颈脊髓损伤的小鼠模型研究后皮损呼吸神经可塑性
1UFR des sciences de la santé – Simone Veil,Université de Versailles Saint-Quentin-en-Yvelines, 2Service de Physiologie – Explorations fonctionnelles,Hôpital Ambroise Paré, 3Services de Physiologie, Explorations Fonctionnelles, Réanimation Médicale et Centre d’Investigation Clinique et d’Innovation Technologique (Unité Inserm 805),Université de Versailles Saint-Quentin-en-Yvelines
Summary
呼吸衰竭是死亡的跟随颈脊髓损伤的首要原因。有呼吸衰竭引起的局部损伤宫颈可重复的，可量化的，可靠的临床前动物模型将有助于了解后续的呼吸和非呼吸神经可塑性，并允许测试假定的维修策略。
Abstract
颈椎脊髓损伤引起永久性瘫痪，并经常导致呼吸窘迫。迄今为止，还没有有效的治疗方法已发展到提高/改善呼吸衰竭以下高位颈脊髓损伤（SCI）。这里我们提出了很高的SCI在宫颈2（C2），同分异构的水平来研究不同的后皮损呼吸神经可塑性小鼠的临床前模型。该技术包括在C2级的外科局部损伤，这将导致隔膜的偏瘫，由于从位于脑干呼吸中枢膈运动神经元的传入神经阻滞。受伤的对侧保持不变，并允许动物恢复。不像影响运动功能（在胸椎和腰椎水平）等的SCI，呼吸功能并不需要动物的动机和赤字/恢复的量化可以容易地进行（隔膜和膈神经记录秒，全身通风）。此临床前C2脊髓损伤模型是一种功能强大，实用，可靠的临床前模型来研究各种呼吸道和非呼吸道神经可塑性的事件在不同层次（分子生理学），并测试不同的推定治疗策略，可以改善呼吸脊髓损伤患者。
Introduction
脊髓损伤是在人群中观察到的与戏剧性的发生率，如永久性瘫痪一个常见的损伤。然而，损伤的严重程度取决于初始损伤的水平和程度。呼吸衰竭是病死率以下上颈段脊髓损伤（SCI）1的首要原因。目前，唯一的治疗治疗是将病人下辅助通气。由于少数患者可以断奶了辅助通气2，由于其发生后皮损延迟自然恢复，需要开发新的创新型非侵入性的治疗是当务之急3。拥有一个良好的标准化的临床前模型来观察颈椎脊髓损伤对呼吸功能不全的影响，因此，研究的推定治疗策略的应用，是必不可少的。
在本技术文章中，我们描述了一个特定的临床前小鼠模型Ø在C2级别F呼吸障碍引起的局部颈脊髓损伤。这种模式是目前使用世界各地的一些实验室（综述：4-13）。然而，在手术过程中的细微差别可以在不同的调查中可以观察到产生这个特殊的宫颈损伤小鼠模型。一个C2脊髓损伤对呼吸输出的影响最早是在1895年14波特描述。颈椎半切引起膈运动神经元的传入神经 阻滞从他们的中央驱动（位于rVRG在脑干， 图1A）上受伤的同侧，导致无声膈神经活动和随后的膈肌麻痹。对侧保持不变，并允许动物生存。不同于位于一个较低的脊柱节段（例如，在C 4级15挫伤性损伤）不同SCI，在两侧膈运动神经元核的完整性被保存下来。一个CERV后iCal的C2伤害，一些自发的活动可以在同侧（膈和隔膜）可以观察到由于它越过脊柱中线在段水平C3-C6（交叉膈途径，CPP， 图1B）对侧沉默突触通路的激活。菲，这是激活，根据定义，一个C2半切结合对侧phrenicotomy从而诱发同侧局部膈神经恢复，可发生从几小时到几周损伤后16-18。这个CPP途径对呼吸复苏的真正有益作用是有限的19，并进一步调查处理应当制定提高自发恢复3的大小。
该协议提供了一个强大的类型的临床前小鼠模型来研究呼吸后皮损可塑性各级（从学前教育和膈运动神经元，的interneurons，分子和cellula呼吸生理r，则前肢为例）的运动，以及一个模型来测试旨在改善以下C2分型颈脊髓损伤的呼吸和运动恢复侵入性和非侵入性的治疗策略。
Protocol
该方案通过卓越（Paris Sud酒店，编号246556赠款协议大学）和大学德凡尔赛圣昆廷 – 烯伊夫林省的RBUCE-UP椅子的伦理委员会。 无菌手术器械1。准备请用实验室的洗涤剂手术器械。 高压灭菌前手术器械。 在外科会话，通过将提示在热珠灭菌10分钟，在180℃下2次手术之间消毒的工具。 2，准备药物准备2×1毫升注射器预麻醉药?…
Representative Results
损伤的程度 成功与这个特殊的实验模型的可重复性依赖于每个机械手/外科医师的经验。呼吸恢复（膈神经活动和膈肌活动）的继C2损伤的后续量相关，其余幸免腹外侧白质21。由于损伤是“手工”和需要一些实践从外科医生，每个损伤的程度要由组织学技术（组织用多聚甲醛4％，冰冻组织切片，甲酚紫染色的固定）进行检查，以确定准确的尺寸的受损组…
Discussion
使得C2损伤模型的技术难点
该C2损伤小鼠模型是研究呼吸后皮损神经可塑性一个有趣的工具。然而，需要产生一个可重复的和可靠的模型的步骤多，而且每一个可能的研究的结果产生影响。例如，在插管过程中，非常小心的是要采取自气管管可产生气管的炎症，其可以导致多种并发症，如阻塞性呼吸衰竭，除了最初的呼吸功能不全，由于损伤本身。此外，在?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是由欧盟第七框架计划（FP7/2007-2013）根据编号246556赠款协议（欧洲项目RBUCE-UP）的资助，HandiMedEx分配由法国公共投资委员会。马塞尔Bonay由Chancellerie德社区学院巴黎（腿华先生），全宗德Dotation RECHERCHE恩桑特Respiratoire和中心D'援助Respiratoire住所德法兰西岛（CARDIF）支持
Materials
| Animal | |||
| Male Sprague Dawley Rat | Janvier | 225-250g | |
| Surgical Instruments | |||
| Student Dumont #5 forceps | Fine Science Tool | 91150-20 | |
| Student Standard Pattern Forceps | Fine Science Tool | 91100-12 | |
| Mayo-Stille Scissors | Fine Science Tool | 14013-15 | Curved |
| Student Vannas Spring Scissors | Fine Science Tool | 91500-09 | Straight |
| Spring Scissors – 8 mm Blades | Fine Science Tool | 15025-10 | Straight Blunt/Blunt |
| Friedman Pearson Rongeur | Fine Science Tool | 16121-14 | Curved |
| Dissecting Knife – Fine Tip | Fine Science Tool | 10055-12 | Straight |
| Olsen-Hegar Needle Holder | Fine Science Tool | 12002-14 | Serrated |
| Weitlaner-Locktite Retractor | Fine Science Tool | 17012-11 | 2×3 Blunt |
| Absorbable surgical sutures | Centravet | BYO001 | |
| Equipment | |||
| Hot Bead Steriliser | Fine Science Tool | 18000-45 | |
| Catheter | Centravet | CAT188 | 16 gauge |
| Laryngoscope | |||
| Guide wire | |||
| Laryngeal mirror | Centravet | MIR011 | |
| Lactated Ringers | Centravet | RIN020 | |
| Syringe | Centravet | ||
| Needle | Centravet | ||
| O2 | Air Liquid | I1001M20R2A001 | |
| 683 RodentT Ventilator 115/230V | Havard Apparatus | 55-0000 | |
| Stand-Alone Vaporizer | WPI | EZ-155 | |
| Thin line heated bed | WPI | EZ-211 | |
| Air canister | WPI | EZ-258 | |
| Drugs | |||
| Carprofen | Centravet | ||
| Rimadyl | Centravet | RIM011 | |
| Buprenorphine | Centravet | BUP001 | |
| Baytril | Centravet | BAY001 | |
| Dexmedetomidine | Centravet | DEX010 | |
| Atipamezole | Centravet | ANT201 | |
| Betadine Solution | Centravet | VET002 | |
| Isoflurane | Centravet | VET066 | |
Referências
- Frankel, H. L., et al. Long-term survival in spinal cord injury: a fifty year investigation. Spinal Cord. 36, 266-274 (1998).
- Ramer, M. S., Harper, G. P., Bradbury, E. J. Progress in spinal cord research – a refined strategy for the International Spinal Research Trust. Spinal Cord. 38, 449-472 (2000).
- Zimmer, M. B., Nantwi, K., Goshgarian, H. G. Effect of spinal cord injury on the respiratory system: basic research and current clinical treatment options. J Spinal Cord Med. 30, 319-330 (2007).
- Mantilla, C. B., Sieck, G. C. Neuromuscular adaptations to respiratory muscle inactivity. Respir Physiol Neurobiol. 169, 133-140 (2009).
- Goshgarian, H. G. The crossed phrenic phenomenon and recovery of function following spinal cord injury. Respir Physiol Neurobiol. 169, 85-93 (2009).
- Nantwi, K. D. Recovery of respiratory activity after C2 hemisection (C2HS): involvement of adenosinergic mechanisms. Respir Physiol Neurobiol. 169, 102-114 (2009).
- Sandhu, M. S., et al. Respiratory recovery following high cervical hemisection. Respir Physiol Neurobiol. 169, 94-101 (2009).
- Lane, M. A., Lee, K. Z., Fuller, D. D., Reier, P. J. Spinal circuitry and respiratory recovery following spinal cord injury. Respir Physiol Neurobiol. 169, 123-132 (2009).
- Seeds, N. W., Akison, L., Minor, K. Role of plasminogen activator in spinal cord remodeling after spinal cord injury. Respir Physiol Neurobiol. 169, 141-149 (2009).
- Alilain, W. J., Horn, K. P., Hu, H., Dick, T. E., Silver, J. Functional regeneration of respiratory pathways after spinal cord injury. Nature. 475, 196-200 (2011).
- Vinit, S. Cervical spinal cord injuries and respiratory insufficiency: a revolutionary treatment. Med Sci (Paris. 28, 33-36 (2012).
- Kastner, A., Gauthier, P. Are rodents an appropriate pre-clinical model for treating spinal cord injury? Examples from the respiratory system). Exp Neurol. 213, 249-256 (2008).
- Vinit, S., Lovett-Barr, M. R., Mitchell, G. S. Intermittent hypoxia induces functional recovery following cervical spinal injury. Physiol Neurobiol. 169, 210-217 (2009).
- Porter, W. T. The Path of the Respiratory Impulse from the Bulb to the Phrenic Nuclei. J Physiol. 17, 455-485 .
- Nicaise, C., et al. Phrenic motor neuron degeneration compromises phrenic axonal circuitry and diaphragm activity in a unilateral cervical contusion model of spinal cord injury. Exp Neurol. 235, 539-552 (2012).
- Goshgarian, H. G. The crossed phrenic phenomenon: a model for plasticity in the respiratory pathways following spinal cord injury. J Appl Physiol. 94, 795-810 (2003).
- Vinit, S., Gauthier, P., Stamegna, J. C., Kastner, A. High cervical lateral spinal cord injury results in long-term ipsilateral hemidiaphragm paralysis. J Neurotrauma. 23, 1137-1146 (2006).
- Fuller, D. D., Johnson, S. M., Johnson, R. A., Mitchell, G. S. Chronic cervical spinal sensory denervation reveals ineffective spinal pathways to phrenic motoneurons in the rat. Neurosci Lett. 323, 25-28 (2002).
- Dougherty, B. J., Lee, K. Z., Lane, M. A., Reier, P. J., Fuller, D. D. Contribution of the spontaneous crossed-phrenic phenomenon to inspiratory tidal volume in spontaneously breathing rats. J Appl Physiol. 112, 96-105 (2012).
- Jou, I. M., et al. Simplified rat intubation using a new oropharyngeal intubation wedge. J Appl Physiol. 89, 1766-1770 (2000).
- Fuller, D. D., et al. Graded unilateral cervical spinal cord injury and respiratory motor recovery. Respir Physiol Neurobiol. 165, 245-253 (2009).
- Vinit, S., Windelborn, J. A., Mitchell, G. S. Lipopolysaccharide attenuates phrenic long-term facilitation following acute intermittent hypoxia. Respir Physiol Neurobiol. 176, 130-135 (2011).
- Ahmad, F., Wang, M. Y., Levi, A. D. Hypothermia for Acute Spinal Cord Injury-A Review. World Neurosurg. , (2013).
- Lovett-Barr, M. R., et al. Repetitive intermittent hypoxia induces respiratory and somatic motor recovery after chronic cervical spinal injury. J Neurosci. 32, 3591-3600 (2012).
- Minor, K. H., Akison, L. K., Goshgarian, H. G., Seeds, N. W. Spinal cord injury-induced plasticity in the mouse–the crossed phrenic phenomenon. Exp Neurol. 200, 486-495 (2006).
- Baussart, B., Stamegna, J. C., Polentes, J., Tadie, M., Gauthier, P. A new model of upper cervical spinal contusion inducing a persistent unilateral diaphragmatic deficit in the adult rat. Neurobiol Dis. 22, 562-574 (2006).
- Golder, F. J., et al. Breathing patterns after mid-cervical spinal contusion in rats. Exp Neurol. 231, 97-103 (2011).
- Lane, M. A., et al. Respiratory function following bilateral mid-cervical contusion injury in the adult rat. Exp Neurol. 235, 197-210 (2012).
- Vinit, S., et al. Axotomized bulbospinal neurons express c-Jun after cervical spinal cord injury. Neuroreport. 16, 1535-1539 (2005).
- Guenther, C. H., Windelborn, J. A., Tubon, T. C., Yin, J. C., Mitchell, G. S. Increased atypical PKC expression and activity in the phrenic motor nucleus following cervical spinal injury. Exp Neurol. 234, 513-520 (2012).
- Mantilla, C. B., Gransee, H. M., Zhan, W. Z., Sieck, G. C. Motoneuron BDNF/TrkB signaling enhances functional recovery after cervical spinal cord injury. Exp Neurol. 247, 101-109 (2013).
- Vinit, S., Darlot, F., Aoulaiche, H., Boulenguez, P., Kastner, A. Distinct expression of c-Jun and HSP27 in axotomized and spared bulbospinal neurons after cervical spinal cord injury. J Mol Neurosci. 45, 119-133 (2011).
- Windelborn, J. A., Mitchell, G. S. Glial activation in the spinal ventral horn caudal to cervical injury. Respir Physiol Neurobiol. 180, 61-68 (2012).
- Vinit, S., Stamegna, J. C., Boulenguez, P., Gauthier, P., Kastner, A. Restorative respiratory pathways after partial cervical spinal cord injury: role of ipsilateral phrenic afferents. Eur J Neurosci. 25, 3551-3560 (2007).
- Dougherty, B. J., et al. Recovery of inspiratory intercostal muscle activity following high cervical hemisection. Respir Physiol Neurobiol. 183, 186-192 (2012).
Citar este artigo
Keomani, E., Deramaudt, T. B., Petitjean, M., Bonay, M., Lofaso, F., Vinit, S. A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity. J. Vis. Exp. (87), e51235, doi:10.3791/51235 (2014).