丘脑底核高频电刺激治疗帕金森病的机制

目的 研究丘脑底核(STN)高频电刺激(HFS)对大鼠黑质-纹状体系统的影响.方法 给予大鼠一侧STN-HFS,应用RT-PCR和蛋白印迹(Western blot)技术观察其对黑质-纹状体TH的影响.结果 RT-PCR检测发现刺激侧黑质-纹状体TH mRNA增高(0.22±0.08 vs 0.07±0.01,P＜0.01;0.41±0.13 vs 0.17±0.09,P＜0.01);Western blot检测发现刺激侧黑质-纹状体TH表达明显增高(0.99±0.14 vs 0.33±0.08,P＜0.01;0.87±0.16 vs 0.19±0.04,P＜0.01).结论 STN-HFS可能通过影响黑质-纹状体多巴胺代谢及其限速酶的水平发挥作用.     

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。     

脂多糖注入黑质对大鼠黑质纹状体单胺类递质和行为的影响

目的　探讨脂多糖 (L ipopolysaccharide,L PS)对大鼠行为学和黑质纹状体单胺类递质的影响。方法　采用立体定位注射 5μg L PS入大鼠脑黑质 ,在不同时间点观察大鼠注射阿朴吗啡后的旋转行为学 ,及采用HPL C测定黑质纹状体单胺类递质的含量变化。结果　 L PS注射后 14、2 1、3 0 d,大鼠出现向注射侧的旋转行为 ,在黑质和纹状体 ,DA及其代谢产物随时间改变呈不同程度下降 (P<0 .0 5) ,而 5-HT仅有短暂下降 ,NA无变化。结论　 L PS注入黑质特异性损害 DA能神经元 ,可降低黑质纹状体 DA及其代谢产物含量 ,诱导大鼠产生旋转行为     

老年大鼠黑质致密部多巴胺能神经元的变化

目的探讨老年大鼠黑质致密部多巴胺(DA)能神经元的变化。方法采用逆转录PCR(RT-PCR)、免疫印迹法、免疫组化染色和电镜技术,观察老年大鼠(≥24月龄)黑质致密部DA能神经元酪氨酸羟化酶(TH)mRNA、蛋白表达水平和TH阳性神经元数及其超微结构,并与成年大鼠(4～5月龄)比较。结果老年组大鼠黑质致密部THmRNA水平(0.66±0.12)明显低于成年组(1.09±0.08)(P<0.05);TH蛋白水平及TH阳性神经元数与成年组差异均无统计学意义(均P>0.05);老年组大鼠黑质致密部尾侧段TH阳性神经元数比成年组明显减少(P<0.05),其超微结构显示细胞器少、脂褐素多、核固缩、线粒体形态异常且数量减少。结论老年大鼠黑质致密部DA能神经元THmRNA水平下降,尾侧DA能神经元明显减少,并呈衰老性改变,这可能是老年黑质纹状体功能降低的生物学基础。     

偏侧猴PD模型STN慢性高频电刺激对脑内多巴胺系统的影响

目的探讨丘脑底核(STN)慢性高频电刺激对猴偏侧帕金森病(PD)模型脑内多巴胺(DA)系统的影响。方法采用单侧颈内动脉注入1-甲基-4-苯基-1,2,3,6四氢吡啶(MPTP)建立猴偏侧PD模型2只,体内植入脑深部电刺激(DBS)系统,行右侧STN慢性高频电刺激。在电刺激前后不同时间点采用微透析技术检测纹状体区细胞外液的多巴胺(DA)及其代谢产物高香草酸(HVA)含量,腰穿取脑脊液标本测量脑脊液中DA、HVA含量,单光子放射计算机断层扫描(SPECT)检测脑内纹状体区巴胺转运体(DAT)及多巴胺D2受体(D2R)的变化。结果猴偏侧PD模型在给予单侧STN慢性高频电刺激后纹状体区DA、HVA明显增高。SPECT显示在有效刺激后纹状体区DAT特异性摄取率增高,D2R特异性摄取率下降。脑脊液中多巴胺及其代谢产物的含量与术前相比无明显差异。结论通过微透析检测提示在给予STN有效慢性高频电刺激后提高了纹状体区DA及其代谢产物的升高,DAT特异性摄取率增高,D2R特异性摄取率下降提示纹状体区的代谢活性有明显升高,这可能是STN-DBS治疗帕金森病的重要机制之一。     

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。     

铁离子对黑质纹状体多巴胺神经元毒性作用的实验研究

目的 探讨铁离子对黑质纹状体多巴胺神经元的毒性作用。方法 采用立体定向偏侧大鼠黑质内注入50μg FeCl3和FeCl2，4周后用阿朴吗啡诱导动物行为学变化，高效液相色谱(HPLC)检测纹状体内多巴胺、去甲肾上腺素、肾上腺素递质含量的变化，免疫组化观察黑质多巴胺神经元和胶质细胞的改变。结果 FeCL3和FeCL2均可引起注射侧纹状体内DA含量选择性降低，而NA、A含量无显著改变；注射侧黑质内DA神经元显著缺失、胶质细胞显著增生；FeCL3组阿朴吗啡诱导大鼠向同侧旋转行为，FeCL2组大鼠于术后即出现特征性自发性对侧旋转行为，阿朴吗啡不能诱发其旋转。结论 铁离子对黑质纹状体多巴胺神经元具有毒性作用，Fe^3 作用最强，胶质细胞的增生可能参与了这一毒性作用过程。     

胶质细胞系源性神经营养因子对帕金森病大鼠黑质钙结合蛋白表达的影响

目的研究胶质细胞系源性神经营养因子(GDNF)对帕金森病(PD)大鼠黑质钙结合蛋白(CB)表达的影响,以及神经细胞黏附分子(NCAM)在其中的作用。方法制作PD模型大鼠36只,分为GD-NF组、NCAM阻断组和对照组,每组12只。采用免疫组化染色和免疫印迹法,检测各组大鼠黑质CB阳性细胞数和CB表达量。结果GDNF组黑质处CB阳性神经元数(46.50±6.28)及表达量(33770.60±6929.76)明显高于对照组[(27.00±8.60)、(18281.00±5266.78)](均P<0.05);与NCAM阻断组[(44.00±13.37)、(30857.00±7484.87)]相比差异无统计学意义(均P>0.05)。结论GDNF可上调PD大鼠黑质CB的表达,从而保护受损的多巴胺能神经元,NCAM对这一作用无明显影响。     

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。     

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。     

High-frequency stimulation of the subthalamic nucleus prolongs the increase in striatal dopamine induced by acute l-3,4-dihydroxyphenylalanine in dopaminergic denervated rats

High-frequency stimulation of the subthalamic nucleus (STN-HFS) is a powerful approach for treating the motor symptoms of Parkinson's disease (PD). It results in clinical improvement in patients with PD, further reducing the l-3,4-dihydroxyphenylalanine (L-DOPA) requirement and thus L-DOPA-induced dyskinesia. However, it remains unclear how STN-HFS modifies the response to L-DOPA. We investigated the effect of STN-HFS on striatal extracellular concentrations of dopamine and its metabolites following acute L-DOPA administration in intact or partially dopaminergic denervated (DA-PL) rats. L-DOPA treatment significantly increased striatal dopamine levels in intact and DA-PL animals, with the maximal effect observed 1 h after L-DOPA injection. This increase was more pronounced in DA-PL rats (ipsilateral to the lesion) than in intact animals. It remained fairly stable 1 h after the maximal effect of L-DOPA and then decreased towards basal values. STN-HFS in intact rats had no effect on the maximal L-DOPA-induced increase in striatal extracellular dopamine concentration or the return to basal values, the profiles observed being similar to those for non-stimulated intact animals. Conversely, STN-HFS amplified the L-DOPA-induced increase in striatal dopamine levels during the stimulation period (1 h) in DA-PL rats and this increase was sustained throughout the post-stimulation period (2.5 h), without the return to basal levels observed in stimulated intact and non-stimulated rats. These new neurochemical data suggest that STN-HFS interferes with L-DOPA effects, probably synergically, by stabilizing dopamine levels in the striatum and shed light on the mechanisms of STN-HFS in PD.     

Placebo-controlled chronic high-frequency stimulation of the subthalamic nucleus preserves dopaminergic nigral neurons in a rat model of progressive Parkinsonism

Chronic high-frequency stimulation (HFS) of the subthalamic nucleus (STN) protects nigral dopaminergic neurons from neurodegeneration in animal models of Parkinson's disease (PD). However, these data are challenged by the lack of control for neuroprotective effects that might be related to tissue damage due to electrode insertion or STN-HFS. Here we report the first placebo-controlled study on continuous STN-HFS in a rat model of PD using an implantable microstimulation system. We found a significant increase of preserved dopaminergic nigral neurons on the lesioned side (expressed as ratio to the non-lesioned side) of approximately 50% in comparison to STN sham-stimulated and STN-naive rats. These data provide evidence for the phenotypic rescue of nigral dopamine neurons by long-term STN-HFS in this animal model of PD.     

Subthalamic nucleus lesions are neuroprotective against terminal 6-OHDA-induced striatal lesions and restore postural balancing reactions

Inactivation of the subthalamic nucleus (STN) by deep brain stimulation or lesioning can ameliorate symptoms in Parkinson' disease (PD) and may alter the underlying progressive degenerative process. We evaluated the effects of STN lesions in a terminal lesion model of PD in rats. Multiple intrastriatal 6-OHDA injections (4 x 7 microg) resulted in a partial loss of striatal TH-positive innervation (-30 to -40%) and nigral dopaminergic neurons (-60%), which was associated with behavioral deficits as observed in drug-induced rotational asymmetry, side-stepping, and postural balancing reactions. Unilateral ibotenic acid lesions of the STN did produce a 50-60% loss of STN neurons based on stereological analysis, which did not induce a functional impairment in rotational asymmetry or spontaneous sensorimotor behaviors. When STN lesions were performed 1 week prior to the 6-OHDA terminal striatal lesions, a significant rescue effect (+23%) on nigral dopaminergic neurons against terminal 6-OHDA neurotoxicity could be demonstrated, whereas striatal TH-positive fiber loss was not attenuated in these animals. In addition, animals with combined STN and striatal lesions exhibited a significant recovery in postural balancing reactions induced by 6-OHDA terminal lesions and did not show a significant impairment in any of the other behavioral parameters examined. Taken together, STN lesions can exert neuroprotective effects on nigral dopamine neurons in a partial lesion model of PD which result in recovery of spontaneous sensorimotor behavior. These findings may therefore provide new insights into the functional interaction between the glutamatergic and the dopaminergic neurotransmitter systems and foster novel therapeutic concepts for the early and middle phases of Parkinson's disease.     

Effects of electrolytic and 6-hydroxydopamine lesions of the lateral hypothalamus on rotation evoked by electrical stimulation of the substantia nigra in rats

Contralateral rotation evoked by electrical stimulation of the left substantia nigra was studied in rats before and after electrolytic or 6-hydroxydopamine (6-OHDA) lesions of the lateral hypothalamus. Electrolytic lesions (2 mA DC, 15 sec) which produced mean ipsilateral striatal dopamine depletion of 58% significantly reduced the rotation at 2 h to 14 days postlesion. 6-OHDA (8 μg in 4 μl) which produced mean ipsilateral striatal dopamine depletion of 93% significantly increased the rotation at 3 to 14 days postlesion. Haloperidol 0.1 and 0.5 mg/kg i.p. partially reduced rotation in both control and lesioned rats in a dose-related manner. Control and lesioned rats showed no sognificant differences in haloperidol sensitivity. If stimulus induced rotation were mediated by activation of dopaminergic neurons, one would have expected lesion effects in the present experiments to parallel those on rotation caused by pharmacologically evoked release of dopamine. The lesion effects we obtained on stimulus induced rotation, however, parallel those on rotation evoked by the predominantly directly acting dopamine agonist, apomorphine, rather than those on rotation evoked by the indirect (presynaptic) action of amphetamine. We suggest that contralateral rotation evoked by electrical stimulation of the substantia nigra may reflect direct activation of neurons postsynaptic to the dopaminergic nigrostriatal neurons.     

Neurochemical mechanisms induced by high frequency stimulation of the subthalamic nucleus: increase of extracellular striatal glutamate and GABA in normal and hemiparkinsonian rats

High frequency stimulation (HFS) (130 Hz) of the subthalamic nucleus (STN) provides beneficial effects in patients suffering from severe parkinsonism, but the mechanisms underlying these clinical results remain to be clarified. To date, very little is known concerning the effects of STN-HFS on neurochemical transmission in the different basal ganglia nuclei and in particular the striatum. This study examines the effects of STN-HFS in intact and hemiparkinsonian rats on extracellular striatal glutamate (Glu) and GABA levels by means of intracerebral microdialysis. Unilateral STN-HFS was found to induce a significant bilateral increase of striatal Glu and GABA both in intact and in dopamine-lesioned animals. In intact rats, these increases were reversed by local administration of the D1 antagonist SCH 23390, but were potentiated by the D2 antagonist sulpiride. Potentiation was also observed after local administration of both D1 and D2 antagonists whose amplitude was similar to that measured in hemiparkinsonian rats. These data furnish the first evidence that STN-HFS influences striatal amino-acid transmission and that this influence is modulated by dopamine. They provide evidence that the effects of STN-HFS are not only restricted to the direct STN targets, but also involve adaptive changes within other structures of the basal ganglia circuitry.     

High frequency stimulation of the STN influences the activity of dopamine neurons in the rat

The effect of high frequency stimulation (HFS) of the subthalamic nucleus (STN) on the spontaneous activity of substantia nigra pars compacta (SNc) dopaminergic neurons was investigated in normal rats and in rats with globus pallidus (GP) lesions. In normal rats, the spontaneous activity of SNc neurons did not significantly differ from that of rats with GP lesions (4.2+/-2.2 versus 4.4+/-2.6 spikes/s). STN-HFS induced an increase of firing rate in the majority of tested cells in normal (76%) and GP-lesioned rats (73%) with an after-effect of 34.4+/-3.4 and 33.2+/-3.1 s, respectively. These results demonstrate that STN-HFS influences the activity of the SNc dopaminergic neurons by increasing their firing rate and that this increase of activity is independent of the globus pallidus.     

High frequency stimulation of the subthalamic nucleus influences striatal dopaminergic metabolism in the naive rat

High frequency stimulation (HFS) of the subthalamic nucleus (STN) can partially alleviate motor symptoms in patients with Parkinson's disease (PD). However, the mechanism of action of HFS is incompletely understood. We investigated the effect of HFS (130 Hz) and low frequency stimulation (LFS, 20 Hz) of the STN on striatal dopaminergic transmission and metabolism using in vivo microdialysis in anaesthetized and freely moving rats. While LFS had no effect, HFS of the STN produced a delayed, stable and intensity-dependent increase of extracellular dopamine metabolites. Striatal extracellular levels of dopamine and 5-HIAA were not influenced by HFS or LFS in the present experimental paradigm. We conclude that HFS of the STN influences striatal dopaminergic metabolism in naive, nonlesioned rats.     

High-frequency stimulation of the subthalamic nucleus modulates the activity of pedunculopontine neurons through direct activation of excitatory fibres as well as through indirect activation of inhibitory pallidal fibres in the rat

Recent data suggest a potential role of pedunculopontine nucleus (PPN) electrical stimulation in improving gait and posture in Parkinson's disease. Because the PPN receives fibres from the subthalamic nucleus (STN), we investigated the effects of STN-high-frequency stimulation (HFS) on PPN neuronal activity in intact rats and in rats bearing either an ibotenate lesion of the entopeduncular nucleus (EP) or a lesion of the substantia nigra (SN). The main response of PPN neurons to STN single-shock stimulations in the three experimental groups was a short latency (4.5 +/- 2.1 ms) and brief (15.3 +/- 6.5 ms) excitation. This response was maintained during 1-5 s of STN-HFS (130 Hz, 60 micros, 100-1000 microA). In EP-lesioned rats the percentage (75.0%) of PPN neurons showing a modulation of activity following STN-HFS was significantly higher compared with that observed in intact (39.7%) and in SN-lesioned rats (35.4%). Furthermore, in EP-lesioned rats the most frequent response of PPN neurons following STN-HFS was a 5-20 s excitation, which was present in 76.6% of responsive neurons in comparison to 15.4% and 9.1% of neurons responsive in intact and in 6-hydroxydopamine-lesioned rats, respectively. Neurons responsive to STN-HFS in the three experimental groups showed either a sharp positively skewed distribution of interspike intervals or multisecond oscillations in autocorrelograms. The results support that STN-HFS modulates the PPN through a balance of excitatory and inhibitory influences, which may be independent from the dopaminergic nigral neurons. In the absence of inhibitory EP fibres, the direct excitatory influence exerted by the STN on the PPN appears to predominate.     

Chronic high-frequency stimulation of the subthalamic nucleus and L-DOPA treatment in experimental parkinsonism: effects on motor behaviour and striatal glutamate transmission

Hyperactivity of striatal glutamatergic synaptic transmission in response to dopamine depletion plays a major role in the pathogenesis of parkinsonian motor symptoms. In the present study we investigated the impact, on this hyperactivity, of chronic dyskinesiogenic L-DOPA treatment, combined or not with high-frequency stimulation (HFS) of the subthalamic nucleus (STN). In vitro patch-clamp recordings were performed from striatal spiny neurons of hemiparkinsonian rats (intranigral 6-OHDA injection). Here we show that dyskinesiogenic L-DOPA treatment exacerbated striatal glutamatergic hyperactivity induced by 6-OHDA lesion. Chronic 5-day STN HFS had the opposite effect, reducing striatal glutamatergic transmission in both parkinsonian and dyskinetic animals. Consistently, chronic HFS stimulation could progressively ameliorate motor parkinsonian signs (akinesia) but, conversely, did not improve L-DOPA-induced dyskinesia (LID). Thus, the effects of L-DOPA and HFS on corticostriatal transmission seem to be dissociated. These data show for the first time that dyskinesiogenic L-DOPA treatment and chronic STN HFS with antiakinetic effects induce opposite plastic rearrangements in the striatum. The interaction between these two treatments provides further evidence that striatal glutamatergic hyperactivity is a pathophysiological correlate of akinesia rather than LID.