本研究由国家自然科学基金（批准号：82174521），湖南中医药大学研究生创新项目（2022CX109）资助

涉及动物的程序已获得湖南中医药大学机构护理和使用委员会的批准。
1.滚动按摩器的组装
<ol><li>选择由橡胶滚轮、叉架、弹簧、限位挡板、调节夹板、螺钉和亚克力手柄组成的按摩器（图 1）。确保胶辊长 3 厘米，直径 1.6 厘米，弹簧长 3 厘米，直径 0.9 厘米，限位挡板长 3 厘米，宽 2 厘米，手柄长 12 厘米，直径 0.9 厘米。</li>
	<li>力控制：根据文献结果23，发现丁氏滚动法向下的压力约为体重的10%，因此在设计前滚时施加的压力约为大鼠体重的10%（0.2-0.3 N）。通过调整限位挡板的角度，测试称重控制器上按摩器的最大压力约为 0.3 N。这种压力要求满足了大鼠的需要。</li>
	<li>回滚时确保最小压力约为 0.08 N（图 2）。确保压力精确符合丁氏横滚法要求，即正反向力比为3：1。</li>
	<li>在处理之前，要求操作人员使用节拍器软件将滚动频率控制在 140 卷/分钟，并在预实验中练习 3 次以上，以确保操作标准化。</li>
</ol>2. 骨骼肌损伤大鼠模型的建立
<ol><li>将 24 只雄性 Sprague-Dawley 大鼠（体重 200-250 g）随机分为三组，每组 8 只大鼠，包括对照组 （C）、notexin （NTX） 和 notexin 与推拿 （NTX + 推拿），并以标准饮食为食。保持在12小时光照/12小时黑暗循环中，在20-25°C和50%-70%湿度下。</li>
	<li>腹膜内注射用1%戊巴比妥钠（40mg / kg）麻醉，然后用脱毛膏去除右下肢的毛发。去除毛皮后，用生理盐水擦拭残留的乳霜。通过脚趾捏合反应确认充分麻醉。在动物麻醉时涂抹眼药膏滋润眼睛。在整个过程中提供热支持。</li>
	<li>注射前用碘伏消毒液和 75% 酒精交替对右下肢进行皮肤消毒。将浸泡在碘伏消毒液中的棉签接触下肢皮肤中心，然后以打圈的方式向外涂抹。用浸有乙醇的棉签重复上述步骤。</li>
	<li>根据参考方法24建立骨骼肌损伤模型。仅在一条腿上注射 notexin（以防止双重 notexin 注射）。用 30G 针头将 200 μL notexin 溶液（通过在 15 mL 离心管中加入 10 μg notexin 加入 10 mL 生理盐水制备的 10 μg/mL notexin 溶液制备）放入 30G 针头的 1 mL 注射器中，并将 notexin 溶液肌肉注射到腓肠肌中以产生肌肉损伤。</li>
	<li>缓慢注射notexin并等待3秒后再拔出针头（以完全注射）。 注意：Notexin 是一种有毒化学物质，接触开放性伤口后需要立即用大量水冲洗，并在必要时及时就医。</li>
	<li>向对照组的大鼠注射200μL生理盐水。将麻醉的大鼠移至空笼子中，并铺有干净的垫料。注意去除老鼠鼻子和嘴巴周围的衬垫，以保持它们的呼吸畅通。目视观察注射结束时的组织颜色和呼吸频率，直到大鼠恢复足够的意识。</li>
	<li>将大鼠放回笼子，通常饲养24小时。</li>
</ol>3. 推拿疗法
<ol><li>将SD大鼠置于俯卧位，头部用黑布覆盖在用75%酒精消毒的实验平台上，以暴露腓肠肌。不要盖得太紧。</li>
	<li>NTX+推拿组使用推拿按摩器：握住按摩器，将滚轮放在大鼠的腓肠肌上，向前滚动，直到弹簧接触限位挡板。然后缩回力并返回其原始位置，从而进行往复运动（图 3）。</li>
	<li>以每分钟 140 卷的速度滚动按摩器，每次操作 3 分钟。连续3天上午和下午各进行一次按摩。</li>
	<li>每次处理后将大鼠放回笼中，并在最后一次处理后禁食8小时。</li>
</ol>4.实验后从大鼠身上采集血液和组织
<ol><li>根据相关动物实验伦理委员会的要求，通过腹腔注射1%戊巴比妥钠（40mg/kg，腹腔注射）麻醉大鼠。通过脚趾捏合反应确认充分麻醉。采血后通过腹主动脉放血对大鼠实施安乐死。</li>
	<li>注射前用碘伏消毒液和75%酒精交替进行皮肤消毒。将浸泡在聚维酮碘中的棉签触摸腹部皮肤的中心，然后以圆周运动向外涂抹。用浸有乙醇的棉签重复上述步骤。重复消毒 3 次。</li>
	<li>让助手使用两个止血钳来抬起腹部中间的皮肤。作为操作者，使用手术刀切开腹部皮肤和肌肉，从臀部到耻骨联合。</li>
	<li>打开腹腔后，用无菌棉球将肠分开，露出腹后壁的腹主动脉。</li>
	<li>找到腹主动脉，将5mL大鼠血液取入采血管中，并在静置血液1小时后以3000× g 离心10分钟，将血浆放入1.5个微管中。将血浆储存在-80°C。</li>
	<li>用手术剪刀沿着下腹部开口向右下肢的外侧切开皮肤，露出下肢肌肉，用镊子小心地分离筋膜后，切开手术刀以去除完整的腓肠肌。</li>
	<li>用无菌盐水清洗腓肠肌，以去除粘附的毛发和血液。</li>
	<li>将去除的腓肠肌放入含有 4% 多聚甲醛的 15 mL 离心管中。</li>
</ol>5. ELISA检测血浆CK和FABP 3水平
<ol><li>计算并确定一次实验所需的预包装板数量。取出所需的板，将它们放入96孔框架中，将剩余的微孔板放回铝箔袋中密封，并将它们储存在4°C。</li>
	<li>将试剂盒和样品在室温（25-28°C）下平衡120分钟，完全平衡至室温。 注意：试剂盒和样品的平衡至关重要，必须平衡到足够的时间。</li>
	<li>设置标准孔、样品孔和空白孔。向标准孔中加入不同浓度（100、50、25、12.5、6.25、0 ng/mL）的 50 μL CK 或 FABP3 标准品。每个标准重复一次，总共占据12个孔。</li>
	<li>用 40 μL 样品稀释剂（0.8 g NaCl、0.02 g KH 2 PO 4、0.29 gNa 2 HPO 4 12H2O、0.02 g KCl、0.01 g NaN3在 100 mL 双蒸水，pH7.4 中）填充样品孔，然后加入 10 μL 待测样品。重复每个样品一次，总共占用 48 个孔。</li>
	<li>除了位于最后一个样品孔后面两个孔的空白孔外，向每个标准品和样品孔中加入 100 μL HRP 标记的抗人 CK 或 FABP3 抗体（酶标记抗体）。</li>
	<li>用密封膜密封孔，并在37°C水浴或恒温器中孵育60分钟。</li>
	<li>丢弃液体，在吸水纸上拍干，用洗涤液填充每个孔，静置 20 秒，甩掉洗涤液，在吸墨纸上拍干，然后重复洗涤板 5 次（或使用洗板机）。</li>
	<li>向每个孔中加入 50 μL 显色剂溶液 A（20 mg 四甲基联苯胺、10 mL 乙醇溶于 100 mL 双蒸水中）和 50 μL 显色剂溶液 B（0.1 M/L 柠檬酸、0.2 M/L 磷酸二氢钠缓冲液，pH 5.0-5.4）。在37°C下避光15分钟。</li>
	<li>对于标准孔、样品孔和空白孔，向每个孔中加入 50 μL 终止溶液，并在 15 分钟内测量每个孔在 450 nm 处的光密度值。</li>
</ol>6. notexin诱导的大鼠腓肠肌损伤的组织学分析
<ol><li>准备用苏木精和伊红染色的 5 μm 厚石蜡切片，用于光学显微镜检查，如25 中所述。</li>
</ol>7. 图像处理和数据分析
<ol><li>使用分析软件读取和分析成像系统捕获的图像。用鼠标将选定的图像视野移动到屏幕中央，单击 40 倍，然后单击“ 拍摄快照”。</li>
	<li>在电子表格中记录ELISA的OD值，并使用标准曲线计算样品中的大鼠CK和FABP3水平。</li>
	<li>使用统计分析软件进行统计分析。将测量值表示为平均值±标准差 （<img alt="Equation 1" src="/files/ftp_upload/65820/65820eq01.jpg" style="margin: auto;">），并通过单因素方差分析分析组间比较，方差均匀时采用 LSD 检验，方差不均匀时采用 Tamhane T2 方法。当 p 值小于 0.05 时，该差异被认为具有统计学意义。</li>
</ol>

基于 Ding's Roll 操作治疗大鼠模型肌肉损伤的按摩器

<ol>
	<li>Lempainen, L., 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=Management+of+anterior+thigh+injuries+in+soccer+players:+practical+guide.">Management of anterior thigh injuries in soccer players: practical guide.</a> BMC Sports Science, Medicine and Rehabilitation. 14 (1), 41 (2022).</li><li>Bayer, M. L., Mackey, A., Magnusson, S. P., Krogsgaard, M. R., Kj&#230;r, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+acute+muscle+injuries+(in+Danish).">Treatment of acute muscle injuries (in Danish).</a> Ugeskrift for Laeger. 181 (8), V11180753 (2019).</li><li>Serner, 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=Progression+of+Strength,+Flexibility,+and+Palpation+Pain+During+Rehabilitation+of+Athletes+with+Acute+Adductor+Injuries:+A+Prospective+Cohort+Study.">Progression of Strength, Flexibility, and Palpation Pain During Rehabilitation of Athletes with Acute Adductor Injuries: A Prospective Cohort Study.</a> The Journal of Orthopaedic and Sports Physical Therapy. 51 (3), 126-134 (2021).</li><li>Gozubuyuk, O. B., Koksal, C., Tasdemir, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rehabilitation+of+a+patient+with+bilateral+rectus+abdominis+full+thickness+tear+sustained+in+recreational+strength+training:+a+case+report.">Rehabilitation of a patient with bilateral rectus abdominis full thickness tear sustained in recreational strength training: a case report.</a> Physiotherapy Theory and Practice. 38 (13), 3216-3225 (2022).</li><li>Hotfiel, T., 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=Current+Conservative+Treatment+and+Management+Strategies+of+Skeletal+Muscle+Injuries.">Current Conservative Treatment and Management Strategies of Skeletal Muscle Injuries.</a> Zeitschrift f&#252;r Orthop&#228;die und Unfallchirurgie. 154 (3), 245-253 (2016).</li><li>de Sire, 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=Pharmacological+Treatment+for+Acute+Traumatic+Musculoskeletal+Pain+in+Athletes.">Pharmacological Treatment for Acute Traumatic Musculoskeletal Pain in Athletes.</a> Medicina. 57 (11), 1208 (2021).</li><li>Connor, D. E., 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=Therapeutic+potential+of+exosomes+in+rotator+cuff+tendon+healing.">Therapeutic potential of exosomes in rotator cuff tendon healing.</a> Journal of Bone and Mineral Metabolism. 37 (5), 759-767 (2019).</li><li>Martins, A. L. L., Giorno, L. P., Santos, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Engineering+Applied+to+Skeletal+Muscle:+Strategies+and+Perspectives.">Tissue Engineering Applied to Skeletal Muscle: Strategies and Perspectives.</a> Bioengineering. 9 (12), 744 (2022).</li><li>Horgan, 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=Clouds+across+the+new+dawn+for+clinical,+diagnostic+and+biological+data:+accelerating+the+development,+delivery+and+uptake+of+personalized+medicine.">Clouds across the new dawn for clinical, diagnostic and biological data: accelerating the development, delivery and uptake of personalized medicine.</a> Diagnosis. , (2023).</li><li>Urits, 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=A+Comprehensive+Review+of+Alternative+Therapies+for+the+Management+of+Chronic+Pain+Patients:+Acupuncture,+Tai+Chi,+Osteopathic+Manipulative+Medicine,+and+Chiropractic+Care.">A Comprehensive Review of Alternative Therapies for the Management of Chronic Pain Patients: Acupuncture, Tai Chi, Osteopathic Manipulative Medicine, and Chiropractic Care.</a> Advances in Therapy. 38 (1), 76-89 (2021).</li><li>Lee, N. 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=Chuna+(or+Tuina)+Manual+Therapy+for+Musculoskeletal+Disorders:+A+Systematic+Review+and+Meta-Analysis+of+Randomized+Controlled+Trials.">Chuna (or Tuina) Manual Therapy for Musculoskeletal Disorders: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.</a> Evidence-based Complementary and Alternative Medicine: eCAM. 2017, 8218139 (2017).</li><li>Xie, J., Deng, D. X., Chen, Y., Peng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+intervention+of+massage+techniques+on+skeletal+muscle+injury.">Progress in the intervention of massage techniques on skeletal muscle injury.</a> Hunan Journal of Traditional Chinese Medicine. 34 (04), 199-201 (2018).</li><li>Yuan, Y., Zhang, H., Zhang, G. H., Xue, X. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+progress+on+microstructure+changes+and+rehabilitation+treatment+of+exercise-induced+skeletal+muscle+injury.">Research progress on microstructure changes and rehabilitation treatment of exercise-induced skeletal muscle injury.</a> Massage and Rehabilitation Medicine. 14 (6), 29-33 (2023).</li><li>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=The+Establishment+of+Famous+Tuina+Master+Ding+Jifeng+and+Wei+Fa+-+Commemorating+the+100th+Anniversary+of+Mr.+Ding+Jifeng's+Birthday.">The Establishment of Famous Tuina Master Ding Jifeng and Wei Fa - Commemorating the 100th Anniversary of Mr. Ding Jifeng's Birthday.</a> Traditional Chinese Medicine Culture. 9 (6), 18-21 (2014).</li><li>Hardy, 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=Comparative+Study+of+Injury+Models+for+Studying+Muscle+Regeneration+in+Mice.">Comparative Study of Injury Models for Studying Muscle Regeneration in Mice.</a> PloS one. 11 (1), e0147198 (2016).</li><li>Plant, D. R., Colarossi, F. E., Lynch, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notexin+causes+greater+myotoxic+damage+and+slower+functional+repair+in+mouse+skeletal+muscles+than+bupivacaine.">Notexin causes greater myotoxic damage and slower functional repair in mouse skeletal muscles than bupivacaine.</a> Muscle &amp; Nerve. 34 (5), 577-585 (2006).</li><li>Tierney, M. T., Sacco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+and+Evaluating+Skeletal+Muscle+Injury+by+Notexin+and+Barium+Chloride.">Inducing and Evaluating Skeletal Muscle Injury by Notexin and Barium Chloride.</a> Methods in Molecular Biology. 1460, 53-60 (2016).</li><li>Torres-Ruiz, J., Alcal&#225;-Carmona, B., Alejandre-Aguilar, R., G&#243;mez-Mart&#237;n, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+myopathies+and+beyond:+The+dual+role+of+neutrophils+in+muscle+damage+and+regeneration.">Inflammatory myopathies and beyond: The dual role of neutrophils in muscle damage and regeneration.</a> Frontiers in Immunology. 14, 1113214 (2023).</li><li>Tu, H., Li, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+balance+in+skeletal+muscle+damage+and+repair.">Inflammation balance in skeletal muscle damage and repair.</a> Frontiers in Immunology. 14, 1133355 (2023).</li><li>Castro, C., Gourley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+treatment+of+inflammatory+myopathy:+issues+and+management.">Diagnosis and treatment of inflammatory myopathy: issues and management.</a> Therapeutic Advances in Musculoskeletal Disease. 4 (2), 111-120 (2012).</li><li>Dabby, R., 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=Asymptomatic+or+minimally+symptomatic+hyperCKemia:+histopathologic+correlates.">Asymptomatic or minimally symptomatic hyperCKemia: histopathologic correlates.</a> The Israel Medical Association Journal: IMAJ. 8 (2), 110-113 (2006).</li><li>Khodabukus, 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=Tissue-Engineered+Human+Myobundle+System+as+a+Platform+for+Evaluation+of+Skeletal+Muscle+Injury+Biomarkers.">Tissue-Engineered Human Myobundle System as a Platform for Evaluation of Skeletal Muscle Injury Biomarkers.</a> Toxicological Sciences. 176 (1), 124-136 (2020).</li><li>Zhou, X. W., Jin, W. D., Zhu, L., Liu, X. H., Zhou, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+observation+on+the+influence+of+different+frequency,+intensity+and+action+time+of+Ding+rolling+manipulation+on+hemodynamics.">Experimental observation on the influence of different frequency, intensity and action time of Ding rolling manipulation on hemodynamics.</a> Shanghai Journal of Traditional Chinese Medicine. (06), 42-44 (1998).</li><li>Pablos, 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=Protective+Effects+of+Foam+Rolling+against+Inflammation+and+Notexin+Induced+Muscle+Damage+in+Rats.">Protective Effects of Foam Rolling against Inflammation and Notexin Induced Muscle Damage in Rats.</a> International Journal of Medical Sciences. 17 (1), 71-81 (2017).</li><li>Wisner, L., Larsen, B., Maguire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+Tumor+Content+through+Tumor+Macrodissection.">Enhancing Tumor Content through Tumor Macrodissection.</a> Journal of Visualized Experiments: JoVE. (180), e62961 (2022).</li><li>Deng, P., 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=Contusion+concomitant+with+ischemia+injury+aggravates+skeletal+muscle+necrosis+and+hinders+muscle+functional+recovery.">Contusion concomitant with ischemia injury aggravates skeletal muscle necrosis and hinders muscle functional recovery.</a> Experimental Biology and Medicine. 247 (17), 1577-1590 (2022).</li><li>Dobek, G. L., Fulkerson, N. D., Nicholas, J., Schneider, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+muscle+crush+injury+of+the+legs.">Mouse model of muscle crush injury of the legs.</a> Comparative Medicine. 63 (3), 227-232 (2013).</li><li>Armstrong, D. M., 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=Sildenafil+citrate+protects+skeletal+muscle+of+ischemia-reperfusion+injury:+immunohistochemical+study+in+rat+model.">Sildenafil citrate protects skeletal muscle of ischemia-reperfusion injury: immunohistochemical study in rat model.</a> Acta Ciru&#769;rgica Brasileira. 28 (4), 282-287 (2013).</li></ol>

作者没有什么可透露的。

在这里，我们描述了推拿治疗大鼠骨骼肌损伤的方案，然后分析了治疗后骨骼肌损伤的程度，以验证该方法的有效性。值得注意的是，大鼠骨骼肌损伤模型，包括但不限于药物诱导（notexin、布比卡因）16、钝挫伤26、挤压 27 和缺血再灌注28，可以用推拿进行干预。通过HE染色观察组织病理学变化和ELISA检测确定骨骼肌损伤标志?...

肌肉损伤是临床和日常生活中常见的创伤性损伤，由外部打击（挫伤）或肌纤维慢性过度拉伤（拉伤）等引起，导致肌肉功能障碍和疼痛，甚至严重影响患者的生活质量1。急性劳损后尽早开始康复是缩短恢复运动时间的关键2 和减轻疼痛 3,4。在现代西医中，肌肉损伤的临床急救遵循休息、冰敷、加压和抬高 （RICE） 原则，以阻止肌肉组织有害出血5 和非甾体抗炎药缓解疼痛6。外泌体7 和组织工程8 等新疗法的发现成为骨骼肌疾病的潜在治疗策略，弥补了先前药物治疗的不足。然而，它也会增加患者的治疗成本，使他们承受巨大的经济压力9.因此，建议使用替代疗法和补充疗法来治疗肌肉骨骼问题10.推拿作为传统医疗方法在中国临床上被广泛使用，因其疗效好、副作用少而受到患者的欢迎。推拿治疗肌肉骨骼疾病可以减轻疼痛并改善功能11,12,13。上海著名推拿法师丁继峰先生创立了丁氏卷法14。它是一种独特的轧碎技术，受力面积大，力均匀柔和，穿透力强。
不同的动物模型基于不同的病因。它们各有利弊，选择正确、合适的动物模型对基础实验具有重要意义，有助于了解骨骼肌损伤后再生和修复的细胞和分子信号通路，为开发治疗骨骼肌疾病的新疗法奠定基础意义。化学诱导的肌肉损伤模型被广泛使用，骨骼肌注射引起肌纤维坏死并产生可以在 2 周内有效再生的再生区域15。notexin 和布比卡因都会导致肌肉损伤。然而，与布比卡因相比，notexin 对骨骼肌的肌毒性损伤更严重，并且自然功能恢复相对较慢16.药物肌内注射成型不仅时间更短，而且对骨骼肌损伤的效果和程度也可控。这种可量化的控制使成功的成型难度降低15,17。
炎症反应是一种重要的生物学反应，已在肌病的背景下进行了广泛研究18,19。在骨骼肌损伤的早期阶段，肌纤维坏死会破坏局部肌肉稳态，许多炎症细胞浸润损伤部位，分泌许多促炎细胞因子19。肌酸激酶 （CK） 是用于评估骨骼肌损伤的传统血清生物标志物。然而，它缺乏组织特异性20 和敏感性21，这限制了其评估药物诱导的肌肉损伤程度和间接报告损伤后肌肉恢复程度的能力。包括脂肪酸结合蛋白 3 （FABP3） 在内的新型生物标志物最近在骨骼肌损伤的啮齿动物模型中显示出相对较高的组织特异性和敏感性。FABP3 是一个结合蛋白家族，主要在心脏和骨骼肌细胞中表达，与脂肪酸代谢、转运和信号传导有关22。因此，我们选择了两种生物标志物CK和FABP3的组合，以评估notexin诱导的骨骼肌损伤和治疗后的恢复程度。
在啮齿类动物中，肌肉较浅，皮肤面积较小，这也决定了啮齿类动物按摩的各种参数不会与人类相同，例如在动物治疗中，按摩治疗师应使用丁氏滚动法以较小的力气对待它们，并且可能由于受伤部位的尺寸较小，不利于该技术的操作， 这最终会导致按摩效果的降低。因此，本实验利用自制的符合丁氏滚动法特点的滚动按摩器，对notexin诱导的大鼠骨骼肌损伤模型的治疗效果进行干预和评价，有助于在实验动物研究中标准化推拿参数，以深入研究推拿的分子作用机制。 一种中医治疗方法，用于肌肉骨骼疾病。

<table><tbody><tr><td>1 mL syringe</td><td>JIANGXI FENGLIN</td><td>20220521</td><td></td></tr><tr><td>1.5 microtubes&amp;nbsp;</td><td>Servicebio</td><td>EP-150X-J</td><td></td></tr><tr><td>15 mL centrifuge tube</td><td>Servicebio</td><td>EP-1501-J</td><td></td></tr><tr><td>30G needle</td><td>CONPUVON</td><td>220318</td><td></td></tr><tr><td>5 mL blood collection tube</td><td>Servicebio</td><td>QX0023</td><td></td></tr><tr><td>Acrylic handle</td><td>Guangdong Guangxingwang Plastic Materials Co., Ltd</td><td>65643645</td><td></td></tr><tr><td>Adjustment splint</td><td>CREROMEM</td><td>20220729</td><td></td></tr><tr><td>Cotton Swab</td><td>INOHV</td><td>22080215</td><td></td></tr><tr><td>Enzyme-labeled Instrument</td><td>Rayto</td><td>RT-6100&amp;nbsp;</td><td></td></tr><tr><td>Ethanol</td><td>INOHV</td><td>211106</td><td></td></tr><tr><td>Fork holder</td><td>Yongkang Kangzhe Health Technology Co., Ltd</td><td>JL001</td><td></td></tr><tr><td>Hair removal cream</td><td>Veet, France</td><td>LOTC190922002</td><td></td></tr><tr><td>Hematoxylin dyeing solution set</td><td>Wuhan Google Biotech</td><td>G1005</td><td></td></tr><tr><td>Imaging system&amp;nbsp;</td><td>Nikon, Japan</td><td>Nikon DS-U3</td><td></td></tr><tr><td>IODOPHOR disfecting solution</td><td>Hale&amp;amp;Hearty</td><td>20221205</td><td></td></tr><tr><td>Light microscope</td><td>Nikon, Japan</td><td>Nikon Eclipse E100</td><td></td></tr><tr><td>Limit baffle</td><td>CREROMEM</td><td>20220724</td><td></td></tr><tr><td>Notexin</td><td>Latoxan S.A.S.</td><td>L8104-100UG</td><td></td></tr><tr><td>Pentobarbital sodium</td><td>Merck KGaA</td><td>P3761</td><td></td></tr><tr><td>Rat creatine kinase (CK) ELISA kit</td><td>LunChangShuoBiotech</td><td>YD-35237</td><td></td></tr><tr><td>Rat fatty acid-binding protein 3 (FABP3) ELISA kit</td><td>LunChangShuoBiotech</td><td>YD-35730</td><td></td></tr><tr><td>Rubber roller</td><td>Hebei Mgkui Chemical Technology Co.,Ltd</td><td>202207</td><td></td></tr><tr><td>Screw</td><td>Weiyan Hardware</td><td>B05Z122</td><td></td></tr><tr><td>Sprague Dawley rats</td><td>Hunan Slake Kingda Laboratory Animal Co.</td><td>SYXK2019-0009</td><td></td></tr><tr><td>Spring</td><td>Bingzhang Hardware</td><td>TH001</td><td></td></tr><tr><td>Surgical blade</td><td>Covetrus</td><td>#23</td><td></td></tr><tr><td>Weigh controller</td><td>Iyoys</td><td>HY-XSQ</td><td></td></tr></tbody></table>

mimicking ding's roll method on notexin-induced muscle injury in rats

丁氏翻滚法是中国传统按摩诊所中最常用的推拿法之一，也是中国当代推拿法中最有影响力的推拿法之一。它以单指禅宗流派中常用的传统卷法为基础，命名为丁氏卷法。由于其抗炎和促进血液循环的作用，丁氏滚动法对肌病有很好的治疗效果。由于施加在人体皮肤上的力面积很大，丁氏滚动法在皮肤面积小的实验动物（如大鼠和兔子）上具有挑战性。此外，推拿应用于人体的强度与应用于实验动物的强度不同，因此在实验过程中可能会发生强度过高或过低而无法达到推拿的治疗效果。该实验旨在根据 Ding 的滚动操作参数（强度、频率、推拿持续时间）创建一种适合大鼠的简单按摩器。该装置可以标准化动物实验中的操作，并减少由于主观因素而施加到不同动物身上的推拿力的变化。建立大鼠骨骼肌损伤模型，采用血浆损伤标志物肌酸激酶（CK）和脂肪酸结合蛋白3（FABP3）评价推拿对骨骼肌损伤的治疗效果。结果表明，该推拿按摩器可降低CK和FABP3的表达水平，减缓骨骼肌损伤的程度。因此，本文介绍的推拿按摩器，模仿丁氏滚动法，有助于实验研究中推拿操作的标准化，对后续推拿治疗肌病分子机制的研究有很大帮助。

为了观察损伤后大鼠骨骼肌的形态学特性，用苏木精和伊红对腓肠肌进行染色，并用方案中描述的分析软件读取染色图像，每组8只大鼠。在notexin诱导的腓肠肌损伤大鼠（NTX组）中，许多肌肉细胞破裂、萎缩、坏死和排列不规则。受影响区域周围也有中性粒细胞和淋巴细胞的高浸润（图4B）。然而，在用滚动按摩器治疗推拿后，NTX+推拿组肌肉细胞的病理状况有所改善，破裂?...

Watch this Scientific Journal Video about Performing Tuina Massage in Rats Using a Self-Made Massager at JoVE.com

作者聚焦：使用自制按摩器对大鼠进行推拿按摩 

该协议描述了一种模仿Ding滚动方法的简单装置，建立了骨骼肌损伤的大鼠模型，并使用苏木精-伊红染色观察受损组织的病理学和酶联免疫吸附测定法来检测血清损伤标志物的变化。

模仿丁氏滚动法治疗大鼠肌损伤

Ding's roll method is one of the most commonly used manipulations in traditional Chinese massage (Tuina) clinics and one of the most influential ...

我们的研究范围是推拿治疗肌肉骨骼疾病的生物学机制。在这里，我们试图制作自制的推拿装置，以标准化并有效地对实验动物进行推拿治疗。我们正在通过创建一个可以模拟大鼠滚动操作的按摩器来解决无法成对进行这种滚动操作的问题，并验证按摩器对肌肉损伤大鼠的有效性。在未来的标准化研究中，我们将重点关注滚动按摩器对不同参数下大鼠骨骼肌损伤的疗效问题。

mimicking-ding-s-roll-method-on-notexin-induced-muscle-injury-in-rats

Research

JoVE Journal

Medicine

Mimicking Ding's Roll Method on Notexin-Induced Muscle Injury in Rats

970 次观看.  Hunan University of Chinese Medicine. 我们的研究范围是推拿治疗肌肉骨骼疾病的生物学机制。在这里，我们试图制作自制的推拿装置，以标准化并有效地对实验动物进行推拿治疗。我们正在通过创建一个可以模拟大鼠滚动操作的按摩器来解决无法成对进行这种滚动操作的问题，并验证按摩器对肌肉损伤大鼠的有效性。在未来的标准化研究中，我们将重点关注滚动按摩器对不同参数下大鼠骨骼肌损伤的疗效?...

视频: 作者聚焦：使用自制按摩器对大鼠进行推拿按摩 

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

一个在体内收缩引起的损伤，非侵入性的监测恢复啮齿动物模型

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

科研

医学

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

一个小鼠模型，通过神经肌肉电刺激肌肉训练

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite cells. Satellite cells are characterized by the expression of the transcription factor Pax7 and their location underneath the basal lamina in the resting skeletal muscle. Upon injury, satellite cells get activated, undergo self-renewal or differentiation to either form new myofibers or to fuse with damaged ones. The functionality of satellite cells in vivo can be investigated using a cardiotoxin based injury model of skeletal muscle. To study the function of one gene during the regeneration of skeletal muscle, transgenic mouse models are mostly used. Here, we present an alternative method to transgenic mice, to investigate the gene function in satellite cells during regeneration, e.g., in cases where transgenic mice are not available. We combine the cardiotoxin mediated injury of a specific skeletal muscle with the injection of a self-delivering siRNA into the regenerating muscle which is then taken up by satellite cells among other cells. Thereby, we provide a method to analyze gene function in satellite cells during regeneration under physiological conditions without the need for transgenic mice.

We describe an in vivo method to investigate the functional loss of a specific gene in satellite cells by using a combination of cardiotoxin mediated injury of skeletal muscle and the injection of a self-delivering siRNA.

通过心肌毒素损伤和在 Vivo 中注射自传递 siRNA 在骨骼肌再生过程中的卫星细胞功能分析

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite ...

遗传学

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

发育生物学

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

慢性刺激在收缩活动性大鼠骨骼肌表型适应性研究中的应用

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

<meta charset="utf8"></head><body>评估推拿疗法对慢性缩窄大鼠的疼痛缓解效果

Characteristics of Pain Changes in Rats with Nerve Injury Within 24 hours After One-Time Tuina Intervention

Tuina, as an external treatment method of traditional Chinese medicine, has been proven to have an analgesic effect on peripheral neuropathic pain (pNP) in clinical and basic research. However, the optimal time point for the analgesic effect of tuina may vary according to different injury sensations, affecting the exploration of the initiation mechanism of tuina analgesia. 
The research used minor chronic constriction injury (minor CCI) model rats to simulate pNP and used the intelligent tuina manipulation simulator to simulate the three methods (point-pressing, plucking, and kneading) and three acupoints (Yinmen BL37, Chengshan BL57, and Yanglingquan GB34) for performing tuina therapy. The study evaluated the changes in pain within 24 h and the optimal time point for the efficacy of tuina analgesia in rats with minor CCI models by testing cold sensitivity threshold (CST), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL). Furthermore, the study evaluated IL-10 and TNF-&#945; expression changes through Elisa detection. The results show that tuina has both immediate and sustained analgesic effects. For the three different injury sensitivity thresholds of CST, MWT, TWL, and two cytokines of IL-10 and TNF-&#945;, the analgesic efficacy of tuina within 24 h after intervention is significantly different at different time points.

<meta charset="utf8"></head><body>作者聚焦：探索神经损伤大鼠“三法三穴”推拿镇痛的启动机制

We present a protocol using the tuina manipulation simulator to perform the "Three-Manipulation and Three-Acupoint" tuina therapy for minor chronic constriction injury rats and evaluate the effective analgesic time points of tuina within 24 h by testing pain changes through behavioral analysis and changes in inflammatory factor expression using enzyme-linked immunosorbent assay.

神经损伤大鼠1次推拿干预后24 h内疼痛变化特征

Tuina, as an external treatment method of traditional Chinese medicine, has been proven to have an analgesic effect on peripheral neuropathic pain (pNP) ...

神经科学

改进大鼠模型中的 FSN 治疗:最大限度地减少不适并最大限度地提高效率

Efficacy of Fu's Subcutaneous Needling on Sciatic Nerve Pain: Behavioral and Electrophysiological Changes in a Chronic Constriction Injury Rat Model

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the mechanisms of action are still not fully understood. During FSN treatment, the FSN needle is inserted and retained in the subcutaneous tissues for a long duration with a swaying movement. However, challenges arise from maintaining a posture while manipulating FSN in animal models (e.g., rats) for researchers. Uncomfortable treatment can lead to fear and resistance to FSN needles, increasing the risk of injury and may even affect research data. Anesthesia may also affect the study results too. Hence, there is a need for strategies in FSN therapy on animals that minimize injury during the intervention. This study employs a chronic constriction injury model in Sprague-Dawley rats to induce neuropathic pain. This model replicates the pain induced by nerve injury observed in humans through surgical constriction of a peripheral nerve, mimicking the compression or entrapment seen in conditions such as nerve compression syndromes and peripheral neuropathies. We introduce an appropriate manipulation for easily inserting an FSN needle into the subcutaneous layer of the animal&#39;s body, including needle insertion and direction, needle retention, and swaying movement. Minimizing the rat&#39;s discomfort prevents the rat from being tense, which causes the muscle to contract and hinder the entry of the needle and improves the study efficiency.

作者聚焦:揭示 FSN 治疗的治疗效果 – 桥接神经性疼痛的研究和临床应用

We present a protocol for using Fu's subcutaneous needling in a chronic constriction injury model to induce sciatic nerve pain in rats.

傅氏皮下针刺治疗坐骨神经疼痛的疗效:慢性缩窄损伤大鼠模型中的行为和电生理变化

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the ...

模仿丁氏滚动法治疗大鼠肌损伤

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神经损伤大鼠1次推拿干预后24 h内疼痛变化特征

神经损伤大鼠1次推拿干预后24 h内疼痛变化特征

神经损伤大鼠1次推拿干预后24 h内疼痛变化特征

傅氏皮下针刺治疗坐骨神经疼痛的疗效:慢性缩窄损伤大鼠模型中的行为和电生理变化

傅氏皮下针刺治疗坐骨神经疼痛的疗效:慢性缩窄损伤大鼠模型中的行为和电生理变化