This work was supported by the Shandong Provincial Traditional Chinese Medicine Science and Technology Project (2021Q080) and the Qilu School of Traditional Chinese Medicine Academic School Inheritance Project [Lu-Wei-Letter (2022) 93].

The study was approved by the ethics committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine (approval number: 2020-29).
1. Experimental animals
<ol>
	<li>Raise 18, 6-month-old normal-grade male New Zealand rabbits (2.75 &#177; 0.25 kg) in standard single cages (12 h light/dark cycle, temperature 20-24 &#176;C, air humidity 40%-60%).</li>
</ol>
2. Grouping method
<ol>
	<li>Select 6 out of the 18 New Zealand rabbits as the sham group using the random number method and assign the remaining 12 rabbits to the modeling group.</li>
	<li>After successful modeling, divide the modeling group rabbits into the model and Tuina groups according to the random number method, with 6 rabbits in each group.</li>
	<li>Perform the Tuina intervention in the Tuina group. Perform the same grasping and fixation in the sham and model group without Tuina. Operate every other day for 4 weeks (Figure 1).</li>
</ol>
3. Establishment of the KOA model
<ol>
	<li>Perform adaptive feeding to rabbits in the standard condition in week 1. Ad libitum access to water and food. Place rabbits on their right sides in the rabbit fixation boxes to calm them down for 15 min a day. Fix their heads on the head-fixing plates. Fix the fastening plates and screws so the rabbits cannot move. Wear protective gloves when grasping and fixing the rabbits (Figure 1).</li>
	<li>On days 1, 4, and 7 of week 2,&#160;place all 18 rabbits on their right sides in the rabbit fixation boxes&#160;(Figure 1).&#160;Perform the below-mentioned operations.</li>
	<li>Inject 3% pentobarbital sodium (1 ml/kg) into the rabbit&#39;s marginal ear vein. Shave the rabbit&#39;s left knee joint with an animal shaver, resulting in no hair on the exposed skin.</li>
	<li>Disinfect the rabbit&#39;s left knee joint from the inside out using medical iodophor and 75% alcohol (Figure 2A).</li>
	<li>Flex the rabbit&#39;s left knee joint at 60&#176;. Insert a needle (22G, 0.7 mm x 30 mm) from Waixiyan. Inject 4% papain solution (0.1 mL/kg, 0.275 mL&#160;on average for a 2.5 kg animal) into the knee joint cavity of the modeling group. Inject an equal amount of 0.9% sodium chloride solution into the sham group. This injection dose is well-tolerated by the animal without causing evidence of pain or distress (Figure 2B). 
	NOTE: Waixiyan (EX-LE5) is located in the patellar ligament&#39;s lateral recess, and Neixiyan (EX-LE4) is located in the medial recess of the patellar ligament20,21,22.</li>
	<li>Press the pinhole for 2 min to avoid solution spillage.</li>
	<li>Place your hands above and below the rabbit&#39;s left knee joint. Gently and passively flex the rabbit&#39;s knee joint and extend 10 times within the physiological range of motion (ROM) to infiltrate the solution into the knee joint cavity evenly15.Observe the rabbit every 8 h for the duration of the modeling. Administer buprenorphine SR (0.18 mg/kg) when the rabbits show signs of hiding, trembling of limbs, shallow and rapid breathing, or even biting and scratching.</li>
	<li>At week 7, observe the rabbit&#39;s left knee in a flexed position as swollen, with increased muscle tone around the knee with nodules and striae, increased local painful irritation response, decreased knee ROM, lame gait, and shift of the center of gravity to the healthy side. This determines the success of the KOA model (Figure 1, Figure 2C)23,24.</li>
</ol>
4. Tuina manipulation
<ol>
	<li>Perform training using the Tuina technique parameter determination instrument before Tuina manipulation. Train for 1 h a day for 1 month by the same professional.
	<ol>
		<li>Perform the rotatory kneading and pressing method with the thumb on the Tuina manipulation simulation platform with a force of 5 N and a frequency of 60 times/min (Figure 3A,C).</li>
		<li>Analyze the force in three directions of X, Y, and Z axes by the Tuina manipulation parameter processing software and check the force&#39;s magnitude, frequency, and time of action displayed on the screen (Figure 3B, D).</li>
		<li>Evaluate the mechanical parameters of the Tuina manipulation and standardize the Tuina manipulation using the software during training. Maintain the standardized rotatory kneading and pressing method with the thumb with a force of 5 N, a frequency of 60 times/min, and a continuous operating time of 10 min. See the standardized quantitative waveform of the manipulation in Figure 3B,D25,26,27.</li>
	</ol>
	</li>
	<li>Place the rabbit on its right side in the rabbit fixation box. Gently stroke the rabbit for 10s to soothe and relax the rabbit21. Then perform the Tuina intervention.</li>
	<li>Perform the rotatory kneading method with the thumb on the rabbit&#39;s left peri-knee muscles stiffness, tendon knots, and patella, with up and down round-trip manipulation at a force of 5 N and a frequency of 60 times/min for 5 min.</li>
	<li>Use the thumb end to press Yanglingquan (GB 34), Yinlingquan (SP 9), Waixiyan (EX-LE5), Neixiyan (EX-LE4), Heding (EX-LE2), Xuehai (SP 10), Liangqiu (ST 34), and Weizhong (BL 40)20,21,22, with a force of 5 N and a frequency of 60 times/min, and operate on each point for 30 s.</li>
	<li>Perform the rotary correction method on the rabbit knee joint and perform this 3x separately for each animal in the group.
	<ol>
		<li>Fix the femur with one hand. Place the other hand behind the knee joint first, then fix the lateral and medial tibial condyles with the thumb and ring fingers, respectively. Fix the popliteal fossa with the index and middle fingers. Apply traction and twisting force.</li>
		<li>Fix the femur with one hand. Fix the patella&#39;s medial and lateral edges with the thumb and little fingers of the other hand. Fix the patellar base with the index, middle, and ring fingers. Apply twisting force.</li>
		<li>Keep the direction of traction force parallel to the long axis of the tibia, and the direction of torsion force in line with the direction of the lower Xiyan. Use fingers to hold the skin in place to avoid friction between the skin and fingers.</li>
	</ol>
	</li>
</ol>
5. Measurement of the knee joint ROM
NOTE: Before measurement, calm the rabbit. The measurement statistician and operator are different from one another.
<ol>
	<li>Measure the mobility of the left knee joint of rabbits of each group at the beginning of the experiment and the end of each week.</li>
	<li>Position the rabbit on its right side in the rabbit fixation box and fix its left femur with one hand.</li>
	<li>Align the center of the circle of the medical arthroscope with the lateral center of the rabbit&#39;s left knee joint. Extend the fixation arm such that it is parallel to the line extension connecting the circle&#39;s center to the greater trochanter. Extend the mobile arm such that it is parallel to the longitudinal axis of the tibia.</li>
	<li>Place the other hand on the longitudinal axis of the tibia, approximately 9 cm from the knee joint. Manually apply approximately 750-850 g torque at an angular velocity of 3&#176;/s28.</li>
	<li>Perform this till the rabbit&#39;s knee joint is no longer moving . Record the number of degrees the goniometer displays when the joint stops moving; this is the knee joint ROM. When reading, ensure the line of sight is perpendicular to the ruler&#39;s surface.</li>
	<li>Measure ROM for each knee 3x and take the average value28.</li>
</ol>
6. Hematoxylin-eosin (HE) staining
<ol>
	<li>Sample collection
	<ol>
		<li>At 1 week from the end of the intervention (Figure 1), place the rabbit on its right side in the rabbit fixation box (rabbits are more likely to remain relaxed when lying on their right side). Inject pentobarbitone (100 mg/kg) into the rabbit&#39;s ear marginal vein for humane euthanasia29,30.</li>
		<li>Open the left knee cavity quickly with a scalpel, scissors, and hemostatic forceps to remove the soft tissue attached around the cartilage of the distal femur.</li>
		<li>Collect an approximately 1 cm x 1 cm cartilage-bone specimen of the distal femur with biting forceps and place it in saline for cleaning.</li>
	</ol>
	</li>
	<li>Fixation and decalcification
	<ol>
		<li>Place the cartilage in 4% paraformaldehyde solution and fix it for 72 h.</li>
		<li>Rinse in running water for 12 h. Decalcify in ethylenediaminetetraacetic acid (EDTA) decalcification solution for 6 weeks. Change the EDTA decalcification solution every 3 days. Determine the end point of decalcification when the bone tissue becomes soft and flexible, can be easily bent, and pierced smoothly with a needle31.</li>
	</ol>
	</li>
	<li>Dehydration of embedded sections
	<ol>
		<li>Place the specimen in an automatic dehydrator for dehydration.</li>
		<li>Place the waxed and trimmed tissue on the bottom of a square container with dissolved paraffin wax for 1 h. Place them in a cooling oven until cooled and solidified into hard blocks. Slice the paraffin-embedded tissue block in a slicer to a thickness of 4 &#181;m.</li>
		<li>Unfold the sections in the bleach machine, then place them on adhesive slides, number them, and dry them with a slice baking machine and oven.</li>
	</ol>
	</li>
	<li>De-waxing and hydration
	<ol>
		<li>Bake the sections at 65 &#176;C for 60 min.</li>
		<li>Soak the sections in xylene for 7 min, followed by 2 more rounds of soaking in fresh xylene for 7 min each.</li>
		<li>Soak the slice in anhydrous ethanol for 5 min, followed by soaking for 2 min each in 95% ethanol, 85% ethanol, and 75% ethanol.</li>
		<li>Soak sections in distilled water for 2 min.</li>
	</ol>
	</li>
	<li>Hematoxylin staining: Stain sections with hematoxylin for 20 s. Rinse sections in running water. Soak sections in hydrochloric acid ethanol fractionation for 3 s. Rinse sections in tap water for 5 min.</li>
	<li>Eosin re-staining: Stain sections with eosin for 30 s. Rinse sections with tap water.</li>
	<li>Dehydration for transparency of the sample
	<ol>
		<li>Place sections in 95% ethanol twice for 3 s each, followed by placing in anhydrous ethanol for 3 s.</li>
		<li>Again, place the slices in anhydrous ethanol for 1 min, followed by 2 rounds of xylene wash for 1 min each.</li>
	</ol>
	</li>
	<li>Sealing of slices: Take out the slices, drop neutral gum sealer, cover with a coverslip, and leave the slices to dry in a fume hood until odorless.</li>
	<li>Photographing the sample: Observe and photograph under the field of view of a light microscope at 100x.</li>
	<li>Evaluation: Evaluate the cartilage tissue by Mankin&#39;s score for each group32.</li>
</ol>
7. Data analysis
<ol>
	<li>Analyze statistically the experimental data using analysis software. When the data were subjected to a normal distribution, compare two groups of samples by t-test and multiple groups by one-way ANOVA.</li>
	<li>Express results as mean &#177; standard deviation (SD). Represent results as statistical plots using commercial software. Differences were statistically significant at p &#60; 0.05.</li>
</ol>

Treating Knee Osteoarthritis with Tuina in a Rabbit Model

The authors declare no potential conflicts of interest.

The design of the experimental protocol is particularly important to investigate the mechanism of Tuina in treating KOA. KOA modeling was performed on rabbits by injection of papain at Waixiyan. Waixiyan is located in the lateral crypt of the patellar ligament, which is easy to locate, and the joint space between the femur and tibia is large here during knee flexion, which makes it easy to inject into the knee joint cavity and prevents damage to the surrounding tissues, so it is easy to establish the KOA model34. During Tuina intervention in KOA rabbits, the model rabbits were appropriately positioned to facilitate Tuina administration. The rabbits were placed in the rabbit fixation box on their healthy side, with their heads fixed, and their emotions were soothed to relax their whole body and not struggle. To standardize Tuina&#39;s strength and frequency and improve Tuina interventions&#39; homogeneity, Tuina is performed by an operator who has undergone rigorous training in Tuina manipulation by the Tuina technique parameter determination instrument.
The key step in this protocol is using Tuina combined with the knee joint rotary correction method to treat KOA. Before the operation, the rabbit&#39;s affected lower extremity was palpated from the hip to the ankle, focusing on the area around the knee joint, probing for tendon knots and muscle stiffness, and then flexing and extending the knee joint to observe the height of Neixiyan and Waixiyan for precise Tuina manipulation. The rotatory kneading and pressing method can release muscle tension and spasm, improve blood circulation in the knee joint, promote the metabolism of inflammatory substances, and reduce swelling and pain35,36. Acupuncture points are the response and treatment points of the disease, and GB 34 and EX-LE2 are points with high sensitivity for the treatment of KOA, and by stimulating them, they can act on mast cells, thus affecting the release of 5-hydroxytryptamine, tryptase, and histamine37. Data mining revealed that GB 34, SP 9, EX-LE5, EX-LE4, EX-LE2, SP 10, ST 34, and BL 40 are all commonly used acupuncture points in clinical practice38,39. These points have the effects of relieving tendons and knots, activating blood circulation, and relieving pain38,39. Studies have shown that stimulation of the above acupoints can reduce serum levels of inflammatory factors such as tumor necrosis factor-&#945; and interleukin-1&#946; and inhibit the destruction of the chondrocyte skeleton, thus treating KOA39,40,41.
The knee joint rotary correction method performed under active resistance exercises with adjustable force levels is easy to perform and avoids the potential risk of injury in regulating the knee structure compared to passive movement techniques such as flexion and compression. Angle affects torque, and an imbalance between the knee joint&#39;s internal closing torque and abduction torque can increase abnormal joint loading, which can trigger KOA42,43,44,45. Some studies have shown that Tuina intervention with regular resistance exercise can improve bone density, release muscle spasms, restore muscle mass and strength, reduce pain, and effectively treat KOA46,47,48,49,50. Because the rabbit knee is smaller than the human knee and cannot perform voluntary resistance movements, the original two-person Tuina was changed to a one-person, two-handed Tuina to ensure better efficacy and ease of operation of the knee joint rotary correction method under resistance12. Now, by fixing the femur of the rabbit with one hand to simulate the resistance movement, the other hand is used to perform the knee joint rotary correction method by applying twisting and traction force to the lower Xiyan so that Waixiyan and Neixiyan are at the same height and the relative height of the medial and lateral tibial plateau is adjusted. The knee joint can also be adjusted inward and outward to regulate its inward and outward torques, promote axial alignment, adjust the alignment of the femorotibial and femoropatellar joints, restore the normal force structure of the knee joint, reduce the knee joint load, increase knee joint stability, and restore the normal mobility and physiological function of the knee joint42,43,44,45.
The team&#39;s previous clinical studies have shown the efficacy of this method in treating KOA, and animal studies have shown that it is more effective than glucosamine sulfate in treating KOA12. Tuina can mediate the interleukin 1&#946; (IL-1&#946;) and extracellular signal-regulated kinase 1/2 (ERK1/2)-nuclear transcription factor &#954;B (NF-&#954;B) signaling pathways, reduce the concentration of IL-1&#946; in rabbit peripheral serum and joint fluid, increase the expression level of B-cell lymphoma-2 (Bcl-2), and decrease the expression levels of ERK1/2, Bcl-2 associated x protein, NF-&#954;B p65, and cysteine aspartate protease 3. This helps regulate the apoptosis and proliferation of chondrocytes and balance the disordered chondrocyte internal environment, thus improving the pathological changes in cartilage12.
The limitation of this method is that the intervention of Tuina is performed by a human rather than a machine, and it is difficult for the operator to achieve complete homogeneity in the strength and frequency of Tuina.
In conclusion, Tuina can effectively reduce the inflammation of knee joints, inhibit the degeneration of knee cartilage, and gradually restore normal physiological mobility, and this study can provide a scientific and feasible research protocol for the mechanism of Tuina treatment of knee joint diseases.

Knee osteoarthritis (KOA) is a degenerative disease of the knee joint, mainly manifested by knee pain, swelling, deformation, and restricted movement, with a high disability rate and a higher prevalence in women, with 527.81 million patients with osteoarthritis worldwide in 2019 and its global prevalence accounting for 60.6% of the total global prevalence of OA1. Clinically, the treatment of KOA is usually divided into non-surgical and surgical therapies. Non-surgical therapies include physiotherapy, pharmacotherapy, and platelet-rich plasma injection therapy2,3. Tuina is a common, safe, reliable, and effective treatment method in Chinese medicine. This study uses Tuina combined with the knee joint rotary correction method to treat KOA. Tuina techniques such as the rotatory kneading and pressing method can balance muscle tissue, reduce pain, adjust inflammatory factor levels, improve tissue metabolism, and inhibit articular cartilage degeneration4,5. The knee joint rotary correction method can adjust the alignment of lower limb bones and joints, improve the knee joint gap, restore the normal force line, and balance the lower limb biomechanics6,7,8,9. Resistance exercises can enhance muscle mass and strength and promote cartilage tissue renewal10,11. A preliminary study found that this Tuina protocol is significantly more effective than oral glucosamine sulfate capsules in treating KOA, with a faster onset of action and significant inhibition of chondrocyte degeneration and repair of damaged cartilage tissue12. In the treatment of KOA, compared to Tuina therapy, non-steroidal anti-inflammatory drugs have adverse effects and unsatisfactory long-term efficacy, relatively high surgical risks and costs, and require certain indications for surgical treatment, with postoperative problems and periprosthetic complications13,14,15. When compared to drug therapy and surgery, Tuina treatment for KOA offers several advantages, including reduced side effects, lower risk, enhanced safety, cost-effectiveness, and longer-lasting efficacy. Additionally, it can effectively alleviate knee joint pain, swelling, popping, and restricted movement6,13,16,17.
However, the mechanism of Tuina for the treatment of KOA needs to be clarified, which limits the improvement and perfection of the treatment protocol for KOA. Therefore, studying the mechanism of Tuina intervention in KOA through animal experiments is an effective method. Rabbits, compared to rats, have a docile temperament and larger knee joints. The anatomical structure and cartilage biochemical indexes are similar to those of humans, so it is a suitable subject for studying the mechanism of knee joint disease by Tuina18. The KOA model established by injecting papain into the knee joint cavity of rabbits has the advantages of short modeling time, reduced trauma, high success rate, high survival rate, and similar pathological mechanism to KOA19. This study aims to establish a scientifically feasible animal experimental protocol for Tuina intervention in KOA and investigate the mechanism of Tuina.

<table><tbody><tr><td>0.9 % sodium chloride injection</td><td>Sichuan Keren Pharmaceutical Co.</td><td>Z22121903</td><td></td></tr><tr><td>-20&amp;deg;C refrigerator</td><td>Haier</td><td>BD-328WL</td><td></td></tr><tr><td>4 % fixative solution</td><td>Solarbio</td><td>P1110</td><td></td></tr><tr><td>4&amp;deg;C refrigerator</td><td>Haier</td><td>SC-315DS</td><td></td></tr><tr><td>Anhydrous ethanol</td><td>Sinopharm</td><td></td><td></td></tr><tr><td>Automatic tissue dewatering machine</td><td>Dakowei (Shenzhen) Medical Equipment Co.</td><td>HP30</td><td></td></tr><tr><td>Blast drying oven</td><td>Shanghai Yiheng Scientific Instruments Co.</td><td>DHG-9070A</td><td></td></tr><tr><td>Coverslip</td><td>Biyuntian</td><td>FCGF50</td><td></td></tr><tr><td>Electric thermostat water bath</td><td>Shanghai Yiheng Scientific Instruments Co.</td><td>HWS-26</td><td></td></tr><tr><td>Embedding freezing table</td><td>Changzhou Paishijie Medical Equipment Co.</td><td>BM450</td><td></td></tr><tr><td>Embedding machine</td><td>Changzhou Paishijie Medical Equipment Co.</td><td>BM450A</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid decalcification solution</td><td>Servicebio</td><td>G1105-500ML</td><td></td></tr><tr><td>Fluorescent inverted microscope</td><td>Leica</td><td>Leica DM IL LED</td><td></td></tr><tr><td>Hematoxylin-eosin staining kit</td><td>Cisco Jet</td><td>EE0012</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Laiyang Economic and Technological Development Zone Fine Chemical Plant</td><td></td><td></td></tr><tr><td>Medical joint goniometer</td><td>KOSLO</td><td></td><td></td></tr><tr><td>Neutral gum</td><td>Cisco Jet</td><td>EE0013</td><td></td></tr><tr><td>Normal-grade male New Zealand rabbit</td><td>Jinan Xilingjiao Breeding and Breeding Center</td><td>SCXK (Lu) 2020 0004</td><td></td></tr><tr><td>Papain(3000 U/mg)</td><td>Bioss</td><td>D10366</td><td></td></tr><tr><td>Pathological tissue bleaching and drying instrument</td><td>Changzhou Paishijie Medical Equipment Co.</td><td>PH60</td><td></td></tr><tr><td>Pet electric clippers</td><td>Codos</td><td>CP-3180</td><td></td></tr><tr><td>Rabbit fixing box</td><td>any brand</td><td></td><td></td></tr><tr><td>Rotating Slicer</td><td>Leica</td><td>531CM-Y43</td><td></td></tr><tr><td>Tuina technique parameter determination instrument</td><td>Shanghai DuKang Instrument Equipment Co. Ltd.</td><td>ZTC-&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/65763/65763eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;</td><td></td></tr><tr><td>Ventilator</td><td>TALY ELECTRIC</td><td>C32</td><td></td></tr><tr><td>Xylene</td><td>Fuyu Reagent</td><td></td><td></td></tr></tbody></table>

tuina intervention in rabbit model of knee osteoarthritis

Knee osteoarthritis (KOA) is mainly characterized by degenerative changes in the knee joint's cartilage and surrounding soft tissues. The efficacy of Tuina in treating KOA has been confirmed, but the underlying mechanism needs to be investigated. This study aims to establish a scientifically feasible KOA rabbit model treated with Tuina to reveal the underlying mechanisms. For this, 18, 6-month-old normal-grade male New Zealand rabbits were randomly divided into sham, model, and Tuina groups, with 6 rabbits in each group. The KOA model was established by injecting 4% papain solution into the knee joint cavity. The Tuina group was intervened with Tuina combined with the knee joint rotary correction method for 4 weeks. Only the standard grasping and fixation were performed in sham and model groups. At the end of the 1-week intervention, the knee joint range of motion (ROM) was observed, and cartilage hematoxylin-eosin (HE) staining was done. The study shows that Tuina could inhibit chondrocyte apoptosis, repair cartilage tissue, and restore knee joint ROM. In conclusion, this study demonstrates the scientific feasibility of Tuina treatment for KOA model rabbits, highlighting its potential application in the study of KOA and similar knee joint-related conditions.

The degree of restricted knee motion and cartilage tissue damage reflects the severity of KOA. The knee joint&#39;s ROM reflects the degree of restriction of knee joint motion. The smaller the knee joint ROM, the more serious the limitation of knee joint motion. On the contrary, the bigger the knee joint ROM, the more normal the degree of knee motion. HE staining to observe the morphology and structure of cartilage tissue reflects the degree of cartilage tissue damage. The more irregular the surface of cartilage tissue, the higher existence of cracks and defects, the lower the number of chondrocytes, the thinner the thickness of the cartilage layer, the more disordered the arrangement of cells, the more uneven the distribution of cells, the more unclear the layers, the less clear and complete the tide line, the higher the Mankin&#39;s score, the more serious the damage to the cartilage tissue of the knee joint, and the other way round, the more normal the cartilage tissue is32. When establishing the KOA model, the success of modeling can be determined by observing the degree of restriction of rabbit knee joint movement23,24. The efficacy of Tuina can be determined by observing the improvement of the degree of limitation of knee joint motion and the degree of cartilage tissue damage when the rabbit knee joint is intervened by Tuina12.
After week 7, comparing the left knee joint of the two groups of rabbits revealed that the muscles in the modeling group were stiffer and movement was limited, with a ROM of 74.67&#176; &#177; 1.21&#176;, which was lower than the 140.17&#176; &#177; 1.33&#176; in the sham group, suggesting successful modeling (Figure 2C, Figure 4).
After the 12th week of measurement and analysis, the knee joint ROM of the sham group, the model group, and the Tuina group were 140.33&#176; &#177; 1.37&#176;, 76.33&#176; &#177; 1.37&#176;, and 134.33&#176; &#177; 1.51&#176;, respectively, and the knee joint mobility of the Tuina group was significantly higher than that of the model group (p &#60; 0.01), indicating that Tuina could improve the knee joint function of KOA rabbits (Figure 5).
HE staining of the cartilage of the left knee joint of rabbits in each group showed that the cartilage tissue surface of the sham group was smooth and intact, the number of chondrocytes was 331.67 &#177; 13.98, the thickness of the cartilage layer was 259.42 &#177; 41.97 &#181;m, the cells were well arranged and evenly distributed, the levels were clear, the tide lines were clear, continuous and complete, and the Mankin&#39;s score was 0.33 &#177; 0.52. Compared to the sham group, the cartilage tissue surface of the model group was irregular with defects and fissures, the number of chondrocytes was 29.50 &#177; 8.04, the thickness of the cartilage layer was 103.15 &#177; 24.64 &#181;m, the cells were disordered, unevenly distributed, the layers were not clear, the tide lines were not clear and incomplete, and the Mankin&#39;s score was 9.33 &#177; 1.03. Compared to the model group, the cartilage of the Tuina group had a regular surface, with fewer defects and fissures, the number of chondrocytes was 291.83 &#177; 8.18, the thickness of cartilage layer was 183.58 &#177; 15.34 &#181;m, the cells were more orderly arranged, slightly unevenly distributed, the layers were clearer, and the tide lines were relatively clear and complete, and the Mankin&#39;s score was 3.00 &#177; 0.63 (Figure 6)15,23,33. The number of cells, cartilage layer thickness, and Mankin&#39;s score in the Tuina group were significantly better than those in the model group (p &#60; 0.001), indicating that Tuina could repair the damaged cartilage tissue.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65763/65763fig01.jpg" /> 
Figure 1: Protocol for the establishment and Tuina of KOA model rabbits. After adaptive feeding of rabbits for 1 week, build the KOA model on rabbits&#39; left knee joint for 6 weeks, with injections of 4% papain solution on days 1, 4, and 7 of the start of modeling. Intervene the rabbits&#39; left knee joint by Tuina for 4 weeks, 1 time every other day. After 1 week of feeding, measure the ROM of the rabbits&#39; left knee joint and take samples. <a href="https://www.jove.com/files/ftp_upload/65763/65763fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65763/65763fig02.jpg" /> 
Figure 2: Establishment of KOA model. (A) The left knee joint of the rabbits was prepared and disinfected. (B) The needle was inserted from Waixiyan, and 4% papain solution and 0.9% sodium chloride solution were injected into the knee joint cavity of modeling and sham groups of rabbits, respectively. (C) The successfully molded KOA model rabbits with limited movement of the left knee joint. <a href="https://www.jove.com/files/ftp_upload/65763/65763fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65763/65763fig03.jpg" /> 
Figure 3: Training for Tuina techniques using the Tuina technique parameter determination instrument. (A) Train for the rotatory kneading method with a thumb. (B) The curve of the rotatory kneading method with a thumb. (C) Train the pressing method with the thumb end. (D) The curve of the pressing method with the thumb end. <a href="https://www.jove.com/files/ftp_upload/65763/65763fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65763/65763fig04.jpg" /> 
Figure 4: Left knee joint ROM in rabbits before and after modeling. The data were processed by t-test about the sham and modeling groups, and the results were expressed as mean &#177; SD. nsp &#62; 0.05,***p &#60; 0.001. <a href="https://www.jove.com/files/ftp_upload/65763/65763fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65763/65763fig05.jpg" /> 
Figure 5: Left knee joint ROM of rabbits. For the sham, model, and Tuina groups, the data were processed by ANOVA, and the results were expressed as mean &#177; SD. At week 1, ROM increased slightly in all three groups. Compared to the sham group, the ROM gradually decreased in the model and Tuina groups at the time of modeling (p &#60; 0.001). In contrast to the model group, ROM gradually increased after intervention in the Tuina group (p &#60; 0.001). <a href="https://www.jove.com/files/ftp_upload/65763/65763fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65763/65763fig06.jpg" /> 
Figure 6: HE staining of the cartilage of rabbit knee joint. (A) The HE staining of the sham group. (B) The HE staining of the model group. (C) The HE staining of the Tuina group. (D) Comparison of the number of chondrocytes between groups. (E) Comparison of cartilage layer thickness between groups. (F) Comparison of Mankin&#39;s score between groups. The data were processed by ANOVA, and the results were expressed as mean &#177; SD. After 12 weeks, as seen by HE staining, the cartilage tissue of the group was structurally intact with normal cell number and arrangement; the cartilage tissue of the model group was structurally destroyed with low cell number and disorderly arrangement; the cartilage tissue of the Tuina group was intact with relatively normal cell number and arrangement. ***p &#60; 0.001. Scale bar = 100 &#181;m. N =6. <a href="https://www.jove.com/files/ftp_upload/65763/65763fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using a Rabbit Model to Explore the Efficacy of Tuina in Treating Knee Osteoarthritis at JoVE.com

Author Spotlight: Using a Rabbit Model to Explore the Efficacy of Tuina in Treating Knee Osteoarthritis 

The protocol describes a method for Tuina intervention in a rabbit model of knee osteoarthritis.

Tuina Intervention in Rabbit Model of Knee Osteoarthritis

Knee osteoarthritis (KOA) is mainly characterized by degenerative changes in the knee joint's cartilage and surrounding soft tissues. The efficacy of ...

tuina-intervention-in-rabbit-model-of-knee-osteoarthritis

Nanyang Technological University

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JoVE Journal

Medicine

1.3K Views.  Shandong University of Traditional Chinese Medicine. The protocol describes a method for Tuina intervention in a rabbit model of knee osteoarthritis.

Video: Author Spotlight: Using a Rabbit Model to Explore the Efficacy of Tuina in Treating Knee Osteoarthritis 

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited effect on the repair of cartilage and on bone regeneration. Platelet-rich plasma (PRP) is an autologous bioactive substance that can repair cartilage injury and accelerate bone regeneration effectively. However, reporting of PRP preparation protocols in clinical studies is highly inconsistent, with the majority of studies providing insufficient information to allow the protocol to be reproduced. We describe a repeatable method of preparing PRP visually, the treatment of KOA using PRP intra-articular injection, and methods of evaluating the outcome. PRP was prepared using manual double centrifugation. The PRP layer was extracted from peripheral blood and used for knee joint cavity injection. Evaluations included assessments of blood platelet concentrations and clinical outcomes. Preparation of PRP by manual centrifugation requires less apparatus and is less costly than plasma filtration or centrifugation using equipment. The centrifugation time of our double centrifugation method was 6 and 5 minutes for the respective centrifugations at forces of 800 and 1400 x g, respectively, to allow for the consistent preparation of standardized PRP. However, a manual method is susceptible to operator error, and PRP batch preparation is not available. Intra-articular injection of PRP proved to be an effective treatment for knee osteoarthritis. The entire treatment procedure took less than 30 minutes, the blood platelet concentration of PRP could be standardized, and treatment was proven to be effective when evaluated by follow-up.

Knee osteoarthritis is frequently seen in the orthopedic department. We introduce in detail the entire knee osteoarthritis treatment process with platelet-rich plasma injection, including preparation, procedures, and evaluation.

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

The Lower Body Positive Pressure Treadmill for Knee Osteoarthritis Rehabilitation

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

Here, based on a clinician&#8217;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in ...

Osteoarthritis Pain Model Induced by Intra-Articular Injection of Mono-Iodoacetate in Rats

The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the pathophysiological changes of human OA. However, as the main symptom in the late stage of OA, pain affects the patients&#39; daily life, and there are not many available models. The mono-iodoacetate (MIA)-induced model is the most widely used OA pain model, mainly used in rodents. MIA is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, which causes chondrocyte death, cartilage degeneration, osteophyte, and measurable changes in animal behavior. Besides, expression changes of matrix metalloproteinase (MMP) and pro-inflammatory cytokines (IL1 &#946; and TNF &#945;) can be detected in the MIA-induced model. Those changes are consistent with OA pathophysiological conditions in humans, indicating that MIA can induce a measurable and successful OA pain model. This study aims to describe the methodology of intra-articular injection of MIA in rats and discuss the resulting pain-related behaviors and histopathological changes.

This study describes the method of intra-articular injection of mono-iodoacetate in rats and discusses the resulting pain-related behaviors and histopathological changes, which provide references for future applications.

The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

Standardized Tuina Manipulation for Knee Osteoarthritis in Rats

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 

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 ...

Targeted Tuina Stimulation Protocol in Lumbar Region of IDD Model Rabbits

Operational and Intervention Effects of Targeted Tuina in Lumbar Intervertebral Disc Degeneration Model Rabbits

Tui Na or massage therapy alleviates symptoms related to intervertebral disc degeneration (IDD). However, precise, repeatable, standardized instructions for Tuina manipulation are lacking. This study establishes IDD model rabbits induced by fibrous ring puncture, creates targeted Tuina stimulation protocols at the acupuncture points in the lumbar region, and describes in detail the operation methods and requirements of kneading, pointing, and flicking. New Zealand male white rabbits (n = 15) were selected and randomly divided into a blank group, a model group, and a Tuina group. The rabbits in the model group and the Tuina group were molded by fibrous ring puncture; the rabbits in the model group were only immobilized on the operating table without treatment. In contrast, the Tuina group used the "8N/10N, 30 cycles/min" prescription for kneading, pointing, and flicking to perform the intervention, using tactile sensory aids to monitor and regulate the intensity of the Tuina operation. Imaging diagnosis and pathological tests were used to assess the effect of Tuina in rabbits, and the results showed improved imaging features and significantly lowered pathology scores of lumbar disc degeneration in the Tuina group compared to the model group (P &lt; 0.01). Targeted Tuina in the lumbar region may be beneficial in the alleviation of lumbar disc degeneration, but further verification is needed. By regularly performing Tuina and recording the mechanical information involved enables reproducible manipulation prescriptions and helps to observe the basic features of the underlying mechanism of Tuina for IDD.

The finger tactile measurement system quantifies tactile pressure on the finger using a highly sensitive electrosurgical pressure sensor, with software displaying and accurately recording the pressure data and video in real time. Two analysis modules unify the data processing for thumb tracking and conditioning during Tuina.

Tui Na or massage therapy alleviates symptoms related to intervertebral disc degeneration (IDD). However, precise, repeatable, standardized instructions ...

Therapeutic Potential of Tuina Massage for Knee Osteoarthritis

Tuina Intervention in Sodium Monoiodoacetate Injection-Induced Rat Model of Knee Osteoarthritis

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable structural damage of the joint. Investigations into the link between articular cartilage, muscles, synovium, and other tissues surrounding the knee joint in KOA are of great importance. Currently, managing KOA includes lifestyle modifications, exercise, medication, and surgical interventions; however, the elucidation of the intricate mechanisms underlying KOA-related pain is still lacking. Consequently, KOA pain remains a key clinical challenge and a therapeutic priority. Tuina has been found to have a regulatory effect on the motor, immune, and endocrine systems, prompting the exploration of whether Tuina could alleviate KOA symptoms, caused by the upregulation of inflammatory factors, and further, if the inflammatory factors in skeletal muscle can augment the progression of KOA.
We randomized 32 male Sprague Dawley (SD) rats (180-220 g) into four groups of eight animals each: antiPD-L1+Tuina (group A), model (group B), Tuina (group C), and sham surgery (group D). For groups A, B, and C, we injected 25 &#181;L of sodium monoiodoacetate (MIA) solution (4 mg MIA diluted in 25 &#181;L of sterile saline solution) into the right knee joint cavity, and for group D, the same amount of sterile physiological saline was injected. All the groups were evaluated using the least to most stressful tests (paw mechanical withdrawal threshold, paw withdrawal thermal latency, swelling of the right knee joint, Lequesne MG score, skin temperature) before injection and 2, 9, and 16 days after injection.

Author Spotlight: Treating Knee Osteoarthritis with Tuina - A New Perspective 

This protocol describes the methods of Tuina intervention in sodium monoiodoacetate injection-induced rat model of knee osteoarthritis (KOA), which provides a reference for the application of Tuina in KOA animal models. This protocol also studies the effective mechanism of Tuina for KOA, and the results will help promote its application.

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable ...