Objective To study the behavioral characteristics of a rat model of the levodopa-induced dyskinesia (LID) and the related factors, and to define clinically the relevant methods for assessing akinesia and dyskinesia in LID rats. Methods Unilaterally lesioned rat model of Parkinson′s disease using 6-hydroxydopamine were treated by levodopa and benserazide once daily for 3 weeks, on the 21st day the acute systemic administration of MK-801 was performed 15 min prior to levodopa treatment to observe the behavior (abnormal involuntary movement, rotation behavior and forelimb stepping) and to estimate the quality of AIM by using the rat AIM rating scale. Immunohistochemical technique was used to measure the number of TH-positive neurons in the substantia nigra (SN) and ventral tegmental area (VTA), which was then correlated to the AIM scores. Results Pulsatile treatment with a subthreshold dose of levodopa gradually induced abnormal involuntary movement (AIM), including stereotypy (limb dyskinesia, axial dystonia and masticatory dyskinesia) towards the side contralateral to the dopamine-denervated striatum and increased rotational behavior. The onset of AIM and motor pattern of each subtype was highly stereotypic across individual rats, and the proportion of each subtype was not consistent among individual rats. The number of TH-positive neurons in the VTA, but not in the SN, was significantly decreased in the LID rats compared with the non-LID rats. MK-801 prevented stereotypy but not rotational behavior. Contralateral forepaw performance was signi-ficantly improved after levodopa treatment, but gradually reduced with more and more severe AIM following repeated levodopa therapy. Conclusion Levodopa-induced rat AIM model of PD demonstrated similar properties with the levodopa-induced dyskinesia (LID) in PD patients, and provided an effective tool for LID study. AIM rating and forelimb stepping test are useful for evaluating the dyskinesia and akinesia of PD rats.

Objective To study the effect of MK801 on behavioral changes and the possible mechanisms. Methods To observe the behavioral changes of levodopa induced dyskinesia (LID) rats during the period of chronic MK801 treatment, immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to measure the changes in expression of FosB, preproenkephalin (PPE) mRNA and prodynorphin (PDyn) mRNA in striatum, respectively. Double labling technique including immunohistochemistry of FosB and retrograde HRP transport tracing was used to observe the cell distribution of FosB. Results Pulsatile treatment with levodopa induced Abnormal involuntary movements (AIM) in PD rats, similar to LID in PD patients. FosB positive neurons and expressions of PPE mRNA and PDyn mRNA in striatum of 6-OHDA-lesioned hemisphere were increased in LID rats, and AIM scores of LID rats were reduced by MK801 treatment(41.9?15.6 vs 7.2?3.0), accompanied by the decrease in expressions of FosB and PDyn mRNA, but not PPE mRNA. Neurons immunoreactive for FosB were mainly located in striatonigral neurons which were labeled by cholera toxin-HRP (CT-HRP) injected in the substantia nigra pars reticulata (SNr). Conclusions MK801 could prevent the occurrence of dyskinesias induced by chronic levodopa treatment. The mechanism might be involved in the high expression of immediate early gene FosB and specific gene PDyn on the direct pathway. It suggests that LID might be related to the abnormal activity of direct pathway.

To observe the effects of heterograft of glomus cells of carotid body on hemiparkinsonian rat models, rats with unilateral 6-hydroxydopamine (6-OHDA)-induced lesions of the right dopaminergic neurons of substantia nigra received intrastriatal glomus cells heterograft. Apomorphine-induced rotation was monitored for 30 min at various time points after grafting. The striata were cut and examined for dopamine content by HPLC and for immunohistochemical staining of tyrosine hydroxylase positive neurons (TH+) at the end of the experiments. The results showed that apomorphine-induced rotational behavior was significantly reduced for 12 weeks and the dopamine contents were significantly elevated after grafting (P < 0.01), and TH+ cells survived better. The present study demonstrates that intrastriatal heterograft of glomus cells within carotid body in rats with 6-OHDA-elicited lesions could reduce apomorphine-induced rotational behavior and elevate the dopamine contents and numbers of TH+ cell surviving within striatum, and can serve as a new and effective alternative for Parkinson disease.
Carotid Body/*cytology ; Carotid Body/transplantation ; *Cell Transplantation ; Dopamine/*metabolism ; Neurons/metabolism ; Parkinson Disease/metabolism ; Parkinson Disease/*surgery ; Random Allocation ; Rats, Sprague-Dawley ; Stereotaxic Techniques ; Transplantation, Heterologous

Objective To explore the protective effect mechanism of minocycline (MC) on cell apoptotic model of Parkinson's disease induced by 1-methyl-4-phenylpyridinium (MPP+ ).Methods Different concentrations of MPP+ (10, 50, 250, 500 ?mol/L) were added into the culture of PC12 cells. The most appropriate concentration of MPP+ (MPP+ group) was selected to establish apoptotic model of dopaminergic neurons. For selecting the most a-ppropriate concentration MC (MC+MPP+ group) of protective effect, MTT was used to assay the viability of model of dopaminergic neurons pretreated by different concentrations MC (0, 10, 50, 100, 200 ?mol/L). The cell apoptosis, apoptosis ratio and caspase-3 mRNA expression of MPP+ group and MC+MPP+ group were assayed by electrophoresis method, flow cytometry and RT-PCR, and compared with control group.Results (1) At concentration of 10 ?mol/L of MPP+, cell parkinsonism model of apoptosis was established. The cell viability of apoptotic model of dopaminergic neurons was the highest when pretreated by 100 ?mol/L of MC ( P

BACKGROUND: 3-nitropropionic acid(3-NP) can inhibit the process of oxidative phosphorylation and injure the energy metabolism of the cell and thereby induce cell injury. However, small dose of 3-NP can excite intrinsic cellular protective factor to protect neurons and increase the tolerance of neurons to ischemic hypoxia through mild inhibiting the process of oxidative phosphorylation. It is unclear whether it also has the similar effect on dopaminergic neurons.OBJECTIVE: To investigate whether 3-NP preconditioning could enhance the tolerance of dopaminergic neurons to MPP+(1-methyl-4-phenylpyridine)toxicity.DESIGN: A randomized controlled exploring research based on neuroblastoma SH-SYSY cell that could secrete dopamine.SETTING: Department of neurology of a university hospital.MATERIALS: The study was conducted in the Laboratory of Pathophysiology of Tongji Medical College between March 2003 and November 2003. SH-SYSY cell was obtained from the Cell Center of Peking Union Medical University.INTERVENTIONS: Cells were randomly divided into 6 groups. MPP+ was added into the cultured dopaminergic neuron SH-SY5Y cells for the establishment of the cell model for Parkinson disease. Before the admission of MPP+ (0.25 mmol/L), 3-NP(0. 2 mmol/L) was added once or repetitive times to form preconditioning. Microculture tetrozolium(MTT) was used to detect cell survival rate, and[3H] DA uptake rate was used to test the anterior synaptic function of dopaminergic neurons.MAIN OUTCOME MEASURES: Major consequence: Cell survival rate;Minor consequence: [3H] DA uptake rate.RESULTS: Cell survival rate of MPP+ group was 54.3%, which was significantly lower than that of blank control group( P ＜ 0.01) . After 3-NP preconditioning, cell survival rate significantly elevated, which was 71.8%(single) or 85.2% (repetitive) . There was significant difference between single preconditioning and repetitive preconditioning( P ＜ 0.05 ). The results of[3H] DA uptake rate were similar to that of cell survival rate. [3H] DA uptake rate was 65.8% (single) or 80. 3% (repetitive), which was significantly higher than 50. 1% of MPP+ group. And moreover, repetitive preconditioning had more favorable effect than single preconditioning. Simple admission of 3-NP had no impact on cells.CONCLUSION: 3-NP preconditioning can significantly enhance the tolerance of dopaminergic neuron to MPP+ toxicity, which has significant protective effects on dopaminergic neuron. Repetitive preconditioning have more significant protective effects.

Objective To investigate the role of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in rotenone-mediated dopaminergic cell damage. Methods Neuronal rat adrenal pheochromecytoma(PC12) cells treated with rotenone were used as the cell model of Parkinson' s disease (PD). Cell viability of PC12 cells after exposure to rotenone was detected by MTT (methyl thiazolyl tetrazolium) method. Immanohistechemistry was used to detect the phosphorylation of ERK1/2 in cells exposed to rotenone. Western blotting was used to verify the phosphorylation of ERK1/2 and to observe the effect of PD98059, an inhibitor of the upstream mitogen activated protein kinase kinase (MEK) that phosphorylates and activates ERK1/2, on rotenone-induced ERK1/2 phosphorylation and cell viability.Results The viability (represented by A570 of PC12 cells exposed to 5 μmoL/L rotenone) declined with the increase of exposure time from 1 h to 24 h (%, 1 h :75.46±5.47, 2 h : 70.42±1.94, 4 h : 65.23± 0.96, 8 h : 59.04 ± 2.85, 24 h :29.64 ± 1.63, comparison between different time points(F=143.014, P=0.000) ; compared with control groups(100.00±2.89), q value: 17.07, 20.58, 24. 19, 28.50, 48.95 respectively, all P <0.01). After exposure to rotenone, phosphorylated ERK1/2 aggregated in the PC12 cells. Western blotting indicated that rotenone induced a biphasic phosphorylation of ERK1/2, which increased from 30 min after rotenone treatment, reached the peak at 1-2 h, decreased at 4 h, and increased again at 8 h, and disappeared after 16 h; PD98059 significantly inhibited ERK1/2 phosphorylation induced by rotenone, and attenuated cell injury examined at 1, 2 and 8 h. Conclusions Our study suggested that ERK1/2 activation plays a detrimental role in rotenone toxicity, and raised the possibility that abnormal patterns of ERK1/2 activation may contribute to dopaminergic neuronal cell death in PD.

Objective To study the effects of dendritic cells (DC) modified with transforming growth factor ?1 (TGF-?1) gene on experimental autoimmune myasthenia gravis (EAMG) in Lewis rats. Methods 30 female Lewis rats were divided randomly into 6 groups: normal group, EAMG group, DCs treatment group, pcDNA_3-TGF-?1-DCs treatment group, pcDNA_3-DCs treatment group and normal saline group. The rats were immunized with the acetylcholine receptor (AChR) protein extracted from electric organ of Narcine timilei and completed Freund’s adjuvant (CFA) in the experiment groups except normal group. 2?106 pcDNA_3-TGF-?1-DCs/rat were injected subcutaneously into the backs of the rats that had been immunized 5 days earlier with AChR+CFA. The rats in DCs treatment group, pcDNA_3-DCs treatment group and normal saline group were injected in parallel with untreated DCs, pcDNA_3-DCs and normal saline respectively. Then the clinical manifestations were observed everyday. And 7 weeks after the first immunization, repetitive nerve stimulation, detection of acetylcholine receptor antibody (AChRab) and ultrastructural study of neuromuscular junction (NMJ) were performed. Results (1) The mild symptoms were observed on 1 or 2 rats in the experiment groups except normal group after a week, which lasted for 2 to 5 days. After about 5 weeks, the rats in EAMG group, DCs treatment group, pcDNA_3-DCs treatment group and normal saline group presented some symptoms at different degree like myasthenia gravis, and only one of the rats in pcDNA_3-TGF-?1-DCs treatment group presented mildly decreased activity. (2) The significant decrement of repetitive nerve stimulation were found in EAMG group, DCs treatment group, pcDNA_3-DCs treatment group and normal saline group(16.75?6.13, 17.75?7.81, 18.25?8.22 and 16.50?7.14, respectively), but there was no attenuation in pcDNA_3-TGF-?1-DCs treatment group and normal group(3.20?3.70 and 5.60?2.70, respectively). The percentage of decrement in pcDNA_3-TGF-?1-DCs treatment group was lower than that in EAMG group(5.60?2.70 and 16.75?6.13, respectively,P0.05). (4) The combined AChRs in NMJ of the rats in pcDNA_3-TGF-?1-DCs group were higher than that in EAMG group, and the structure changes of the synapse were relieved.Conclusion It suggests that DCs, transfected with pcDNA_3-TGF-?1, when injected subcutaneously into Lewis rats with incipient EAMG, could inhibit the production of AChR-Ab, relieve the pathologic changes in NMJ and ameliorate the development of EAMG.

To study the effects of 6-hydroxydopamine (6-OHDA) and reduced glutathione (GSH) on the nigral dopaminergic neurons in brain slices in vitro, immolunohistochemical technique was used to observe the changes of TH-stained neurons, including cell bodies and the dendrites, in the substantia nigra (SN) of midbrain slices of rats after incubation for 1 h in the presence of GSH 15 min before and during the period of incubation with 6-OHDA. The results showed that cell bodies remained intact but dendrites were fragmented and truncated after treatment with 6-OHDA. The antioxidant GSH alone did not significantly affect the dendrites of SN neurons but prevented 6-O-HDA-induced damage of dendrites. It was concluded that glutathione may prevent 6-OHDA-induced dopaminergic neurodegeneration and play a protective role in dopaminergic neurons.
Glutathione/*therapeutic use ; Neurons/pathology ; Oxidopamine ; Parkinson Disease, Secondary/chemically induced ; Parkinson Disease, Secondary/*drug therapy ; Random Allocation ; Rats, Sprague-Dawley ; Substantia Nigra/pathology ; Tyrosine 3-Monooxygenase/metabolism

In order to investigate the neurotoxicity of lipopolysaccharide (LPS) on the dopaminergic neurons of substantia nigra and the pathogenesis of Parkinson disease, LPS was stereotaxically infused into substantia nigra (SN). At different dosages and different time points with 5 microg LPS, the damage of the dopaminergic neurons in SN was observed by using tyrosine-hydroxylase (TH) immunohistochemical staining. The results showed that 14 days after injection of 0.1 microg to 10 microg LPS into the rat SN, TH-positive (TH+) neurons in the SN were decreased by 5%, 15%, 20%, 45 %, 96% and 99% respectively. After injection of 5 microg LPS, as compared with the control groups, TH+ neurons began to decrease at 3rd day and obviously decrease at 14th day, only 5% of total cells, and almost disappeared 30 days later. The results suggested that LPS could induce the degeneration of dopaminergic neurons in the SN in a dose- and time-dependent manner.
Dopamine/metabolism ; Dose-Response Relationship, Drug ; Lipopolysaccharides/*toxicity ; *Nerve Degeneration ; Neurons/pathology ; Parkinson Disease, Secondary/*chemically induced ; Random Allocation ; Rats, Sprague-Dawley ; Substantia Nigra/*pathology

BACKGROUND: Mainly pathological changes of Parkinson disease (PD)are related to irreversible degeneration and reduction of dopamine neurons of substantia nigra in midbrain; however, oxidative stress reaction plays an important role in onset of PD. 3-nitropropionie acid (3-NP) is an inhibitor of mitochondria compound I, and it can inhibit oxidative phosphorylation so as to restrain energy metabolism. However, professor Riepe from Germany found that small dose of 3-NP can increase the tolerance of neurons to ischemic hypoxia. It is unclear whether it can also strengthen the tolerance of dopamine neurons to neurotoxin.OBJECTIVE: To investigate the possible mechanism and prevention of repetitively preconditioning 3-NP for treating PD.DESIGN: Controlled observational animal study. SETTING: Department of Neurology, Union Hospital affiliated to TongjiMedical College, Huazhong University of Science and Technology. MATERIALS: The experiment was carried out at the Neurological Lab oratory, Union Hospital affiliated to Tongji Medical College, Huazhong U niversity of Science and Technology from March to July 2004. A total of48 C57BL mice, weighing 18-20 g, aged 2-3 months, of both genders, were randomly divided into 6 groups with 8 in each group. ① Blank con trol group: Mice were not medicated. ② 3-NP single administrationgroup: Mice were intraperitoneally injected with 3-NP once. ③ 3-NPrepetitively administrations group: Mice were intraperitoneally injectedwith 3-NP every 5 days for 5 times in total. ④ Neurotoxin group: Micewere intraperitoneally injected with neurotoxin once every day for 5 timesin total. ⑤ 3-NP single preconditioning group: Mice were intraperitoneal ly injected with 3-NP once, and 3 days later, they were intraperitoneallyinjected with neurotoxin once every day for 5 times in total. ⑥ 3-NPrepetitively preconditionings group: Mice were intraperitoneally injectedwith 3-NP and repetitively every 5 days for 5 times in total; 3 days later, mice were intraperitoneally injected with neurotoxin once every day for5 times in total. Dosages of 3-NP and neurotoxin were 20 mg/kg and30 mg/kg, respectively. METHODS: Motor coordination of mice was scored with pole test andtraction test before experiment and at 3 days after the last injection ofneurotoxin. Three days after complete injection, mice were sacrificed rapid ly to measure the contents of malondialdehyde (MDA) and reduced glu tathione (GSH) in the substantia nigra of midbrain. MAIN OUTCOME MEASURES: ① Motor and behavior scores; ② con tent of MDA; ③ content of GSH.~ULTS: All 48 mice were involved in the final analysis. ① Scores of pole test and traction test were decreased in neurotoxin group as compared with those in control group (P＜0.01); but the scores were increased after 3-NP single/repetitively preconditionings, and there were significant difference (P＜0.05, P＜0.01). Meanwhile, there was also significant differencebetween 3-NP repetitively preconditionings group and 3-NP single preconditioning group (P＜0.05). ② Content of MDA was increased in neurotoxin group as compared with that in control group, and there was significant difference (P＜0.01); content of MDA was decreased after 3-NP single preconditioning as compared with that in neurotoxin group, and there was significant difference (P＜0.05); content of MDA was remarkably decreased after 3-NP repetitively preconditionings as compared with that in neurotoxin group, and there was greatly significant difference (P＜0.01); meanwhile, there was also significant difference between 3-NP repetitively preconditionings group and 3-NP single preconditioning group (P＜0.05). ③As compared with that in blank control group, content GSH in 3-NP single administration group was not changed; content of GSH in 3-NP repetitively administrations group was increased (P＜0.05); content of GSH in neurotoxin group was decreased as compared with that in blank control group (P＜0.01); content of GSH in 3-NP single preconditioning group was not changed as compared with that in neurotoxin group (P＞0.05); content of GSH was increased after 3-NP repetitively preconditionings, and there was significant difference (P＜0.05); meanwhile, there was significant difference between 3-NP repetitively preconditionings group and 3-NP single preconditioning group (P＜0.05).CONCLUSION: 3-NP repetitively preconditionings can activate synthesis of GSH, protect dopamine neurons through decreasing production of MDA.