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

Summary

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.

Abstract

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)-α 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.

Introduction

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 women^2,3. In addition, KOA might bring a serious economic burden on both the individual and the society. A survey sho....

Protocol

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

1. Animal Preparation
   1. Rear a total of 10 healthy SPF SD female rats of 200-220 g at room temperature (18-21 °C), humidity 40%-50%, 12 h: 12 h circadian rhythm alternations. Conduct pain-related animal experiments in strict compliance with the relevant provisions of animal ethical guidelines and guid....

Results

Pain behavior tests
The MWT results showed that the MWT of the right hind limb after modeling was significantly lower than before (p<0.05). Compared with the control group, the MWT of rats was significantly elevated after Tuina (p<0.05; Figure 5 and Table 2).

Discussion

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's important to choose an appropriate method for inducing the KOA model. There are various.......

Materials

List of materials used in this article

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