Influence of endogenous ciliary neurotrophic factor on neural differentiation of adult rat hippocampal progenitors

Ciliary neurotrophic factor is the only known neurotrophic factor that can promote differentiation of hippocampal neural progenitor cells to glial cells and neurons in adult rats. This process is similar to spontaneous differentiation. Therefore, ciliary neurotrophic factor may be involved in spontaneous differentiation of neural stem cells. To verify this hypothesis, the present study isolated neural progenitor cells from adult male rats and cultured them in vitro. Results showed that when neural progenitor cells were cultured in the absence of mitogen fibroblast growth factor-2 or epidermal growth factor, they underwent spontaneous differentiation into neurons and glial cells. Western blot and immunocytochemical staining showed that exogenous ciliary neurotrophic factor strongly induced adult hippocampal progenitor cells to differentiate into neurons and glial cells. Moreover, passage 4 adult hippocampal progenitor cells expressed high levels of endogenous ciliary neurotrophic factor, and a neutralizing antibody against ciliary neurotrophic factor prevented the spontaneous neuronal and glial differentiation of adult hippocampal progenitor cells. These results suggest that the spontaneous differentiation of adult hippocampal progenitor cells is mediated partially by endogenous ciliary neurotrophic factor.

14-3-3 gamma and zeta protein expression in active microglia Immune response mechanisms of Parkinson’s disease

BACKGROUND： The progressive degeneration of dopaminergic neurons in Parkinson＇s disease is associated with an activated glial reaction, combined with an inflammatory process. These responses lead to the production of cytokines, such as interferon- γ, tumor necrosis factor- α （TNF- α ）, and interleukin-1 β. In addition, 14-3-3 protein is a component of Lewy bodies in Parkinson＇s disease. OBJECTIVE： To observe the expression of 14-3-3 γ and ζ protein, as well as TNF-α, in mouse microglia, as well as changes after lipopolysaccharide （LPS） activation. To investigate possible mechanisms of dopaminergic neuronal injury due to activated microglia. To and clarify the immune response mechanisms of Parkinson＇s disease. DESIGN： Randomized controlled observation, cell study.SETTING： Laboratory of Department of Neurology, the Affiliated Union Hospital of Tongji Medical College, Huazhong University of Science and Technology. MATERIALS： The BV-2 immortalized murine microglia cell line was purchased from China Unit cell center. LPS was provided by Sigma Company. Cell cultures were purchased from Gibco. Phospho-（Ser） 14-3-3 binding motif antibody was purchased from Santa Cruz Biotechnologies. FITC was provided by Linfei Biotechnology, Wuhan, China. TNF- α ELISA was provided by Jingmei Biotech Co, Wuhan, China. The flow cytometer was provided by Becton Dickinson, Canada. METHODS： The present experiment was performed at the Laboratory of Department of Neurology, the Affiliated Union Hospital of Tongji Medical College, Huazhong University of Science and Technology from April to December 2006. The microglial cell line, BV-2, was cultured in vitro and stimulated with LPS for 2, 6, 12, and 24 hours. BV-2 cultures without LPS were used as controls. MAIN OUTCOME MEASURES： Expression of 14-3-3 γ protein was detected by flow cytometry. 14-3-3 ζ percentage expression and the mean fluorescence intensity was detected by immunofluorescence. TNF- α expression was detected by ELISA. RESULTS： 14-3-3 γ protein expression analysis： following LPS-induction in BV-2 cells, the fluorescence intensity of the 14-3-3 γ proteins gradually decreased. The 12 and 24 hours groups exhibited significantly lower expression than the normal control group （P 〈 0.05）. 14-3-3 ζ percentage expression and the mean fluorescence intensity： the percentage of 14-3-3 ζ protein expression gradually decreased with LPS stimulation. The mean fluorescence intensity from the 6, 12, and 24 hours groups was significantly less than the control group （P 〈 0.05）. TNF-α expression： resting BV-2 cells did not express TNF-α. Following 2 hours of LPS stimulation, TNF-α was highly expressed in BV-2 cells, but decreased again by 24 hours. CONCLUSION： Dopaminergic neuronal injury, due to activated microglial cells, might be related to the participation of 14-3-3 proteins and the release of TNF-α.

Minocycline protects the apoptosis of PC12 cells induced by 1-methyl-4-phenylpyridinium

Objective： To explore the protective effect of minocycline on the apoptosis of cellular parkinsonism models induced by MPP^＋ . Methods： Using PC12 cells as the apoptotic model of dopaminergic neurons, MC and MPP^＋ were added into the culture medium of PC12 cells, and using MTr to assay the cell viability and metabolic state; The cells apoptosis was assayed by electrophoresis method and using flow cytometry FACS to assay the apoptosis ratio. Results： Added the MPP^＋ to get the concentration of 10μmol/L, the cellular parkinsonism model of apoptosis had been prepared. The pre-treatment of MC （ 100/μmol/L） could significantly increase the PC12 cell viability. The apoptosis ratio of MC＋MPP^＋ group was significantly lower than that of MPP^＋ group, but was still significantly higher than that of control group. Conclusion： MC may protect the cell apoptosis induced by MPP^＋ to some extent.

Effect and mechanism of homocysteine on Parkinson's disease induced by 6-OHDA

Objective： To study the effects and mechanism of homocysteine（Hcy） on Parkinson＇ s disease（PD） induced by 6-hydroxydopamine （6-OHDA） in vivo. Methods：Forty rats were divided into 4 groups. 6-OHDA or the solvent of 6-OHDA was focally administrated to induce PD, 2 h later Hcy or 0.9% sodium chloride was administrated in the ipsolateral substantial nigra（SN）. We Used behavioral testing, Immolunohistochemical techniques, biochemistry techniques to detect the injury of SN. Results：The rotary turns of PD rats induced by 6-OHDA showed significant increase after treatment with Hcy compared with the controls（P 〈 0.05）. Also the numbers of tyrosine hydroxylase（TH）-stained neurons were decreased, and dendrites were fragmented and truncated. Free radicals were increased and antioxidaat enzymes decreased. Conclusion：Focal infusion of Hcy into the SN increased the vulnerability of the doparninergic neurons to 6-OHDA-induced degeneration, it seems that the endangering effect of Hcy is due to exacerbating oxidative stress.

Study of apoptosis pattern of dopaminergic neurons and neuroprotective effect of nicotine in MPTP mouse model

Objective：To investigate the apoptosis of dopaminergic neurons and the protective effect of nicotine in 1-methyl-4-phenyl- 1, 2,3,6-tetrahydropyridine （MPTP）-induced mouse model of Parkinson＇s disease. Methods：The mouse model of Parkinson＇s disease were formed by MPTP （30 mg/kg/d×7, i.p. ）; and the loss and apoptosis of dopaminergic neurons was observed by Tyrosine Hydroxylase（TH） and TUNEL stains.In ＂Nicotime plus MPTP＂ group, mice were pretreated with nicotine before MPTP injection. The putative protective effect of nicotine was analyzed. Results：The number of TH-positive cells decreased during MPTP treatment. Apoptotic neurons began to appear after three injections of MPTP and peaked on the 8th day. In the MPTP-intoxicated mice treated with nicotine, the loss of TH-positive cells was significantly less than that of MPTP- treated group （30 mg/kg/d×7）（P 〈 0.05）. Conclusion：The chronic treatment of MPTP can induce the apoptosis of dopaminergic neurons in substantia nigra, and nicotine might have a neuroprotecitve effect on dopaminergic neurons against MPTP toxicity.

Functional localization of epileptic foci in cats using manganese-enhanced MRI

Objective： To determine the encephalic region correlated with epilepsy by manganese-enhanced MRI （MEMRI） and determine the correlation of epilepsy with calcium overloading. Methods： The cats were divided into two groups. The first group underwent EEG examination and ethological observation. The second group underwent MEMRI measurement. Signal enhanced encephalic regions were sectioned. Results： The achievement ratio of convulsive cats intramusclelarly injected with PTZ was 80%. MEMRI showed diffuse signal enhancement in the cerebral cortex of the cats with generalized tonic-clonic convulsive seizures compared with control animals. The enhancement rate of frontal-parietal-occipital lobe was 34.6% and 22.9% in temporl lobe compared with the control groups. Signal enhancement on frontal-parietal lobe persisted for 24 h after epileptic seizures were induced. The neurons of enhanced encephalic regions showed obvious degeneration and necrosis. Conclusion： Seizures can be induced in cats by intramuscular injection of PTZ （55 mg/kg）. Frontal-parietal lobe is the correlated encephalic regions of epilepsy. MEMRI plays an important role in localizing and revealing pathogenesis of epileptic seizures.

Changes of dynamical balance of free radicals induced by levodopa in rat glia-containing mesencephalic culture fluid

BACKGROUND ： Parkinson disease is neurodegenerative disorders, characterized by a progressive and selective degeneration of nigrostdatal dopaminergic pathway. Its main clinical symptoms include bradykinesia, dgidity, rest tremor and disturbances in balance. Levodopa （L-DOPA） is the ＂gold standard＂ for the symptomatic treatment of Parkinson disease, but L-DOPA is toxic to dopaminergic neurons and the chronic administration of L-DOPA often causes the side effects of motor such as ＂on-off ＂, etc., and its mechanism still has not been completely clarified. OBJECTIVE ： To observe the changes of the content of glutathione （GSH）, activities of glutathione peroxidase （GSH-Px） and superoxide dismutase （SOD） and content of malondialdehyde （MDA） in gila-containing mesencephalic culture fluid after L-DOPA of different concentrations were added. DESIGN： A comparative observation. SETTINGS ： Department of Neurology, Affiliated Hospital of Taishan Medical University; Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS ： The experiment was carried out in the Basic Research Institute, Taishan Medical University from December 2003 to September 2004. Ten postnatal newborn SD rats （within 2 days） were selected, either male or female. Fetal bovine serum （DMEM/F12） was from Gibco Company; L-DOPA and glial fibrillary acidic protein （GFAP） from Sigma Company. Kits for determination of GSH, GSH-Px, SOD and MDA were purchased from Nanjing Jiancheng Bioengineering Research Institute. METHODS ： Gila-containing mesencephalic culture fluid were placed in 24-well culture plate, and L-DOPA of 50, 100 and 500 μmot/L was added to each group, the blank control group was also set. The glia-containing mesencephalic culture fluid selected at 4, 24, 48 and 72 hours respectively to determine the GSH content with colorimetdc quantitative technique, GSH-Px activity with colorimetdc method, SOD activity with xanthine oxidase method and MDA content with thiol-barbituric acid method. MAIN OUTCOME MEASURES ： GSH and MDA contents, GSH-Px and SOD activities in the glia-containing mesencephalic culture fluid at 4, 24, 48 and 72 hours after L-DOPA of different concentrations were added. RESULTS ： In the glia-containing mesencephalic culture fluid after 100 μmol/L L-DOPA was added for 24, 48 and 72 hours, the GSH contents were lower than those in the blank control group [（174.14±39.28）, （161.55± 40.79）, （144.97±57.59） mg/L; （220.66±32.61）, （221.10±32.98）, （220.43±31.98） mg/L, P 〈 0.05]; The GSH-Px activities were lower than those in the blank control group [（4.03±1.05）, （3.99±1.12）, （3.47±1.00） μmol/L; （5.45±1.14）, （5.69±1.21）, （5.49±1.28） μmol/L, P 〈 0.05]; The SOD activities were also lower than those in the blank control group [（42.02±5.08）, （39.38±5.34）, （38.87±5.75）kNU/L; （51.35±8.87）, （51.78±8.96）, （50.99± 9.09） kNU/L, P 〈 0.05]; Whereas the MDA contents were higher than those in the blank control group [（3.51 ± 1.05）, （3.99±1.03）, （4.45±1.58） μmol/L; （2.09±1.13）, （2.18±1.29）, （2.01±1.05） μmol/L, P〈 0.05]. In the gli- a-containing mesencephalic culture fluid after 100 μmol/L L-DOPA was added for 4, 24, 48 and 72 hours, the GSH contents were （172.27±26.07）, （140.15±61.44）, （137.30±50.87）, （121.09±66.07） mg/L, the GSH-Px activities were （3.89±1.20）, （3.56±1.23）, （3.38±1.18）, （3.01±1.09） μmol/L, the SOD activities were （38.18±6.75）, （35.23±7.85）, （4.59±1.24）, （31.42±7.01） kNU/L, which were all lower than those in the blank control group （P 〈 0.05-0.01 ）; The MDA contents were （3.65±0.86）, （3.87±1.14）, （4.59±1.24）, （4.79±1.32） μmol/L, which were higher than those in the blank control group （P 〈 0.05-0.01）. In the gila-containing mesencephalic culture fluid added by 50 μmol/L L-DOPA, the GSH and MDA contents, GSH-Px and SOD activities at each time point were all close to those in the blank control group （P〉 0.05）. CONCLUSION ： L-DOPA of certain concentration can destroy the dynamical balance of free radicals in glia-containing mesencephalic culture fluid, and accelerate the degeneration of neurons, which are in concentration- and time-dependent manners.

The recommendations of Chinese Parkinson’s disease and movement disorder society consensus on therapeutic management of Parkinson’s disease

Background:Parkinson’s disease(PD)is a chronic,progressive and debilitating disease,which affects over 2.5 million people in China.PD is characterized clinically by resting tremor,muscular rigidity,bradykinesia and postural instability.As the disease progresses,additional complications can arise such as non-motor and neurobehavioral symptoms.Pharmacological treatment and surgical intervention for PD have been implemented in China.Until 10 years ago,there was lack of standardization for the management of PD in different regions and among different physicians,leading to different treatment levels in different regions and different physicians.Since then,the Chinese Parkinson’s Disease and Movement Disorder Society have published three versions of guidelines for the management of PD in China,in 2006,2009 and 2014,respectively.Correspondingly,the overall level of treatment for PD in China improved.Objectives:To update the treatment guidelines based on current foreign and domestic practice guidelines and clinical evidence,and to improve the treatment options available to physicians in the management of PD.Summary:A variety of treatment recommendations in the treatment guidelines have been proposed,including physical activity and disease-modifying medication,which should be initiated at the early-stage of the disease.The principles of dosage titration should be followed to avoid acute adverse reactions to the drugs,to achieve a satisfactory clinical effect with a low dose and to reduce the incidence of long-term motor complications.Moreover,different treatment strategies should be considered at different stages of the disease.Importantly,treatment guidelines and personalized treatments should be valued equally.A set of treatment recommendations has been developed to assist physicians to improve and optimize clinical outcomes for patients with PD in China.

Efficacy and safety of rasagiline in Chinese patients with early Parkinson’s disease:a randomized, double-blind,parallel,placebo-controlled,fixed-dose study

Background:Rasagiline is a monoamine oxidase-B inhibitor used for Parkinson’s disease(PD)treatment,but its effectiveness on Chinese patients is unclear.This study aimed to evaluate the efficacy and safety of rasagiline monotherapy in Chinese patients with early PD.Methods:A 26-weeks,randomized,double-blind,placebo-controlled study has been performed at 15 sites in China and enrolled outpatients(≥35 years old)with idiopathic PD without a history of using any dopaminergic drugs.Participants were randomized 1:1 to receive rasagiline 1 mg once daily or placebo.The primary endpoint was the change of the Unified Parkinson’s Disease Rating Scale(UPDRS)total score from baseline to 26 weeks treatment.Secondary endpoints included changes in UPDRS subscale scores from part Ⅰ to Ⅲ.Health status was assessed with the PD Questionnaire(PDQ)-39 and EuroQol-Five-Dimension(EQ-5D)questionnaire.Safety profile was collected until 30 weeks after randomization.Results:A total of 130 patients(n=65/group)were recruited,and 127(rasagiline,n=64;placebo,n=63)were included in the full analysis set.Baseline characteristics were comparable between the two groups.The decrease in the mean UPDRS total score was greater in the rasagiline group than in the placebo group(−3.18±0.95 vs.−0.18±0.98,P=0.025),and the mean UPDRS part I non-motor symptoms score(−0.54±0.15 vs.-0.08±0.15,P=0.003)were significantly decreased in the rasagiline group compared with placebo treated patients.An improvement trend was observed in the active treatment group for the subscales evaluation with parts Ⅱ and Ⅲ,while the difference to placebo was not statistically significant.Life quality assessed by the EQ-5D visual analog scale improved in the rasagiline group but worsened in placebo treated patients.The overall incidence of treatment-emergent adverse events(AEs)was slightly lower in the rasagiline group(41.5%)than in the placebo group(46.2%).Conclusions:Rasagiline is effective,safe,and well tolerated as monotherapy for the treatment of Chinese PD patients.

The efficacy and safety of pramipexole ER versus IR in Chinese patients with Parkinson’s disease: a randomized, double-blind, double-dummy, parallel-group study

Objective:To evaluate the non-inferiority of pramipexole extended-release(ER)versus immediate-release(IR)in Chinese patients with Parkinson’s disease(PD)in a double-blind,randomized,parallel-group study.Methods:Subjects were Chinese patients with idiopathic PD with diagnosis≥2 years prior to trial,age≥30 years old at diagnosis,and Modified Hoehn and Yahr score 2-4 during‘on’-time.Subjects received treatment with pramipexole ER(n=234)or IR(n=239).Non-inferiority was based on the primary endpoint,the change from baseline to end of maintenance(week 18)in the UPDRS(Parts II+III)total score.Results:For the primary endpoint,the adjusted mean changes(standard error)of UPDRS Parts II+III at week 18 were−13.81(0.655)and−13.05(0.643)for ER and IR formulations,respectively,using ANCOVA adjusted for treatment and centre(fixed effect)and baseline(covariate).The adjusted mean between group difference was 0.8 for the 2-sided 95%CI(−1.047,2.566).Since the lower limit of the 2-sided 95%CI(−1.047)for treatment difference was higher than the non-inferiority margin of−4,non-inferiority between pramipexole ER and IR was demonstrated.The incidence of adverse events(AEs)was 68.8%in the ER arm and 73.6%in the IR arm with few severe AEs(ER:2.1%;IR:3.8%).Conclusion:Based on the UPDRS II+III score,pramipexole ER was non-inferior to pramipexole IR.The safety profiles of pramipexole ER and IR were similar.These results were based on comparable mean daily doses and durations of treatment for both formulations.

Adjunct rasagiline to treat Parkinson’s disease with motor fluctuations:a randomized,double-blind study in China

Background:The use of adjunct rasagiline in levodopa-treated patients with Parkinson’s disease and motor fluctuations is supported by findings from large-scale clinical studies.This study is to investigate the efficacy and safety of adjunct rasagiline in Chinese patients with Parkinson’s disease,as a product registration study.Methods:This 16-week,randomized,double-blind,parallel-group,multicenter,placebo-controlled study of rasagiline 1 mg/day included levodopa-treated patients with Parkinson’s disease and motor fluctuations.The primary efficacy endpoint was mean change from baseline in total daily OFF time over 16 weeks.Secondary endpoints were Clinical Global Impressions–Improvement(CGI-I),and change in Unified Parkinson’s Disease Rating Scale(UPDRS)Activities of daily living(ADL)and Motor scores.Patient well-being(EQ-5D),and the frequency of adverse events were also assessed.Results:In total,324 levodopa-treated patients were randomized to rasagiline 1 mg/day(n=165)or placebo(n=159).Over 16 weeks,rasagiline statistically significantly reduced the mean[95% confidence interval]total daily OFF time versus placebo(−0.5 h[−0.92,−0.07];p=0.023).There were also statistically significant improvements versus placebo in CGI-I(−0.4 points[−0.61,−0.22];p<0.001),UPDRS-ADL OFF(−1.0 points[−1.75,−0.27];p=0.008),and UPDRS-Motor ON(−1.6 points[−3.05,−0.14];p=0.032)scores,as well as the EQ-5D utility index(p<0.05).Rasagiline was safe and well tolerated.Conclusions:In levodopa-treated Chinese patients with Parkinson’s disease and motor fluctuations,adjunct rasagiline 1 mg/day statistically significantly reduced OFF time,and improved daily function and overall well-being,versus placebo.Consistent with findings in other countries,adjunct rasagiline was proven efficacious and well tolerated in Chinese patients.