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Culture of motor neurons from Newborn Rat Spinal Cord

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2009年第4期29卷 413-416页

作者：程时刚石瑛海波韩晓敏陈朝晖李兵肖传国 Department of Urology Union HospitalTongji Medical CollegeHuazhong University of Science and Technology

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.

关键词： newborn rats spinal cord motor neurons

Protective Effect of Interleukin-1β on motor neurons after Sciatic Nerve Injury in Rats

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2004年第1期24卷 71-74页

作者：翁雨雄巴拉特洪光祥王发斌陈振斌黄启顺 Department of Hand Surgery

Protective effect of interleukin 1β (IL 1β) on motor neurons was studied after peripheral nerve injury. Twenty Wistar rats were divided into 2 groups randomly. The right sciatic nerve of each rat was resected. After silicon tubulization of sciatic nerve in rat, 15 μl 1 ng/ml IL 1β and PBS solution were injected into the silicon capsule respectively. Enzyme histochemistry was performed to show acetyle cholesterase (AchE) and nitric oxide staining (NOS) activity of spinal α motor neurons in spinal segments 2 weeks later. neurons were counted and the diameter and cross sectional (c/s) area of neurons were analyzed by using computer image analysis system. The results showed that as compared with the normal side, both enzyme activities significantly changed in motor neurons in PBS group. The diameter and c/s area of both neurons changed significantly too ( Pmotor neurons from degeneration and necrosis after peripheral nerve injury.

关键词： protective effect interleukin 1β motor neurons nerve injury

Proteomic difference between spinal cord stem cells and motor neurons

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Proteomic difference between spinal cord stem cells and moto...

Chx10+V2a interneurons in spinal motor regulation and spinal cord injury

中国神经科学学会第四次会员代表大会暨第八届全国学术会议

作者： Peng-Fei LI,Chun-Fang WANG~*,Fei WANG,Hong-En WEI Department of Biotechnology,Shanxi Medical University,Taiyuan 030001,China

＜正＞Objective motor neuron damage always occurs in amyotrophic lateral sclerosis and spinal cord injury.Current research offers hope that neural stem cells（NSCs） may be donors to replace lost motor neurons.In vitro,we proved that NSCs can differentiate into cholinergic neurons（motor neuron is a type of cholinergic neuron） in a special condition. Others have shown that some factors could induce mouse embryonic stem（ES） cells to differentiate into motor neurons. Since proteomic approaches are most appropriate to determine protein content,stability and subcellular localization,many people used the 2-DE comparative proteome analysis to study the differentiation process of NSCs.Despite some learned details of the differentiation process,questions that whether these differentiated cells are really the ones which we want remain unanswered.So,before inducing NSCs into motor neurons,we should find the difference between them.In this paper,we used the proteomic analysis to gain more insight into molecular mechanisms of the difference between NSCs and motor neurons.Spinal cord stem cells（SCSCs） are a subclass of NSCs and as such are restricted to generating neural tissues,which is a notable advantage over embryonic stem cells,and have the further advantage of being regionally specified to produce spinal cord progeny.Methods SCSCs and motor neurons obtained from the same 16-day-old rat fetus spinal cord,this can make our result have more obviously biological statistical significance.Specifically,we employed the method of difference gel electrophoresis（DIGE）,which allows protein differences between two samples to be analyzed on the same gel.Since the transiently expressed p75-NGFR was chosen for motor neuron isolation from embryonic day 15 to postnatal day 15 rat spinal cords,we used CELLection Pan Mouse IgG Kit to separate motor neurons,and we used the rest spinal cord after motor neurons separation to SCSCs culture.After cell lysate preparation,we did 2D gel analysis according to the process of DIGE.Following scanning the ge

关键词： neural stem cells motor neurons proteome

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Neural Regeneration Research 2023年第5期18卷 933-939页

作者： Wen-Yuan Li Ling-Xiao Deng Feng-Guo Zhai Xiao-Yu Wang Zhi-Gang Li Ying Wang Institute of Neural Tissue Engineering Mudanjiang College of MedicineMudanjiangHeilongjiang ProvinceChina Spinal Cord and Brain Injury Research Group Stark Neurosciences Research InstituteIndiana University School of MedicineIndianapolisINUSA Department of Pharmacy Mudanjiang College of MedicineMudanjiangHeilongjiang ProvinceChina Department of General Surgery Hongqi HospitalMudanjiang College of MedicineMudanjiangHeilongjiang ProvinceChina

Chx10-expressing V2 a(Chx10+V2 a) spinal interneurons play a large role in the excitatory drive of motoneurons. Chemogenetic ablation studies have demonstrated the essential nature of Chx10+V2 a interneurons in the regulation of locomotor initiation, maintenance, alternation, speed, and rhythmicity. The role of Chx10+V2 a interneurons in locomotion and autonomic nervous system regulation is thought to be robust, but their precise role in spinal motor regulation and spinal cord injury have not been fully explored. The present paper reviews the origin, characteristics, and functional roles of Chx10+V2 a interneurons with an emphasis on their involvement in the pathogenesis of spinal cord injury. The diverse functional properties of these cells have only been substantiated by and are due in large part to their integration in a variety of diverse spinal circuits. Chx10+V2 a interneurons play an integral role in conferring locomotion, which integrates various corticospinal, mechanosensory, and interneuron pathways. Moreover, accumulating evidence suggests that Chx10+V2 a interneurons also play an important role in rhythmic patterning maintenance, leftright alternation of central pattern generation, and locomotor pattern generation in higher order mammals, likely conferring complex locomotion. Consequently, the latest research has focused on postinjury transplantation and noninvasive stimulation of Chx10+V2 a interneurons as a therapeutic strategy, particularly in spinal cord injury. Finally, we review the latest preclinical study advances in laboratory derivation and stimulation/transplantation of these cells as a strategy for the treatment of spinal cord injury. The evidence supports that the Chx10+V2 a interneurons act as a new therapeutic target for spinal cord injury. Future optimization strategies should focus on the viability, maturity, and functional integration of Chx10+V2 a interneurons transplanted in spinal cord injury foci.

关键词： axons central nervous system central pattern generator Chx10 differentiation interneurons locomotion motor neurons propriospinal spinal cord injuries therapy transcription factor transplantation V2a neuron

Alexa Fluor 488-conjugated cholera toxin subunit B optimally labels neurons 3-7 days after injection into the rat gastrocnemius muscle

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Neural Regeneration Research 2022年第10期17卷 2316-2320页

作者： Jing-Jing Cui Jia Wang Dong-Sheng Xu Shuang Wu Ya-Ting Guo Yu-Xin Su Yi-Han Liu Yu-Qing Wang Xiang-Hong Jing Wan-Zhu Bai Institute of Acupuncture and Moxibustion China Academy of Chinese Medical SciencesBeijingChina

Neural tract tracing is used to study neural pathways and evaluate neuronal regeneration following nerve injuries.However,it is not always clear which tracer should be used to yield optimal results.In this study,we examined the use of Alexa Fluor 488-conjugated cholera toxin subunit B(AF488-CTB).This was injected into the gastrocnemius muscle of rats,and it was found that motor,sensory,and sympathetic neurons were labeled in the spinal ventral horn,dorsal root ganglia,and sympathetic chain,respectively.Similar results were obtained when we injected AF594-CTB into the tibialis anterior muscle.The morphology and number of neurons were evaluated at different time points following the AF488-CTB injection.It was found that labeled motor and sensory neurons could be observed 12 hours post-injection.The intensity was found to increase over time,and the morphology appeared clear and complete 3-7 days post-injection,with clearly distinguishable motor neuron axons and dendrites.However,14 days after the injection,the quality of the images decreased and the neurons appeared blurred and incomplete.Nissl and immunohistochemical staining showed that the AF488-CTB-labeled neurons retained normal neurochemical and morphological features,and the surrounding microglia were also found to be unaltered.Overall,these results imply that the cholera toxin subunit B,whether unconjugated or conjugated with Alexa Fluor,is effective for retrograde tracing in muscular tissues and that it would also be suitable for evaluating the regeneration or degeneration of injured nerves.

关键词： Alexa Fluor-conjugated cholera toxin subunit B calcitonin gene-related peptide microglia motor neurons neural tract tracing optimal time window sensory neurons somatotopic organization sympathetic neurons tibialis anterior muscle

Three-dimensional in vitro models of neuromuscular tissue

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Neural Regeneration Research 2022年第4期17卷 759-766页

作者： Paolo Raffa Maria Easler Anna Urciuolo Institute of Pediatric Research IRP PadovaItaly Molecular Medicine Department University of PadovaPadovaItaly

Skeletal muscle is a dynamic tissue in which homeostasis and function are guaranteed by a very defined three-dimensional organization of myofibers in respect to other nonmuscular components,including the extracellular matrix and the nervous network.In particular,communication between myofibers and the nervous system is essential for the overall correct development and function of the skeletal muscle.A wide range of chronic,acute and genetic-based human pathologies that lead to the alteration of muscle function are associated with modified preservation of the fine interaction between motor neurons and myofibers at the neuromuscular junction.Recent advancements in the development of in vitro models for human skeletal muscle have shown that three-dimensionality and integration of multiple cell types are both key parameters required to unveil pathophysiological relevant phenotypes.Here,we describe recent achievement reached in skeletal muscle modeling which used biomaterials for the generation of three-dimensional constructs of myotubes integrated with motor neurons.

关键词： 3D organization bioengineering biomaterials motor neurons neuromuscular junction skeletal muscle models

A perspective on therapies for amyotrophic lateral sclerosis:can disease progression be curbed?

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Translational Neurodegeneration 2021年第3期10卷 367-384页

作者： Xiaojiao Xu Dingding Shen Yining Gao Qinming Zhou You Ni Huanyu Meng Hongqin Shi Weidong Le Shengdi Chen Sheng Chen School of Medicine University of Electronic Science and Technology of ChinaChengdu 610054China Department of Neurology Ruijin HospitalShanghai Jiaotong University School of MedicineShanghai 200020China Department of Neurology Xinrui HospitalWuxi 214028China Institute of Neurology Sichuan Academy of Medical Sciences-Sichuan Provincial HospitalChengdu 610031China Center for Clinical Research on Neurological Diseases the First Affiliated HospitalDalian Medical UniversityDalian 116021China

Amyotrophic lateral sclerosis(ALS)is a progressive neurodegenerative disease involving both upper and lower motor neurons,leading to paralysis and eventually death.Symptomatic treatments such as inhibition of salivation,alleviation of muscle cramps,and relief of spasticity and pain still play an important role in enhancing the quality of life.To date,riluzole and edaravone are the only two drugs approved by the Food and Drug Administration for the treatment of ALS in a few countries.While there is adequate consensus on the modest efficacy of riluzole,there are still open questions concerning the efficacy of edaravone in slowing the disease progression.Therefore,identification of novel therapeutic strategies is urgently needed.Impaired autophagic process plays a critical role in ALS pathogenesis.In this review,we focus on therapies modulating autophagy in the context of ALS.Furthermore,stem cell therapies,gene therapies,and newly-developed biomaterials have great potentials in alleviating neurodegeneration,which might halt the disease progression.In this review,we will summarize the current and prospective therapies for ALS.

关键词： Amyotrophic lateral sclerosis motor neurons Autophagy Stem cells Gene editing

A progressive compression model of thoracic spinal cord injury in mice： function assessment and pathological changes in spinal cord

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Neural Regeneration Research 2017年第8期12卷 1365-1374页

作者： Guo-dong Sun Yan Chen Zhi-gang Zhou Shu-xian Yang Cheng Zhong Zhi-zhong Li Department of Orthopedics First Affiliated HospitalJinan UniversityGuangzhouGuangdong ProvinceChina Biomedical Translational Research Institute and Guangdong Province Key Laboratory of Molecular Immunology and Antibody Engineering Jinan UniversityGuangzhouGuangdong ProvinceChina Department Of Traumatology and-Plastic Surgery The Affiliated Jiangmen Traditional Chinese Medicine Hospital of Jinan UniversityJiangmenGuangdong ProvinceChina Department of Orthopedics Heyuan People's Hospital(Heyuan Affiliated Hospital of Jinan University)HeyuanGuangdong ProvinceChina

Non-traumatic injury accounts for approximately half of clinical spinal cord injury, including chronic spinal cord compression. However, previous rodent spinal cord compression models are mainly designed for rats, few are available for mice. Our aim is to develop a thoracic progressive compression mice model of spinal cord injury. In this study, adult wild-type C57BL/6 mice were divided into two groups： in the surgery group, a screw was inserted at T9 lamina to compress the spinal cord, and the compression was increased by turning it further into the canal（0.2 mm） post-surgery every 2 weeks up to 8 weeks. In the control group, a hole was drilled into the lamina without inserting a screw. The results showed that Basso Mouse Scale scores were lower and gait worsened. In addition, the degree of hindlimb dysfunction in mice was consistent with the degree of spinal cord compression. The number of motor neurons in the anterior horn of the spinal cord was reduced in all groups of mice, whereas astrocytes and microglia were gradually activated and proliferated. In conclusion, this progressive compression of thoracic spinal cord injury in mice is a preferable model for chronic progressive spinal cord compression injury.

关键词： nerve regeneration progressive spinal cord compression injury pathological changes Basso Mouse Scale scores gait motor evokedpotentials astrocytes microglia motor neurons hindlimb dysfunction neural regeneration

Electroacupuncture promotes the recovery of motor neuron function in the anterior horn of the injured spinal cord

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Neural Regeneration Research 2015年第12期10卷 2033-2039页

作者： Jian-hui Yang Jian-guo Lv Hui Wang Hui-yong Nie Rehabilitation Center First Affiliated Hospital of Health Science CenterXi’an Jiaotong University

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.

关键词： nerve regeneration spinal cord injury electroacupuncture spine injury secondaryinjury acupoint motor neurons acetylcholinesterase glial cell line-derived neurotrophic factor inclined board test Basso, Beattie and Bresnahan locomotor rating scale functional recovery neuralregeneration