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.     

转基因成肌细胞脑内移植后帕金森病大鼠纹状体多巴胺含量的变化

为评价转鼠酪氨酸羟化酶（TH）基因的成肌细胞脑内移植对偏侧帕金森病（PD）大鼠模型纹状体区多巴胺及代谢产物含量的影响，应用6－羟多巴胺损毁SD大鼠单侧黑质制备偏侧帕金森病臣模型。模型稳定必个月后，移植转TH基因的成肌细胞（n＝24）或未转基国的成肌细胞（n＝10）于偏侧PD鼠损毁侧纹状体。移植治疗后6个月，用高效液相色谱电化学法（HPLC－ECD）检测偏侧PD鼠模型纹状体区多巴胺、3，4－二羟苯乙酸（DOPAC）以及高香草酸（HVA）含量。结果转TH基因成肌细胞植入组PD鼠纹状体区多巴胺及代谢产物含量明显增高（P＜0．01），其中多巴胺含量从治疗前平均39．20Pg／mg提高至治疗后的985．71Pg／mg，相当于正常侧纹状体的49．99％．对照组植入未转基因的成肌细胞后纹状体多巴胺及代谢产物含量无明显变化（P＞0．05）。可见转鼠TH基因的成肌细胞脑内移植能够部分改善偏侧PD鼠模型纹状体区多巴胺的缺乏，成肌细胞是PD基因治疗的合适靶细胞之一。     

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。     

6-羟基多巴定向注射建立帕金森病大鼠模型的实验研究

目的探讨立体定向间隔注射6-羟基多巴(6-OHDA)毁损黑质致密部(SNc)和中脑腹侧被盖(VTA)建立类似于人类帕金森病(PD)中晚期的PD大鼠模型方法。方法近交系Wistar大鼠50只,脑立体定向将6-OHDA注入大鼠右侧VTA及SNc,间隔两周,对阿朴吗啡(Apo)诱发旋转后旋转不明显或无稳定左侧旋转模型再次制模,并观察大鼠行为学改变,免疫组化检测黑质多巴胺(DA)能神经元的数量以及高效液相-荧光法检测黑质纹状体中DA含量的变化。结果(1)50只大鼠中有41只经APO诱导表现为恒定左侧旋转且结果稳定,旋转圈数>210r/30min,视为成功PD大鼠模型,部分大鼠伴有震颤、活动迟缓、嗅探、觅食、竖尾等异常行为改变,并且可持续存在16周;(2)免疫组化结果:PD大鼠模型毁损侧黑质区多巴胺能神经元较对侧及对照组减少大于90%;(3)PD大鼠右侧黑质纹状体中DA含量较左侧及对照组减少90%以上。结论6-OHDA毁损SNc及VTA间隔注射法可有效建立模拟人类PD中晚期的大鼠PD模型。     

6-羟基多巴胺单侧纹状体注射对大鼠双侧多巴胺能神经元的影响

目的探讨帕金森病大鼠模型中6-羟基多巴胺（6-OHDA）单侧纹状体注射对双侧黑质纹状体多巴胺能神经元的影响。方法大鼠随机分成模型组和对照组，模型组自一侧纹状体注射6-OHDA，对照组注射PBS；用免疫组织化学方法分别检测大鼠双侧黑质和纹状体区酪氨酸羟化酶（TH）阳性细胞和纤维的表达；高效液相色谱检测双侧纹状体多巴胺（DA）及其代谢产物3，4-二羟基苯乙酸（DOPAC）和高香草酸（HVA）含量。结果模型组大鼠双侧（毁损侧与其对侧）黑质致密区TH阳性细胞数量均少于对照组（P〈0．01），模型组双侧纹状体区TH阳性纤维密度均低于对照组；模型组大鼠双侧纹状体区DA含量均低于对照组（P〈0．01）；双侧DOPAC和HVA含量也降低。结论6-羟多巴胺单侧纹状体注射制作的帕金森病大鼠模型的对侧黑质纹状体也有损伤。     

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。     

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。     

酪氨酸羟化酶cDNA移植治疗帕金森病的实验研究

帕金森病(PD)的主要病因是黑质多巴胺能神经元损伤,表达的酪氨酸羟化酶(TH)减少。本文在应用6-羟多巴胺(6-OHDA)制备偏侧PD大鼠模型的前提下,首次将TH cDNA移植到PD大鼠纹状体内,模型动物的旋转行为明显改善,用免疫组化法及PCR法证实了外源性THcDNA可以进入脑细胞内,并表达出有生物活性的TH。     

帕金森病基因治疗的实验研究

帕金森病(PD)的主要病理特征是中脑黑质多巴胺能神经元变性,表达的酪氨酸羟化酶(TH)减少或者活性降低。目前外源性多巴是最有效的抗PD药物,但常在数年后失去其有效的治疗作用。用胚胎脑细胞移植虽有效果,但胚胎脑来源困难。因此需要探索新的有效治疗方法。本研究将遗传修饰的成肌细胞植入偏侧PD鼠模型纹状体进行基因治疗。移植转TH基因的成肌细胞(治疗组,n=24)和未经遗传修饰的成肌细胞(对照组,n=10)于偏侧PD鼠损毁侧纹状体。用阿朴吗啡(APO)诱发旋转行为,RT-PCR、TH免疫组化检测TH基因的表达和TH蛋白的合成以及HPLC-ECD检测纹状体多巴胺及代谢产物含量以此评估基因治疗的效果。治疗组移植治疗后APO诱发的旋转行为明显改善(P<0.01),且可持续13个月,而对照组APO诱发的旋转行为无改善(P>0.05),应用RT-PCR、TH免疫组化和HPLC-ECD在治疗组移植部位检测到TH基因的表达、TH蛋白的合成和移植侧纹状体部多巴胺及其代谢产物含量增高。遗传修饰的成肌细胞能够在体内长时间、有效地表达TH,并改善PD鼠的病理行为,是PD基因治疗的合适靶细胞之一。     

Vitamin D(3) attenuates 6-hydroxydopamine-induced neurotoxicity in rats

Previous reports have demonstrated that exogeneous administration of glial cell line-derived neurotrophic factor (GDNF) reduces ventral mesencephalic (VM) dopaminergic (DA) neuron damage induced by 6-hydroxydopamine (6-OHDA) lesioning in rats. Recent studies have shown that 1,25-dihydroxyvitamin D(3) (D3) enhances endogenous GDNF expression in vitro and in vivo. The purpose of present study was to investigate if administration of D3 in vivo and in vitro would protect against 6-OHDA-induced DA neuron injury. Adult male Sprague-Dawley rats were injected daily with D3 or with saline for 8 days and then lesioned unilaterally with 6-OHDA into the medial forebrain bundle. Locomotor activity was measured using automated activity chambers. We found that unilateral 6-OHDA lesioning reduced locomotor activity in saline-pretreated animals. Pretreatment with D3 for 8 days significantly restored locomotor activity in the lesioned animals. All animals were sacrificed for neurochemical analysis 6 weeks after lesioning. We found that 6-OHDA administration significantly reduced dopamine (DA), 3,4-dihydroxy-phenylacetic acid (DOPAC) and homovanilic acid (HVA) levels in the substantia nigra (SN) on the lesioned side in the saline-treated rats. D3 pretreatment protected against 6-OHDA-mediated depletion of DA and its metabolites in SN. Using primary cultures obtained from the VM of rat embryos, we found that 6-OHDA or H(2)O(2) alone caused significant cell death. Pretreatment with D3 (10(-10) M) protected VM neurons against 6-OHDA- or H(2)O(2)-induced cell death in vitro. Taken together, our data indicate that D3 pretreatment attenuates the hypokinesia and DA neuronal toxicity induced by 6-OHDA. Since both H(2)O(2) and 6-OHDA may injure cells via free radical and reactive oxygen species, the neuroprotection seen here may operate via a reversal of such a toxic mechanism.     

In vivo measurement of spontaneous release and metabolism of dopamine from intrastriatal nigral grafts using intracerebral dialysis

Spontaneous release and metabolism of dopamine (DA) from intrastriatal grafts of fetal mesencephalic DA neurons was measured by intracerebral dialysis. Mesencephalic DA cell suspensions were implanted into the head of the caudate-putamen in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the mesostriatal DA pathway. Four months later, when tests for amphetamine-induced turning behaviour showed that the grafts had become functional, loops of dialysis tubing were implanted into the striatum on the grafted side and the contralateral non-lesioned side of the grafted rats, and in a similar position in the denervated caudate-putamen of 6-OHDA lesioned control rats. Dialysis perfusates collected from the 6-OHDA lesioned striata showed a reduction of about 95-98% in DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). In the grafted animals these levels had recovered to about 40% of control for DA and to 12-16% of control for HVA and DOPAC. In addition, the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) was increased in the grafted striata compared to both the lesioned and non-lesioned controls. Amphetamine had little or no effect on DA release in the 6-OHDA lesioned rats, but caused a marked increase in DA release in the grafted rats, this response being proportional to that seen in intact striata. Since the subsequent histochemical analysis showed that the dialysis probe had been located in the transplant-reinnervated part of the caudate-putamen, the results provide additional evidence that the grafted DA neurons exert their functional effects through a continuous active transmitter release from their newly-established terminals in the reinnervated host target.     

Vitamin D3 attenuates 6-hydroxydopamine-induced neurotoxicity in rats

Previous reports have demonstrated that exogeneous administration of glial cell line-derived neurotrophic factor (GDNF) reduces ventral mesencephalic (VM) dopaminergic (DA) neuron damage induced by 6-hydroxydopamine (6-OHDA) lesioning in rats. Recent studies have shown that 1,25-dihydroxyvitamin D₃ (D3) enhances endogenous GDNF expression in vitro and in vivo. The purpose of present study was to investigate if administration of D3 in vivo and in vitro would protect against 6-OHDA-induced DA neuron injury. Adult male Sprague–Dawley rats were injected daily with D3 or with saline for 8 days and then lesioned unilaterally with 6-OHDA into the medial forebrain bundle. Locomotor activity was measured using automated activity chambers. We found that unilateral 6-OHDA lesioning reduced locomotor activity in saline-pretreated animals. Pretreatment with D3 for 8 days significantly restored locomotor activity in the lesioned animals. All animals were sacrificed for neurochemical analysis 6 weeks after lesioning. We found that 6-OHDA administration significantly reduced dopamine (DA), 3,4-dihydroxy-phenylacetic acid (DOPAC) and homovanilic acid (HVA) levels in the substantia nigra (SN) on the lesioned side in the saline-treated rats. D3 pretreatment protected against 6-OHDA-mediated depletion of DA and its metabolites in SN. Using primary cultures obtained from the VM of rat embryos, we found that 6-OHDA or H₂O₂ alone caused significant cell death. Pretreatment with D3 (10⁻¹⁰ M) protected VM neurons against 6-OHDA- or H₂O₂-induced cell death in vitro. Taken together, our data indicate that D3 pretreatment attenuates the hypokinesia and DA neuronal toxicity induced by 6-OHDA. Since both H₂O₂ and 6-OHDA may injure cells via free radical and reactive oxygen species, the neuroprotection seen here may operate via a reversal of such a toxic mechanism.     

Intraventricular microdialysis: a new method for determining monoamine metabolite concentrations in the cerebrospinal fluid of freely moving rats

A new method is described to estimate the cerebrospinal fluid (CSF) concentrations of monoamine metabolites (dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA] in the lateral ventricle of freely moving rats by use of in vivo microdialysis. Both the baseline concentrations of these metabolites and the rate of dopamine (DA) turnover (estimated by the accumulation of total DA metabolites after 200 mg/kg probenecid) were within the range reported when other methods were used to sample CSF. A series of preliminary studies were conducted to demonstrate that this method can be used to repeatedly sample CSF, and to show that the method is sensitive to local changes in dopaminergic activity induced by lesions, drugs or grafts. (1) Unilateral 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra produced a significant decrease in the CSF concentrations of DOPAC and HVA ipsilateral to the lesion, relative to the contralateral side or to concentrations in animals without lesions. (2) When left and right lateral ventricles were sampled simultaneously in animals with a unilateral 6-OHDA lesion, haloperidol induced an increase in DOPAC and HVA concentrations in CSF on both sides of the brain. Interestingly, the haloperidol-induced increase in CSF concentrations of DA metabolites was greater adjacent to the intact striatum of rats with unilateral 6-OHDA lesions than in animals with no lesion. (3) Finally, in animals with adrenal medulla tissue grafted into the lateral ventricle there was an increase in the CSF concentration of DOPAC compared to pregraft values or to those of animals with control grafts.     

Treadmill running combined with microdialysis can evaluate motor deficit and improvement following dopaminergic grafts in 6-OHDA lesioned rats

To evaluate the physiological role of striatal dopamine (DA) during exercise and the mechanism of functional recovery mediated by grafted DAergic neurons, the locomotor ability (treadmill running) and DA turnover were investigated using treadmill running combined with in vivo microdialysis in the intact control rats, 6-hydroxydopamine (6-OHDA) lesioned rats (hemi-parkinsonian model rats) and DAergic cell grafted rats. The 3 groups of rats were trained to run on a straight treadmill at a speed of 1,800 cm/min for 20 min every day for 7 consecutive days. If the rats could not follow the speed they got electrostimulation (ES) from the grid behind the treadmill belt. The numbers of ES rats received during treadmill running were counted to quantify the locomotor ability. Control rats could keep up with the treadmill easily (0-1 ES/10 min), whereas lesioned rats could not follow the speed (80-100 ES/10 min). Most of the grafted rats received only a few ES, but a few received over 100 ES/10 min. Extracellular DA and its metabolites, dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA), were measured by in vivo microdialysis and high-performance liquid chromatography (HPLC) during and after treadmill running. In control rats the basal levels of DA, DOPAC and HVA were 2.3 fmol/μl, 1,109.8 fmol/μl and 612.2 fmol/μl, respectively. They increased up to 130%, 140% and 160% by running. In 6-OHDA lesioned rats basal values of DA, DOPAC and HVA were less than 10% of controls. We did not perform microdialysis in these rats since they got too much ES during running. In grafted rats that showed good recovery in locomotor ability, DA returned to almost control level (1.9 fmol/μl), but those of DOPAC (127.8 fmol/μl) and HVA (100.2 fmol/μl) were still low. DA, DOPAC and HVA increased up to 130%, 130% and 150% by running in a similar pattern as in intact rats. These results suggest that grafted neurons can release and metabolize DA in the host striatum both tonically and phasically in relation with internal and external stimuli and also suggest that treadmill running ability is a good indicator of DA turnover in the striatum. Thus, the treadmill running test with microdialysis is useful for quantitative evaluation of motor function in grafted animals.     

L-DOPA modulation of corpus striatal dopamine and dihydroxyphenylacetic acid output from intact and 6-OHDA lesioned rats

Summary In the present report we examined the differences in in vitro dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) efflux from the corpus striatum (CS) of intact versus 6-hydroxydopamine (6-OHDA) lesioned (in substantia nigra) male rats in response to different doses of two pulse infusions of L-dihydroxyphenylalanine (L-DOPA). In the first experiment, we tested the effects of two 20-min infusions of 5 uM L-DOPA. In the second experiment we repeated this protocol using 50 uM L-DOPA. There was an overall significantly greater output of DA for intact versus 6-OHDA lesioned rats for both doses. Moreover, in Experiment 1, the 5 uM L-DOPA produced a peak DA response to the second infusion which was significantly higher than that of the first infusion in the intact, but not lesioned rats. In Experiment 2, the 50 uM L-DOPA group showed no significant differences in DA output between the two infusions for both intact and lesioned rats. In contrast to DA responses, there were no overall significant differences in DOPAC output between intact and 6-OHDA lesioned rats for both doses. However, for both doses tested, the peak DOPAC output from the second infusion was significantly increased in lesioned, but not intact rats. These data demonstrate that L-DOPA evoked DA and DOPAC output are differentially modulated in intact and 6-OHDA lesioned striatum. The lesions of the striatal dopaminergic system may alter these responses through changes in intraneuronal storage and metabolism of DA following L-DOPA infusion.     

Autoradiographic study of dopamine transporter in rat Model of Parkinson' s disease with 125I-β-CIT

Objective To evaluate the value of iaaging for dopamine transpter(DAT) wi th 125I- β-CIT. Methods The partial and complete lesioned rat models of hemiparkinsonism were rendered with 6- hydroxy-dopamine (6-OHDA). Each rat was injected intravenously with 1251-β-CIT containing 40 μ Ci. Coronal t issue sections were imaged by autoradiography. The levets of dopamine (DA)and its metabolites were measured by high performance 1iquid choromatography and electro-chemical detection (HPLC-ECD). The t yros i nc hydroxylase(Tll)-positive cells and fibres in substantia nigra and striatum of the rats were observed by immunohistochemieal staining. Results The radioactivities in the lesioned striatum of both partial and complete lesioned hemiparkinsonian rats were 2.67±0.25 and O. 98±0.29 respectively , and were singificantly decreased by.18% and 72% respectively, as compared with those of unlesioned side. The levels of DA in the lesioned striatum of partial and complete lesioned models were decreased by 39% and 98% respectively. The loss of TH-positive eells and fibres in the substantia nigra and striatum was found in the lesioned striatum of both partial and complete-lesioned models. Conclusion The imaging study of DAT may be helpful for the early diagnosis of Parkinson's disease and for the monitor of the progression of this discaose;.     

An experimental study on intracerebroventricular transplantation of human amniotic epithelial cells in a rat model of Parkinson's disease

Abstract

Objectives: Human amniotic epithelial (HAE) cells are formed from amnioblasts, separated from the epiblast at about the eighth day after fertilization. In the present study, we attempt to investigate the effects of intracerebroventricular transplantation of HAE cells on Parkinson's disease (PD) rats.

Methods: A PD rat model was induced by 6-OHDA injections. Then the rats were transplanted intracerebroventricularly with HAE cells. Apomorphin-induced turns were used to assess the neurobehavioral deficit in rats. Immunofluorescence cytochemistry was used to track the survival of HAE cells. Tyrosinehydroxylase (TH) immunohistochemistry was used to determine the density of TH-positive cells in rat substantia nigra and the differentiation of HAE cells. High performance liquid chromatography (HPLC) was used to measure the levels of dopamine (DA) and its metabolites 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat striatum. HVA levels in the cerebrospinal fluid of rats were also determined by HPLC.

Results: The results showed that transplanted HAE cells can survive for at least 10 weeks and differentiate into TH-positive cells in PD rats. The grafts significantly ameliorated apomorphine-induced turns in PD rats. TH immunohistochemistry showed that HAE cells attenuated the loss of TH-positive cells in rat substantia nigra. In addition, HAE cells prevented the fall of DA and its metabolites DOPAC and HVA in PD rats. Increased HVA levels in the cerebrospinal fluid of PD rats were also observed.

Conclusion: These results demonstrate that HAE cells have beneficial effect on 6-OHDA-induced PD rats, which may be due to the neurotrophic factors secrete by HAE cells.