隔膜的神经支配膈运动神经元的功能和形态评估
Summary
Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.
Abstract
This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.
Introduction
肌萎缩性侧索硬化症（ALS）是与两个上和下运动神经元和随后的肌肉麻痹的损失相关联的衰弱运动神经元疾病。经诊断，病人的生存是唯一的2-5年平均1。膈运动神经元（PhMN）损失是ALS发病机制的一个重要组成部分。患者最终死于PhMN支配膈肌，灵感2,3的主要肌肉损失。外伤性脊髓损伤（SCI）也与相关联的呼吸困难的严重问题。每年4约12,000 SCI新病例发生是由于脊髓损伤创伤。尽管相对于位置，类型和严重程度的异质性疾病，多数SCI案件涉及外伤的脊髓型颈椎病，其结果往往是虚弱和持续性呼吸道妥协。除了ALS和脊髓损伤，其他中枢神经系统（CNS）的疾病可以关联瓦特第i膈肌呼吸功能障碍5,6。
膈神经是传出运动神经支配是同侧半隔膜和源自位于同侧颈脊髓的C3-C5水平PhMN细胞体。 PhMN输出由在称为延髓腹侧呼吸组（rVRG）7的区域降序从脑干bulbospinal输入控制。所述rVRG-PhMN隔膜电路是中央的吸气呼吸，以及其他非通气隔膜行为的控制。各种外伤和神经退行性疾病，影响该电路可导致呼吸功能及患者的生活质量产生深远的下降。下行输入从rVRG，PhMN生存，膈神经的完整性和适当神经支配的肌肉隔膜接头（NMJ）PhMNs都是必要的正常功能隔膜。它采用的技术，因此重要的是，可以定量地评价本电路在体内在ALS，脊髓损伤等中枢神经系统疾病的啮齿动物模型。
与此协议中，目标是描述实验工具用于评估在两个电生理学和形态学水平膜片PhMN支配。复合肌肉动作电位（的CMap）通过刺激一个给定的运动神经的所有传出的运动神经元的轴突，然后分析所述目标肌纤维的诱发的去极化应答被记录。此技术可用于在体内麻醉大鼠和小鼠的量化半膈由PhMNs 8的官能支配。由于这样的事实，的CMap表示同时记录所有（或至少许多/大部分）的整个半膈，它也检查个别运动轴突和肌纤维在膜片NMJ的表型，以便跟踪疾病是有用的肌纤维 – 和治疗相关的形态变化，如局部和完井德失神经支配，再生出芽及神经再支配。这可以通过在隔膜的整个贴装免疫组织化学（IHC）来完成，接着个体NMJs的详细的形态学评估整个肌肉9。结合的CMap和NMJ分析提供了一个强大的方法定量研究膈神经支配的中枢神经系统和PNS疾病的啮齿动物模型。
Protocol
Representative Results
Discussion
由于呼吸功能受到损害双方在外伤性脊髓损伤和ALS，开发针对呼吸和具体隔膜神经支配疗法是临床相关5,6。为了全面考察呼吸功能，应使用相结合的办法方法。的CMap测量膜片由外部膈神经的刺激方式的功能性神经支配的程度，但不是内源bulbospinal呼吸驱动8。此外，这些录音不允许检查在NMJ形态变化，特别是在个人的运动轴突和突触后乙酰胆碱受体的水平。通过使用所描述的染色?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the NINDS (grant #1R01NS079702 to A.C.L.) and the SURP Program at Thomas Jefferson University (M.M.).
Materials
| Paraformaldehyde | Fisher | T353-500 | Make 10% solution first in de-ionized distilled water; make 4% with 1X PBS, adjust pH to 7.4 |
| 1X Phosphate Buffered Saline, pH 7.4 | Invitrogen | 10010049 | |
| 2% Bovine serum albumin (2% BSA) | Sigma-Aldrich | A3059-100g | Dissolve 2g BSA into 100mL of 1X PBS |
| 0.2% Triton X100 in 2% BSA/PBS (Blocking Buffer) | Sigma-Aldrich | T9284-100mL | Dissolve 0.2ml/100mL 2% BSA/PBS |
| 0.1M Glycine | Sigma-Aldrich | G-7126 | Add 0.185g to 25mL of 2% BSA/PBS |
| α-bungarotoxin | Invitrogen | T1175 | Concentration 1:400 |
| SMI-312 | Sternberger Monoclonals | SMI312 | Concentration 1:1,000 |
| SV2 | Developmental Studies Hybridoma Bank | SV2-Supernatant | Concentration 1:10 |
| FITC goat anti-mouse IgG1 | Roche | 3117731001 | Concentration 1:100 |
| Silicone rubber | Sylgard, Dow Corning | Part # 184 | Follow instructions that come with kit: can use multiple sized culture dish (30mm, 60mm, 100mm) depending on needs |
| Vectashield fluorescent mounting medium | Vector laboratories | H-1000 | This is not a hard-set medium. You will need to secure the cover slip with clear nail polish. |
| Small Spring Scissors | Fine Science Tools | 15002-08 | |
| Dissection forceps | Fine Science Tools | 11295-51 | |
| Software for CMAP recordings | Scope 3.5.6; ADI | ||
| Disk surface electrodes | Natus neurology | 019-409000 | |
| Subdermal needle electrodes | Natus neurology | 019-453100 | |
| Conductive gel | Aquasonic | 122-73720 | |
| Stimulator/recording system for CMAP recordings | ADI Powerlab 8SP stimulator | ||
| Amplifier for CMAP recordings | BioAMP |
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