脊髄、運動細胞、筋肉 Spinal cord Motor Neurons and Muscle
P3-1-99 融合糖タンパク質組換え体レンチウイルスベクターによる運動ニューロンへの高頻度逆行性遺伝子導入 Highly efficient retrograde gene delivery into motor neurons by lentiviral vector pseudotyped with fusion glycoprotein ○平野雅1, 加藤成樹1, 小林憲太2, 岡田知明3, 八木沼洋行3, 小林和人1 ○Miyabi Hirano1, Shigeki Kato1, Kenta Kobayashi2, Tomoaki Okada3, Hiroyuki Yaginuma3, Kazuto Kobayashi1 福島医大・医・生体機能1, 生理研・ウイルスベクター開発室2, 福島医大・医・神経解剖・発生3 Dept. Mol. Genet., Fukushima Med. Univ., Fukushima, Japan1, Section of Viral Vector Dev., NIPS, Okazaki, Japan2, Neuroanat. & Embryol., Fukushima Med. Univ., Fukushima, Japan3 Approaches for therapy to prevent nerve degeneration and repair neuronal damage have not been established. One useful technique for these therapeutic approaches is to introduce the genes that promote the survival and protection of neuronal cells into specific target areas. We have recently developed a vector with highly efficient retrograde gene transfer (HiRet) by pseudotyping a human immunodeficiency virus type 1 (HIV-1)-based vector with fusion glycoprotein, in which the cytoplasmic domain of RV-G was replaced by the corresponding part of vesicular stomatitis virus glycoprotein (VSV-G). The HiRet vector possesses the high efficiency of retrograde gene transfer into different neuronal populations in the brain. In addition, we developed another lentiviral vector for neuron-specific retrograde gene transfer (NeuRet) by using a different type of fusion glycoprotein. The NeuRet vector shows highly efficient retrograde gene delivery and transduces only neuronal cells around the injection sites. In the present study, we investigated the efficiency of retrograde gene transfer of the HiRet, NeuRet, and RV-G pseudotyped vectors into motor neurons in the spinal cord and medulla oblongata in mice by injecting these vectors into gastrocnemius muscle and lingual muscle. As a result, the HiRet vector displayed the capability for the most efficient retrograde gene transfer into both spinal cord and medulla oblongata motor neurons after intramuscular injection. The HiRet vector system will provide a powerful tool for gene therapy of motor neuron diseases by introducing the genes involved in prevention of nerve degeneration and repair of neuronal damage.	P3-1-100 損傷後早期の運動負荷によるBDNFの上昇は，KCC2発現増加を介して長期的な疼痛と痙性の抑制に寄与する BDNF expression secondary to treadmill training contributes to the long-term suppression of allodinia and spasticity through functional KCC2 increase in lumbar enlargement after thoracic cord injury ○田代祥一1,2, 篠崎宗久3, 向野雅彦1,5, ルノウ ミハラフランソワ3, 岩井宏樹4, 西村空也4, 里宇明元1, 中村雅也4, 岡野栄之3 ○Syoichi Tashiro1,2, Munehisa Shinozaki3, Masahiko Mukaino1,5, Francois Renault-Mihara3, Hiroki Iwai4, Soraya Nishimura4, Meigen Liu1, Masaya Nakamura4, Hideyuki Okano3 慶應義塾大・医・リハ医学1, 多磨全生園2, 慶應義塾・医・生理学3, 慶應義塾・医・整形外科4, 旭川医大・医・リハ医学5 Dept Rehabil Med, KEIO univ, Tokyo1, TAMA Zensho-en2, Dept Physiol, KEIO univ, Tokyo3, Dept Orthopedics surg, KEIO univ, Tokyo4, Dept Rehabili, Asahikawa med univ, Hokkaido5 Background: Spasticity and allodinia are major sequelae after spinal cord injury (SCI). Several studies have shown treadmill training (TT) improves them, however, the mechanism still remains unclear. So far, those are reported that BDNF is induced by training, that KCC2, Kaliume-Chloride cotrainsporter 2, is induced by BDNF administration after SCI, and that allodinia and spasticity are exacerbated by KCC2 down-regulation, separately. Here we investigated how TT effect on post SCI spasticity and allodinia, focusing on BDNF and KCC2 using rat contusive SCI model.Material and Method: In 50 Female 8-weeks-old Sprague-Dawley rats, moderate contusive SCI was induced at Th10 level using an IH impactor (200 Kdynes). The animals were divided into 2 groups at random. In training group, TT was performed using robotic device from 7 to 21 post-operative days. In control group, no exercise was performed. Spasticity, allodinia and locomotor function were assessed up to 7 weeks after SCI. Transition of BDNF and KCC2 expression were assayed in lumbar enlargement. In other 9 SCI model rats, intrathecal TrkB/Fc chimera administration was performed with implanted osmotic pump to inhibit BDNF during TT period. Then TT was performed to randomly selected 5 out of 9. For these animals, functional and biological assessments were employed in the same way.Result: Spasticity and sensory function were significantly improved in training group. Interestingly, this trend continued at least following 4 weeks. In biological assay, phospho-KCC2 expression took parallel transition with the improvement of spasticity and allodinia. Increase in BDNF expression was observed prior to that in KCC2. And surprisingly, beneficial effect of TT was invalidated by TrkB/Fc chimera administration.Conclusion: Present data indicates that TT would contribute to suppression of spasticity and allodinia through inducing BDNF expression and consequently functional KCC2 expression.
P3-1-101 ラットの傷害顔面運動ニューロンにおけるコリン作動性機能の低下 Cholinergic down-regulation of axotomized rat facial motoneurons ○中嶋一行1,2, 島川大輝1, 一宮俊文1, 山本伸一1, 菊池麗香1, 高坂新一2 ○Kazuyuki Nakajima1,2, Daiki Shimakawa1, Toshihumi Ichimiya1, Shinichi Yamamoto1, Reika Kikuchia1, Shinichi Kohsaka2 創価大・工・ 生命情報1, （独）国立精神・神経医療研究センター、代謝研究部2 Dept. of Bioinfo, Soka Univ, Tokyo, Japan1, Dept. of Neurochemistry,National Institute of Neuroscience,Tokyo, Japan2 Functional alterations in injured motoneurons were quantitatively analyzed in axotomized rat facial nuclei. Choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAchT) and m2 muscarinic acetylcholine receptor (m2MAchR) were chosen as indicators of motoneuron function. Immunoblotting showed that the amounts of ChAT in the ipsilateral facial nucleus significantly decreased to below 20% from 3 to 14 days after transection. The decreased level of ChAT in injured motoneurons was ascertained by immunohistochemical study. However, at 4-5 weeks after transection the level of ChAT was restored to that of control side. The amounts of VAchT in the transected nucleus were observed to decrease to below 20% in the first 14 days after transection. The down-regulated levels of VAchT in injured motoneurons were confirmed by immunohistochemical results. The reduced VAchT levels returned to the control levels at 4-5 weeks following insult. The level of m2MAchR in the ipsilateral nucleus was recognized to decrease to below 10% starting on the 5th day after insult, and the low levels were sustained for 5 weeks. Nissl staining at 5 days and 12 days after insult revealed that facial motoneurons in the transected nucleus were almost all alive. Altogether, these results indicate that transected adult rat facial motoneurons are functionally depressed with down-regulated levels of ChAT, VAchT and m2MAchR during the first 14 days after insult, and during Weeks 4 to 5 ChAT and VAchT levels are restored while the levels of m2MAchR remain low.	P3-1-102 ラット発達期頸髄運動ニューロンの形態解析：四肢筋、体幹筋支配運動ニューロン間の異同を中心にして Analysis of dendritic morphology of cervical motoneuron in the rat at an early developmental stage: the difference of motoneurons projecting to arm and trunk muscles ○福田諭1, 前田仁士1, 吉岡昇1, 亀田浩司1, 村部直之1, 桜井正樹1 ○Satoshi Fukuda1, Hitoshi Maeda1, Noboru Yoshioka1, Hiroshi Kameda1, Naoyuki Murabe1, Masaki Sakurai1 帝京大学医学部生理学1 Dept Physiol, Teikyo Univ Sch Med, Tokyo1 It is generally believed that dendritic morphology of spinal motoneuron is "star-shaped". However, the analysis of their dendritic pattern at an early stage has not been done. Thus we investigated the shape of rat cervical motoneurons in an early developmental age (P6 - 8). The motoneurons were identified by retrograde labeling with cholera toxin B subunit conjugated with Alexa Fluor 488 which was injected into the forelimb or trunk (pectral or serratus anterior muscles) muscle groups. Following a 48-hour survival period, transverse slices of the cervical cord were prepared. Whole cell recordings were made from retrogradely-labeled fluorescence-positive motoneurons. NeurobiotinTM was injected from the recording pipettes which were visualized with Texas Red-avidin. Double-labeled neurons were imaged with two-photon laser scanning microscope and traced with NeurolucidaTM system. Some motoneurons projecting to a forelimb muscle received direct synapses with corticospinal (CS) axons (CS-EPSC-positive). The somata of motoneurons were located in conventional Rexed IX layer. The total lengths of dendrites showed no significant difference among motoneuron pools (CS-EPSC-positive forelimb 3.33 ± 0.63 mm (mean ± SE), CS-EPSC-negative forelimb 3.37 ± 0.42 mm, pectral 2.67 ± 0.78 mm, serratus anterior 2.34 ± 0.65 mm) . The motoneurons projecting to serratus anterior muscle extended their long dendrites dorso-medially and ventro-lateally with much shorter dendrites for the other directions. Most motoneurons in the other groups also had relatively large gaps between their dendrites fields. There are little or no motoneurons which extend their dendrites nearly equally to every direction, instead they have considerable gaps between their major dendritic branches. These observations mean that the dendritic pattern of most motoneurons is greatly deviated from a typical "star-shaped" pattern at least at an early developmental stage.
P3-1-103 脊髄損傷からの回復過程における損傷部位付近での増殖性細胞の分布 Distribution of proliferating cells around lesion during functional recovery after spinal injury in medaka fish ○川崎隆史1, 出口友則1, 弓場俊輔1 ○Takashi Kawasaki1, Tomonori Deguchi1, Shunsuke Yuba1 Health Research Institute, AIST, Amagasaki, Japan1 Rehabilitative training is currently used for a treatment to promote functional recovery after spinal injury. However, it is very difficult to design the most effective training, consequently the training do not necessarily result in beneficial therapeutic value, because the mechanism of the promotion of the recovery with the training remains to be elucidated. In contrast to mammals having very low capacity to regenerate in spinal cord, fish have the high capacity. Therefore, studies using the model animal may give insightful information. Recently, we established the methods evaluating the functional recovery after spinal injury using animal model prepared with medaka fish. As the results of the analysis, athletic training promoted the functional recovery after spinal injury. The effectiveness of training was affected by timing for start of the training in a manner, in which the recovery was promoted by the training started with definite delay after spinal injury. This result should be important from the point of view of "window of opportunity" for rehabilitative training. Various types of cells would be involved in the functional recovery after spinal injury. Proliferating cell are one of the most interesting candidates of the cells relating to the recovery. In this study, we analyzed distribution of proliferating cells around lesion during the recovery process following spinal injury, using spinal injury model of medaka fish, and speculated these cells were involved in the recovery.	P3-1-104 脊髄路損傷モデルザルにおける大脳皮質運動野から脊髄への越シナプス的入力様式 Organization of multisynaptic cortical input through the indirect corticospinal pathway in macaques ○二宮太平1,4, 中川浩2,4, 上野将紀2,4, 西村幸男3, 大石高生1,4, 山下俊英2,4, 高田昌彦1,4 ○Taihei Ninomiya1,4, Hiroshi Nakagawa2,4, Masaki Ueno2,4, Yukio Nishimura3, Takao Oishi1,4, Toshihide Yamashita2,4, Masahiko Takada1,4 京大・霊長研・統合脳1, 阪大・医・分子神経科学2, 生理研・認知行動発達3 Sys Neurosci, Primate Res Inst, Kyoto Univ, Inuyama, Aichi, Japan1, Dept Mol Neurosci, Grad Sch Med, Osaka Univ, Osaka, Japan2, Dept Dev Physiol, Natl Inst Physiol Sci, Okazaki, Aichi, Japan3, JST CREST, Tokyo, Japan4 Several studies have suggested that the indirect corticospinal (CS) pathway substantiates dexterous motor control following hemisection of the direct CS pathway. The indirect CS pathway is a poorly understood fiber tract of which the cortical origin has yet to be determined. Revealing the cortical origin of this pathway is an important step for understanding any potential rehabilitative mechanism following spinal cord injury. In order to investigate the cortical origin of the indirect CS pathway, we injected rabies virus, a retrograde transsynaptic tracer, into C6-T1 of the spinal cord where motoneurons of the hand muscles are located. Three days after rabies injections, neuronal labeling appeared in the primary motor cortex, the supplementary motor area, and the premotor cortex, indicating that all these regions give rise to the indirect CS pathway. Neuronal labeling was also observed in the upper cervical segments (C2-C4), suggesting that the corticopropriospinal pathway implicates the multisynaptic inputs from these motor areas to C6-T1 of the spinal cord. The corticopropriospinal pathway constitutes a functional pathway to relay the same signals as the direct CS pathway, given that its cortical origin is the same as the direct CS pathway.
P3-1-105 感覚入力が脊髄介在ニューロンの活動特性に及ぼす影響 Effect of afferent input to activity of spinal interneuron ○戸松彩花1, 金祉希1, 武井智彦1, 関和彦1,2 ○Saeka Tomatsu1, Geehee Kim1, Tomohiko Takei1, Kazuhiko Seki1,2 国立精神・神経医療研究センター　神経研究所　モデル動物開発部門1, さきがけ2 National Institute of Neuroscience, National Center of Neurology and Psychiatry1, PRESTO, JST2 In the spinal cord, sensory afferent inputs encounter efferent motor command during movement execution. However, it is not well known how much effect the two lines of inputs have toward activity of single spinal neuron. To know the answer, we recorded activity of 149 spinal interneurons (INs) during movement execution from three monkeys trained to perform extension and flexion of right wrist joint both volitionally and passively. The recorded spinal INs were identified as the first-order neurons from at least one of three sensory afferent nerves in the right hand and forearm; superficial radial nerve (SR, cutaneous nerve from extensor side), radial nerve (DR, muscle nerve from extensor side), and median nerve (M, mixed nerve from flexor side). We compared their activity between flexion and extension (i.e., polarity) of volitional and passive movements and classified them into two groups. Spinal INs in the first group exhibited same polarity during voluntary and passive movement, and the second group exhibited different polarity between voluntary and passive movement. Among 149 neurons tested, 62 neurons (42%) were categorized into the first group. Of these, only SR-INs showed biased modulation for extension (p < 0.05) and DR- and M-INs showed equivalent activity to both movement directions. On the other hand, 86 neurons (58%) were in the second group. Of these, DR-INs showed biased activity for volitional extension and passive flexion (p < 0.01), although SR- and M-INs showed equivalent to both movement directions. These results suggest that the way afferent (sensory) and efferent (motor) inputs converged at single spinal neuron could be characterized by the pattern of afferent input to each neuron. These categorized neurons may act as a functional unit for the sensorimotor integration at the spinal level.	P3-1-106 脳梗塞後痙縮発症マウスへのwheel exercise負荷が、痙縮軽減および脊髄運動神経細胞膜に局在するKCC2発現量回復に及ぼす影響 Wheel running exercise may improve spasticity and recover the KCC2 expression levels in plasma membrane of motoneuron in spastic mice after the stroke ○戸田拓弥1, 石田和人1, 李佐知子1 ○Takuya Toda1, Kazuto Ishida1, Sachiko Lee1 名古屋大院・医学系研究科・リハ1 Dept of physical Therapy, Nagoya University, Nagoya1 The spasticity causes motor dysfunctions and limitation of physical activity for the affected individuals after the central nerve injury. There is no effective treatment for spasticity. Therefore, to understand the intricate mechanism of spasticity is the important clues to find a better therapeutic approach. One of the main mechanisms has been hypothesized to be responsible for spasticity is increased motoneuron excitability in the ventral horn. Recently, we revealed a possibility that the potassium-chloride cotransporter 2 (KCC2) expression in plasma membrane of motoneuron downregulated after the stroke. KCC2 is known to work mainly for maintaining low intracellular chloride concentration. Reduction of KCC2 expression or the function causes function of inhibitory receptor, namely γ-aminobutyric acid type A and glycine receptors, to change from inhibition to excitation, as a result aggravating spasticity. Some studies have reported that a rhythmical-cyclic movement gave effects to improve spastic limbs. In this study, we investigated whether the spasticity was improved and whether KCC2 expression in plasma membrane of motoneuron was recovered by a wheel running exercise, as a cyclic movement. Rate-Dependent Depressions (RDDs) of Hoffmann reflex using the measurement of motoneuron hyperexcitability is reduced in the spasticity individuals and animals. Here, we showed RDDs were decreased in non-exercise stroke groups at 7 days after stroke. By contrast, RDDs levels in exercise stroke groups were same with non exercise and exercise sham groups at 7 days after stroke. And we detected the recovery of KCC2-positive areas at the plasma membrane in exercise stroke mice comparing to that of non-exercise stroke mice at 7 days after the stroke using immunohistochemical analysis. These results suggest that a wheel running exercise may enhance KCC2 expression in motoneuron membranes and the spasticity after the stroke may be improved by exercise.

上部に戻る

前に戻る