在小鼠小腿上应用一致的按摩样扰动，并监测由此产生的肌肉压力变化
1Department of Rehabilitation for Motor Functions,National Rehabilitation Center for Persons with Disabilities, 2Graduate School of Sport Sciences,Waseda University, 3Faculty of Sport Sciences,Waseda University, 4Department of Orthopaedic Surgery, Graduate School of Medicine,The University of Tokyo, 5Frontier Research Institute for Interdisciplinary Sciences,Tohoku University, 6University of Cincinnati College of Medicine, 7Department of Assistive Technology,National Rehabilitation Center for Persons with Disabilities, 8Department of Clinical Research,National Rehabilitation Center for Persons with Disabilities
Summary
在这里，我们描述了将定义的机械载荷应用于小鼠小腿和监测伴随的肌肉压力变化的协议。我们开发的实验系统对于研究体育锻炼和按摩的有益作用背后的机制非常有用。
Abstract
按摩被普遍认为有助于缓解疼痛和炎症。虽然以前的研究已经报道了按摩对骨骼肌肉的抗炎作用，但背后的分子机制却知之甚少。我们最近开发了一种简单的装置来应用局部周期性压缩（LCC），它可以产生不同振幅的肌肉压力波。使用该装置，我们已经证明LCC调节巨噬细胞的原位炎症反应，并减轻固定引起的肌肉萎缩。在这里，我们描述了LCC的优化和应用方案，作为一种按摩样干预固定引起的炎症和小鼠后肢骨骼肌肉萎缩。我们开发的协议可用于研究运动和按摩的有益作用的机理。我们的实验系统提供了分析方法的原型，以阐明肌肉平衡的机械调节，虽然需要进一步的发展，以进行更全面的研究。
Introduction
按摩被普遍认为对减轻疼痛和提高竞技运动员和非运动员的身体表现都是有益的。事实上，以前的研究表明，按摩抑制局部炎症3，并促使从运动后肌肉损伤4，5恢复。按摩有益作用的分子机制在很大程度上仍不为人所知。
按摩机械学调查的一个困难是，通过实验技术对按摩样干预进行试验的可重复性。在以前的研究中，模仿按摩的实验程序主要涉及使用练习者身体部位（如手掌和手指6、7、8）进行身体干预。这使得很难精确再现其大小、频率、持续时间和模式。
许多设备已被开发用于将定义的机械载荷应用于目标组织。例如，曾等人开发了一种气动系统，用于对大鼠的后肢进行长度机械加载，王等人也开发了一种机电一体化装置，可以将按摩般的机械载荷应用于大鼠和兔子的后肢。实时反馈控制10。与它们相比，我们的局部循环压缩 （LCC） 系统要简单得多，对施工成本的要求要低得多。尽管如此，我们可以重现在轻度肌肉收缩期间产生的肌肉压力变化。利用该装置，我们已经成功地证明，按摩样的机械干预调节局部间质流体动力学，缓解固定引起的肌肉萎缩11。
在这里，我们描述了我们的设备和协议的细节，这可能有助于探索运动和按摩的积极作用背后的分子机制。该协议的原理图作为补充图1提出。
Protocol
所有动物实验都是经国家残疾人康复中心动物护理和使用委员会批准进行的。 1. 固定小鼠双边后肢 注：雄性C57BL/6小鼠在适应后至少7天11-12周时用于实验。 使用五巴比妥钠（50毫克/千克i.p.）对小鼠进行充分麻醉。确保小鼠对后肢脚趾捏没有反应。注：在上午10时至下午7时之间进行固定程序，以尽量减少对小鼠进食活动的可能影响。 将?…
Representative Results
与我们以前的观察12一致，胃肠肌纤维的CSA因后肢固定而显著减少（图2A，B）。此外，我们的免疫荧光染色分析表明，表达MCP-1和TNF-α的细胞在调节炎症过程方面起着关键作用13，14，在胃肠肌中显著增加固定后肢组织（MCP-1：图2C，F，H;TNF-α：图2</stro…
Discussion
我们描述了一种应用按摩样机械刺激的方法，这种方法具有抗炎作用。与之前报告的系统相比，我们的系统具有以下优势。首先，以前的研究没有定量定义施加的机械力2或根据人体表面的测量确定其幅度，而不是组织10内。相比之下，我们使用血压遥测仪测量肌肉压力。其次，我们设备的简单结构（图1B）使我们能够以相对较低的成本构?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢中桥K.、Hamamoto、N.Kume和K.Tsurumi在整个项目过程中一贯给予的支持。这项工作部分得到了日本厚生劳动省内部研究基金的支持;日本科学促进协会提供的科学研究援助赠款;MEXT支持私立大学战略研究基金会项目，2015-2019年，由日本教育、文化、体育、科学和技术部（S1511017）提供。
Materials
| Aluminum wire | DAISO JAPAN | B028 | An aluminum wire is used to avoid escaping restriction by the wire |
| Blood pressure telemeter | Millar | SPR-671 | A blood pressure telemeter is used to mesure intramuscular pressure. |
| DAPI | Thermo Fisher Scientific | D1306 | DAPI is a fluorescent probe which is commonly used to stain DNA for fluorescent microscopy. |
| Goat anti-rabbit Alexa Fluor 488 (Dilution ratio, 1:500) | Invitrogen | A11034 | Antibody for immunohistochemical staining. |
| Goat anti-rat Alexa Fluor 568 (Dilution ratio, 1:500)) | Invitrogen | A11077 | Antibody for immunohistochemical staining. |
| ImageJ | NIH | N/A | Analysis software for image |
| LabChart8 | ADInstrumens | Analysis software for acquiring biological signals. | |
| Prolong gold | Thermo Fisher Scientific | P36930 | Prolong gold is for mounting stained samples. |
| Protein Block Serum-Free | Dako | X090930-2 | For blocking non-specific background staining in immunohistochemical procedures. |
| Rat monoclonal anti-laminin-2 antibody (Dilution ratio, 1:1000) | Sigma Aldrich | L0663 | Antibody for immunohistochemical staining. |
| Rat monoclonal anti-F4/80 antibody (Dilution ratio, 1:500) | Abcam | ab6640 | Antibody for immunohistochemical staining. |
| Rabbit polyclonal anti-MCP-1 antibody (Dilution ratio, 1:1000) | Abcam | ab25124 | Antibody for immunohistochemical staining. |
| Rabbit polyclonal anti-TNF-α antibody (Dilution ratio, 1:1000) | Abcam | ab66579 | Antibody for immunohistochemical staining. |
| Surgical tape | 3M Japan | 1530EP-0 | Surgical tape is used to restrict joint movement. |
References
- Furlan, A. D., Imamura, M., Dryden, T., Irvin, E. Massage for low back pain: an updated systematic review within the framework of the Cochrane Back Review Group. Spine. 34 (16), 1669-1684 (2009).
- Robertson, A., Watt, J. M., Galloway, S. D. R. Effects of leg massage on recovery from high intensity cycling exercise. British Journal of Sports Medicine. 38 (2), 173-176 (2004).
- Waters-Banker, C., Butterfield, T. A., Dupont-Versteegden, E. E. Immunomodulatory effects of massage on nonperturbed skeletal muscle in rats. Journal of Applied Physiology. 116 (2), 164-175 (2014).
- Haas, C., et al. Massage timing affects postexercise muscle recovery and inflammation in a rabbit model. Medicine & Science in Sports & Exercise. 45 (6), 1105-1112 (2013).
- Crane, J. D., et al. Massage therapy attenuates inflammatory signaling after exercise-induced muscle damage. Science Translational Medicine. 4 (119), 119ra113 (2012).
- Bove, G. M., Harris, M. Y., Zhao, H., Barbe, M. F. Manual therapy as an effective treatment for fibrosis in a rat model of upper extremity overuse injury. Journal of the Neurological Sciences. 361, 168-180 (2016).
- Andrzejewski, W., et al. Increased skeletal muscle expression of VEGF induced by massage and exercise. Folia Histochemica et Cytobiologica. 53 (2), 145-151 (2015).
- Mantovani Junior, N., et al. Effects of massage as a recuperative technique on autonomic modulation of heart rate and cardiorespiratory parameters: a study protocol for a randomized clinical trial. Trials. 19 (1), 459 (2018).
- Zeng, H., Butterfield, S., Agarwal, F., Haq, T., Zhao, Y. An engineering approach for quantitative analysis of the lengthwise strokes in massage therapies. Journal of Medical Devices. 2 (4), (2008).
- Wang, Q., et al. A mechatronic system for quantitative application and assessment of massage-like actions in small animals. Annals of Biomedical Engineering. 42 (1), 36-49 (2014).
- Saitou, K., et al. Local cyclical compression modulates macrophage function in situ and alleviates immobilization-induced muscle atrophy. Clinical Science. 132 (19), 2147-2161 (2018).
- Onda, A., et al. A New mouse model of skeletal muscle atrophy using spiral wire immobilization. Muscle Nerve. 54 (4), 788-791 (2016).
- Luster, A. D. Chemokines–chemotactic cytokines that mediate inflammation. The New England Journal of Medicine. 338, 436-445 (1998).
- Reid, M. B., Li, Y. P. Tumor necrosis factor-α and muscle wasting: a cellular perspective. Respiratory Research. 2 (5), 269-272 (2001).
- Baumann, J. U., Sutherland, M. D., Hangg, A. Intramuscular pressure during walking: An experimental study using the wick catheter technique. Clinical Orthopaedics Related Research. 145, 292-299 (1979).
- Lee, I., et al. Effect of physical inactivity on major non-communicable diseases worldwide: an analysis of burden of disease and life expectancy. Lancet. 380, 219-229 (2012).
Cite This Article
Sakitani, N., Maekawa, T., Saitou, K., Suzuki, K., Murase, S., Tokunaga, M., Yoshino, D., Sawada, K., Takashima, A., Nagao, M., Ogata, T., Sawada, Y. Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes. J. Vis. Exp. (151), e59475, doi:10.3791/59475 (2019).