Correlations of transcranial sonography of substantia nigra characteristics with MRI iron deposition on substantia nigra in patients with Parkinson disease

Objective To explore the correlations of transcranial sonography of substantia nigra(SN-TCS)characteristics with MRI iron deposition on substantia nigra in patients with Parkinson disease(PD).Methods Data of SN-TCS and craniocerebral MRI in 120 PD patients were retrospectively analyzed.The patients were divided into iron deposition positive group(positive group,n=46)and iron deposition negative group(negative group,n=74)according to quantitative susceptibility mapping(QSM)value.Then parameters of SN-TCS and MRI were compared between groups(both P<0.05),and correlation analysis were also performed.Results The proportion of high echo positive,strong echo area and QSM value of substantia nigra,as well as of hyper-substantia nigra area/midbrain area(S/M)in positive group were all higher than those in negative group(all P<0.001).No significant difference of midbrain area was found between groups(P>0.05).Strong echo area of substantia nigra and S/M based on SN-TCS were both low-medium positively correlated with substantia nigra QSM value showed on MRI(r=0.497,0.529,both P<0.001).Conclusion SN-TCS characteristics of PD patients were correlated with MRI iron deposition on substantia nigra,among which strong echo area and S/M were valuable for evaluating iron deposition on substantia nigra.

Protective effects of kidney-tonifying Chinese herbal preparation on substantia nigra neurons in a mouse model of Parkinson's disease

The Chinese herbs Herba Epimedii, Fructus Ligustri Lucidi and Rhizoma Polygonati were injected into Parkinson＇s disease mice established via intraperitoneal injection of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine hydrochloride. The selective monoamine oxidase B inhibitor selegiline was used as a positive control drug. After successive administration for 4 weeks, Herba Epimedii could downregulate the expression of caspase-3 and increase the brain-derived neurotrophic factor level, as well as increase tyrosine hydroxylase activity in the substantia nigra of Parkinson＇s disease mouse models. Rhizoma Polygonaticould downregulate the expression of caspase-3 and FasL, and increase neural growth factor and brain-derived neurotrophic factor levels. Fructus Ligustn Lucidi could downregulate caspase-3 expression. Rhizoma Polygonati and Fructus Ligustn Lucidi did not produce obvious effects on tyrosine hydroxylase activity. Herba Epimedii and Fructus Ligustri Lucidi yielded similar effects on apoptosis-promoting factors to those elicited by selegiline. Herba Epimedii and Rhizoma Polygonati significantly increased the levels of neurotrophic factors compared with selegiline. Herba Epimedii significantly increased tyrosine hydroxylase activity compared with selegiline. It is indicated that the kidney-tonifying Chinese herbal preparation can downregulate the expression of apoptosis-promoting factors, increase neurotrophic factors levels in the substantia nigra and striatum, as well as increase tyrosine hydroxylase activity in the substantia nigra of Parkinson＇s disease mouse models, thereby exerting a stronger or similar neuroprotective effects compared with selegiline.

Anti-apoptotic effect of Shudipingchan granule in the substantia nigra of rat models of Parkinson＇s disease

Levodopa is the gold-standard treatment for Parkinson＇s disease. However, although it alleviates the clinical symptoms, it cannot delay the progressive apoptosis of dopaminergic neurons or prevent motor complications in the long term. In the present study, we investigated the effect of Shudipingchan granule on neuronal apoptosis in a rat model of Parkinson＇s disease, established by injecting 6-hydroxydopamine into the substantia nigra pars compacta and ventral tegmental area. We then administered levodopa （20 mg/kg intraperitoneally, twice daily） with or without Shudipingchan granule （7.5 mL/kg intragastrically, twice daily）, for 4 weeks. The long-term use of levodopa accel- erated apoptosis of nigral cells and worsened behavioral symptoms by activating the extracellular signal-regulated kinase pathway and downstream apoptotic factors. However, administration of Shudipingchan granule with levodopa reduced expression of phosphorylated extracellular signal-regulated kinase 1/2 and Bax, increased tyrosine hydroxylase and Bcl-2, reduced apoptosis in the substantia nigra, and markedly improved dyskinesia. These findings suggest that Shudipingchan granule suppresses neuronal apoptosis by inhibiting the hyper- phosphorylation of extracellular signal-regulated kinase and downregulating expression of anti-apoptotic genes. Shudipingchan granule, used in combination with levodopa, can effectively reduce the symptoms of Parkinson＇s disease.

Signal Alteration of Substantia Nigra on 3.0T Susceptibility-weighted Imaging in Parkinson's Disease and Vascular Parkinsonism

Recent researches have found that 7 Tesla SWI can detect the alteration of substantia nigra hyperintensity in Parkinson's disease(PD),multiple system atrophy(MSA),and progressive supranuclear palsy(PSP).The aim of this study was to investigate whether 3 Tesla SWI(3T SWI)can visualize anatomical alterations occurring in a hyperintense structure of the substantia nigra in PD and vascular parkinsonism(VP),and whether the evaluation of abnormal signal can be used as a factor in the differential diagnosis of PD and VP.Using 3 Tesla MRI,we evaluated 38 healthy subjects,33 patients with PD and 34 patients with VP.Two blinded readers independently assessed the images.We found that the dorsolateral nigral hyperintensity was absent in 31 of 33 patients with PD and 15 of 34 patients with VP.The dorsolateral nigral hyperintensity was present in 19 of 34 patients with VP and 35 of 38 healthy controls.Group comparisons of absence of dorsolateral nigral hyperintensity revealed significant differences between the patients with PD and those with VP(P<0.001).The sensitivity of SWI for PD was 93.9%and the specificity was 92.1%.Visual assessment of dorsolateral nigral hyperintensity on high-field SWI scans may serve as a new simple diagnostic imaging marker for PD.And our study results indicate that 3T SWI can be used as a tool to identify PD and VP.

Experimental Study of Serum Substantia Nigra Neuron Autoantibody and Its Effect in Parkinson Disease Patients

To investigate the serum substantia nigra neuron autoantibody and its effect in the patients with Parkinson disease (PD), substantia nigra slices and a rat model of injection of serum from PD patients in unilateral side substantia nigra were applied. The results showed that the positive rate of substantia nigra neuron autoantibody in PD patients was significantly higher than in the healthy control group (36.67 % vs 6.67 %, P <0.01), but no significant difference was found between PD group and myasthenia gravis (MG) group (26.67 %, P >0.05). The sera from PD patients positive for substantia nigra neuron autoantibody could decrease the number of the dopaminergic neurons more seriously than those from MG and the healthy once respectively (both P <0.01). The results suggested that the immunological mechanism might partly play a role in the development of PD.

Enriched environment elevates expression of growth associated protein-43 in the substantia nigra of SAMP8 mice

An enriched environment protects dopaminergic neurons from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine（MPTP）-induced neuronal injury, but the underlying mechanism for this is not clear. Growth associated protein-43（GAP-43） is closely associated with neurite outgrowth and axon regeneration during neural development. We speculate that an enriched environment can reduce damage to dopaminergic neurons by affecting the expression of GAP-43. This study is designed to test this hypothesis. Three-month-old female senescence-accelerated mouse prone 8（SAMP8） mice were housed for 3 months in an enriched environment or a standard environment. These mice were then subcutaneously injected in the abdomen with 14 mg/kg MPTP four times at 2-hour intervals. Morris water maze testing demonstrated that learning and memory abilities were better in the enriched environment group than in the standard environment group. Reverse-transcription polymerase chain reaction, immunohistochemistry and western blot assays showed that m RNA and protein levels of GAP-43 in the substantia nigra were higher after MPTP application in the enriched environment group compared with the standard environment group. These findings indicate that an enriched environment can increase GAP-43 expression in SAMP8 mice. The upregulation of GAP-43 may be a mechanism by which an enriched environment protects against MPTP-induced neuronal damage.

Electroacupuncture-regulated neurotrophic factor mRNA expression in the substantia nigra of Parkinson's disease rats

Acupuncture for the treatment of Parkinson＇s disease has a precise clinical outcome. This study investigated the effect of electroacupuncture at Fengfu （GV16） and Taichong （LR3） acupoints in rat models of Parkinson＇s disease induced by subcutaneous injection of rotenone into rat neck and back. Reverse transcription-PCR demonstrated that brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression was significantly increased in the substantia nigra of rat models of Parkinson＇s disease, and that abnormal behavior of rats was significantly improved following electroacupuncture treatment. These results indicated that electroacupuncture treatment upregulated brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression in the substantia nigra of rat models of Parkinson＇s disease. Thus, electroacupuncture may be useful in the treatment of Parkinson＇s disease.

Differential protein expression in substantia nigra induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine in a mouse model of chronic Parkinson’s disease

BACKGROUND： To date, a complete protein expression profile of the midbrain substantia nigra in a mouse model of chronic Parkinson＇s disease, induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine （MPTP）, does not exist. In addition, there are no reports of analysis of differential protein expression. OBJECTIVE： To separate and evaluate MPTP-induced differential protein expression through the use of proteomics in the substantia nigra of a mouse model of chronic Parkinson＇s disease. DESIGN： Randomized controlled animal study. SETTING： Department of Neurology, the First Affiliated Hospital, Chongqing Medical University. MATERIALS： Sixteen 8-10-week old, healthy, male, C57BL mice, weighing 20-25 g, and of clean grade, were provided by the Experimental Animal Center of Chongqing Medical University. The experimental animals were disposed according to ethical criteria. MPTP was provided by Sigma Company, USA; Pdquest 2D image analysis software and gelatum/irradiance image analysis system （ChemiDoc XRS） by Bio-Rad, USA; and Voyager DE-PROMALD1-TOF-MS mass spectroscopy analyzer by AB1 Company, USA. METHODS： This study was performed in Chongqing Neurological Laboratory between November 2006 and July 2007. Mice were randomly divided into model and control groups, with 8 mice in each group. Mice in the model group were received a subcutaneous injection of MPTP （25 mg＆g）, twice a week, for five successive weeks, to establish a chronic Parkinson＇s disease model. Mice in the control group received the same volume of a subcutaneous saline injection at the same time points. Mice were sacrificed by anesthesia to rapidly obtain the midbrain for protein separation of the substantia nigra. MAIN OUTCOME MEASURES： （1） 2-ED handbook （Bio-Rad Company） was referenced for two-dimensional electrophoresis, （2） PDQUEST8,0 analytical electrophoresis pattern was adopted to evaluate differential protein expression. （3） Peptide mass finger print map and data were retrieved on http：//www.prospector.ucsf.edu to compare differential substantia nigral protein expression in the two groups. RESULTS： Two-dimensional gel electrophoresis of substantia nigra tissue indicated that there were 33 differential protein expressions between the two groups. Three new proteins were evaluated, including α -enolase, which exhibited regulated expression, tumor necrosis factor ligand superfamily member 4, and cyclin-dependent kinase inhibitor 1B. CONCLUSION： There are three proteins that exhibit differential expression in the substantia nigra- α -enolase, tumor necrosis factor ligand superfamily member 4, and cyclin-dependent kinase inhibitor 1B.

Mechanisms of secondary damage to the thalamic nucleus and substantia nigra in an adult hypertensive rat model following middle cerebral artery occlusion

BACKGROUND： Following ischemia, apoptosis is observed at the ipsilateral ventropostenor thalamic nucleus and substantia nigra, which are distant from, but connected to, the ischemic cerebral cortex, in animals with normotension. However, secondary brain damage in hypertension has not been clearly investigated. OBJECTIVE： The present study determined whether neuronal apoptosis is associated with neuronal loss in the ipsilateral ventroposterior thalamic nucleus and substantia nigra following cortical ischemia in adult hypertensive rats. Results should provide options for determining a time window for anti-apoptotic therapy. DESIGN, TIME AND SETTING： All experimental procedures in this randomized, controlled trial were conducted at the Neurological Laboratory of the First Affiliated Hospital of Sun Yat-sen University of China between October 2006 and July 2008. MATERIALS： Monoclonal primary antibodies specific to mouse anti-rat microtubule-associated protein 2 and glial fibrillary acidic protein were respectively purchased from Sigma Chemical, USA and BD Pharmingen, USA. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling （TUNEL） detection kits were purchased from Roche Applied Science, Switzerland and Upstate, USA, respectively. METHODS： A total of 64 male, Sprague Dawiey rats, aged 60-90 days, were equally and randomly divided into middle cerebral artery occlusion and sham surgery groups. Renovascular hypertension was established in both groups by renal artery occlusion. Right distal middle cerebral artery occlusion was performed by electrocoagulation in the middle cerebral artery occlusion group. MAIN OUTCOME MEASURES： Microtubule-associated protein 2 and glial fibrillary acidic protein were detected by immunohistochemistry, and apoptotic cells were observed by TUNEL assay. The stainings were separately detected in the ipsilateral ventroposterior thalamic nucleus and substantia nigra. RESULTS： During the 4 weeks following distal middle cerebral artery occlusion in renovascular hypertensive rats, microtubule-associated protein 2 expression gradually, but significantly, decreased （P 〈 0.05）. Expression of glial fibrillary acidic protein increased significantly in the ipsilateral ventroposterior thalamic nucleus and substantia nigra （P 〈 0.05） and reached a peak at 4 weeks. In addition, number of apoptotic cells was significantly increased in both areas compared with the sham controls （P 〈 0.05）, with a peak at 2 weeks. CONCLUSION： Results suggested that neuronal loss in the ipsilateral ventroposterior thalamic nucleus and substantia nigra following distal middle cerebral artery occlusion in hypertensive rats could be a secondary event resulting from apoptosis. The temporal apoptosis profile provides options for determining a time window for anti-apoptotic therapy at 2 weeks after stroke.

Effects of Zhichan powder on signal transduction and apoptosis-associated gene expression in the substantia nigra of Parkinson's disease rats

Previous studies have shown that Zhichan powder elevated immunity and suppressed oxidation in mice. Rat models of Parkinson＇s disease were induced by stereotaxically injecting 6-hydroxydopamine into the substantia nigra. The rat models were intragastrically treated with Zhichan powder, which is composed of milkvetch root, ginseng, bunge swallowwort root, himalayan teasel root. Magnolia officinalis, Ligustrum lucidum Ait. and szechwan Iovage rhizome. Immunohistochemistry and reverse transcription-PCR results demonstrated that mRNA and protein expression of tumor necrosis factor receptor 1, Fas, caspase-8, cvtochrome C, Bax, casDase-3, and p53 significantly increased, but Bcl-2 expression significantly decreased in the substantia nigra of rats with Parkinson＇s disease. Following Zhichan powder administration, mRNA and protein expression of tumor necrosis factor receptor 1, Fas, caspase-8, cytochrome C, Bax, caspase-3, and p53 diminished, but Bcl-2 expression increased in the rat substantia nigra. These results indicate that Zhichan powder regulates signal transduction protein expression, inhibits apoptosis, and exerts therapeutic effects on Parkinson＇s disease.

Expression of calbindin D28K in substantia nigra of model rats with Parkinson disease

BACKGROUND： Previous researches suggested that expression level of calbindin D28K mRNA decreased in substantia nigra （SN） of model rats with Parkinson disease （PD）, and this might be related to the decrease of anti-degeneration potentials of dopaminergic neurons. OBJECTIVE： To observe expression changes of calbindin D28K in SN dopaminergic neurons during their degeneration and death in midbrain of PD model rats. DESIGN： Completely randomized grouping design SETTING： Department of Neurobiology, Xuzhou Medical College MATERIALS＂ A total of 92 healthy male SD rats, with the age of 3 months, weighing 200-250 g, were selected from Experimental Animal Center of Xuzhou Medical College [certification： SCXK （su） 2003-0003]. Calbindin D28K（CB）, tyroxine hydroxylase （TH）, ABC kit, 6-hydroxydopamine （6-OHDA） and Nissl dyes were provided by Sigma Company, and sheep serum was provided by Beijing Zhongshan Company. METHODS： The experiment was carried out in the Neurobiological Center of Xuzhou Medical College from October 2003 to October 2004. ① With lot method, rats were divided into blank control group （n=28）, experimental control group （n=-28） and experimental group （n=36）. Rats in experimental group were injected with 6-OHDA at right corpus striatum for PD modeling; rats in experimental control group were injected with saline at the same site; rats in blank control group did not give any injections.② On the 7^th, 14^th, 21^st and 28^th days, SN segments on right midbrain from every 5 rats in experimental group were fixed, embedded with paraffin and cut into successively coronary pieces. Rats in other two groups were treated with the same methods and then stained with Nissl to show neuronal form. Meanwhile, CB and TH antibodies staining with immunohistochemistry were used to show CB containing dopaminergic neurons and dopaminergic neurons, and cells were calculated and observed under optic microscope. ③ On the 14^th and 28^th days, every 4 rats in experimental group and every 4 rats in control group were selected to obtain their brains and separate SN on the injured side. Western blot was used to detect expression of calbindin D28K, protein band was scanned with imaging equipment, and data were analyzed with LabWorks software. MAIN OUTCOME MEASURES：① On the 7^th, 14^th, 21^st and 28^th days, Nissl staining results of SN neurons and immunohistochemical staining results of CB and TH antibodies; ② On the 14^th and 28^th days, Western blot results of calbindin D28K in SN neurons, RESULTS; Among 92 rats, 2 rats in experimental group died after 1 day due to 6-OHDA injection and other 90 rats were involved in the final analysis. ①Nissl staining results： On the 7^th day of 6-OHDA injection, most neuronal somas on right SN pars compacta were shown as deep pycnosis or lysis breakage; on the 14^th and 21^st days, amount of neurons was decreased remarkably; on the 28^th day, most neurons in SN pars compacta disappeared. ② Results of immunohistochemical staining： Amount of positive neurons of calbindin D28K in right SN pars compacta was not changed on the 7^th day after 6-OHDA injection; on the 14^th day, the amount was higher in experimental group than that in both control groups （P 〈 0.01） and was decreased till the 21^st day, but it was still higher than that in the two control groups （P 〈 0.05）; on the 28^th day, positive neurons of calbindin D28K nearly disappeared, and the amount was lower than that in the two control groups （P 〈 0.01）. In addition, on the 7^th day after 6-OHDA injection into corpus striatum, positive TH neurons decreased 24% in right SN, and there was significant difference from that in control groups; on the 14^th, 21^st and 28^th days, positive TH neurons decreased 37%, 46% and 64%, respectively. ③ Western blot results： On the 14＇h day after 6-OHDA injection into corpus striatum, expression of calbindin D28K in right SN was higher in experimental group than that in both control groups （P 〈 0.05）; however, on the 28^th day, the expression was lower than that in the two control groups （P 〈 0.01 ）. CONCLUSION ： During degeneration and death of dopaminergic neurons, CB expression in SN pars compacta increases firstly and decreased gradually.

N-cadherin expression in the midbrain substantia nigra of perinatal rats

BACKGROUND： Cellular adhesion molecule mediates the effects of glial cell line-derived neurotrophic factor in dopaminergic neurons during nervous system development. It is assumed that cellular adhesion molecule plays an important role in dopaminergic neuron development. OBJECTIVE： To investigate N-cadherin expression in the midbrain substantia nigra during rat development. DESIGN, TIME AND SETTING： The developmental biology, controlled study was performed at the Laboratory of Research Center of Neurobiology, Xuzhou Medical College, China from April to October 2007. MATERIALS： Sprague Dawley rats （embryonic days 16, 18, and 20, as well as postnatal days 1, 3, 7, 14, and 21） were selected for this study. N-cadherin antibody was purchased from Santa Cruz, USA. METHODS： N-cadherin expression in the midbrain substantia nigra of perinatal rats was detected using reverse transcription polymerase chain reaction, Western blot, and immunohistochemistry methods. N-cadherin-positive cells were counted. MAIN OUTCOME MEASURES： N-cadherin mRNA, protein, and positive cells in the rat midbrain substantia nigra were quantified. RESULTS： The results of reverse transcription polymerase chain reaction and Western blot demonstrated that altered N-cadherin mRNA levels were similar to protein levels in the midbrain substantia nigra. N-cadherin mRNA and protein expression was low in the midbrain substantia nigra of embryonic day 16 rats but gradually increased, and reached a peak at postnatal day 1. N-cadherin mRNA and protein levels were still high at postnatal days 3 and 7, but significantly decreased at postnatal day 14. There was no significant difference in N-cadherin mRNA and protein expression between postnatal days 14 and 21 （P 〉 0.05）. The immunohistochemistry results demonstrated that there was no significant difference in the quantity of N-cadherin-positive cells per area in rats from embryonic day 16 to postnatal day 7 （P 〉 0.05）. The quantity of N-cadherin-positive cells per area was significantly decreased in rats at postnatal day 14. There was no significant difference in the number of N-cadherin-positive cells per area between postnatal days 14 and 21 （P 〉 0.05）. CONCLUSION： N-cadherin expression was reduced during embryonic stages, but reached a peak at postnatal days 1-7, and then gradually decreased in the rat midbrain substantia nigra.

Directional induction of dopaminergic neurons from neural stem cells using substantia nigra homogenates and basic fibroblast growth factor

To date, complex components of available reagents have been used for directional induction of neural stem cells into dopaminergic neurons, resulting in a poor ability to repeat experiments. This study sought to investigate whether a homogenate of the substantia nigra of adult rats and/or basic fibroblast growth factor could directionally induce neural stem cells derived from the subventricular zone of embryonic rats to differentiate into dopaminergic neurons. Tyrosine hydroxylase-positive cells were observed exclusively after induction with the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor for 48 hours in vitro. However, in the groups treated with homogenate supernatant or basic fibroblast growth factor alone, tyrosine hydroxylase expression was not observed. Moreover, the content of dopamine in the culture medium of subventricular zone neurons was significantly increased at 48 hours after induction with the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor. Experimental findings indicate that the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor could directionally induce neural stem cells derived from the subventricular zone of embryonic rats to differentiate into dopaminergic neurons in the substantia nigra with the ability to secrete dopamine.

Intracerebroventricularly-administered 1-methyl-4-phenylpyridinium ion and brain-derived neurotrophic factor affect catecholaminergic nerve terminals and neurogenesis in the hippocampus,striatum and substantia nigra

Parkinson＇s disease is a progressive neurological disease characterized by the degeneration of dopaminergic neurons in the substantia nigra.A highly similar pattern of neurodegeneration can be induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine（MPTP） or 1-methyl-4-phenylpyridinium ion（MPP＋）,which cause the death of dopaminergic neurons.Administration of MPTP or MPP＋ results in Parkinson＇s disease-like symptoms in rodents.However,it remains unclear whether intracerebroventricular MPP＋ administration affects neurogenesis in the substantia nigra and subgranular zone or whether brain-derived neurotrophic factor alters the effects of MPP＋.In this study,MPP＋（100 nmol） was intracerebroventricularly injected into mice to model Parkinson＇s disease.At 7 days after administration,the number of bromodeoxyuridine（Brd U）-positive cells in the subgranular zone of the hippocampal dentate gyrus increased,indicating enhanced neurogenesis.In contrast,a reduction in Brd U-positive cells was detected in the substantia nigra.Administration of brain-derived neurotrophic factor（100 ng） 1 day after MPP＋ administration attenuated the effect of MPP＋ in the subgranular zone and the substantia nigra.These findings reveal the complex interaction between neurotrophic factors and neurotoxins in the Parkinsonian model that result in distinct effects on the catecholaminergic system and on neurogenesis in different brain regions.

MPTP对C57BL/6小鼠SNpc和VTA脑区多巴胺能神经元的差异性损伤研究

目的:观察MPTP对小鼠黑质致密部(SNpc)和腹侧被盖区(VTA)多巴胺(DA)能神经元损伤的差异。方法:按20 mg/kg的剂量每2 h腹腔注射MPTP 0.1 mL,共注射4次制作小鼠急性损伤模型。(1)行为学测试:通过爬杆实验和悬杆实验,分别检测给药后不同时间点小鼠运动迟缓症状和肢体僵硬症状的程度;(2)免疫荧光:检测小鼠SNpc和VTA脑区的DA能神经元标志物酪氨酸羟化酶(TH)。结果:MPTP急性损伤后小鼠出现运动迟缓和肢体僵硬的表现;SNpc脑区的DA能神经元在各时间点均有减少,其中1 d组减少最显著,达69%(P<0.001),其DA能神经元突起亦变短或消失;VTA脑区的DA能神经元在各时间点亦有所减少,只是其幅度(23%)比SNpc脑区小。结论:小鼠急性损伤模型能选择性损伤SNpc和VTA脑区的DA能神经元,损伤快且严重,并在行为学上也有改变,能较好地模拟人类PD的神经病理学改变。

Ginsenoside Rb1 protects dopaminergic neurons from inflammatory injury induced by intranigral lipopolysaccharide injection

Accumulating studies suggest that neuroinflammation characterized by microglial overactivation plays a pivotal role in the pathogenesis of Parkinson’s disease.As such,inhibition of microglial overactivation might be a promising treatment strategy to delay the onset or slow the progression of Parkinson’s disease.Ginsenoside Rbl,the most active ingredient of ginseng,reportedly exerts neuroprotective effects by suppressing inflammation in vitro.The present study aimed to evaluate the neuroprotective and anti-inflammatory effects of ginsenoside Rbl in a lipopolysaccharide-induced rat Parkinson’s disease model.Rats were divided into four groups.In the control group,sham-operated rats were intraperitoneally administered normal saline for 14 consecutive days.In the ginsenoside Rbl group,ginsenoside Rb1(20 mg/kg)was intraperitoneally injected for 14 consecutive days after sham surgery.In the lipopolysaccharide group,a single dose of lipopolysaccharide was unilaterally microinjected into the rat substantial nigra to establish the Parkinson’s disease model.Lipopolysaccharide-injected rats were treated with normal saline for 14 consecutive days.In the ginsenoside Rbl +lipopolysaccharide group,lipopolysaccharide was unilaterally microinjected into the rat substantial nigra.Subsequently,ginsenoside Rbl was intraperitoneally injected for 14 consecutive days.To investigate the therapeutic effects of ginsenoside Rbl,behavioral tests were performed on day 15 after lipopolysaccharide injection.We found that ginsenoside Rbl treatment remarkably reduced apomorphine-induced rotations in lipopolysaccharide-treated rats compared with the lipopolysaccharide group.To investigate the neurotoxicity of lipopolysaccharide and potential protective effect of ginsenoside Rbl,contents of dopamine and its metabolites in the striatum were measured by high-performance liquid chromatography.Compared with the lipopolysaccharide group,ginsenoside Rbl obviously attenuated the lipopolysaccharide-induced depletion of dopamine and its metabolites in the striatum.To further explore the neuroprotective effect of ginsenoside Rbl against lipopolysaccharide-induced neurotoxicity,immunohistochemistry and western blot assay of tyrosine hydroxylase were performed to evaluate dopaminergic neuron degeneration in the substantial nigra par compacta.The results showed that lipopolysaccharide injection caused a large loss of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra and a significant decrease in overall tyrosine hydroxylase expression.However,ginsenoside Rb1 noticeably reversed these changes.To investigate whether the neuroprotective effect of ginsenoside Rbl was associated with inhibition of lipopolysaccharide-induced microglial activation,we examined expression of the microglia marker Iba-1.Our results confirmed that lipopolysaccharide injection induced a significant increase in Iba-1 expression in the substantia nigra;however,ginsenoside Rbl effectively suppressed lipopolysaccharide-induced microglial overactivation.To elucidate the inhibitory mechanism of ginsenoside Rb1,we examined expression levels of inflammatory mediators(tumor necrosis factor-a,interleukin-1β,inducible nitric oxide synthase,and cyclooxygenase 2)and phosphorylation of nuclear factor kappa B signaling-related proteins(IκB,IKK)in the substantia nigra with enzyme-linked immunosorbent and western blot assays.Our results revealed that compared with the control group,phosphorylation and expression of inflammatory mediators IκB and IKK in the substantia nigra of lipopolysaccharide group rats were significantly increased;whereas,ginsenoside Rbl obviously reduced lipopolysaccharide-induced changes on the lesioned side of the substantial nigra par compacta.These findings confirm that ginsenoside Rbl can inhibit inflammation induced by lipopolysaccharide injection into the substantia nigra and protect dopaminergic neurons,which may be related to its inhibition of the nuclear factor kappa B signaling pathway.This study was approved by the Experimental Animal Ethics Committee of Shandong University of China in April 2016(approval No.KYLL-2016-0148).

Discovery of nigral dopaminergic neurogenesis in adult mice

Parkinson＇s disease is characterized by the loss of dopaminergic neurons in the substantia nigra. As a result, intensive efforts have focused upon mechanisms that facilitate the death of mature dopaminergic neurons. Unfortunately, these efforts have been unsuccessful in providing an effective treatment to address neurodegeneration in this disease. Therefore, alternative theories of pathogenesis are being explored. Adult neurogenesis of dopaminergic neurons is an attractive concept that would provide a possible mechanism of neurodegeneration as well as offer an endogenous means to replenish affected neurons. To determine whether dopaminergic neurons experience neurogenesis in adult mice we developed a novel cell lineage tracing model that permitted detection of neurogenesis without many of the issues associated with popular techniques. Remarkably, we discovered that dopaminergic neurons are replenished in adult mice by Nestin＋/Sox2- progenitor cells. What＇s more, the rate of neurogenesis is similar to the rate of dopaminergic neuron loss reported using a chronic, systemic inflammatory response mouse model. This observation may indicate that neuron loss in Parkinson＇s disease results from inhibition of neurogenesis.

Nigral dopaminergic neuron replenishment in adult mice through VE-cadherin-expressing neural progenitor cells

The function of dopaminergic neurons in the substantia nigra is of central importance to the coordination of movement by the brain＇s basal ganglia circuitry. This is evidenced by the loss of these neurons, resulting in the cardinal motor deficits associated with Parkinson＇s disease. In order to fully understand the physiology of these key neurons and develop potential therapies for their loss, it is essential to determine if and how dopaminergic neurons are replenished in the adult brain. Recent work has presented evidence for adult neurogenesis of these neurons by Nestin＋/Sox2 neural progenitor cells. We sought to further validate this finding and explore a potential atypical origin for these progenitor cells. Since neural progenitor cells have a proximal association with the vasculature of the brain and subsets of endothelial cells are Nestin＋, we hypothesized that dopaminergic neural progenitors might share a common cell lineage. Therefore, we employed a VE-cadherin promoter-driven CREERT2：TIlox/Tlox transgenic mouse line to ablate the tyrosine hydroxylase gene from endothelial cells in adult animals. After 26 weeks, but not 13 weeks, following the genetic blockade of tyrosine hydroxylase expression in VE-cadherin＋ cells, we observed a significant reduction in tyrosine hydroxylase＋ neurons in the substantia nigra. The results from this genetic lineage tracing study suggest that dopaminergic neurons are replenished in adult mice by a VE-cadherin＋ progenitor cell population potentially arising from an endothelial lineage.

GABA Attenuates L-DOPA-Induced Striatal and Nigral ERK1/2 Signaling in a Rat Model of Parkinson’s Disease

L-DOPA is the primary drug used to treat Parkinson’s disease (PD) symptoms, but motor side effects limit its long term use. Previous experimental studies show that L-DOPA acts on supersensitive D1 receptors in the basal ganglia to induce extracellular signal-regulated kinases 1 and 2 (ERK1/2), a pair of MAP-kinase proteins that may be involved in induction of motor side effects. Since GABA is known to be intimately involved in basal ganglia function, we investigated whether elevating GABA levels via a GABA-transaminase (GABA-T) inhibitor affects the L-DOPA-induced ERK1/2 phosphorylation in the striatum and substantia nigra (SN) using a rat model of PD. Unilateral dopaminergic lesions of median forebrain bundle neurons were done using the neurotoxin 6-hydroxydopamine. Rats were prescreened for the extent of the lesion by apomorphine-induced rotation test. Lesioned rats were treated with aminooxyacetic acid (AOAA, a GABA-T inhibitor), L-DOPA, or in combination. Immunohistochemistry of tyrosine hydroxylase (TH, a direct indicator of dopaminergic lesion), substance P (SP, an indirect marker that decreases after lesion), and phospho-ERK1/2 was done using slices at the level of striatum and SN. Unilateral dopaminergic lesioned rats, as expected, exhibited >90% TH loss and a modest SP loss in the striatum and SN. L-DOPA alone induced a 343% and 330% increase in phospho-ERK1/2 in the striatum and SN, respectively. We report here a novel finding that pretreatment with AOAA attenuated the L-DOPA induced increase in phospho-ERK1/2 by 62% and 68% in the striatum and SN, respectively, suggesting a DA-GABA-ERK1/2 link in the therapeutic and/or side effects of L-DOPA.

Dopamine and GABA Interaction in Basal Ganglia: GABA-A or GABA-B Receptor Stimulation Attenuates L-DOPA-Induced Striatal and Nigral ERK1/2 Signaling in a Rat Model of Parkinson’s Disease

Parkinson’s disease (PD) is characterized by degeneration of nigrostriatal dopamine (DA) neurons. The primary drug used to treat PD symptoms is L-DOPA, but side effects such as dyskinesias limit its use. Previous findings show that L-DOPA treatment induces extracellular signal-regulated kinase (ERK1/2), a MAP-kinase protein. γ-aminobutyric acid (GABA) is intimately involved in basal ganglia function. Our previous study using a unilaterally lesioned rat model of PD indicated that elevating GABA levels by GABA transaminase inhibitor, aminooxyacetic acid significantly attenuated L-DOPA-induced ERK phosphorylation in the striatum and substantia nigra (SN). The aim of the present study was to assess the role of GABA-A and GABA-B receptor by using a selective agonists, muscimol and baclofen respectively, on L-DOPA-induced ERK phosphorylation in the striatum and SN. Unilaterally 6-OHDA-lesioned rats were prescreened by apomorphine induced rotation test for the extent of DA loss. Lesioned rats were treated with L-DOPA alone or after muscimol or baclofen pretreatment. Appropriate control groups were used. Phospho-ERK levels, tyrosine hydroxylase (to ascertain DA loss) and substance P (an indirect marker for DA loss) levels were assessed by immunohistochemistry using coronal slices at the level of striatum and SN. L-DOPA administration induced a robust increase (>300%) in phospho-ERK1/2 levels in the striatum and SN. Muscimol as well as baclofen pretreatment attenuated the L-DOPA-induced increase in phospho-ERK1/2 levels by >60% in the striatum and SN. Muscimol and baclofen pretreatment also greatly reduced the number of L-DOPA induced phospho-ERK1/2 stained cells in the striatum as well as the contralateral rotational behavior. The present data taken together with our previous study indicate that the L-DOPA induced increase in ERK1/2 is attenuated by GABA via a GABA-A and GABA-B receptor linked mechanism. The study provides further insight into a dopamine-GABA-ERK interaction in the therapeutic and/or side effects of L-DOPA in the basal ganglia.