免疫组化染色观察黑质细胞α-synuclein表达和酪氨酸羟化酶(TH)阳性细胞数;测定纹状体区多巴胺和高香草酸含量。结果NS组大鼠未见行为异常;Lactacystin组大鼠出现进行性的运动迟缓、少动、震颤、头向健侧倾斜,注射阿朴吗啡后出现向健侧旋转运动;给药后3周黑质部TH阳性细胞数较NS组减少了83.29%(P<0.01),部分黑质细胞内出现α-synuclein免疫反应呈强阳性的Lewy体;纹状体多巴胺和高香草酸含量(154.82±37.17,98.66±18.81)较NS组明显减少(822.87±131.25,617.77±95.74)(均P<0.01);6-OHDA组大鼠出现与Lactacystin组类似的行为变化,黑质细胞亦显著减少,但未见Lewy体。结论利用蛋白酶体抑制剂Lactacystin阻碍α-synuclein的降解可以建立有Lewy体的PD大鼠模型。     

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。     

铜离子引起大鼠黑质多巴胺能神经元的退行性变

目的探讨黑质（substantia nigra,SN）内注射不同剂量的CuSO45H2O对大鼠黑质纹状体系统多巴胺能神经元的影响。方法实验用Wistar大鼠,分成对照组和左侧SN内分别注射10nmol、50nmol、200nmol CuSO。组,7天后采用高效液相色谱法（high performance lipid chromotophotography,HPLC）检测纹状体内多巴胺（dopamine,DA）及其代谢产物的含量;酪氨酸羟化酶（tyrosine hydroxylase,TH）免疫组织化学法检测纹状体内TH免疫阳性纤维的改变;半定量RT-PCR法检测黑质内TH,Caspase-3mRNA的表达量：用生化试剂盒分析大鼠中脑内超氧化物岐化酶（superoxide dismutase,SOD）活性的改变。结果在10nmol CuSO4注射组中,DA及其代谢产物的含量与对照组相比没有统计学差别。但是从50nmol组开始,损毁侧纹状体内DA含量随注射CuSO4剂量的增加而逐渐减少,显示出明显的剂量依赖关系（F=34．16,P〈0．01）。注射50nmol CuSO4组大鼠纹状体内TH免疫阳性纤维明显少于对照组和未损毁侧（F=121．9,P〈0．01）。注射50nmol CuSO4组大鼠SN内THmRNA的表达与对照组相比下降（t=3．12,P〈0．01）,但Caspase-3mRNA的表达量与对照组相比却明显增加（t=8．96,P〈0．01）。在注射50nmolCuSO4组中,大鼠损伤侧中脑内SOD的活性与对照组相比下降（t=2．33,P〈0．01）。结论铜离子可以导致大鼠黑质内多巴胺能神经元的损伤,该损伤作用可能是通过破坏抗氧化保护系统和促进细胞凋亡而实现的。     

The dual dopamine‐glutamate phenotype of growing mesencephalic neurons regresses in mature rat brain

Coexpression of tyrosine hydroxylase (TH) and vesicular glutamate transporter 2 (VGLUT2) mRNAs in the ventral tegmental area (VTA) and colocalization of these proteins in axon terminals of the nucleus accumbens (nAcb) have recently been demonstrated in immature (15‐day‐old) rat. After neonatal 6‐hydroxydopamine (6‐OHDA) lesion, the proportion of VTA neurons expressing both mRNAs and of nAcb terminals displaying the two proteins was enhanced. To determine the fate of this dual phenotype in adults, double in situ hybridization and dual immunolabeling for TH and VGLUT2 were performed in 90‐day‐old rats subjected or not to the neonatal 6‐OHDA lesion. Very few neurons expressed both mRNAs in the VTA and substantia nigra (SN) of P90 rats, even after neonatal 6‐OHDA. Dually immunolabeled terminals were no longer found in the nAcb of normal P90 rats and were exceedingly rare in the nAcb of 6‐OHDA‐lesioned rats, although they had represented 28% and 37% of all TH terminals at P15. Similarly, 17% of all TH terminals in normal neostriatum and 46% in the dopamine neoinnervation of SN in 6‐OHDA‐lesioned rats were also immunoreactive for VGLUT2 at P15, but none at P90. In these three regions, all dually labeled terminals made synapse, in contradistinction to those immunolabeled for only TH or VGLUT2 at P15. These results suggest a regression of the VGLUT2 phenotype of dopamine neurons with age, following normal development, lesion, or sprouting after injury, and a role for glutamate in the establishment of synapses by these neurons. J. Comp. Neurol. 517:873–891, 2009. © 2009 Wiley‐Liss, Inc.     

谷胱甘肽对黑质多巴胺能神经元保护作用的研究

目的　探讨谷胱甘肽对多巴胺能神经元保护作用。方法　用还原型谷胱甘肽和 6 羟多巴胺孵育大鼠脑片 1小时 ,用抗酪氨酸羟化酶免疫组织化学法观察黑质阳性神经元胞体及突起的变化。结果　 6 羟多巴胺使脑片黑质酪氨酸羟化酶阳性神经元突起明显减少 ,而胞体不变 ;谷胱甘肽单独不影响多巴胺能神经元 ,但却阻止 6 羟多巴胺引起的神经元突起减少。结论　谷胱甘肽能阻止 6 羟多巴胺导致的多巴胺能神经元的变性 ,对多巴胺能神经元具有保护作用     

Quantitative analyses of GFRalpha-1 and GFRalpha-2 mRNAs and tyrosine hydroxylase protein in the nigrostriatal system reveal bilateral compensatory changes following unilateral 6-OHDA lesions in the rat

Copy numbers of mRNAs for GFRalpha-1 and GFRalpha-2, the preferred receptors for glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) were determined by real-time quantitative RT-PCR (QRT-PCR). Receptor expression was assessed in striatum (ST) and substantia nigra (SN) of normal rats and rats acutely or progressively lesioned by 6-OHDA injected into the medial forebrain bundle or ST, respectively. GFRalpha-1 mRNA was clearly detected in normal ST. In normal SN, significantly higher expression of both receptors was observed. At 4 weeks after acute lesion, GFRalpha-2 mRNA was markedly decreased in SN bilaterally, whereas GFRalpha-1 mRNA in SN and ST was not affected. A progressive lesion resulted in a progressive decrease of GFRalpha1 mRNA in ST bilaterally. In SN, levels of GFRalpha-1 mRNA were not significantly affected by a progressive lesion, whereas GFRalpha-2 mRNA was markedly decreased bilaterally. Quantitative western blotting standardized against tyrosine hydroxylase (TH) protein from PC12 cells revealed the expected decrease in TH protein in lesioned SN, but also significant increases in TH protein in contralateral, unlesioned SNs at 4 weeks after both acute and progressive lesions. These data suggest that previously unrecognized compensatory changes in the nigrostriatal system occur in response to unilateral dopamine depletion. Since the changes observed in receptor expression did not always parallel loss of dopamine neurons, cells in addition to the nigral dopamine neurons appear to be affected by a 6-OHDA insult and are potential targets for the neurotrophic factors, GDNF and NTN.     

MPTP处理的小鼠和6-OHDA损毁的大鼠帕金森病模型病理变化的比较

目的比较ＭＰＴＰ处理的小鼠与６－ＯＨＤＡ损毁大鼠ＰＤ模型的病理变化。方法应用免疫组化方法观察两种ＰＤ模型的中脑腹侧多巴胺能神经元,星形胶质细胞的变化。结果免疫组化方法结果表明：Ｃ５７ＢＬ小鼠在ＭＰＴＰ处理后,脑内酪氨酸羟化酶阳性神经元的数目从第４ｄ开始有所减少;黑质区域的ＧＦＡＰ免疫阳性星形胶质细胞数目从第１ｄ起即有增加。大鼠ＰＤ模型较早出现损毁侧ＴＨ免疫阳性神经元明显减少的现象,损毁２月后几乎完全消失;而在黑质区的ＧＦＡＰ免疫阳性星形胶质细胞数目增多现象出现较晚。结论两种动物模型从不同侧面反映了帕金森病的病理特征,６－ＯＨＤＡ损毁中脑ＤＡ能神经元制成的大鼠ＰＤ模型比ＭＰＴＰ处理的Ｃ５７ＢＬ小鼠ＰＤ模型更接近反映ＰＤ的病理变化。     

Age-related decreases in Nurr1 immunoreactivity in the human substantia nigra

Nuclear receptor-related factor 1 (Nurr1), a member of the nuclear receptor superfamily, is associated with the induction of dopaminergic (DA) phenotypes in developing and mature midbrain neurons. It is well established that dopaminergic nigrostriatal function decreases with age. Whether age-related deficits in DA phenotypic markers are associated with alterations in Nurr1 expression is unknown. The present study found that virtually all of tyrosine hydroxylase-immunoreactive (TH-ir) neurons within the young adult human substantia nigra were Nurr1-immunoreactive (Nurr1-ir) positive. Stereologic counts revealed a significant reduction in the number of Nurr1-ir nigral neurons in middle-aged (23.13%) and aged (46.33%) individuals relative to young subjects. The loss of Nurr1-ir neurons was associated with a similar decline in TH-ir neuron number. In this regard, TH-ir neuronal number was decreased in middle-aged (11.10%) and in aged (45.97%) subjects, and this loss of TH-ir neurons was highly correlated (r = 0.92) with the loss of Nurr1-ir neurons. In contrast, the number of melanin-containing nigral neuron number was generally stable across age groups, indicating that changes in Nurr1 and TH reflect phenotypic age-related changes and not frank neuronal degeneration. In support of this concept, confocal microscopic analyses of Nurr1-ir and TH-ir fluorescence intensity revealed parallel decreases in Nurr1- and TH-immunofluorescence as a function of age. These data demonstrate that age-related decline of DA phenotypic markers is associated with down-regulation of Nurr1 expression in the SN.     

Retinoid X receptor α acts as a negative regulator in Nurr1-induced dopaminergic differentiation in rat neural precursor cells

To investigate the role of retinoid X receptor (RXRα)–Nurr1 heterodimers in tyrosine hydroxylase (TH) expression, we observed retrovirus-induced RXRα–Nurr1 heterodimer interactions with, and transactivation of, the TH promoter region in cultured rat embryonic neural precursor cells. Interestingly, forced expression of RXRα with Nurr1 remarkably reduced Nurr1 activity in TH+ dopaminergic neuron generation and significantly down-regulated TH promoter activity. These regulatory activities were altered in both Nurr1dim- and RXRαdim- that disrupted dimeric binding, verifying that the Nurr1–RXRα heterodimer represses TH promoter activity. Therefore, a plausible explanation for the inhibitory role of RXRα in Nurr1-induced TH expression is that RXRα differentially affects an inhibitory element of the TH promoter.     

经颅重复性磁刺激后大鼠纹状体FosB蛋白的表达

观察经颅重复性低频磁刺激（rTMS)对大鼠纹状体FosB蛋白表达的影响。6－OHDA单侧损毁纹状体边缘区,磁刺激器给予大鼠头部1Hz,100mT的重复性刺激,14d后用免疫组织化学ABC法检测纹状体FosB免疫阳性产物。rTMS后能够引起大鼠纹状体区域明显的FosB蛋白表达,这种表达广泛分布于尾壳核及苍白球,尤以腹侧纹状体及尾侧的壳核明显。6－OHDA损毁后予以rTMS,损毁侧Fos蛋白表达未出现减少,且尾壳核的背外侧部表达明显上调。对照组动物仅损毁侧有少量的FosB表达外,纹状体内未见FosB阳性标记。经颅重复性低频磁刺激能够激活纹状体内神经元,推测rTMS可能在增加多巴胺释放的同时,又能够激活多巴胺受体的活性,故在帕金森病等的治疗上有一定的意义。