止颤汤干预神经干细胞移植帕金森病大鼠脑内多巴胺及其代谢产物的变化*

背景：如何促进脑内多巴胺含量的增加以及减少多巴胺的代谢,是治疗帕金森病的热点所在。 目的：从多巴胺代谢途径角度观察止颤汤对神经干细胞移植帕金森病大鼠的脑黑质中多巴胺及其代谢产物含量的变化。 方法：以大鼠脑立体定位和1-甲基-4-苯基-1,2,3,6-四氢吡啶建立帕金森病大鼠模型。应用高效液相色谱法测定帕金森病大鼠中脑多巴胺及其代谢产物的含量。 结果与结论：止颤汤可以提高神经干细胞移植后帕金森病大鼠中脑多巴胺及其代谢产物双羟苯乙酸的含量,但对代谢产物高香草酸无明显影响。通过促进帕金森病大鼠干细胞移植后神经干细胞的存活,使之定向分化为多巴胺能神经元并分泌多巴胺,同时抑制多巴胺分解达到治疗作用。     

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.     

Activation of the Subthalamic Nucleus and Pedunculopontine Tegmentum: Does it Affect Dopamine Levels in the Substantia Nigra,Nucleus Accumbens and Striatum?

Parkinson's disease is a neurodegenerative disorder, of which the most prominent morphological feature is the progressive loss of dopaminergic nigrostriatal neurons. Increased glutamatergic transmission in the basal ganglia has been implicated in the pathophysiology of Parkinson's disease (PD). This study investigated whether death of substantia nigra (SN) dopaminergic neurons could be caused by the hyperactivity of afferent pathways resulting in the release of a toxic dose of excitatory amino acids in the SN. Twice-daily unilateral stimulation of the subthalamic nucleus (STN) for 21 days, using two different pulse frequencies and current strengths, significantly increased amphetamine-induced rotation, whereas sham stimulated rats showed significantly reduced rotation. Striatal and SN dopamine (DA) levels were unaffected when compared to naı̈ve and sham stimulated rats. However, levels of the DA metabolite, homovanillic acid (HVA), were significantly higher in the ipsilateral anterior striata of rats that had been stimulated at high frequency (100 Hz) and low current (100 μA) as compared to sham treated animals. Stimulation of the pedunculopontine tegmentum (PPT), using a single kainic acid injection, did not affect DA concentration in the ipsilateral striatum and nucleus accumbens when compared to sham-treated rats. DA levels in the contralateral striatum and nucleus accumbens of lesioned rats were significantly higher than ipsilateral levels. DOPAC/DA ratios were lower in the contralateral striatum and nucleus accumbens, suggesting decreased DA turnover. Glutamic acid decarboxylase activity was significantly higher in the ipsilateral than the contralateral SN. The physical manifestations of PD require a large reduction in caudate and putamen DA levels and no such depletion was measured in this study. These results, therefore, do not support the hypothesis that Parkinson's disease may result from an overstimulation of substantia nigral DAneurons by glutamate afferents originating from the STN or PPT.     

Substantia nigra 6-hydroxydopamine lesions alter dopaminergic synaptic markers in the nucleus basalis magnocellularis and striatum of rats

Recent findings have demonstrated the existence of dopaminergic (DA) markers in the nbM of the human brain and a reduction of these markers in both the nbM and the striatum of patients suffering from Alzheimer's disease (AD). To investigate the source of the DA synaptic markers found in the nbM, rats received unilateral 6-OHDA lesions of the substantia nigra pars compacta (SNc). The SNc lesions caused significant reductions in DA and DOPAC but not HVA in the nbM and the striatum; 3H-sulpiride binding to D2 receptors ipsilateral to the SNc lesion was significantly increased in the striatum (16%), consistent with denervation supersensitivity, but single-point analysis showed no significant changes in the nbM. These data suggest that the decreases in DA and 3H-spiperone binding levels observed in the nbM of AD patients may be due to partial destruction of DA nbM afferent projections from the brainstem.     

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。     

Behavior, neurochemistry and histology after intranigral lipopolysaccharide injection

Inflammation and neuronal degeneration of the substantia nigra (SN) occur in Parkinson's disease (PD). We studied the effects of intranigral lipopolysaccharide (LPS) injection on adult Sprague-Dawley rats. Locomotor activity measurement, neurotransmitter determination and perfusion fixation for immunohistochemistry were done on the 7th day. Bilateral LPS injection increased locomotor activity 2- to 3-fold. In the SN, dopamine (DA) and serotonin (5-HT) decreased but the ratios dihydroxyphenylacetic acid (DOPAC)/DA, homovanillic acid (HVA)/DA and 5-hydroxyindole-acetic acid (5-HIAA)/5-HT increased. In the striatum, DA, DOPAC, HVA, 3-methoxytyramine and epinephrine decreased but HVA/DA and 5-HIAA/5HT ratios increased. Unilateral LPS decreased dopamineric neurons ipsilaterally but increased contralaterally. This study provides the first evidence of behavioral hyperactivity, epinephrine suppression and neuronal plasticity in the LPS model of PD.     

Effects of intranigral administration of dopamine agonists and antagonists and baclofen on concentrations of dopac and dopamine in the striatum and substantia nigra of the rat

Summary Systemic administration of haloperidol to rats failed to alter the concentration of dopamine but increased the concentration of 3, 4-dihydroxy-phenylacetic acid (DOPAC), the major metabolite of dopamine, in both the striatum and substantia nigra. These haloperidol-induced changes in DOPAC were prevented by an intranigral microinjection of baclofen, a drug which reduces nigrostriatal dopaminergic neuronal activity.It has been proposed that nigrostriatal neurons are regulated, in part, by the activation of autoreceptors in substantia nigra. However, intranigral microinjections of high concentrations of several dopaminergic antagonists failed to mimic the biochemical effects resulting from systemic administration of these drugs; instead, they slightly increased the concentrations of both dopamine and DOPAC in the striatum and decreased dopamine in substantia nigra. Intranigral microinjections of the dopamine agonist, apomorphine, failed to alter the concentration of DOPAC in the striatum, but reduced both dopamine and DOPAC in the nigra. If DOPAC concentrations reflect nigrostriatal dopaminergic neuronal activity, the present results suggest that dopaminergic agonists and antagonists acting in substantia nigra do not exert a major influence on the activity of these neurons.     

Chronic restraint stress triggers dopaminergic and noradrenergic neurodegeneration: Possible role of chronic stress in the onset of Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and, to a lesser extent, in the noradrenergic neurons of the locus coeruleus (LC). Most cases of PD are idiopathic and sporadic and are believed to be the result of both environmental and genetic factors. Here, to the best of our knowledge, we report the first evidence that chronic restraint stress (8 h/day, 5 days/week) substantially reduces nigral DA and LC noradrenergic neuronal cell numbers in rats. Loss of DA neurons in the SNpc was evident after 2 weeks of stress and progressed in a time-dependent manner, reaching up to 61% at 16 weeks. This reduction was accompanied by robust microglial activation and oxidative stress and was marked by nitrotyrosine in the SNpc and LC of the midbrain. These results indicate that chronic stress triggers DA and noradrenergic neurodegeneration by increasing oxidative stress, and that activated microglia in the substantia nigra and LC may play an important role in modulating the neurotoxic effects of oxidative stress. Taken together, these data suggest that exposure to chronic stress triggers DA and noradrenergic neurodegeneration, which is a cause of PD.     

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。     

Glial responses associated with dopaminergic striatal reinnervation following lesions of the rat substantia nigra

Lesioning of dopaminergic substantia nigra pars compacta (SNpc) neurons leads to depletion of dopamine (DA) and dopaminergic axons in the dorsal striatum, followed by subsequent compensatory sprouting of dopaminergic fibers and striatal reinnervation. In this study, the response of striatal glia (microglia and astroglia) was compared with the degeneration and regeneration of dopaminergic axons following SNpc lesions. Following partial SNpc lesions, density of dopamine transporter (DAT) immunoreactive (-ir) terminals in the dorsal striatum returned to normal within 16 weeks of injury, suggesting that dopaminergic reinnervation of the striatum was complete. In conjunction, the glial responses in the dorsal striatum consisted of two peaks. The first peak in glial density occurred immediately after lesioning, peaking at 7 days, implying that it was likely to be associated with removal of debris from degenerating terminals. The second glial response commenced 8 weeks after lesioning and peaked some time after 16 weeks. The time of onset of the second peak suggests that it may be associated with the establishment of synapses rather than with axonal guidance.     

Taurine infused intrastriatally elevates,but intranigrally decreases striatal extracellular dopamine concentration in anaesthetised rats

Summary In the present study we infused taurine (50,150 or 450mM, 2l/min for 4h) into the dorsal striatum or into the substantia nigra via microdialysis probe and estimated the extracellular concentrations of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in the dorsal striatum of anaesthetised rats. Intrastriatal infusion of taurine elevated striatal dopamine at all concentrations studied. At the 450 mM concentration taurine elevated the extracellular dopamine 10-fold, but only in the first 30min sample after starting the taurine infusion. At 50 and 150mM taurine elevated dopamine throughout the 4h infusion maximally up to 3–4-fold the control level. Extracellular DOPAC was increased by 150 and 450mM taurine (up to about 150–160% of the control level), whereas at all three concentrations taurine decreased HVA to about 85% of the control; however, the decrease caused by 450mM taurine was short-lasting. At all three concentrations taurine infused into the substantia nigra decreased the extracellular dopamine in the ipsilateral striatum to about 40–50% of the control, and increased extracellular DOPAC and HVA maximally to about 150% and 170% of the control, respectively. These results show that the effects of taurine on the concentrations of extracellular dopamine and its metabolites depend on its administration site on nigrostriatal dopaminergic neurons. It elevates the extracellular dopamine when given into the striatum, but when given into the cell body region of the nigrostriatal dopaminergic pathway it decreases the extracellular dopamine in the ipsilateral striatum.     

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.     

Effects of homocysteine on the dopaminergic system and behavior in rodents

Long-term treatment of levodopa for Parkinson's disease (PD) patients is known to elevate homocysteine level in their plasma. The present study was designed to examine the possible neurotoxic effects of the increased homocysteine level on the dopaminergic system. Homocysteine was administered into Sprague-Dawley male rats intracerebroventricularly or C57BL/6 mice intraperitoneally. Following homocysteine injection the locomotor activities, the levels of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and immunohistochemistry of dopaminergic neurons were examined. The results obtained indicate that homocysteine administration (1 or 2 micromol, i.c.v.) into the rat brains for 5 days significantly decreased the locomotor activities and dopamine as well as its metabolites, DOPAC and HVA, in the rat striatal regions. Two different doses of homocysteine (50 and 100mg/100g, i.p. daily) were administered into mice for 36 days to evaluate the effect of systemic treatment of homocysteine on the dopaminergic neurons of the brain. The intraperitoneal injections of two doses of homocysteine significantly increased homocysteine levels in the striatal regions of mouse brains by 21.5 and 39.2%, while reducing dopamine turnover rates in the striatal regions by decreasing (DOPAC+HVA)/DA, 23.7 and 51.6%, respectively. Accordingly, homocysteine decreased locomotor activities significantly by decreasing movement time by 29 and 38%, total distance by 32 and 42%, and numbers of movement by 28 and 41%, respectively. Moreover, homocysteine decreased tyrosine hydroxylase immunoreactivity in substantia nigra of mouse brain. The data obtained indicate that the potential of homocysteine to be toxic to the dopaminergic system. Consequently, long-term levodopa therapy for PD may accelerate the progression of PD, at least in part by elevated homocysteine.     

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     

Triptolide protects dopaminergic neurons from inflammation-mediated damage induced by lipopolysaccharide intranigral injection

Converging lines of evidence suggest that neuroinflammatory processes may account for the progressive death of dopaminergic neurons in Parkinson's disease (PD). Therefore, anti-inflammatory strategies have attracted much interest for their potential to prevent further deterioration of PD. Our previous study showed that triptolide, a traditional Chinese herbal compound with anti-inflammatory and immunosuppressive properties, protected dopaminergic neurons from lipopolysaccharide (LPS)-induced damage in primary embryonic midbrain cell cultures. To examine further if triptolide can protect dopaminergic neurons from inflammation-mediated damage in vivo, microglial activation and injury of dopaminergic neurons were induced by LPS intranigral injection, and the effects of triptolide treatment on microglial activation and survival ratio and function of dopaminergic neurons were investigated. Our results demonstrated that microglial activation induced by a single intranigral dose of 10 mug of LPS reduced the survival ratio of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the substantia nigra pars compacta (SNpc) to 29% and the content of dopamine (DA) in striatum to 37% of the non-injected side. Intriguingly, treatment with triptolide of 5 mug/kg for 24 days once per day dramatically improved the survival rate of TH-ir neurons in the SNpc to 79% of the non-injected side. Meanwhile, treatment with triptolide of 1 or 5 mug/kg for 24 days once per day significantly improved DA level in striatum to 70% and 68% of the non-injected side, respectively. Complement receptor 3 (CR3) immunohistochemical staining revealed that triptolide treatment potently inhibited LPS-elicited deleterious activation of microglia in SNpc. The excessive production of cytokines, such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta, was significantly abolished by triptolide administration. These results, together with our previous data in vitro, highly suggest the effectiveness of triptolide in protecting dopaminergic neurons against inflammatory challenge.     

Pharmacokinetic,neurochemical, stereological and neuropathological studies on the potential effects of paraquat in the substantia nigra pars compacta and striatum of male C57BL/6J mice

The pharmacokinetics and neurotoxicity of paraquat dichloride (PQ) were assessed following once weekly administration to C57BL/6J male mice by intraperitoneal injection for 1, 2 or 3 weeks at doses of 10, 15 or 25 mg/kg/week. Approximately 0.3% of the administered dose was taken up by the brain and was slowly eliminated, with a half-life of approximately 3 weeks. PQ did not alter the concentration of dopamine (DA), homovanillic acid (HVA) or 3,4-dihydroxyphenylacetic acid (DOPAC), or increase dopamine turnover in the striatum. There was inconsistent stereological evidence of a loss of DA neurons, as identified by chromogenic or fluorescent-tagged antibodies to tyrosine hydroxylase in the substantia nigra pars compacta (SNpc). There was no evidence that PQ induced neuronal degeneration in the SNpc or degenerating neuronal processes in the striatum, as indicated by the absence of uptake of silver stain or reduced immunolabeling of tyrosine-hydroxylase-positive (TH⁺) neurons. There was no evidence of apoptotic cell death, which was evaluated using TUNEL or caspase 3 assays. Microglia (IBA-1 immunoreactivity) and astrocytes (GFAP immunoreactivity) were not activated in PQ-treated mice 4, 8, 16, 24, 48, 96 or 168 h after 1, 2 or 3 doses of PQ.In contrast, mice dosed with the positive control substance, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 10 mg/kg/dose × 4 doses, 2 h apart), displayed significantly reduced DA and DOPAC concentrations and increased DA turnover in the striatum 7 days after dosing. The number of TH⁺ neurons in the SNpc was reduced, and there were increased numbers of degenerating neurons and neuronal processes in the SNpc and striatum. MPTP-mediated cell death was not attributed to apoptosis. MPTP activated microglia and astrocytes within 4 h of the last dose, reaching a peak within 48 h. The microglial response ended by 96 h in the SNpc, but the astrocytic response continued through 168 h in the striatum.These results bring into question previous published stereological studies that report loss of TH⁺ neurons in the SNpc of PQ-treated mice. This study also suggests that even if the reduction in TH⁺ neurons reported by others occurs in PQ-treated mice, this apparent phenotypic change is unaccompanied by neuronal cell death or by modification of dopamine levels in the striatum.     

Inhibition of striatal energy metabolism produces cell loss in the ipsilateral substantia nigra

This study examined whether damage to dopamine (DA) nerve terminals via inhibition of energy metabolism in the striatum would result in the retrograde loss of cell bodies in the substantia nigra. Infusion of 2 μmol malonate into the left striatum of rats resulted in a 67% loss of striatal DA and a 40% loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra. No change in the number of Nissl-positive-TH-negative neurons was observed. These findings demonstrate the retrograde destruction of DA cell bodies in the substantia nigra resulting from energy impairment at their terminal projection site.