Name: application of consistent massage-like perturbations on mouse calves and monitoring the resulting intramuscular pressure changes
Uploaded: 2019-09-20T13:00:00
Description: Watch this Scientific Journal Video about Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes at JoVE.com

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。

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。
<ol><li>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。</li>
	<li>将?...

<ol>
	<li>Furlan, A. D., Imamura, M., Dryden, T., Irvin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massage+for+low+back+pain:+an+updated+systematic+review+within+the+framework+of+the+Cochrane+Back+Review+Group.">Massage for low back pain: an updated systematic review within the framework of the Cochrane Back Review Group.</a> Spine. 34 (16), 1669-1684 (2009).</li><li>Robertson, A., Watt, J. M., Galloway, S. D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+leg+massage+on+recovery+from+high+intensity+cycling+exercise.">Effects of leg massage on recovery from high intensity cycling exercise.</a> British Journal of Sports Medicine. 38 (2), 173-176 (2004).</li><li>Waters-Banker, C., Butterfield, T. A., Dupont-Versteegden, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+effects+of+massage+on+nonperturbed+skeletal+muscle+in+rats.">Immunomodulatory effects of massage on nonperturbed skeletal muscle in rats.</a> Journal of Applied Physiology. 116 (2), 164-175 (2014).</li><li>Haas, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massage+timing+affects+postexercise+muscle+recovery+and+inflammation+in+a+rabbit+model.">Massage timing affects postexercise muscle recovery and inflammation in a rabbit model.</a> Medicine &amp; Science in Sports &amp; Exercise. 45 (6), 1105-1112 (2013).</li><li>Crane, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massage+therapy+attenuates+inflammatory+signaling+after+exercise-induced+muscle+damage.">Massage therapy attenuates inflammatory signaling after exercise-induced muscle damage.</a> Science Translational Medicine. 4 (119), 119ra113 (2012).</li><li>Bove, G. M., Harris, M. Y., Zhao, H., Barbe, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+therapy+as+an+effective+treatment+for+fibrosis+in+a+rat+model+of+upper+extremity+overuse+injury.">Manual therapy as an effective treatment for fibrosis in a rat model of upper extremity overuse injury.</a> Journal of the Neurological Sciences. 361, 168-180 (2016).</li><li>Andrzejewski, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+skeletal+muscle+expression+of+VEGF+induced+by+massage+and+exercise.">Increased skeletal muscle expression of VEGF induced by massage and exercise.</a> Folia Histochemica et Cytobiologica. 53 (2), 145-151 (2015).</li><li>Mantovani Junior, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+massage+as+a+recuperative+technique+on+autonomic+modulation+of+heart+rate+and+cardiorespiratory+parameters:+a+study+protocol+for+a+randomized+clinical+trial.">Effects of massage as a recuperative technique on autonomic modulation of heart rate and cardiorespiratory parameters: a study protocol for a randomized clinical trial.</a> Trials. 19 (1), 459 (2018).</li><li>Zeng, H., Butterfield, S., Agarwal, F., Haq, T., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+engineering+approach+for+quantitative+analysis+of+the+lengthwise+strokes+in+massage+therapies.">An engineering approach for quantitative analysis of the lengthwise strokes in massage therapies.</a> Journal of Medical Devices. 2 (4), (2008).</li><li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechatronic+system+for+quantitative+application+and+assessment+of+massage-like+actions+in+small+animals.">A mechatronic system for quantitative application and assessment of massage-like actions in small animals.</a> Annals of Biomedical Engineering. 42 (1), 36-49 (2014).</li><li>Saitou, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+cyclical+compression+modulates+macrophage+function+in+situ+and+alleviates+immobilization-induced+muscle+atrophy.">Local cyclical compression modulates macrophage function in situ and alleviates immobilization-induced muscle atrophy.</a> Clinical Science. 132 (19), 2147-2161 (2018).</li><li>Onda, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+mouse+model+of+skeletal+muscle+atrophy+using+spiral+wire+immobilization.">A New mouse model of skeletal muscle atrophy using spiral wire immobilization.</a> Muscle Nerve. 54 (4), 788-791 (2016).</li><li>Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines--chemotactic+cytokines+that+mediate+inflammation.">Chemokines--chemotactic cytokines that mediate inflammation.</a> The New England Journal of Medicine. 338, 436-445 (1998).</li><li>Reid, M. B., Li, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor-&#945;+and+muscle+wasting:+a+cellular+perspective.">Tumor necrosis factor-&#945; and muscle wasting: a cellular perspective.</a> Respiratory Research. 2 (5), 269-272 (2001).</li><li>Baumann, J. U., Sutherland, M. D., Hangg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramuscular+pressure+during+walking:+An+experimental+study+using+the+wick+catheter+technique.">Intramuscular pressure during walking: An experimental study using the wick catheter technique.</a> Clinical Orthopaedics Related Research. 145, 292-299 (1979).</li><li>Lee, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+physical+inactivity+on+major+non-communicable+diseases+worldwide:+an+analysis+of+burden+of+disease+and+life+expectancy.">Effect of physical inactivity on major non-communicable diseases worldwide: an analysis of burden of disease and life expectancy.</a> Lancet. 380, 219-229 (2012).</li></ol>

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?...

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。

<table>
<tbody>
<tr>
<td>Aluminum wire</td>
<td>DAISO JAPAN</td>
<td>B028</td>
<td>An aluminum wire is used to avoid escaping restriction by the wire</td>
</tr>
<tr>
<td>Blood pressure telemeter</td>
<td>Millar</td>
<td>SPR-671</td>
<td>A blood pressure telemeter is used to mesure intramuscular pressure.</td>
</tr>
<tr>
<td>DAPI</td>
<td>Thermo Fisher Scientific</td>
<td>D1306</td>
<td>DAPI is a fluorescent probe which is commonly used to stain DNA for fluorescent microscopy.</td>
</tr>
<tr>
<td>Goat anti-rabbit Alexa Fluor 488 (Dilution ratio, 1:500)</td>
<td>Invitrogen </td>
<td>A11034</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>Goat anti-rat Alexa Fluor 568 (Dilution ratio, 1:500))</td>
<td>Invitrogen </td>
<td>A11077</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>ImageJ</td>
<td>NIH</td>
<td>N/A</td>
<td>Analysis software for image</td>
</tr>
<tr>
<td>LabChart8</td>
<td>ADInstrumens</td>
<td>&nbsp;</td>
<td>Analysis software for acquiring biological signals.</td>
</tr>
<tr>
<td>Prolong gold</td>
<td>Thermo Fisher Scientific</td>
<td>P36930</td>
<td>Prolong gold is for mounting stained samples.</td>
</tr>
<tr>
<td>Protein Block Serum-Free</td>
<td>Dako</td>
<td>X090930-2</td>
<td>For blocking non-specific background staining in immunohistochemical procedures.</td>
</tr>
<tr>
<td>Rat monoclonal anti-laminin-2 antibody (Dilution ratio, 1:1000)</td>
<td>Sigma Aldrich</td>
<td>L0663</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>Rat monoclonal anti-F4/80 antibody (Dilution ratio, 1:500)</td>
<td>Abcam</td>
<td>ab6640</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>Rabbit polyclonal anti-MCP-1 antibody (Dilution ratio, 1:1000)</td>
<td>Abcam</td>
<td>ab25124</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>Rabbit polyclonal anti-TNF-&alpha; antibody (Dilution ratio, 1:1000)</td>
<td>Abcam</td>
<td>ab66579</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>Surgical tape</td>
<td>3M Japan</td>
<td>1530EP-0</td>
<td>Surgical tape is used to restrict joint movement.</td>
</tr>
</tbody>
</table>

application of consistent massage-like perturbations on mouse calves and monitoring the resulting intramuscular pressure changes

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。

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...

Watch this Scientific Journal Video about Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes at JoVE.com

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

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。

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

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

我们的协议在系统和机制中非常有用。正确的信息显示了身体压力和按摩的效果。我们当地的循环完成系统要简单得多。本地插入不需要常规方法。在膝关节延长且脚踝关节弯曲时，将手术胶带涂抹在支撑位置的双边后肢上。将一根铝线放在 L4-5 脊柱水平的后备箱上，将钢丝绕在后肢周围，螺旋的每个转弯之间有五毫米的间隙。为了尽量减少从线路中逃逸的可能性，请手动调整导线配置，以在 90 度诱拐位置固定臀部关节。然后将鼠标返回到其家庭笼子与监控，直到完全恢复。测量肌肉内肌肉压力，将麻醉小鼠放在容易的位置，并剃须、脱毛和半消毒后小腿的皮肤。使用手术刀在消毒的皮肤中做两毫米切口。在皮肤表面以 150 到 170 度的角度将 20 量表的内井针插入胃部肌肉。接下来，使用针头的塑料护套作为指导，在胃肌的中腹放置一个血压遥测传感器。用 4-O 尼龙缝合线封闭皮肤。然后，使用几个不同的圆柱形单位重量对小腿肌肉应用局部圆柱形压缩一分钟。并使用适当的生物信号分析软件监测肌肉内压力。对于局部周期性压缩治疗，麻醉小鼠脱离后肢接线。将动物放在容易的位置。调整后肢，使膝盖关节延长，脚踝关节与小腿朝上弯曲。垂直移动一个圆柱形的重量单位，覆盖着一个缓冲垫，在小腿肌肉上以一赫兹，每天30分钟，每天7天。每次压缩后，在将动物返回到家笼之前，将鼠标后肢重新布线。在实验结束时，将后小腿表面脱皮，并做一个皮肤切口，使胃部肌肉与骨质肉骨分离。在最佳的切割温度复合溶液中快速冻结收获的肌肉组织。并使用低温恒温器在玻璃滑梯上获取胃部肌肉的部分。用于免疫组织标记解冻和脱水部分在室温下空气干燥，然后使用疏水笔在每张幻灯片上的组织部分周围绘制一个环。接下来，将幻灯片放在湿度室中。并在每侧添加 100 微升阻塞缓冲区。室温下30分钟后，用两次5分钟的PBS-T洗涤，然后用100微升的原抗体在室温下过夜地标记这些部分。第二天早上，在PBS-T中清洗幻灯片3次，每次洗涤5分钟。并在室温下用适当的二次抗体标记样品1小时，防止光线。在孵化结束时，用新鲜的PBS-T清洗幻灯片3次，如所证明的。在黑暗中用100微升DAPI染色3分钟。然后，再洗三次幻灯片。使用兼容的安装介质和盖板将样品安装。对于组织形态分析，将幻灯片放在荧光显微镜的舞台上，并在适当的过滤器中以 20-X 的主观性查看样品。然后，在图像分析软件中测量每个真菌的横截面面积，并计算表示实验标记的细胞数。胃骨质纤维的横截面面积因后肢固定而显著减少。此外，免疫荧光染色分析显示，在固定后肢的胃部肌肉组织中，表达MCP-1和TNF-alpha的细胞显著增加。同时，随着积极染色的F4/80细胞的增加，后肢固定似乎引发小腿肌肉萎缩，包括局部炎症反应，包括巨噬细胞积累。在通过改变圆柱形单位重量而测试的几种不同的局部周期性压缩幅度中，对应于50毫升汞肌肉间压力波的压缩，似乎最有效地缓解了肌纤维横截面面积的固定性诱发的减少，增加了胃细胞肌肉中的巨噬素积累。66克局部周期性压缩，每天以1赫兹30分钟，为期7天，显著缓解观察到的胃细胞肌肉横截面面积的固定性减少。更多，局部周期性压缩部分缓和固定诱导三头肌肌肉收缩力下降。此外，局部周期性压缩可抑制F4/80阳性、TNF-alpha阳性和MCP-1一个阳性细胞在固定后肢的胃细胞肌肉组织中增加。我们的方法将为患者肌肉增加和我们潜在的预防程序策略提供科学依据。老鼠发现缺乏身体活动是有限的。使用我们的系统，我们已经开始评估大多数患者缺乏机械师的病人在病人。适度的本地工业方式做到这一点，使一个完美的固定诱导肌肉萎缩。

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Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

监测改变膜极性，膜完整性和细胞内离子浓度<em&gt;肺炎链球菌</em&gt;使用荧光染料

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

监视分泌细菌毒力因子的组装使用站点特定的交联

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

在肠系膜淋巴管空心​​大鼠模型：应用肠淋巴药物运输的评估

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

流式细胞仪为基础的检测方法的NK细胞功能的监控

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

表征形态变化的议定书<em&gt;艰难梭菌</em&gt;应对抗生素治疗

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...