Neuronal quantity and ultrastructure in extracorticospinal tract of newborn rabbits with cerebral palsy

BACKGROUND: The liver enzyme system and blood brain barrier function of newborn rabbits are incomplete. High level of bilirubin can lead to cerebral palsy (CP) of newborn rabbits. The pathological and ultrastructural changes of brains of immature rabbits may differ from those of mature rabbits. OBJECTIVE: To observe the changes in dopaminergic neuron amount and ultrastructure in the extracorticospinal tract of animal models of CP induced by hyperbilirubinemia. DESIGN: A randomized controlled observation. SETTING: Rehabilitation Medical College of Jiamusi University; Laboratory for Pediatric Neurology Rehabilitation of Jiamusi University. MATERIALS: Thirty newborn 2- to 5- day inbred Japanese rabbits, weighing about 50 g, of either gender, were provided by Experimental Animal Center, Jilin University. Bilirubin and tyrosine hydroxylase (TH) antibody were purchased from Sigma Corporation, USA. METHODS: This experiment was carried out in the laboratory for children neuro-rehabilitation, Jiamusi University between September 2002 and December 2005. ①Thirty rabbits were randomly divided into 2 groups: control group (n =10) and model group (n =20). ②Bilirubin of 100 mg/kg was given to the rabbits of model group through intraperitoneal injection, once every other day, 3 times totally; The same amount of normal saline was given to the rabbits of control group. After injection, the newborn rabbits had been fed by maternal milk for 45 days. ③TH-positive neurons in substantia nigra and corpora striatum in each area of 0.15 mm×0.15 mm were counted. Ultrastructures of substantia nigra and corpora striatum were observed under the electron miscroscope. The concentration of bilirubin in brain tissue of CP rabbits of model group was measured with high performance liquid chromatogram. MAIN OUTCOME MEASURES: Quantitative analysis results and ultrastructure of TH-positive neurons in substantia nigra and corpora striatum in two groups. RESULTS: Thirty newborn rabbits were involved, 6 rabbits from model group died and 4 were failed in modeling, finally, 20 rabbits were involved in the result analysis. ① The concentration of bilirubin in brain tissue of newborn rabbits of model group was （59.9±17.04） mg/L, which was significantly higher than that of control group [（42.79±3.57） mg/L, P < 0.01]. ② TH positive neurons amount in the substantia nigra and corpora striatum of newborn rabbits in the model group was 30.30±9.58, 4.70±2.79, which was significantly less than that in the control group respectively （45.20±12.74, 11±2.45, P < 0.01). ③The concentration of bilirubin in brain tissue was significantly negatively correlated with TH-positive neurons in substantia nigra（r =-0.69, P < 0.01）; The concentration of bilirubin in brain tissue was also significantly negatively correlated with TH-positive neurons in corpora striatum（r =-0.59, P < 0.01）. TH-positive neurons in substantia nigra were positively correlated with those in corpora striatum （r =0.77, P < 0.01）.④ Under the electron microscope, the nerve cells in substantia nigra and corpora striatum of newborn rabbits of model group were significantly changed in ultrastructure as compared with control group. CONCLUSION: Ultrastructure of extracorticospinal tract of newborn rabbits with CP is significantly changed, presenting the injury of dopaminergic neuron and decrease of neuronal amount.     

Neuronal quantity and ultrastructure in extracorticospinal tract of newborn rabbits with cerebral palsy

BACKGROUND: The liver enzyme system and blood brain barrier function of newborn rabbits are incomplete. High level of bilirubin can lead to cerebral palsy (CP) of newborn rabbits. The pathological and ultrastructural changes of brains of immature rabbits may differ from those of mature rabbits. OBJECTIVE: To observe the changes in dopaminergic neuron amount and ultrastructure in the extracorticospinal tract of animal models of CP induced by hyperbilirubinemia. DESIGN: A randomized controlled observation. SETTING: Rehabilitation Medical College of Jiamusi University; Laboratory for Pediatric Neurology Rehabilitation of Jiamusi University. MATERIALS: Thirty newborn 2- to 5- day inbred Japanese rabbits, weighing about 50 g, of either gender, were provided by Experimental Animal Center, Jilin University. Bilirubin and tyrosine hydroxylase (TH) antibody were purchased from Sigma Corporation, USA. METHODS: This experiment was carried out in the laboratory for children neuro-rehabilitation, Jiamusi University between September 2002 and December 2005. ①Thirty rabbits were randomly divided into 2 groups: control group (n =10) and model group (n =20). ②Bilirubin of 100 mg/kg was given to the rabbits of model group through intraperitoneal injection, once every other day, 3 times totally; The same amount of normal saline was given to the rabbits of control group. After injection, the newborn rabbits had been fed by maternal milk for 45 days. ③TH-positive neurons in substantia nigra and corpora striatum in each area of 0.15 mm×0.15 mm were counted. Ultrastructures of substantia nigra and corpora striatum were observed under the electron miscroscope. The concentration of bilirubin in brain tissue of CP rabbits of model group was measured with high performance liquid chromatogram. MAIN OUTCOME MEASURES: Quantitative analysis results and ultrastructure of TH-positive neurons in substantia nigra and corpora striatum in two groups. RESULTS: Thirty newborn rabbits were involved, 6 rabbits from model group died and 4 were failed in modeling, finally, 20 rabbits were involved in the result analysis. ① The concentration of bilirubin in brain tissue of newborn rabbits of model group was (59.9±17.04) mg/L, which was significantly higher than that of control group [(42.79±3.57) mg/L, P < 0.01]. ② TH positive neurons amount in the substantia nigra and corpora striatum of newborn rabbits in the model group was 30.30±9.58, 4.70±2.79, which was significantly less than that in the control group respectively (45.20±12.74, 11±2.45, P < 0.01). ③The concentration of bilirubin in brain tissue was significantly negatively correlated with TH-positive neurons in substantia nigra(r =-0.69, P < 0.01); The concentration of bilirubin in brain tissue was also significantly negatively correlated with TH-positive neurons in corpora striatum(r =-0.59, P < 0.01). TH-positive neurons in substantia nigra were positively correlated with those in corpora striatum (r =0.77, P < 0.01).④ Under the electron microscope, the nerve cells in substantia nigra and corpora striatum of newborn rabbits of model group were significantly changed in ultrastructure as compared with control group. CONCLUSION: Ultrastructure of extracorticospinal tract of newborn rabbits with CP is significantly changed, presenting the injury of dopaminergic neuron and decrease of neuronal amount.     

Distribution of nitric oxide synthase positive neurons in the substantia nigra of rats with liver cirrhosis

BACKGROUND: Nitrogen monoxide plays an important role in the physiological activity and pathological process of striatum in substantia nigra, and the nitric oxide synthase in substantia nigra may have characteristic changes after liver cirrhosis. OBJECTIVE: To observe the distribution and forms of nitric oxide synthase (NOS) positive neurons and fibers in substantia nigra of rats with liver cirrhosis. DESIGN: A comparative observational experiment. SETTINGS: Beijing Friendship Hospital; Capital Medical University. MATERIALS: Twenty 4-month-old male Wistar rats (120–150 g) of clean grade, were maintained in a 12-hour light/dark cycle at a constant temperature with free access to standard diet and water. Cryostat microtome (LEICA, Germany); All the reagents were purchased from Sigma Company. METHODS: The experiment was carried out in the Department of Anatomy (key laboratory of Beijing city), Capital Medical University from July 2000 to March 2002. The rats were randomly divided into normal group (n =10) and liver fibrosis group (n =10). Rats in the liver fibrosis group were subcutaneously injected with 60% CCl4 oil at a dose of 5 mL/kg for the first time, and 3 mL/kg for the next 14 times, twice a week, totally 15 times. Liver fibrosis of grades 5–6 was taken as successful models. Whereas rats in the normal group were not given any treatment. Four months after CCl4 treatment, all the rats were anesthetized to remove brain, and frontal frozen serial sections were prepared. The expressions of nitric oxide synthase positive neurons in substantia nigra of rats were observed under inverted microscope. The number and gray scale of cell body of nitric oxide synthase positive neurons in substantia nigra were detected with NADPH-diaphorase staining. MAIN OUTCOME MEASURES: ① Number and gray scale of cell body of nitric oxide synthase positive neurons in substantia nigra; ② Expressions of nitric oxide synthase positive neurons in substantia nigra. RESULTS: All the 20 rats were involved in the analysis of results. ① The nitric oxide synthase positive neurons in substantia nigra were obviously fewer in the liver cirrhosis group than in the normal group (P < 0.01), and the gray scale of the positive cell body was higher in the liver cirrhosis group than in the normal group (P < 0.05). ② Abundant nitric oxide synthase positive neurons were observed in substantia nigra of normal rats, the cell body of nitric oxide synthase positive neurons was clear and transparent, with short own cloudy processes. In substantia nigra of rats with liver cirrhosis, the body of nitric oxide synthase positive neurons were observed shrink obviously, less fibrin than normal. CONCLUSION: Rats with liver cirrhosis may suffer from the physiological dysfunction of neurons due to lack of fibers. The nitric oxide synthase positive neurons in substantia nigra can shrink and reduce.     

Neuroprotective effects of subthalamic nucleus high-frequency stimulation on substantia nigra neurons in a Parkinson''s disease rat model*☆◇

BACKGROUND： Deep-brain stimulation has proven to be beneficial in the treatment of Parkinson＇s disease （PD） patients. OBJECTIVE： To investigate the effects of high-frequency stimulation （HFS） to the subthalamic nucleus （STN） on neuronal apoptosis and apoptosis-related gene expression in the substantia nigra pars compacta, and to analyze the neuroprotective effect of HFS-STN. DESIGN, TIME AND SETTING： Neuronal morphology experiments were performed in the Beijing Neurosurgical Institute from May to December in 2005. MATERIALS： Forty healthy, adult, Sprague Dawley rats were used to establish a PD model with a unilateral microinjection of 6-hydroxydopamine into two target areas of the right medial forebrain bundle. 6-hydroxydopamine was purchased from Sigma （USA）; high-frequency electrical stimulator was produced by World Precision Instruments （USA）; Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） kit was a product of Nanjing Jiancheng Technology Co., Ltd. （China）; and Bcl-2 and Bax protein assay kit were purchased from Wuhan Boster Bioengineering Co., Ltd. （China）. METHODS： Forty rats were randomly divided into three groups. The stimulation group （n = 15） received HFS-STN on the day of PD modeling. The PD model group （n = 15） was used to establish the PD model. The control group （n = 10） was injected with normal saline containing 0.2 g/L ascorbic acid into two areas of the right medial forebrain bundle. MAIN OUTCOME MEASURES： Survival of dopaminergic neurons in the substantia nigra pars compacta was determined using Nissl staining. Apoptosis of dopaminergic neurons was detected using TUNEL techniques. Expression of anti-apoptotic protein, Bcl-2, and pro-apoptotic protein, Bax, were assayed by immunohistochemistry. RESULTS： Following 6-hydroxydopamine injection, the number of substantia nigra pars compacta neurons was reduced in the stimulation and PD model groups, compared to the control group. At 2 and 4 weeks post-surgery, the grey va     

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.     

骨髓间充质干细胞对1-甲基-4-苯基吡啶离子损伤的离体纹状体-黑质脑片多巴胺能神经元的保护作用

BACKGROUND： To date, the use of bone marrow-derived mesenchymal stem cells （MSCs） for the treatment of Parkinson’s disease have solely focused on in vivo animal models. Because of the number of influencing factors, it has been difficult to determine a consistent outcome.
OBJECTIVE： To establish an injury model in brain slices of substantia nigra and striatum using 1-methyl-4-phenylpytidinium ion （MPP＋）, and to investigate the effect of MSCs on dopaminergic neurons following MPP＋ induced damage.
DESIGN, TIME AND SETTING： An in vitro, randomized, controlled, animal experiment using I mmunohistochemistry was performed at the Laboratory of the Department of Anatomy, Fudan University between January 2004 and December 2006.
MATERIALS： Primary MSC cultures were obtained from femurs and tibias of adult Sprague Dawley rats. Organotypic brain slices were isolated from substantia nigra and striatum of 1-day-old Sprague Dawley rat pups. Monoclonal antibodies for tyrosine hydroxylase （TH, 1：5 000） were from Santa Cruz （USA）; goat anti-rabbit IgG antibodies labeled with FITC were from Boster Company （China）.
METHODS： Organotypic brain slices were cultured for 5 days in whole culture medium supplemented with 50% DMEM, 25% equine serum, and 25% Tyrode’s balanced salt solution. The medium was supplemented with 5 μg/mL Ara-C, and the culture was continued for an additional 5 days. The undergrowth of brain slices was discarded at day 10. Eugonic brain slices were cultured with basal media for an additional 7 days. The brain slices were divided into three groups： control, MPP＋ exposure, and co-culture. For the MPP＋ group, MPP＋ （30 μmol/L） was added to the media at day 17 and brain slices were cultured for 4 days, followed by control media. For the co-culture group, the MPP＋ injured brain slices were placed over MSCs in the well and were further cultured for 7 days.
MAIN OUTCOME MEASURES： After 28 days in culture, neurite outgrowth was examined in the brain slices under phase-contrast microscopy. The percent of area containing dead cells in each brain slice was calculated with the help of propidium iodide fluorescence. Brain slices were stained with antibodies for TH to indicate the presence of dopaminergic neurons. Transmission electron microscopy was applied to determine the effect of MSCs on neuronal ultrastructure.
RESULTS： Massive cell death and neurite breakage was observed in the MPP＋ group. In addition, TH expression was significantly reduced, compared to the control group （P 〈 0.01）. After 7 days in culture with MSCs, the co-culture group presented with less cell damage and reduced neurite breakage, and TH expression was increased. However, these changes were not significantly different from the MPP＋ group （P 〈 0.01）. Electron microscopy revealed reduced ultrastructural injury to cells in the brain slices. However, vacuoles were present in cells, with some autophagic vacuoles.
CONCLUSION： Bone marrow-derived MSCs can promote survival of dopaminergic neurons following MPP＋-induced neurotoxicity in co-cultures with substantia nigra and striatum brain slices.     

Differences in brain pathological changes between rotenone and 6-hydroxydopamine Parkinson's disease models

Rotenone and 6-hydroxydopamine are two drugs commonly used to generate Parkinson's disease animal models.They not only achieve degenerative changes of dopaminergic neurons in the substantia nigra,but also satisfy the requirements for iron deposition.However,few studies have compared the characteristics of these two models by magnetic resonance imaging.In this study,rat models of Parkinson's disease were generated by injection of 3 μg rotenone or 10 μg 6-hydroxydopamine into the right substantia nigra.At 1,2,4,and 6 weeks after injection,coronal whole-brain T2-weighted imaging,transverse whole-brain T2-weighted imaging,and coronal diffusion tensor weighted imaging were conducted to measure fractional anisotropy and T2* values at the injury site.The fractional anisotropy value on the right side of the substantia nigra was remarkably lower at 6 weeks than at other time points in the rotenone group.In the 6-hydroxydopamine group,the fractional anisotropy value was decreased,but T2* values were increased on the right side of the substantia nigra at 1 week.Our findings confirm that the 6-hydroxydopamine-induced model is suitable for studying dopaminergic neurons over short periods,while the rotenone-induced model may be appropriate for studying the pathological and physiological processes of Parkinson's disease over long periods.     

Effect of moxibustion on mTOR-mediated autophagy in rotenone-induced Parkinson's disease model rats

Defects in autophagy-mediated clearance of α-synuclein may be one of the key factors leading to progressive loss of dopaminergic neurons in the substantia nigra. Moxibustion therapy for Parkinson's disease has been shown to have a positive effect, but the underlying mechanism remains unknown. Based on this, we explored whether moxibustion could protect dopaminergic neurons by promoting autophagy mediated by mammalian target of rapamycin(m TOR), with subsequent elimination of α-syn. A Parkinson's disease model was induced in rats by subcutaneous injection of rotenone at the back of their necks, and they received moxibustion at Zusanli(ST36), Guanyuan(CV4) and Fengfu(GV16), for 10 minutes at every point, once per day, for 14 consecutive days. Model rats without any treatment were used as a sham control. Compared with the Parkinson's disease group, the moxibustion group showed significantly greater tyrosine hydroxylase immunoreactivity and expression of light chain 3-Ⅱ protein in the substantia nigra, and their behavioral score, α-synuclein immunoreactivity, the expression of phosphorylated m TOR and phosphorylated ribosomal protein S6 kinase(p-p70S6K) in the substantia nigra were significantly lower. These results suggest that moxibustion can promote the autophagic clearance of α-syn and improve behavioral performance in Parkinson's disease model rats. The protective mechanism may be associated with suppression of the m TOR/p70S6K pathway.     

X-box-binding protein 1-modified neural stem cells for treatment of Parkinson’s disease

X-box-binding protein 1-transfected neural stem cells were transplanted into the right lateral ventricles of rats with rotenone-induced Parkinson’s disease. The survival capacities and differentiation rates of cells expressing the dopaminergic marker tyrosine hydroxylase were higher in X-box-binding protein 1-transfected neural stem cells compared to non-transfected cells. Moreover, dopamine and 3,4-dihydroxyphenylacetic acid levels in the substantia nigra were significantly increased, α-synuclein expression was decreased, and neurological behaviors were significantly ameliorated in rats following transplantation of X-box-binding protein 1-transfected neural stem cells. These results indicate that transplantation of X-box-binding protein 1-transfected neural stem cells can promote stem cell survival and differentiation into dopaminergic neurons, increase dopamine and 3,4-dihydroxyphenylacetic acid levels, reduce α-synuclein aggregation in the substantia nigra, and improve the symptoms of Parkinson’s disease in rats.     

Detecting dopaminergic neuronal degeneration using diffusion tensor imaging in a rotenone-induced rat model of Parkinson's disease: fractional anisotropy and mean diffusivity values

Dopamine content in the basal ganglia is strongly associated with the degree of dopaminergic neuron loss in the substantia nigra pars com-pacta. Symptoms of Parkinson's disease might not arise until more than 50% of the substantia nigra pars compacta is lost and the dopamine content in the basal ganglia is reduced by more than 80%. Greater diagnostic sensitivity and specificity would allow earlier detection of Parkinson's disease. Diffusion tensor imaging is a recently developed magnetic resonance imaging technique that measures mean diffusiv-ity and fractional anisotropy, and responds to changes in brain microstructure. When the microscopic barrier (including cell membranes, microtubules and other structures that interfere with the free diffusion of water) is destroyed and extracellular fluid volume accumulates, the mean diffusivity value increases; when the integrity of the microstructure (such as myelin) is destroyed, fractional anisotropy value decreases. However, there is no consensus as to whether these changes can reflect the early pathological alterations in Parkinson's disease. Here, we established a rat model of Parkinson's disease by injecting rotenone (or sunflower oil in controls) into the right substantia nigra. Diffusion tensor imaging results revealed that in the stages of disease, at 1, 2, 4, and 6 weeks after rotenone injection, fractional anisotropy value decreased, but mean diffusivity values increased in the right substantia nigra in the experimental group. Fractional anisotropy values were lower at 4 weeks than at 6 weeks in the right substantia nigra of rats from the experimental group. Mean diffusivity values were mark-edly greater at 1 week than at 6 weeks in the right corpus striatum of rats from the experimental group. These findings suggest that mean diffusivity and fractional anisotropy values in the brain of rat models of Parkinson's disease 4 weeks after model establishment can reflect early degeneration of dopaminergic neurons. The change in fractional anisotropy values after destruction of myelin integrity is likely to be of greater early diagnostic significance than the change in mean diffusivity values.     

Transcranial alternating current stimulation rescues motor deficits in a mouse model of Parkinson's disease via the production of glial cell line-derived neurotrophic factor

BackgroundTherapeutic effects of transcranial alternating current stimulation (tACS) for treating Parkinson's disease (PD) are limited to modulating abnormally synchronized oscillations; however, long-lasting tACS effects may involve non-neuronal mechanisms like the regulation of neurotrophic factors.Objectives/HypothesisWe investigated whether tACS exerts neuroprotective effects on dopaminergic neurons in a mouse model of PD by regulating endogenous glial cell line-derived neurotrophic factor (GDNF).MethodsRepeated high-definition tACS (HD-tACS, 20 min, 89.1 μA/mm²) was administered over the primary motor cortex of C57BL/6J 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice. Behavioral tests assessing motor function, immunohistochemistry, western blots, enzyme-linked immunosorbent assays, and flow cytometric analyses were performed to examine suitable tACS conditions and its underlying mechanisms.ResultsStimulation at representative frequencies (theta to gamma; 20-Hz beta frequency, in particular) attenuated motor dysfunction and protected the dopaminergic neurons with increased GDNF production. Beta-frequency (20 Hz) tACS application significantly attenuated motor deficits to levels comparable with those of levodopa treatment. Moreover, beta-frequency tACS induced the survival of dopaminergic neurons in the substantia nigra with upregulated production of endogenous GDNF in striatal parvalbumin-positive interneurons. An inhibitor of the GDNF receptor-associated rearranged during transfection (RET) kinase suppressed most aspects of the tACS-induced behavioral recovery, dopaminergic cell survival, and GDNF production. Beta-frequency tACS activated RET-related survival signaling for dopaminergic neurons in the substantia nigra.ConclusionsApplication of tACS over the primary motor cortex may exert protective effects on dopaminergic neurons in the substantia nigra via activation of endogenous GDNF production by striatal parvalbumin-positive interneurons and its survival signaling.     

全反式维甲酸通过增强调节性T细胞的抑炎效应发挥对多巴胺能神经元的保护作用

目的探讨全反式维甲酸(ATRA)对帕金森病(PD)小鼠多巴胺能神经元的作用及其机制。方法将野生型小鼠随机分为MPTP对照组与ATRA+MPTP组,造模前后进行行为学检测,流式细胞学技术检测外周T淋巴细胞亚群,实时定量PCR法检测脉络丛黏附分子和中脑黑质区炎症因子的表达,免疫荧光法检测黑质区胶质细胞活化及多巴胺能神经元存活情况。结果经ATRA预处理的PD小鼠外周调节性T细胞(Tregs)数目增多[(2.3±0.21)%;P0.05]。PD1表达上调[CD4+:(4.65±0.61)%;CD8+:(3.50±0.31)%;P0.05]。黏附分子(Icam-1:0.37±0.13;Vcam-1:0.32±0.06)及炎症因子(IL-6:0.16±0.05;TNF-α:0.29±0.12)相对表达均下调(P0.05)。黑质区胶质细胞活化减少,多巴胺能神经元存活率提高。小鼠的协调能力[转棒停留总时间:(157.5±24.5)s]及自主运动能力[运动总距离:(42.58±2.96)m]均得到改善(P0.05)。结论 ATRA可能通过增强Tregs的抑炎效应,间接发挥多巴胺能神经元保护作用。     

Glial cell line‐derived neurotrophic factor enhances in vitro differentiation of mid‐/hindbrain neural progenitor cells to dopaminergic‐like neurons

Parkinson's disease (PD) affects the motor system through the degeneration of the dopaminergic neurons of the substantia nigra. The use of human embryonic stem cell (hESC)‐derived human neural progenitor (hNP) cells provides a potential cell source for cell therapies and drug screens for future treatments. Glial cell line‐derived neurotrophic factor (GDNF) is a known dopaminergic neuroprotectant agent; however, its potential role in neural differentiation remains largely unknown. Addition of 25 ng/ml GDNF to hNP cell differentiation media, over a 21‐day period, induced a significantly (P < 0.05) greater portion of hNP cells to differentiate into dopaminergic neurons than non‐GDNF cultures, 50% compared with 2.9% of cells expressing tyrosine hydroxylase (TH), respectively. The hNP cells exposed to GDNF selectively expressed dopamine receptors 1, 4, and 5 and were evoked to release dopamine with KCl. This is the first report of GDNF and leukemia inhibitory factor enriching hESC‐derived hNP cells toward dopaminergic‐like neurons. © 2010 Wiley‐Liss, Inc.     

Glial cell line-derived neurotrophic factor supports survival of injured midbrain dopaminergic neurons

Glial cell-lined derived neurotrophic factor (GDNF) has been shown to promote survival of developing mesencephalic dopaminergic neurons in vitro. In order to determine if there is a positive effect of GDNF on injured adult midbrain dopaminergic neurons in situ, we have carried out experiments in which a single dose of GDNF was injected into the substantia nigra following a unilateral lesion of the nigrostriatal system. Rats were unilaterally lesioned by a single stereotaxic injection of 6-hydroxydopamine (6-OHDA; 9 μg/4 μl normal saline with 0.02% ascorbate) into the medial forebrain bundle and tested weekly for apomorphine-induced (0.05 mg/kg s. c. ) contralateral rotation behavior, Rats that manifested >300 turns/hour received a nigral injection of 100 μg GDNF, or cytochrome C as a control, 4 weeks following the 6-OHDA lesion, Rotation behavior was quantified weekly for 5 weeks after GDNF. Rats were subsequently anesthetized, transcardially perfused, and processed for tyrosine hydroxylase immunohistochemistry. It was found that 100 μg GDNF decreased apomorphine-induced rotational behavior by more than 85%. Immunohistochemical studies revealed that tyrosine hydroxylase immunoreactivity was equally reduced in the striatum ipsilateral to the lesion in both cytochrome C and GDNF-injected animals. In contrast, large increments in tyrosine hydroxylase immunoreactivity were observed in the substantia nigra of animals treated with 100 μg of GDNF, with a significant increase in numbers of tyrosine hydroxylase-immunoreactive cell bodies and neurites as well as a small increase in the cell body area of these neurons. The results suggest that GDNF can maintain the dopaminergic neuronal phenotype in a number of nigral neurons following a unilateral nigrostriatal lesion in the rat.     

CX3CR1 Disruption Differentially Influences Dopaminergic Neuron Degeneration in Parkinsonian Mice Depending on the Neurotoxin and Route of Administration

Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic neurons accompanied by an inflammatory reaction. The neuron-derived chemokine fractalkine (CX3CL1) is an exclusive ligand for the receptor CX3CR1 expressed on microglia. The CX3CL1/CX3CR1 signaling is important for sustaining microglial activity. Using a recently developed PD model, in which the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxin is delivered intranasally, we hypothesized that CX3CR1 could play a role in neurotoxicity and glial activation. For this, we used CX3CR1 knock-in mice and compared results with those obtained using the classical PD models through intraperitonal MPTP or intrastriatal 6-hydroxydopamine (6-OHDA). The striatum from all genotypes (CX3CR1^+/+, CX3CR1^+/GFP and CX3CR1-deficient mice) showed a significant dopaminergic depletion after intranasal MPTP inoculation. In contrast to that, we could not see differences in the number of dopaminergic neurons in the substantia nigra of CX3CR1-deficient animals. Similarly, after 6-OHDA infusion, the CX3CR1 deletion decreased the amphetamine-induced turning behavior observed in CX3CR1^+/GFP mice. After the 6-OHDA inoculation, a minor dopaminergic neuronal loss was observed in the substantia nigra from CX3CR1-deficient mice. Distinctly, a more extensive neuronal cell loss was observed in the substantia nigra after the intraperitoneal MPTP injection in CX3CR1 disrupted animals, corroborating previous results. Intranasal and intraperitoneal MPTP inoculation induced a similar microgliosis in CX3CR1-deficient mice but a dissimilar change in the astrocyte proliferation in the substantia nigra. Nigral astrocyte proliferation was observed only after intraperitoneal MPTP inoculation. In conclusion, intranasal MPTP and 6-OHDA lesion in CX3CR1-deficient mice yield no nigral dopaminergic neuron loss, linked to the absence of astroglial proliferation.     

漆黄素对帕金森病小鼠模型的保护作用及机制研究

目的 探讨漆黄素对帕金森病的神经保护作用及具体机制。方法 采用MPTP腹腔注射复制亚急性PD小鼠模型，漆黄素灌胃给药，实验分为3组：control组、MPTP组、漆黄素+MPTP组。通过旷场实验、爬杆实验、悬挂实验等行为学指标评估小鼠的运动行为。采用Western blotting和免疫荧光技术检测纹状体中TH水平和黑质中TH阳性神经元数量。采用尼氏染色检测黑质区神经元的损伤状况。通过检测纹状体区GSH、SOD、T-AOC、MDA含量，评估脑组织中氧化应激水平。结果 与MPTP组相比，漆黄素+MPTP组，小鼠的运动总距离及运动速度提高（P<0.05）；爬杆总时间及转头时间缩短（P<0.05）。悬挂实验评分提高（P<0.05）。尼氏染色结果发现，漆黄素可缓解MPTP小鼠黑质区神经元损伤（P<0.05）。TH免疫印迹及免疫荧光实验发现漆黄素可改善MPTP诱导的小鼠TH表达量水平下降及阳性神经元丢失（P<0.05）。同时，漆黄素处理后，提高了MPTP小鼠GSH、SOD、T-AOC水平，降低了MPTP小鼠MDA的含量（P<0.05）。结论 漆黄素能有效改善帕金森病模型小鼠的运动功能，缓解黑质－纹状体多巴胺能神经元损伤，其机制可能与漆黄素的抗氧化作用有关。     

Astrocyte delivery of glial cell line-derived neurotrophic factor in a mouse model of Parkinson's disease

Primary astrocytes were genetically modified ex vivo to express recombinant glial cell line-derived neurotrophic factor (GDNF) and subsequently were tested for their ability to provide neuroprotection to dopaminergic neurons in a 6-hydroxydopamine (6-OHDA) mouse model of Parkinson's disease. A replication-defective retrovirus was constructed, which contained the rat GDNF sequence and a sequence encoding a beta-galactosidase (beta-gal)/neomycin phosphotransferase fusion protein, linked via an internal ribosomal entry site. Murine astrocytes transduced with this vector secreted GDNF into the culture media at the rate of 115 +/- 34 pg/24 h/10(5) cells and expressed cytoplasmic beta-gal, whereas control nontransduced astrocytes were negative for GDNF production and cytoplasmic beta-gal expression. Mice that received implants of GDNF-producing astrocytes into the striatum or nigra displayed elevated levels of GDNF compared to mice that received control nontransduced astrocytes. In addition, tissue content of GDNF was increased bilaterally and in brain regions both proximal and distal to the graft, even though astrocyte migration away from the graft site did not occur. Importantly, GDNF-producing astrocytes provided marked neuroprotection of nigral dopaminergic perikarya, and partial protection of striatal dopaminergic fibers, when implanted into the midbrain 6 days prior to a retrograde 6-OHDA lesion, as assessed by tyrosine hydroxylase immunohistochemistry. Similarly, GDNF-producing astrocytes prevented the acquisition of amphetamine-induced rotational behavior in 6-OHDA-treated mice and completely prevented dopamine depletion within the substantia nigra, as assessed by high-performance liquid chromatography. These results indicate that continuous exposure to low levels of GDNF provided by transgenic astrocytes provides marked neuroprotection of nigral dopaminergic neurons. (c)2002 Elsevier Science (USA).     

百草枯致小鼠黑质部多巴胺能神经细胞凋亡的作用

目的:探讨百草枯致小鼠黑质细胞死亡的方式及神经毒性机制。方法:C57BL小鼠口服百草枯不同时间后采用酪氨酸羟化酶(TH)免疫组化及TUNEL法、流式细胞仪、透射电镜检测黑质部多巴胺能神经元的凋亡现象。结果:TH免疫组化及TUNEL双标染色检查示C57BL小鼠暴露于百草枯后2周后黑质部多巴胺能神经细胞即有凋亡样改变,流式细胞仪检测显示小鼠于暴露百草枯1个月时凋亡细胞数最多,与对照组相比,P<0.001;2个月时有所减少,但仍多于对照组(P<0.01)。结论:小鼠暴露于百草枯一定时间后可引起黑质部多巴胺能神经元发生凋亡,这可能是百草枯的神经毒性作用机制之一。     

PD模型中GDNF与星形胶质细胞对黑质DA能神经元的影响

目的探讨星形胶质细胞和胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)在帕金森病(Parkinson's disease,PD)中对多巴胺(dopamine neurons,DA)能神经元损伤的影响。方法成年大鼠右侧前脑侧束注射6羟多巴胺(6-OHDA)制备PD模型。PD模型右侧黑质内注射GDNF,于注射后第6周采用免疫组织化学方法观察星形胶质细胞神经纤维酸性蛋白(glial fibrillary acidic protein,GFAP)以及多巴胺能神经元酪氨酸羟化酶(tyrosine hydroxylasa,TH)的变化。结果模型组、PBS和GDNF组注射侧与非注射侧星形胶质细胞相比,均发现GFAP阳性细胞明显增多,DA能神经元数量明显减少(P<0.05)。GDNF组与模型组相比,发现GFAP阳性细胞明显增多,同时残存的DA能神经元数量有所增加(P<0.05)。结论黑质内注射GDNF可能通过激活的星形胶质细胞保护PD大鼠模型黑质DA能神经元。