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。     

丘脑底核高频电刺激对偏侧帕金森猴苍白球内侧部中氨基酸神经递质含量的影响

目的通过微透析-高效液相神经递质分析技术观察丘脑底核(subthalamic nucleus,STN)脑深部电刺激(deep brain stimulation,DBS)对偏侧帕金森(parkinson’s disease,PD)模型猴苍白球内侧部(globus pallidusinternus,GPi)氨基酸类神经递质水平的影响。探讨PD的发病机制及DBS治疗PD的可能作用机制。方法成功制备的偏侧PD模型猴4只,微透析方法收集未注药侧GPi的细胞外液以及注药侧STN高频电刺激(highfrequncy stimulation,HFS)过程中以及刺激前后同侧GPi的细胞外液,应用高效液相-荧光法分析谷氨酸(gluta-mate,Glu)和γ-氨基丁酸(gamma-aminobutyric acid,GABA)的水平变化。结果 MPTP注药侧GPi细胞外液中Glu含量较未注药侧升高,而GABA含量却下降。高频电刺激偏侧PD猴模型的STN后,同侧的GPi细胞外液中Glu的含量明显上升,而GABA的含量无明显变化。结论无法用单一的神经元抑制机制解释STN-HFS的作用机制,STN-HFS的作用不只是局限于STN核团本身,对整个基底节环路都有影响。     

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。     

星形胶质细胞在帕金森大鼠中的作用机制研究

目的观察移植星形胶质细胞后的PD大鼠行为学改变和单胺类神经递质含量的时空变化及相互关系,旨在分析星形胶质细胞在帕金森大鼠中的作用机制。方法偏侧两点法建立PD模型大鼠后,将星形胶质细胞移入PD大鼠纹状体中,2 w后对比PD组,观察行为学改变,检测单胺类神经递质(DA、DOPAC、HVA)含量变化。结果 PD+T2As组的行为学从第4周开始较PD组有显著改善(P 0. 01),单胺类神经递质(DA、DOPAC、HVA)含量与PD组比较第2周无明显区别(P0. 05),从第3周开始显著高于PD组(P 0. 01)。结论星形胶质细胞在一定程度上保护了受损的DA能神经细胞,对PD大鼠的黑质-纹状体通路具有修复作用。     

丘脑底核电刺激对猴偏侧帕金森病纹状体神经递质的影响

目的 探讨应用微透析技术在慢性STN—DBS对纹状体细胞外液多巴胺及代谢产物的影响。方法 选择已经成功安装脑深部刺激电极的偏侧PD猕猴模型2只，分别在打开脉冲发生器前、后的不同时间点取样（开机后8h、1周、1个月、2个月）。应用高效液相电化学方法检测开机前后的尾状核和壳核细胞外液的多巴胺（DA）及其代谢产物含量。结果 电极侧壳核和尾状核的DA在开机后8h、1周、1个月、2个月相应地分别较各自开机前的DA含量增高了39％、91％、111％、114％和31％、91％、106％、102％（P〈0．05）。电极侧壳核和尾状核HVA／DA在开机后8h分别较各自开机前增高了186％和91％（P〈0．05），而开机后1周、1个月、2个月HVA／DA较开机前无明显变化（P〉0．05）。电极侧的多巴胺周转率在开机后的各时间点均显著低于非电极侧（P〈0．01）。结论 STN—DBS可有效的改善猴偏侧PD模型的症状，应用微透析取样技术结合高效液相色谱测，定法发现在给予有效电刺激后可增加刺激侧纹状体细胞外液的多巴胺及其代谢产物含量。为STN—DBS治疗帕金森病提供理论依据。     

CCK-8及损毁MFB对腹侧被盖区和伏核多巴胺、CCK-8含量的影响

应用荧光分光光度法和放射免疫法,在以6-羟基多巴胺(6-OHDA)单侧损毁内侧前脑束(MFB)制备的偏侧帕金森病(PD)大鼠模型身上,测定了腹侧被盖区(VTA)和伏核(Acb)中多巴胺(DA)和八肽胆囊收缩素(CCK-8)的含量 ,并观察了VTA和Acb区微量注射CCK-8对正常大鼠DA含量的影响.结果如下:PD大鼠模型损毁侧VTA和Acb的DA和CCK-8的含量与健侧及对照组相比均减少(P<0.01);而健侧VTA 和Acb的DA和CCK-8的含量同正常大鼠相比,差别无显著性(P>0.05).正常大鼠Acb微量注射CCK-8能使Acb的DA含量增高(P<0.05);而VTA微量注射CCK-8则不影响VTA和A cb区的DA含量(P>0.05).结果表明,6-OHDA单侧损毁MFB后既降低中脑DA能系统中VT A和Acb的DA含量,又降低该系统中VTA和Acb区与DA共存的CCK-8含量;并提示Acb区外源性C CK-8可增加该区的DA含量.     

NMDA受体和GABA受体拮抗剂对小鼠脑内多巴胺的影响

目的 观察兴奋性氨基酸受体拮抗剂和GABA受体拮抗剂对帕金森病（PD）模型动物全脑多巴胺（DA）含量的影响，为PD发病机制研究提供理论依据。方法 采用MPTP腹腔注射建立C57 BL小鼠PD模型，同时分别腹腔注射兴奋性氨基酸NMDA受体拮抗剂ketamine和GABA受体拮抗剂bicucullin．采用荧光分光光度计法测定各组小鼠全脑DA的含量。结果 ketamine＋MPTP组、bicucullin＋MPTP组与MPTP组及NS组比较，DA含量差异有统计学意义。结论 NMDA受体拮抗剂可抑制由MPTP引起的DA神经递质减少，GABA受体拮抗剂可增强MPTP引起的DA神经递质减少。     

3-硝基丙酸诱发肌张力障碍大鼠的相关脑区细胞外神经递质变化

目的 研究3-硝基丙酸(3-NP)诱发肌张力障碍大鼠相关核团的神经递质变化.方法 24只SD大鼠随机分为对照组和实验组,每组12只.实验组大鼠尾壳核注射3-NP4000 μmol,对照组尾壳核注射生理盐水4μl,3 d后对两组大鼠进行行为学评分,然后对尾壳核、苍白球内侧、苍白球外侧和丘脑底核行微透析,用高效液相色谱法测定透析液中的神经递质含量.结果 实验组尾壳核天冬氨酸及谷氨酸较对照组明显增加(P＜0.05),而甘氨酸及γ-氨基丁酸较对照组明显减少(P＜0.01);苍白球内侧细胞外神经递质较对照组均明显减少(P＜0.01);丘脑底核细胞外神经递质较对照组均明显增加(P＜0.05);苍白球外侧细胞外神经递质含量较对照组均无明显差异(P＞0.05).结论 3-NP可引起尾壳核神经递质变化,并通过直接和间接通路引起苍白球内侧及丘脑底核神经递质变化,从而诱发肌张力障碍.     

PD大鼠健侧纹状体多巴胺能系统的功能性改变

应用快速周期伏安法(FCV)在体监测电刺激内侧前脑束(MFB)诱发的正常和帕金森病(PD)大鼠健侧及损毁侧纹状体(Str)内多巴胺(DA)的释放,并结合高效液相色谱电化学检测法(HPLC-ECD)测定Str内DA及其代谢产物的含量,从在体和离体水平分别对PD大鼠健侧及损毁侧Str区DA的释放及代谢进行观察及评价.实验采用自身对照,先用FCV监测DA的释放,后行HPLC-ECD离体测定Str内DA及其代谢产物的含量.结果表明:(1)在PD大鼠损毁侧,用FCV技术几乎不能监测到DA的释放,而在健侧Str区监测到的DA释放量远大于正常对照(P《0.01);(2)高效液相色谱测定结果为:PD大鼠健侧Str区DA,DOPAC和HVA的含量均在正常范围内,但DA的更新率与正常大鼠相比升高(P《0.05).损毁侧Str内DA及其代谢产物的含量均降低(以DA的减少最明显),而DA的更新速度加快(P《0.01).结果提示:6-OHDA单侧损毁的PD大鼠损毁侧DA能系统的功能性改变对健侧有影响.     

PD大鼠健侧纹状体多巴胺能系统的功能性改变

应用快速周期伏安法 (FCV)在体监测电刺激内侧前脑束 (MFB)诱发的正常和帕金森病 (PD)大鼠健侧及损毁侧纹状体 (Str)内多巴胺 (DA)的释放 ,并结合高效液相色谱电化学检测法 (HPLC ECD)测定Str内DA及其代谢产物的含量 ,从在体和离体水平分别对PD大鼠健侧及损毁侧Str区DA的释放及代谢进行观察及评价。实验采用自身对照 ,先用FCV监测DA的释放 ,后行HPLC ECD离体测定Str内DA及其代谢产物的含量。结果表明 :(1)在PD大鼠损毁侧 ,用FCV技术几乎不能监测到DA的释放 ,而在健侧Str区监测到的DA释放量远大于正常对照 (P <0 .0 1) ;(2 )高效液相色谱测定结果为 :PD大鼠健侧Str区DA ,DOPAC和HVA的含量均在正常范围内 ,但DA的更新率与正常大鼠相比升高 (P <0 .0 5 )。损毁侧Str内DA及其代谢产物的含量均降低 (以DA的减少最明显 ) ,而DA的更新速度加快 (P <0 .0 1)。结果提示 :6 OHDA单侧损毁的PD大鼠损毁侧DA能系统的功能性改变对健侧有影响。     

In vivo changes of catecholamines in hemiparkinsonian monkeys measured by microdialysis.

In monkeys, unilateral intracarotid infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces a useful model of hemiparkinsonism. To evaluate MPTP-induced neurochemical changes in vivo, brain microdialysis was employed to measure extracellular levels of dopamine and its metabolites in the neostriatum of normal and hemiparkinsonian rhesus monkeys (Macaca mulatta). The microdialysis probes were implanted bilaterally into the caudate nucleus and putamen at coordinates determined from magnetic resonance imaging. Dopamine and its metabolites were depleted in the MPTP-lesioned side versus the unlesioned side in hemiparkinsonian monkeys. Tyrosine hydroxylase immunocytochemistry revealed a complete unilateral denervation in the caudate nucleus and putamen and a total loss of tyrosine hydroxylase-immunoreactive cells in the substantia nigra pars compacta in those monkeys. Baseline levels of amines in the neostriatum in normal monkeys were not significantly different from those in the normal (non-MPTP-treated) side in hemiparkinsonian monkeys. These data demonstrate that brain microdialysis is a valuable tool for measuring in vivo neurochemical changes in nonhuman primate brains.     

In vivo characterization of extracellular GABA release in the caudate nucleus and prefrontal cortex of the rhesus monkey

Extracellular gamma amino butyric acid (GABA) levels were measured in the caudate nucleus and the prefrontal cortex of the rhesus monkey brain using in vivo microdialysis under isofluorane gas anesthesia. Evoked GABA release was investigated for voltage sensitivity and calcium (Ca²⁺) dependency. There was a multifold increase in extracellular GABA levels following local perfusion with: (1) high potassium (50 mM, KCl), (2) veratridine (10 μM), and (3) the GABA releasing agent and uptake blocker, (−) nipecotic acid (1 mM). Release of GABA was significantly reduced when veratridine or (−) nipecotic acid were coinfused in Ca²⁺-free cerebrospinal fluid (CSF). Coinfusion of nipecotic acid with TTX (10 μM) also resulted in attenuation of evoked GABA release. These results suggest that GABA levels recovered using in vivo microdialysis, from the caudate nucleus and the prefrontal cortex in the rhesus monkey, derive in significant part from vesicular pools and the exocytotic process is both Ca²⁺ -dependent and voltage-sensitive. Synapse 25:285–292, 1997. © 1997 Wiley-Liss, Inc.

1 This article is a US government work and, as such, is in the public domain in the United States of America.

     

Formation of extracellular glutamate from glutamine: exclusion of pyroglutamate as an intermediate

A 4.6-fold increase in interstitial glutamate was observed following the reverse microdialysis of 5 mM glutamine into the rat hippocampus. Two possible mechanisms of glutamine hydrolysis were examined: (a) an enzymatic glutaminase activity and (b) a non-enzymatic mechanism. Injection of 14C-glutamine at the site of microdialysis followed by microdialysis with artificial cerebral spinal fluid allowed isolation of 14C-glutamine (63%), 14C-glutamate (14%), and a compound tentatively identified as pyroglutamate (22%). In this study, we determined if non-enzymatic pyroglutamate formation from glutamine contributed to the synthesis of glutamate. Pyroglutamate is in chemical equilibrium with glutamate, although under physiological conditions, the chemical equilibrium is strongly in the direction of pyroglutamate. In vitro stability studies indicated that 14C-glutamine and 14C-pyroglutamate are not subject to significant non-enzymatic breakdown at pH 6.5-7.5 at 37 degrees C for up to 8 h. Reverse microdialysis with 1 mM pyroglutamate did not increase interstitial glutamate levels. Following injection of 14C-pyroglutamate and microdialysis, radioactivity was recovered in 14C-pyroglutamate (88%) and 14C-glutamine (11%). Less than 1% of the radioactivity was recovered as glutamate. Our data do not support a role of pyroglutamate as an intermediate in the formation of extracellular glutamate following the infusion of glutamine. However, it confirms that pyroglutamate, a known constituent in brain, is actively metabolized in brain cells and contributes to glutamine in the interstitial space.     

Extracellular glutamate and dopamine measured by microdialysis in the rat striatum during blockade of synaptic transmission in anesthetized and awake rats

We investigated the effect of high dose tetrodotoxin (TTX) on microdialysis measurements of extracellular striatal glutamate and dopamine in normal female rats. Both halothane-anesthetized rats with acutely implanted microdialysis probes and awake rats with microdialysis probes implanted for 24 h were tested. Glutamate levels in awake rats were 45% higher than those of anesthetized rats. Extracellular glutamate remained TTX-insensitive irregardless of TTX concentration, anesthesia, or time lapsed after probe implantation. In contrast, TTX reduced dialysate dopamine in all TTX concentrations tested. We speculate that the lower glutamate levels in anesthetized rats reflect the effect of anesthesia. Because glutamate is involved, either as a reactant or a product in a variety of reactions critical to intermediary metabolism in the brain, basal dialysate glutamate levels might indirectly reflect brain metabolism. Further, we conclude that extracellular glutamate collected during non-stimulated conditions is TTX-insensitive. The fact that glutamate levels are TTX-independent does not rule out that glutamate is synaptic in origin but rather demonstrates that it is not nerve impulse-dependent. However, the brain interstitial glutamate pool accessible to the microdialysis probe during control conditions is most likely isolated from the synapse, and therefore does not impose a neurotoxic potential.     

Evaluation of an osmotic pump for microdialysis sampling in an awake and untethered rat

The feasibility of using an osmotic pump in place of a syringe pump for microdialysis sampling in rat brain was investigated. The use of an osmotic pump permits the rat to be free from the constraints of the standard tethered system. The in vitro flow rates of a microdialysis syringe pump (set at 10.80 μl/h) and the osmotic pump (pump specifications were 11.35 μl/h) with no probe attached were compared, yielding results of 10.87 μl/h ± 1.7% and 10.95 μl/h ± 8.0%, respectively. The average of four flow rate experiments in vivo yielded R.S.D.s less than 10% and an average flow rate of 11.1 μl/h. Following the flow rate studies, in vivo sampling of neurotransmitters was accomplished with the osmotic pump coupled to a microdialysis probe implanted in the brain. Finally, after determination of basal levels of 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindole-3-acetic acid (5-HIAA) in the rats, the rats were dosed with benserazide followed by l-3,4-dihydroxyphenylalanine (l-DOPA). The results from the dosing study showed at least a 10-fold increase in compounds in the l-DOPA metabolic pathway (DOPAC and HVA) and a slight or no increase in 5-HIAA (serotonin metabolic pathway.) These results indicate that the osmotic pump is a viable alternative to the syringe pump for use in microdialysis sampling.     

Autoradiographic studies of 5-HT1A-receptor-stimulated [35S]GTPgammaS-binding responses in the human and monkey brain

G-protein activation mediated by serotonin 5-HT1A receptors in human and monkey brain was investigated by using quantitative autoradiography of agonist-stimulated [35S]GTPgammaS binding to whole-hemisphere brain sections. [35S]GTPgammaS binding was stimulated by the mixed 5-HT1A/1B/1D agonist L 694247 (10 microm) in human brain regions enriched in 5-HT1A binding sites [e.g. hippocampus (132-137%), superficial layers of the neocortex (37-61%), and cingulate and entorhinal cortex (34 and 32%, respectively)]. L 694247 caused virtually no stimulation in regions with 5-HT1B/1D receptors, such as substantia nigra, caudate nucleus and putamen. Similar results were obtained with monkey brain sections. The L 694247-mediated [35S]GTPgammaS-binding responses in human and monkey brain sections were antagonized by the selective, silent 5-HT1A antagonist WAY 100635 (10 microm). The 5-HT1B inverse agonist SB 224289 (10 microm) did not affect the [35S]GTPgammaS-binding response of L 694247. The distribution pattern of the [35S]GTPgammaS-binding response and the antagonist profile suggest the L 694247-induced response in human and monkey brain is mediated by 5-HT1A receptors. A weak stimulation of [35S]GTPgammaS binding was also observed in human hippocampus with either 10 microm 8-OH-DPAT (25 +/- 4%) or naratriptan (42 +/- 2%) compared with that obtained with L 694247. In conclusion, G-protein activation by 5-HT1A receptors can be measured in human and monkey brain sections. L 694247 appears to possess higher efficacy at 5-HT1A receptors compared with 8-OH-DPAT and naratriptan.     

Determination of extracellular release of neurotensin in discrete rat brain regions utilizing in vivo microdialysis/electrospray mass spectrometry

In vivo microdialysis was used together with structure-specific high sensitivity nano-flow capillary liquid chromatography/micro-electrospray mass spectrometry to quantify and compare extracellular neurotensin from discrete regions of the rat brain. Microdialysis probes were implanted in the hypothalamus or globus pallidus/ventral pallidum in unanesthetized freely moving animals. Utilizing this specific methodology, recovered basal levels of neurotensin were detectable in hypothalamus and globus pallidus/ventral pallidum. The basal level of neurotensin in these regions were slightly higher in hypothalamus (101+/-11 amol/10 microl, n=6) compared to those in the globus pallidus/ventral pallidum region (74+/-12 amol/10 microl, n=8) in samples collected for 30 min at a flow-rate of 0.4 microl/min 150-180 min after the microdialysis probe implantation. After a pulse of 1.0 microl of 100 mM KCl-containing artificial cerebrospinal fluid during the next 30-min sampling period (180-210 min), the recovered neurotensin increased in hypothalamus and globus pallidus/ventral pallidum by 544% (548+/-90 amol/10 microl) and 674% (499+/-99 amol/10 microl), respectively. The basal levels of endogenously released neurotensin in the hypothalamus and globus pallidus/ventral pallidum were lower in the present study compared to those previously reported in the rat brain using in vivo microdialysis and radioimmunoassays. Our data demonstrate the effectiveness of combining in vivo microdialysis and structure-specific micro-electrospray mass spectrometry for the quantitation of basal and stimulated in vivo levels of endogenous neurotensin (NT) in different brain areas.     

Lentiviral gene transfer to the nonhuman primate brain

Lentiviral vectors infect quiescent cells and allow for the delivery of genes to discrete brain regions. The present study assessed whether stable lentiviral gene transduction can be achieved in the monkey nigrostriatal system. Three young adult Rhesus monkeys received injections of a lentiviral vector encoding for the marker gene beta galatosidase (beta Gal). On one side of the brain, each monkey received multiple lentivirus injections into the caudate and putamen. On the opposite side, each animal received a single injection aimed at the substantia nigra. The first two monkeys were sacrificed 1 month postinjection, while the third monkey was sacrificed 3 months postinjection. Robust incorporation of the beta Gal gene was seen in the striatum of all three monkeys. Stereological counts revealed that 930,218; 1,192,359; and 1,501,217 cells in the striatum were beta Gal positive in monkeys 1 (n = 2) and 3 (n = 1) months later, respectively. Only the third monkey had an injection placed directly into the substantia nigra and 187,308 beta Gal-positive cells were identified in this animal. The injections induced only minor perivascular cuffing and there was no apparent inflammatory response resulting from the lentivirus injections. Double label experiments revealed that between 80 and 87% of the beta Gal-positive cells were neurons. These data indicate that robust transduction of striatal and nigral cells can occur in the nonhuman primate brain for up to 3 months. Studies are now ongoing testing the ability of lentivirus encoding for dopaminergic trophic factors to augment the nigrostriatal system in nonhuman primate models of Parkinson's disease.     

Use of microdialysis to measure brain noradrenergic receptor function in vivo

The present studies were undertaken to explore the use of intracerebral microdialysis to measure brain adrenergic receptor activity in vivo using the efflux of cyclic adenosine monophosphate (cAMP) in the extracellular fluid as an index of receptor function. An initial study with brain slices showed that extracellular levels of cAMP were highly correlated with intracellular levels after noradrenergic receptor activation. Detectable levels of extracellular cAMP were obtained from microdialysis probes implanted in the frontal cortex. Stable levels (3.5 ± 2.7pmol/ml) were obtained between 1.5 and 7 h after implantation. Perfusion of probes with NE (10⁻⁵ to 10⁻³ M) led to dose-dependent increases in cAMP efflux. The response to NE was blocked by infusion of the β-antagonist, timolol, indicating that it reflected β-receptor activation. Similar responses were obtained at 2 and 24 h after implantation indicating that the responses were not affected by acute traumatic effects of implantation. The finding show that the microdialysis technique can be successfully applied to the in vivo study of central adrenoceptor function. This suggests that the in vivo measurement of second messengers by microdialysis will provide a valuable new neurochemical and neuropharmacological technique.