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 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.
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 ± $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)-β 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)-α, 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.
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
2. Modeling of animals
Table 1. OA cartilage histopathology grade assessment. Grade is depth progression into cartilage. Total score = Grade x Staging. 0 for normal joints, 24 for severe arthritis. Please click here to download this Table.
Figure 1. Rats immobilized in plaster. After the rats were anesthetized, their right lower limbs were wrapped with plaster bandages, fixed in the hyperextended position, and covered with a layer of denture base materials outside. Please click here to view a larger version of this figure.
3. Tuina manipulation
Figure 2. Acupoints position. SP10 is located 5 mm above the inner knee joint in rats. ST34 is located 5 mm above the outer knee joint in rats. EX-LE4 is located in the medial side of the knee ligament in rats. ST35 is located in the lateral side of the knee ligament in rats. BL40 is located at the midpoint of the transverse popliteal stripe. Please click here to view a larger version of this figure.
Figure 3. Tuina manipulation applied on rats. The rats were kept in a black bag with their hind limbs exposed. The performer held the rat's tail with the left hand while the right hand performed the manipulation. Please click here to view a larger version of this figure.
Figure 4. Finger pressure recordings. A device that records the force and frequency of finger pressure is used for real-time feedback on the intensity and frequency in the process of Tuina manipulation. (A) Pressure sensor and transmission equipment. (B) Finger pressure recordings. (C) The force recorded during Tuina manipulation. Please click here to view a larger version of this figure.
4. Pain Behavior Tests
5. Sample preparation
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).
Figure 5. Results of the mechanical withdrawal threshold test (, Newton). There were 5 rats each in the Tuina group and the control group. The MWT of the rats at different time points is shown in the figure. After the modeling, the MWT of the rats decreased significantly, suggesting that the pain of the rats was aggravated, and the KOA model was successfully prepared. Subsequently, the MWT gradually improved, suggesting pain relief. The generalized estimation equation was used for statistical calculation. The difference in MWT between the Tuina group and the control group was statistically significant at day 21 compared to that after modeling. The comparison between the two groups was statistically significant at D7, D14 and D21, *p<0.05. Moreover, rats in Tuina group have higher MWT than that in the control group. Please click here to view a larger version of this figure.
Table 2. Mechanical Withdrawal Threshold ( , N). Comparison before and after modeling, #p<0.05. Comparison between Tuina group and control group, *p<0.05. Please click here to download this Table.
The PWL results showed that the PWL of the right hind limb after modeling was significantly shorter than before (p<0.05). Compared with the control group, the MWT of rats was significantly prolonged after Tuina (p<0.001; Figure 6 and Table 3).
Figure 6. Results of the paw withdrawal latency test (, second). There were 5 rats each in the Tuina group and the control group. The PWL of the rats at different time points is shown in the figure. After the modeling, the PWL of the rats decreased significantly, suggesting that the pain of the rats was aggravated, and the KOA model was successfully prepared. At first, the improvement of PWT in the Tuina group was slower than that in the control group. After D7, rats in the Tuina group improved rapidly and surpassed the control group at D21. The generalized estimation equation was used for statistical calculation. The difference in MWT between the Tuina group and the control group was statistically significant at day 21 compared to that after modeling. The comparison between the two groups was statistically significant at D7, D14 and D21, ***p<0.001. Please click here to view a larger version of this figure.
Table 3. Paw Withdrawal Latency (, s). Comparison before and after modeling, #p<0.05. Comparison between the Tuina group and the control group, ***p<0.001. Please click here to download this Table.
Histomorphological assay
Upon analyzing the synovial membrane in the control group, inflammatory cell infiltration and fibrous tissue hyperplasia were seen in synovial tissue. The synovial cells were disorganized, and a small amount of capillary hyperplasia was seen around the synovial tissue (Figure 7).
Figure 7. Microscopic observation of the synovial tissue. (A) Synovial tissue in the control group. The disorganized arrangement of synovial cells is seen in the figure. The proliferating vessels are marked with red arrows, and inflammatory cells are marked with black arrows. (B) Synovial tissue in Tuina group. Synovial cells were more neatly arranged. Inflammatory cells are mainly distributed at the edges rather than infiltrating inwards. The proliferating vessels are marked with red arrows, and inflammatory cells are marked with black arrows. Please click here to view a larger version of this figure.
In the Tuina group, synovial cells were neatly arranged, with a small amount of inflammatory cell infiltration, fibrous tissue hyperplasia, and a small amount of capillary hyperplasia visible at the tissue margins.
Upon analyzing the cartilage, it was seen that the TUNEL staining positive area in the Tuina group was significantly smaller than that in the control group, indicating that there were fewer apoptotic chondrocytes in the Tuina group (Figure 8).
Figure 8. TUNEL staining of the cartilage. (A) Cartilage in the control group. The red part of the figure is the positive area of TUNEL staining. (B) Cartilage in Tuina group. The red part of the figure is the positive area of TUNEL staining. The positive area in the Tuina group is significantly smaller than that in the control group. Please click here to view a larger version of this figure.
Immunohistochemistry
For TNF-α, there was a significant difference in synovial TNF-α expression between the two groups, and the expression in the Tuina group was significantly lower than that in the control group (Table 4).
Table 4. TNF-α Expression in Synovium (, *10-2). Two independent sample t-test was used for statistical analysis, *p<0.05. Please click here to download this Table.
For IL-1β, there was no significant difference in the amount of synovial IL-1β expression between the two groups, as measured by the statistics of Image J. However, the mean value of the Tuina group was subtly lower, indicating less expression (Table 5).
Table 5. IL-β Expression in Synovium (, *10-2). Two independent sample t-test was used for statistical analysis. Please click here to download this Table.
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 methods to induce KOA model, including joint cavity injection25, intra-articular surgery26,27, joint braking method28,29, etc. Since KOA models induced by invasive methods would leave wounds near the joints, which may interfere with the effect of Tuina manipulation, we chose the non-invasive method of joint immobilization. The joint immobilization method can be divided into hyper-extension and hyper-flexion fixation. Shang et al. compared the effects of these two fixation methods and found there was little difference in the effect of cartilage apoptosis23. However, their experimental subjects were rabbits, which are larger in size and relatively easy to fix compared to rats. Fixed plaster in a flexion position is more likely to fall off under rats gnawing. Therefore, we chose the immobilization method of hyper-extension fixation to induce KOA. We referred to He et al. and used the plaster bandage with a tongue depressor as the fixation device30. However, the gnawing ability of rats was stronger than expected, and the device could not serve as a fixation. Later we found a kind of denture base material, which can form a moldable but hard shell on the outside of the plaster, and effectively reduce the wear and tear of the fixation device by rats gnawing. A tight fixation would affect the blood circulation of the lower limbs, while too loose fixation would be easy to fall off. Therefore, the circulation of the lower limbs of the rats should be observed daily during the 3 weeks of modeling. The fixation should be released when the lower limbs become swollen and purplish. Re-install the fixation when it is loose, and the rats can flex their knees. During this study, the rats were not restricted from socializing during modeling, but it was found that they would chew each other's fixation to help each other break free. Perhaps isolating the rats would have resulted in better modeling. However, isolation might contribute to the rats' depression and stereotypical behavior.
We consider that the key point of Tuina manipulation is to keep the consistency of the intensity and frequency. Previous research concluded that the optimal intensity for animal Tuina should be 80% of its maximum tolerable intensity31. At this point, the rats should show signs of receiving mechanical stimulation, but with no signs of pain or paw retraction. We tested the maximum tolerable intensity of rats, which is around 5-8 N. So, we set the pushing intensity to 3-5 N, which may lead to better efficacy. The frequency of the manipulation is 2 Hz, according to the textbook of Tuina. Previous studies have not set a standardization for Tuina manipulation, and the intensity and frequency might vary after operating different acupoints and altering the left and right hands during the experiment, which can affect the accuracy of the results. The finger pressure recordings used in this experiment provide real-time observation of the intensity and frequency of the manipulation during the experiment, which can keep the consistency of the manipulation. Some scholars use machines to simulate Tuina manipulation and apply it on animals, which has the advantage of standardization of the manipulation32.
For the selection of acupoints, we referred to the study of Wang and Liu and chose five acupoints around the knee joint to facilitate manipulation33,34. We chose the plaster-immobilized method to induce KOA, which mostly ends up with muscle atrophy and joint stiffness35. ST34 and ST35 belong to the foot Yangming stomach meridian which is critical in treating atrophy (mostly manifested as weakness and muscle atrophy) in traditional Chinese medicine theory and has good adaptability for KOA induced by the plaster-immobilized method. EX-LE4 is a common acupoint for the clinical treatment of KOA, mostly used together with ST35. Tan et al. found that acupuncture and moxibustion on ST35, ST36, and EX-LE4 could reduce inflammatory factor expression in the synovial membrane36. SP10 and BL40 are the core acupoints of clinical Tuina treatment for KOA37,38, and SP10 is most frequently used in Tuina prescriptions for KOA39.
A difficult part of Tuina manipulation is the fixation of rats. Rats may struggle or avoid manipulation, which increases the difficulty of performing the experiment. Although rat immobilizers can immobilize rats and expose their hind limbs, the development of KOA limits hind limb flexion and extension. Therefore, settling rats with immobilizers may lead to pain and incomplete exposure of the hind limbs, which may affect Tuina manipulation. We designed a single-exit cloth bag based on the rat's preference for dark environments and its tendency to burrow. Close the cloth bag when the rat enters, which is, the head and forelimbs are located in the bag, and its hind limbs are exposed outside. This method can effectively reduce the struggle of rats and keep them quiet during Tuina.
The results of the pre-experiment suggest that Tuina manipulation may reduce the inflammatory response of synovial tissue and decrease the apoptosis of chondrocytes, thus acting as a treatment for KOA. However, more studies are needed to verify this.
There are several shortcomings of this protocol. First, the small size of rats makes it difficult to locate the acupoints precisely. The operator's fingers are too large compared to the acupoints on rats, which may affect the effect of Tuina manipulation. We had considered adding a rubber particle of about 2 mm in diameter on the fingertip to increase the accuracy of stimulating acupoints. But the particles might lead to higher intensity of pressure and increase the difficulty in controlling the consistency. In addition, it was more likely to damage the finger pressure recordings, so we did not choose this method. Second, our study ignored the change of inflammatory factors in the synovial fluid and chondrocyte apoptosis, which may weaken the credibility of the study. We will intensify research in this area in the future. Third, press and kneading manipulation is one of the Tuina manipulations, and cannot encompass the effects of Tuina in treating KOA. Other Tuina manipulations implemented in animals need to be further explored, including joint movement manipulations, and rubbing manipulation. Moreover, positive control would better show the effectiveness of the manipulation, but it was not designed in this study, and we will add this in a follow-up study.
Overall, this study provides a standardized protocol for Tuina manipulation on KOA rats, and the effectiveness of the manipulation has been verified by pain behavior tests and microscopical findings. The protocol preliminary proves that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis. More research is needed to explore the mechanism of Tuina for KOA.
The authors have nothing to disclose.
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).
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 |