The purpose of this study is to determine whether synapses can be formed between spinal motor neurons(SMNs)and major pelvic ganglion(MPG)neurons of a rat in vitro.The green fluorescent protein(GFP)-labelled MPG cells ...The purpose of this study is to determine whether synapses can be formed between spinal motor neurons(SMNs)and major pelvic ganglion(MPG)neurons of a rat in vitro.The green fluorescent protein(GFP)-labelled MPG cells were cultured together with SMNs in a specific medium.The synaptic-like contacts established between SMNs and MPG neurons were studied in co-cultures using morphologic and immunocytochemistry approaches.Phase-contrast observation of co-cultures showed apparent SMNs-MPG neurons contacts as early as three or four days in vitro.We demonstrate some evidence of synaptic contacts between SMNs and MPG neurons in vitro by immunostaining with antibody directed against postsynaptic density protein 95(PSD-95).We describe the development process of a defined SMNs-MPG neurons co-culture system.The results suggest that the hybrid synapse formation that may occur between SMNs and MPG neurons in vitro played an essential role in the mechanisms of a regenerated bladder with an artificial somatic-autonomic reflex arc.展开更多
A protocol for the isolation, purification and culture of motor neurons from newborn rat spinal cord was described and the effect of glial cell line-derived neurotrophic factor （GDNF） on the growth of neurite of mot...A protocol for the isolation, purification and culture of motor neurons from newborn rat spinal cord was described and the effect of glial cell line-derived neurotrophic factor （GDNF） on the growth of neurite of motor neurons was investigated in vitro. Spinal motor neurons （SMNs） were dissociated from ventral spinal cord of postnatal day 1 rats. The culture system for SMNs was established by density gradient centrifugation, differential adhesion, and use of serum-free defined media and addition of exogenous GDNF. After 72-h culture, the cells displayed the characteristic morphology of motor neurons, exhibited extensive neuritic processes and were positive for choline acetyl- transferase （CHAT） expression. The neurite length of SMNs in GDNF groups was significantly longer than that in control group （P〈0.05）. This protocol can be adapted for various postnatal motor neurons studies.展开更多
BACKGROUND: Sodium valproate (VPA) is used to be an effective anti-epileptic drug. VPA possesses the characteristics of penetrating rapidly through the blood-brain barrier (BBB) and increasing levels of Bcl-2 and grow...BACKGROUND: Sodium valproate (VPA) is used to be an effective anti-epileptic drug. VPA possesses the characteristics of penetrating rapidly through the blood-brain barrier (BBB) and increasing levels of Bcl-2 and growth cone-associated protein (GAP) 43 in spinal cord. OBJECTIVE: To observe the effect of VPA on Bcl-2 expression and motor neuronal apoptosis in spinal cord of rats following sciatic nerve transection. DESIGN: Randomized controlled experiment. SETTING: Department of Hand Surgery and Microsurgery, Wuhan Puai Hospital. MATERIALS: A total of 30 male healthy SD rats of clean grade and with the body mass of 180-220 g were provided by Experimental Animal Center of Medical College of Wuhan University. Sodium Valproate Tablets were purchases from Hengrui Pharmaceutical Factory, Jiangsu. METHODS: The experiment was performed in the Central Laboratory of Wuhan Puai Hospital and Medical College of Wuhan University from February to May 2006. Totally 30 rats were randomly divided into two groups: treatment group (n =15) and model group (n =15). Longitudinal incision along backside of right hind limbs of rats was made to expose sciatic nerves, which were sharply transected 1 cm distal to the inferior margin of piriform muscle after nerve liberation under operation microscope to establish sciatic nerve injury rat models. Sodium Valproate Tablets were pulverized and diluted into 50 g/L suspension with saline. On the day of operation, the rats in the treatment group received 6 mL/kg VPA suspension by gastric perfusion, once a day, whereas model group received 10 mL/kg saline by gastric perfusion, once a day. L4-6 spinal cords were obtained at days 1, 4, 7, 14 and 28 after operation, respectively. Terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) technique and immunohistochemical method (SP method) were used to detect absorbance (A) of neurons with positive Bcl-2 expression. Apoptotic rate of cells (number of apoptotic cells/total number of cells×100%) was calculated. MAIN OUTCOME MEASURES: A value of neurons with positive Bcl-2 expression and apoptotic rate in spinal cord of rats in the two groups. RESULTS: A total of 30 SD rats were involved in the result analysis. ①expression of positive Bcl-2 neurons: A value of positive Bcl-2 neurons were 0.71±0.02, 0.86±0.04, 1.02±0.06 at days 4, 7 and 14, respectively after operation in the treatment group, which were obviously higher than those in the model group (0.62±0.03, 0.71±0.05, 0.89±0.04, t = 3.10-4.50, P < 0.05). ②apoptotic result of motor neurons: Apoptotic rate of motor neurons in spinal cord was (6.91±0.89)% and (15.12±2.34)% at days 7 and 14 in the treatment group, which was significantly lower than those in the model group [(9.45±1.61)%, (19.35±0.92)%, t = 2.39, 3.03. P < 0.05]. CONCLUSION: VPA can increase expression of Bcl-2 in spinal cord and reduce neuronal apoptosis in rats following sciatic nerve injury, and has protective effect on motor neuron in spinal cord of rats.展开更多
Acupuncture has been shown to lessen the inflammatory reaction after acute spinal cord injury and reduce secondary injury.However,the mechanism of action remains unclear.In this study,a rat model of spinal cord injury...Acupuncture has been shown to lessen the inflammatory reaction after acute spinal cord injury and reduce secondary injury.However,the mechanism of action remains unclear.In this study,a rat model of spinal cord injury was established by compressing the T8-9 segments using a modified Nystrom method.Twenty-four hours after injury,Zusanli（ST36）,Xuanzhong（GB39）,Futu（ST32）and Sanyinjiao（SP6）were stimulated with electroacupuncture.Rats with spinal cord injury alone were used as controls.At 2,4 and 6 weeks after injury,acetylcholinesterase（ACh E）activity at the site of injury,the number of medium and large neurons in the spinal cord anterior horn,glial cell line-derived neurotrophic factor（GDNF）m RNA expression,and Basso,Beattie and Bresnahan locomotor rating scale scores were greater in the electroacupuncture group compared with the control group.These results demonstrate that electroacupuncture increases ACh E activity,up-regulates GDNF m RNA expression,and promotes the recovery of motor neuron function in the anterior horn after spinal cord injury.展开更多
In a previous study, heat shock protein 27 was persistently upregulated in ventral motor neurons following nerve root avulsion or crush. Here, we examined whether the upregulation of heat shock protein 27 would increa...In a previous study, heat shock protein 27 was persistently upregulated in ventral motor neurons following nerve root avulsion or crush. Here, we examined whether the upregulation of heat shock protein 27 would increase the survival rate of motor neurons. Rats were divided into two groups： an avulsion-only group （avtflsion of the L4 lumbar nerve root only） and a crush-avulsion group （the L4 lumbar nerve root was crushed 1 week prior to the avulsion）. Immunofluores- cent staining revealed that the survival rate of motor neurons was significantly greater in the crush-avulsion group than in the avulsion-only group, and this difference remained for at least 5 weeks after avulsion. The higher neuronal survival rate may be explained by the upregulation of heat shock protein 27 expression in motor neurons in the crush-avulsion group. Further- more, preconditioning crush greatly attenuated the expression of nitric oxide synthase in the motor neurons. Our findings indicate that the neuroprotective action of preconditioning crush is mediated through the upregulation of heat shock protein 27 expression and the attenuation of neuronal nitric oxide synthase upregulation following avulsion.展开更多
Bone marrow mesenchymal stem cell transplantation has been shown to be therapeutic in the repair of spinal cord injury. However, the low survival rate of transplanted bone marrow mesen- chymal stem cells in vivo remai...Bone marrow mesenchymal stem cell transplantation has been shown to be therapeutic in the repair of spinal cord injury. However, the low survival rate of transplanted bone marrow mesen- chymal stem cells in vivo remains a problem. Neurotrophin-3 promotes motor neuron survival and it is hypothesized that its transfection can enhance the therapeutic effect. We show that in vitro transfection of neurotrophin-3 gene increases the number of bone marrow mesenchymal stem cells in the region of spinal cord injury. These results indicate that neurotrophin-3 can promote the survival of bone marrow mesenchymal stem cells transplanted into the region of spinal cord injury and potentially enhance the therapeutic effect in the repair of spinal cord injury.展开更多
目的利用室间质量评价(EQA)分析上海地区临床实验室运动神经元存活(survival motor neuron,SMN)基因外显子缺失项目的检测能力。方法选用含有不同SMN外显子拷贝数的细胞株基因组DNA作为EQA样本,2023年间共两次,每次随机选取5份发放给上...目的利用室间质量评价(EQA)分析上海地区临床实验室运动神经元存活(survival motor neuron,SMN)基因外显子缺失项目的检测能力。方法选用含有不同SMN外显子拷贝数的细胞株基因组DNA作为EQA样本,2023年间共两次,每次随机选取5份发放给上海地区各参评实验室,要求其在规定时间内进行检测并上传结果,依据回报结果统计分析并评价其检测能力。结果两次EQA中SMN1判定结果回报全部正确的实验室分别占100%(38/38)和97.22%(35/36),SMN1第7,8号外显子拷贝数检测的总体符合率分别为96.92%(315/325)和98.18%(324/330),SMN2第7,8号外显子拷贝数检测的总体符合率分别为100%(65/65)和96.56%(43/45)。回报错误结果中包括1例结果判定错误和4例拷贝数检测错误。结论通过筛选出具有不同SMN外显子拷贝数的细胞株基因组DNA可有效模拟临床样本并应用于SMN外显子缺失EQA活动中。回报结果分析显示上海地区临床实验室SMN外显子缺失项目整体符合率较高,但个别实验室检测能力尚待提高。展开更多
基金Supported by Key Natural Scienceof Hubei Province(2013CFA069)。
文摘The purpose of this study is to determine whether synapses can be formed between spinal motor neurons(SMNs)and major pelvic ganglion(MPG)neurons of a rat in vitro.The green fluorescent protein(GFP)-labelled MPG cells were cultured together with SMNs in a specific medium.The synaptic-like contacts established between SMNs and MPG neurons were studied in co-cultures using morphologic and immunocytochemistry approaches.Phase-contrast observation of co-cultures showed apparent SMNs-MPG neurons contacts as early as three or four days in vitro.We demonstrate some evidence of synaptic contacts between SMNs and MPG neurons in vitro by immunostaining with antibody directed against postsynaptic density protein 95(PSD-95).We describe the development process of a defined SMNs-MPG neurons co-culture system.The results suggest that the hybrid synapse formation that may occur between SMNs and MPG neurons in vitro played an essential role in the mechanisms of a regenerated bladder with an artificial somatic-autonomic reflex arc.
基金supported by a grant from National Research Program of Basic Medicine of China(No.2003CB515304)
文摘A protocol for the isolation, purification and culture of motor neurons from newborn rat spinal cord was described and the effect of glial cell line-derived neurotrophic factor （GDNF） on the growth of neurite of motor neurons was investigated in vitro. Spinal motor neurons （SMNs） were dissociated from ventral spinal cord of postnatal day 1 rats. The culture system for SMNs was established by density gradient centrifugation, differential adhesion, and use of serum-free defined media and addition of exogenous GDNF. After 72-h culture, the cells displayed the characteristic morphology of motor neurons, exhibited extensive neuritic processes and were positive for choline acetyl- transferase （CHAT） expression. The neurite length of SMNs in GDNF groups was significantly longer than that in control group （P〈0.05）. This protocol can be adapted for various postnatal motor neurons studies.
文摘BACKGROUND: Sodium valproate (VPA) is used to be an effective anti-epileptic drug. VPA possesses the characteristics of penetrating rapidly through the blood-brain barrier (BBB) and increasing levels of Bcl-2 and growth cone-associated protein (GAP) 43 in spinal cord. OBJECTIVE: To observe the effect of VPA on Bcl-2 expression and motor neuronal apoptosis in spinal cord of rats following sciatic nerve transection. DESIGN: Randomized controlled experiment. SETTING: Department of Hand Surgery and Microsurgery, Wuhan Puai Hospital. MATERIALS: A total of 30 male healthy SD rats of clean grade and with the body mass of 180-220 g were provided by Experimental Animal Center of Medical College of Wuhan University. Sodium Valproate Tablets were purchases from Hengrui Pharmaceutical Factory, Jiangsu. METHODS: The experiment was performed in the Central Laboratory of Wuhan Puai Hospital and Medical College of Wuhan University from February to May 2006. Totally 30 rats were randomly divided into two groups: treatment group (n =15) and model group (n =15). Longitudinal incision along backside of right hind limbs of rats was made to expose sciatic nerves, which were sharply transected 1 cm distal to the inferior margin of piriform muscle after nerve liberation under operation microscope to establish sciatic nerve injury rat models. Sodium Valproate Tablets were pulverized and diluted into 50 g/L suspension with saline. On the day of operation, the rats in the treatment group received 6 mL/kg VPA suspension by gastric perfusion, once a day, whereas model group received 10 mL/kg saline by gastric perfusion, once a day. L4-6 spinal cords were obtained at days 1, 4, 7, 14 and 28 after operation, respectively. Terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) technique and immunohistochemical method (SP method) were used to detect absorbance (A) of neurons with positive Bcl-2 expression. Apoptotic rate of cells (number of apoptotic cells/total number of cells×100%) was calculated. MAIN OUTCOME MEASURES: A value of neurons with positive Bcl-2 expression and apoptotic rate in spinal cord of rats in the two groups. RESULTS: A total of 30 SD rats were involved in the result analysis. ①expression of positive Bcl-2 neurons: A value of positive Bcl-2 neurons were 0.71±0.02, 0.86±0.04, 1.02±0.06 at days 4, 7 and 14, respectively after operation in the treatment group, which were obviously higher than those in the model group (0.62±0.03, 0.71±0.05, 0.89±0.04, t = 3.10-4.50, P < 0.05). ②apoptotic result of motor neurons: Apoptotic rate of motor neurons in spinal cord was (6.91±0.89)% and (15.12±2.34)% at days 7 and 14 in the treatment group, which was significantly lower than those in the model group [(9.45±1.61)%, (19.35±0.92)%, t = 2.39, 3.03. P < 0.05]. CONCLUSION: VPA can increase expression of Bcl-2 in spinal cord and reduce neuronal apoptosis in rats following sciatic nerve injury, and has protective effect on motor neuron in spinal cord of rats.
基金supported by a grant from the Shaanxi Province Scientific and Technological Project in China,No.2014TM4193
文摘Acupuncture has been shown to lessen the inflammatory reaction after acute spinal cord injury and reduce secondary injury.However,the mechanism of action remains unclear.In this study,a rat model of spinal cord injury was established by compressing the T8-9 segments using a modified Nystrom method.Twenty-four hours after injury,Zusanli（ST36）,Xuanzhong（GB39）,Futu（ST32）and Sanyinjiao（SP6）were stimulated with electroacupuncture.Rats with spinal cord injury alone were used as controls.At 2,4 and 6 weeks after injury,acetylcholinesterase（ACh E）activity at the site of injury,the number of medium and large neurons in the spinal cord anterior horn,glial cell line-derived neurotrophic factor（GDNF）m RNA expression,and Basso,Beattie and Bresnahan locomotor rating scale scores were greater in the electroacupuncture group compared with the control group.These results demonstrate that electroacupuncture increases ACh E activity,up-regulates GDNF m RNA expression,and promotes the recovery of motor neuron function in the anterior horn after spinal cord injury.
基金supported by a grant from Education Ministry of Jiangsu Province,No.08KJB310002Excellent Discipline of Jiangsu Province,No.JX10131801096
文摘In a previous study, heat shock protein 27 was persistently upregulated in ventral motor neurons following nerve root avulsion or crush. Here, we examined whether the upregulation of heat shock protein 27 would increase the survival rate of motor neurons. Rats were divided into two groups： an avulsion-only group （avtflsion of the L4 lumbar nerve root only） and a crush-avulsion group （the L4 lumbar nerve root was crushed 1 week prior to the avulsion）. Immunofluores- cent staining revealed that the survival rate of motor neurons was significantly greater in the crush-avulsion group than in the avulsion-only group, and this difference remained for at least 5 weeks after avulsion. The higher neuronal survival rate may be explained by the upregulation of heat shock protein 27 expression in motor neurons in the crush-avulsion group. Further- more, preconditioning crush greatly attenuated the expression of nitric oxide synthase in the motor neurons. Our findings indicate that the neuroprotective action of preconditioning crush is mediated through the upregulation of heat shock protein 27 expression and the attenuation of neuronal nitric oxide synthase upregulation following avulsion.
基金supported by Scientific Research Fund of Xinxiang Medical University,No.2013ZD120Science and Technology Innovation Talents in Universities in Ministry of Education of Henan Province in 2010,No.2010HASTIT036
文摘Bone marrow mesenchymal stem cell transplantation has been shown to be therapeutic in the repair of spinal cord injury. However, the low survival rate of transplanted bone marrow mesen- chymal stem cells in vivo remains a problem. Neurotrophin-3 promotes motor neuron survival and it is hypothesized that its transfection can enhance the therapeutic effect. We show that in vitro transfection of neurotrophin-3 gene increases the number of bone marrow mesenchymal stem cells in the region of spinal cord injury. These results indicate that neurotrophin-3 can promote the survival of bone marrow mesenchymal stem cells transplanted into the region of spinal cord injury and potentially enhance the therapeutic effect in the repair of spinal cord injury.
文摘目的利用室间质量评价(EQA)分析上海地区临床实验室运动神经元存活(survival motor neuron,SMN)基因外显子缺失项目的检测能力。方法选用含有不同SMN外显子拷贝数的细胞株基因组DNA作为EQA样本,2023年间共两次,每次随机选取5份发放给上海地区各参评实验室,要求其在规定时间内进行检测并上传结果,依据回报结果统计分析并评价其检测能力。结果两次EQA中SMN1判定结果回报全部正确的实验室分别占100%(38/38)和97.22%(35/36),SMN1第7,8号外显子拷贝数检测的总体符合率分别为96.92%(315/325)和98.18%(324/330),SMN2第7,8号外显子拷贝数检测的总体符合率分别为100%(65/65)和96.56%(43/45)。回报错误结果中包括1例结果判定错误和4例拷贝数检测错误。结论通过筛选出具有不同SMN外显子拷贝数的细胞株基因组DNA可有效模拟临床样本并应用于SMN外显子缺失EQA活动中。回报结果分析显示上海地区临床实验室SMN外显子缺失项目整体符合率较高,但个别实验室检测能力尚待提高。
