This work was supported by Shanghai Critical Clinical Specialties Construction Project (Grant Number: Shslczdzk04001); the Sailing program of Shanghai Science and Technology Commission (Grant Number: 22YF1444300); Projects within the budget of Shanghai University of Traditional Chinese Medicine (Grant Number: 2021LK091).

This study has passed the animal ethics review conducted by the experimental animal ethics committee of Yueyang Hospital of integrated traditional Chinese and western medicine affiliated with Shanghai University of Traditional Chinese Medicine (YYLAC-2022-166).
1. Experimental animal preparation and grouping
<ol>
	<li>Animal Preparation
	<ol>
		<li>Rear a total of 10 healthy SPF SD female rats of 200-220 g at room temperature (18-21 &#176;C), humidity 40%-50%, 12 h: 12 h cir...

Standardized Tuina Manipulation for Knee Osteoarthritis in Rats

<ol>
	<li>Joint Surgery Group of Chinese Orthopaedic Association, Chinese Association of Orthopaedic Surgeons, National Clinical Research Center for Geriatric Diseases, Chinese Journal of Orthopaedics. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chinese+Osteoarthritis+Treatment+Guidelines+(2021+Edition).">Chinese Osteoarthritis Treatment Guidelines (2021 Edition).</a> Chinese Journal of Orthopaedics. 41 (18), 24 (2021).</li><li>David, S., 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=Epidemiology+of+knee+osteoarthritis+in+general+practice:+a+registry-based+study.">Epidemiology of knee osteoarthritis in general practice: a registry-based study.</a> BMJ Open. 10 (1), 031734 (2020).</li><li>Callahan, L. F., Cleveland, R. J., Allen, K. D., Golightly, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Racial/Ethnic,+Socioeconomic,+and+Geographic+Disparities+in+the+Epidemiology+of+Knee+and+Hip+Osteoarthritis.">Racial/Ethnic, Socioeconomic, and Geographic Disparities in the Epidemiology of Knee and Hip Osteoarthritis.</a> Rheumatic Disease Clinics of North America. 47 (1), 1-20 (2021).</li><li>Wang, K., Dong, X., Lin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+Medical+Costs+of+Disease+In+Patients+With+Osteoarthritis+of+the+Knee+Joint.">Investigation of Medical Costs of Disease In Patients With Osteoarthritis of the Knee Joint.</a> National Medical Journal of China. 97 (1), 4 (2017).</li><li>Perlman, 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=Efficacy+and+Safety+of+Massage+for+Osteoarthritis+of+the+Knee:+a+Randomized+Clinical+Trial.">Efficacy and Safety of Massage for Osteoarthritis of the Knee: a Randomized Clinical Trial.</a> Journal of General Internal Medicine. 34 (3), 379-386 (2019).</li><li>Xing, H., 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+massage+for+knee+osteoarthritis:+a+systematic+review+and+meta-analysis+of+randomized+controlled+trials.">Therapeutic massage for knee osteoarthritis: a systematic review and meta-analysis of randomized controlled trials.</a> Journal of Acupuncture and Tuina Science. 19 (5), 354-363 (2021).</li><li>Seo, B. 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=Skeletal+muscle+regeneration+with+robotic+actuation-mediated+clearance+of+neutrophils.">Skeletal muscle regeneration with robotic actuation-mediated clearance of neutrophils.</a> Science translational medicine. 13 (614), (2021).</li><li>Wu, J. H., Zhang, C., Dong, S. J., Yin, H. <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+with+#34;Relaxing+Tendons&amp;#34;+Technique+on+the+Interleukin-1B+and+5-hydroxytryptamine+Levels+in+the+Joint+Fluid+of+a+Rabbit+Knee+Osteoarthritis+Model.">Effects of Massage with #34;Relaxing Tendons&amp;#34; Technique on the Interleukin-1B and 5-hydroxytryptamine Levels in the Joint Fluid of a Rabbit Knee Osteoarthritis Model.</a> Chinese General Practice. 21 (6), 688-693 (2018).</li><li>Luo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+IL-1B,IL-6,IL-13,IL-26,TNF-a+by+patellar+manipulations+on+rabbit+knee+osteoarthritis+model.">The effects of IL-1B,IL-6,IL-13,IL-26,TNF-a by patellar manipulations on rabbit knee osteoarthritis model.</a> Guangxi University of Traditional Chinese Medicine. , (2017).</li><li>Qiu, F., Li, C., Wu, X., Liu, Y., Zhang, X. <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+Massage+on+the+Function+of+Foot-Yangming+Meridian-Muscle+in+Patients+with+Knee+Osteoarthritis.">Effect of Massage on the Function of Foot-Yangming Meridian-Muscle in Patients with Knee Osteoarthritis.</a> Acta Chinese Medicine. 36 (3), 649-655 (2021).</li><li>Yang, B., Li, S. <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+of+Massage+in+Improving+Biomechanical+Indexes+of+Knee+Osteoarthritis.">Research Progress of Massage in Improving Biomechanical Indexes of Knee Osteoarthritis.</a> Acta Chinese Medicine. 37 (12), 2571-2576 (2022).</li><li>Li, C., Qiu, F., Ding, J., Hu, G., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curative+Observation+of+Retaining+of+Heated+Needle+at+Trigger+Points+Combined+with+Manipulation+for+Muscles+along+Meridians+in+Treating+Knee+Osteoarthritis.">Curative Observation of Retaining of Heated Needle at Trigger Points Combined with Manipulation for Muscles along Meridians in Treating Knee Osteoarthritis.</a> Journal of Guangzhou University of Traditional Chinese Medicine. 37 (11), 2157-2162 (2020).</li><li>Ding, X., Zhang, X., Hou, Y., Zhao, Z., Ye, X. <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+massage+manipulation+on+joint+stiffness+of+knee+osteoarthritis+patients+based+on+&amp;#34;spine-pelvis-knee&amp;#34;+holistic+diagnosis+and+treatment+pattern.">Effect of massage manipulation on joint stiffness of knee osteoarthritis patients based on &amp;#34;spine-pelvis-knee&amp;#34; holistic diagnosis and treatment pattern.</a> Shanghai Journal of Traditional Chinese Medicine. 55 (08), 54-57 (2021).</li><li>Fu, Y., Gong, L., Li, 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+clinical+studies+of+Rolling+combined+with+Pulling+Manipulation+on+Early+and+Middle-term.">The clinical studies of Rolling combined with Pulling Manipulation on Early and Middle-term.</a> Jilin Journal of Chinese Medicine. 40 (07), 958-962 (2020).</li><li>Jiang, J., Hu, X., Tang, R., Qiu, F., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Change+of+lower+limb+force+line+in+treating+knee+osteoarthritis+with+manipulation.">Change of lower limb force line in treating knee osteoarthritis with manipulation.</a> Journal of Changchun University of Chinese Medicine. 34 (01), 129-132 (2018).</li><li>Liang, Y., 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=Some+issues+on+animal+experiments+standardization+of+acupuncture+and+moxibustion.">Some issues on animal experiments standardization of acupuncture and moxibustion.</a> Lishizhen Medicine and Materia Medica Research. 25 (09), 2299-2300 (2014).</li><li>Guo, Y., Liu, Y., Liu, Q., Chen, Z., Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+System+of+Chinese+Acupuncture+Standard.">Basic System of Chinese Acupuncture Standard.</a> Chinese Acupuncture &amp; Moxibustion. 31 (6), 549-550 (2011).</li><li>YAN, X., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+the+Standardization+of+Classification+of+Tuina+Manipulation.">Study on the Standardization of Classification of Tuina Manipulation.</a> Acta Chinese Medicine. 32 (5), 875-878 (2017).</li><li>Aikebaier, G., Lu, X., Liu, J., Liu, l., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+on+Manipulation+and+Acupoint+Selection+Laws+of+Massage+for+Treatment+of+Knee+Osteoarthritis+Based+on+Data+Mining+Technology.">Analysis on Manipulation and Acupoint Selection Laws of Massage for Treatment of Knee Osteoarthritis Based on Data Mining Technology.</a> Chinese Journal of Information on Traditional Chinese Medicine. 29 (5), 23-29 (2022).</li><li>Gong, L., Wuquan, S., Zhang, H., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+of+Yan+Juntao's+Academic+Experiences+of+Differential+Treatment+and+Manipulation+for+Treating+of+Knee+Osteoarthritis.">Research of Yan Juntao's Academic Experiences of Differential Treatment and Manipulation for Treating of Knee Osteoarthritis.</a> Chinese Journal of Traditional Medical Traumatology &amp; Orthopedics. 24 (7), 16-19 (2016).</li><li>Qian, J., Xing, X., Liang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+Methods+to+Establish+Rat+Model+of+Osteoarthritis+of+the+Knee.">Two Methods to Establish Rat Model of Osteoarthritis of the Knee.</a> Research and Exploration in Laboratory. 33 (11), 23-27 (2014).</li><li>Liu, J., 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=Experimental+study+of+a+modified+Videman+method+for+replicating+knee+osteoarthritis+on+rabbit.">Experimental study of a modified Videman method for replicating knee osteoarthritis on rabbit.</a> Rehabilitation Medicine. 30 (03), 212-219 (2020).</li><li>Moskowitz, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis+cartilage+histopathology:+grading+and+staging.">Osteoarthritis cartilage histopathology: grading and staging.</a> Osteoarthritis and Cartilage. 14 (1), 1-2 (2005).</li><li>Shen, M., Li, Z., Shen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+Exploration+of+Experiment+Teaching+on+Experiment+Acupuncture+Science.">Preliminary Exploration of Experiment Teaching on Experiment Acupuncture Science.</a> Chinese Medicine Modern Distance Education of China. 7 (02), 130-131 (2009).</li><li>Jeong, J., 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=Anti-osteoarthritic+effects+of+ChondroT+in+a+rat+model+of+collagenase-induced+osteoarthritis.">Anti-osteoarthritic effects of ChondroT in a rat model of collagenase-induced osteoarthritis.</a> BMC complementary and alternative medicine. 18 (1), 131 (2018).</li><li>Hulth, A., Lindberg, L., Telhag, 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+osteoarthritis+in+rabbits.+Preliminary+report.">Experimental osteoarthritis in rabbits. Preliminary report.</a> Acta orthopaedica Scandinavica. 41 (5), 522-530 (1970).</li><li>Tawonsawatruk, T., Sriwatananukulkit, O., Himakhun, W., Hemstapat, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+pain+behaviour+and+osteoarthritis+progression+between+anterior+cruciate+ligament+transection+and+osteochondral+injury+in+rat+models.">Comparison of pain behaviour and osteoarthritis progression between anterior cruciate ligament transection and osteochondral injury in rat models.</a> Bone &amp; Joint Research. 7 (3), 244-251 (2018).</li><li>Zhou, 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=Cartilage+matrix+changes+in+contralateral+mobile+knees+in+a+rabbit+model+of+osteoarthritis+induced+by+immobilization.">Cartilage matrix changes in contralateral mobile knees in a rabbit model of osteoarthritis induced by immobilization.</a> BMC musculoskeletal disorders. 16, 224 (2015).</li><li>Zeng, J., 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=Establishment+and+identification+of+experimental+rabbit+model+of+knee+osteoarthritis.">Establishment and identification of experimental rabbit model of knee osteoarthritis.</a> Chinese Journal of Clinical Research. 29 (5), 679-682 (2016).</li><li>Shang, 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=Comparison+with+two+kind+osteoarthritis+animal+models+reduced+by+plaster+immobilization+in+extend+excessive+position+and+bend+excessive+position+respectively.">Comparison with two kind osteoarthritis animal models reduced by plaster immobilization in extend excessive position and bend excessive position respectively.</a> Orthopaedic Biomechanics Materials and Clinical Study. (1), 11-14 (2006).</li><li>He, Y., 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=Evaluation+of+the+effect+of+improved+cast+immobilization+method+on+rabbit+knee+osteoarthritis+model.">Evaluation of the effect of improved cast immobilization method on rabbit knee osteoarthritis model.</a> Chinese Imaging Journal of Integrated Traditional and Western Medicine. 18 (2), 198-219 (2020).</li><li>Pengfei, S., 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=Possible+mechanism+underlying+analgesic+effect+of+Tuina+in+rats+may+involve+piezo+mechanosensitive+channels+within+dorsal+root+ganglia+axon.">Possible mechanism underlying analgesic effect of Tuina in rats may involve piezo mechanosensitive channels within dorsal root ganglia axon.</a> Journal of Traditional Chinese Medicine. 38 (6), 834-841 (2018).</li><li>Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Massage+Mediating+Chondrocyte+Apoptosis+in+Knee+Osteoarthritis+Through+Piezo+1/JAK2+Signaling+Pathway.">Mechanisms of Massage Mediating Chondrocyte Apoptosis in Knee Osteoarthritis Through Piezo 1/JAK2 Signaling Pathway.</a> Shandong University of Traditional Chinese Medicine. , (2019).</li><li>Liu, J. <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+Massage+on+TLR4/MyD88+Signal+Transduction+Pathway+in+Rat+Knee+Osteoarthritis+Model.">Effect of Massage on TLR4/MyD88 Signal Transduction Pathway in Rat Knee Osteoarthritis Model.</a> Guangzhou University of Traditional Chinese Medicine. , (2019).</li><li>Yu, S., Zhou, J., Pang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+induced+animal+models+of+knee+osteoarthritis.">Advances in induced animal models of knee osteoarthritis.</a> Journal of Guangxi University of Chinese Medicine. 25 (5), 50-55 (2022).</li><li>Tan, 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=Acupuncture+combined+with+moxibustion+regulates+the+expression+of+circadian+clock+protein+in+the+synovium+of+rats+with+osteoarthritis.">Acupuncture combined with moxibustion regulates the expression of circadian clock protein in the synovium of rats with osteoarthritis.</a> Chinese Journal of Tissue Engineering Research. 26 (11), 1714-1719 (2022).</li><li>Zhang, Z., 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+Review+of+Massage+Therapy+for+Knee+Osteoarthritis.">A Review of Massage Therapy for Knee Osteoarthritis.</a> Henan Traditional Chinese Medicine. 39 (1), 146-149 (2019).</li><li>Gong, L., Sun, W., Zhang, H., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+of+Yan+Juntao's+Academic+Experiences+of+Differential+Treatment+and+Manipulation+for+Treating+of+Knee+Osteoarthritis.">Research of Yan Juntao's Academic Experiences of Differential Treatment and Manipulation for Treating of Knee Osteoarthritis.</a> Chinese Journal of Traditional Medical Traumatology &amp; Orthopedics. 24 (7), 16-19 (2016).</li><li>Chen, C., Zhang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+on+the+rules+of+acupoint+selection+for+the+treatment+of+knee+osteoarthritis+with+massage+based+on+data+mining.">Research on the rules of acupoint selection for the treatment of knee osteoarthritis with massage based on data mining.</a> Hainan Medical Journal. 29 (18), 2617-2619 (2018).</li></ol>

The authors declare that they have no competing interests.

This study provides a protocol for Tuina manipulation on KOA rats. Through pain behavior tests and histomorphological findings, it suggested that such a series of Tuina manipulation applied to KOA rats could reduce synovial inflammation and cartilage apoptosis, which could be a reference of Tuina manipulation on animal models of KOA.
There are several critical procedures during the protocol. First, it&#39;s important to choose an appropriate method for inducing the KOA model. There are various...

Knee osteoarthritis (KOA) is a degenerative disease mainly manifested in joint pain. Fibrosis, cracking, ulceration, and loss of articular cartilage are the main causes of this disease1. KOA has a high prevalence and may result in a profound impact on the daily life of patients, causing disability in severe cases. Among people aged 45-84 years, the prevalence of KOA increases with age, and the prevalence among people aged 85 years and above is 15%, with a predominance in women2,3. In addition, KOA might bring a serious economic burden on both the individual and the society. A survey showed that direct health care costs on KOA per capita reached up to $8,858 &#177; $5,120 per year4. With the aging of society, KOA has become a worldwide health problem and a major social issue, as well as a topical issue for scientific research.
Evidence-based studies have shown the effectiveness of Tuina manipulation in treating KOA5. Tuina manipulation could relieve pain and improve dysfunction in KOA patients, the mechanism of which is related to anti-inflammatory effects6,7. Scholars found that Tuina manipulation effectively inhibited the expression of inflammatory factors interleukin (IL)-&#946; and 5-hydroxytryptamine and slowed down the degeneration of articular cartilage in a rabbit KOA model8. It suggested that Tuina could promote blood circulation and metabolism at the lesion site, which helped clear inflammatory factors such as IL-1, IL-6 and tumor necrosis factor (TNF)-&#945;, thereby alleviating the clinical symptoms of KOA9. In addition, the passive movement of the joint through Tuina manipulation can promote the penetration and diffusion of synovial fluid into the articular cartilage and improves tissue nutrient metabolism10. Other studies suggested that Tuina manipulation can effectively improve the biomechanical indexes in KOA patients11. Manipulations applied on soft tissues can improve stress distribution over limbs and enhance balance function12,13. At the same time, with some joint adjustment manipulations, the alignment of the lower limbs can also be adjusted to correct abnormal gaits14,15.
The mechanism of action of Tuina manipulation in treating KOA remains to be explored, and therefore, an experimental study is necessary. The key to the application of Tuina in experimental animals is the standardization of modeling, animal fixation and intervention methods16. The modeling method determines whether the experimental animal can exhibit the characteristics of the disease. Meanwhile, appropriate fixation methods can facilitate the intervention of the Tuina manipulation and better reflect the effect of Tuina. The standardization of intervention methods is the most difficult part of Tuina manipulation. In 2010, the basic system of Chinese acupuncture standards mentioned the acupuncture point standards for experimental animals, providing the possibility for acupuncture and Tuina operations in animal experiments17. However, there are still difficulties in standardizing Tuina manipulation. There are multiple types of Tuina manipulation18. The choice of the specific manipulation mainly depends on the disease to be treated and the therapeutic theories the performer prefers. In the study of Tuina for KOA, more attention has been paid to the point-pressing manipulation (pressing the specific acupoints with the thumb or elbow), Yizhichan pushing manipulation (a pushing manipulation by wiggling the thumb), and press and kneading manipulation (which refers to pressing and kneading the specific area of the body surface by finger or palm)19. Press and kneading manipulation is one of the most widely used Tuina manipulations, which combines pressing and kneading to move the subcutaneous tissue20. Press and kneading manipulation applied on acupoints can promote blood circulation and relieve pain and represents the therapeutic effect of Tuina on KOA19.
In this protocol, the operation of press and kneading manipulation on KOA rats will be described in detail, including selected acupoints, intensity and frequency of the manipulation and the body position of the rat, so as to provide a reference for future research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>absolute ethanol</td>
			<td>Supelco</td>
			<td>PHR1070</td>
			<td>For making specimen</td>
		</tr>
		<tr>
			<td>ALMEMO admeasuring apparatus</td>
			<td>ahlborn</td>
			<td>2450-1</td>
			<td>For Mechanical Withdrawal Threshold test</td>
		</tr>
		<tr>
			<td>Anti-Digoxin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>SAB4200669</td>
			<td>For HE stain IHC or TUNEL</td>
		</tr>
		<tr>
			<td>Anti-IL-1 beta</td>
			<td>abcam</td>
			<td>ab283818</td>
			<td>For HE stain IHC or TUNEL</td>
		</tr>
		<tr>
			<td>DAB Substrate kit</td>
			<td>Solarbio</td>
			<td>DA1010</td>
			<td>For HE stain IHC or TUNEL</td>
		</tr>
		<tr>
			<td>Denture base materials</td>
			<td>Shanghai New Century</td>
			<td>20000356</td>
			<td>For model making</td>
		</tr>
		<tr>
			<td>eosin</td>
			<td>bioswamp&#160;</td>
			<td>PAB180016&#160;</td>
			<td>For HE stain IHC or TUNEL</td>
		</tr>
		<tr>
			<td>Finger pressure recordings</td>
			<td>Suzhou Changxian Optoelectronic Technology</td>
			<td>CX1003w</td>
			<td>For Tuina manipulation</td>
		</tr>
		<tr>
			<td>formic acid solution</td>
			<td>Sigma-Aldrich</td>
			<td>695076</td>
			<td>For decalcification</td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma-Aldrich</td>
			<td>386790-M</td>
			<td>For HE stain IHC or TUNEL</td>
		</tr>
		<tr>
			<td>hematoxylin</td>
			<td>bioswamp&#160;</td>
			<td>PAB180015</td>
			<td>For HE stain IHC or TUNEL</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Shanghai Yuyan Scientific Instrument Company</td>
			<td>S10010533</td>
			<td>For gas anesthesia</td>
		</tr>
		<tr>
			<td>neutral resins</td>
			<td>bioswamp&#160;</td>
			<td>PAB180017</td>
			<td>For HE stain IHC or TUNEL</td>
		</tr>
		<tr>
			<td>Paraformaldehyde Fix Solution</td>
			<td>Sigma-Aldrich</td>
			<td>100496</td>
			<td>For histology</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>P3813</td>
			<td>For HE stain IHC or TUNEL</td>
		</tr>
		<tr>
			<td>Plantar Test Apparatus</td>
			<td>IITC Life Science</td>
			<td>/</td>
			<td>For Paw Withdrawal Latency test</td>
		</tr>
		<tr>
			<td>plaster of Paris bandage</td>
			<td>WANDE</td>
			<td>20150023</td>
			<td>For model making</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td>124568</td>
			<td>For HE stain IHC or TUNEL</td>
		</tr>
		<tr>
			<td>TNF Alpha Monoclonal antibody</td>
			<td>Proteintech</td>
			<td>60291-1-Ig</td>
			<td>For HE stain IHC or TUNEL</td>
		</tr>
		<tr>
			<td>TUNEL&#160;</td>
			<td>Servicebio</td>
			<td>GDP1042</td>
			<td>For HE stain IHC or TUNEL</td>
		</tr>
		<tr>
			<td>Wax</td>
			<td>Sigma-Aldrich</td>
			<td>327204</td>
			<td>For making specimen</td>
		</tr>
		<tr>
			<td>xylene</td>
			<td>Shanghai Sinopharm Group</td>
			<td>100092</td>
			<td>For making specimen</td>
		</tr>
	</tbody>
</table>

tuina manipulation to reduce inflammation and cartilage loss in knee osteoarthritis rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Author Spotlight: Understanding the Mechanism of Tuina Manipulation in Rats 

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

We studied the mechanism of Tuina manipulation in treating knee osteoarthritis. We found that Tuina is effective for pain relieving and improving motor functions. However, there is still standard protocols for Tuina of KOA, and the conclusions are not reliable enough, so we are trying to build a standard protocol.Recently, many teams have begun to develop Tuina on massage robots, which can output special mechanical wave forms for manipulation to achieve standardization of techniques. In addition, there are some cell mechanic loading devices, which can now put the manual force for the cell for cellular research. We believe that the difficulty of Tuina in animals is how to maintain the standardization of manipulation and how to accurately locate acupuncture points.We preliminarily verified the effect of Tuina on inhibiting synovial inflammation and the cartilage of proptosis through preliminary experiments, which will point out the direction for future research on the mechanism of Tuina for KOA. In the future, we'll focus on the mechanism of Tuina for KOA, mainly related to the mechanical signal transduction pathway, and how to inhibit synovial inflammation and cartilage apoptosis.

Pain behavior tests 
The MWT results showed that the MWT of the right hind limb after modeling was significantly lower than before (p&#60;0.05). Compared with the control group, the MWT of rats was significantly elevated after Tuina (p&#60;0.05; Figure 5 and Table 2).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65451/65451fig05.jpg" /> 
Figure...

Watch this Scientific Journal Video about Understanding the Mechanism of Tuina Manipulation in Rats at JoVE.com

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...

tuina-manipulation-to-reduce-inflammation-cartilage-loss-knee

Research

JoVE Journal

Medicine

Experimental Modeling of Knee Osteoarthritis in Rats

After anesthetizing the rat, use a shaving machine to remove the hair of the right hind limb. Then, place a medical cotton pad between the right ankle and hip joint. Fix the right knee joint at 180 degree extension using five to six layers of wet plaster bandage evenly.Spiral wrap the plaster bandage starting from the ankle and covering one-third of the previous one. Use a hairdryer to dry and harden the plaster. After the plaster bandage dries and hardens, mix the denture base materials to make it sticky.And externally wrap the plaster with denture based materials to fix and prevent it from gnawing. Once the mixture hardens, turn off the anesthesia machine and supervise the blood circulation in the terminal limb. Then, wait for the rat to wake up naturally.After three weeks of continuous immobilization, using surgical scissors, cut off the denture base material outside and plaster bandage. Rinse the lower limb of the rat with saline and dry it with gauze.

Tuina Manipulation, Pain Behavior Tests, and Statistical Analysis

Select five acupoints on the right hind limb of the rat according to the principles of acupoint positioning. Before the Tuina manipulation, gently pull the rat tail to make it burrow into the bag and expose its hind limbs outside the bag. For Tuina manipulation, using one hand, place the rat in the prone position, holding its tail and hind leg, then place the performer's thumb on the selected acupuncture point to perform rhythmic press and kneading, driving the skin and subcutaneous tissue together in a circular motion ensuring consistent intensity and frequency of manipulation.For the next mechanical withdrawal threshold, or MWT, stimulate the rat with electronic Von Frey fiber at the center of the foot and withdraw the fiber when the rat shows obvious movements, like leg grazing and avoidance. The machine automatically records the maximum pressure value in Newtons. After measurement, delete the values with large differences and take the average of the remaining three values as the MWT.Next, for paw withdrawal latency, or PWL, focus the spotlight on the center of the rat's foot and press the start button. Monitor the rat for behaviors such as foot retraction or paw licking, and record the time before pressing the stop button. After measurement, delete the values with large differences and take the average of the remaining three values as the PWL.For statistical analysis, open the analysis software. If the data conforms to a normal distribution, perform a T-test. Alternatively, analyze repeated measurement data by generalized estimating equations.The MWT results showed that the MWT of the right hind limb after modeling was significantly lower than before. Compared with the control group, the MWT of rats was significantly elevated after Tuina. The PWL results showed that the PWL of the right hind limb after modeling was significantly shorter than before.Compared with the control group, the MWT of rats was significantly prolonged after Tuina.

1.1K Views.  Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Shanghai University of Traditional Chinese Medicine.  We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

Standardized Tuina Manipulation for Knee Osteoarthritis in Rats

Summary