Tuina Intervention in Rabbit Model of Knee Osteoarthritis

Summary

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

Abstract

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.

Introduction

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 therapy^2,3. 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 degeneration^4,5. 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 biomechanics^6,7,8,9. Resistance exercises can enhance muscle mass and strength and promote cartilage tissue renewal^10,11. 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 complications^13,14,15. 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 movement^6,13,16,17.

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.

Protocol

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

1. Raise 18, 6-month-old normal-grade male New Zealand rabbits (2.75 ± 0.25 kg) in standard single cages (12 h light/dark cycle, temperature 20-24 °C, air humidity 40%-60%).

2. Grouping method

1. 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.
2. 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.
3. 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).

3. Establishment of the KOA model

1. 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).
2. On days 1, 4, and 7 of week 2, place all 18 rabbits on their right sides in the rabbit fixation boxes (Figure 1). Perform the below-mentioned operations.
3. Inject 3% pentobarbital sodium (1 ml/kg) into the rabbit's marginal ear vein. Shave the rabbit's left knee joint with an animal shaver, resulting in no hair on the exposed skin.
4. Disinfect the rabbit's left knee joint from the inside out using medical iodophor and 75% alcohol (Figure 2A).
5. Flex the rabbit's left knee joint at 60°. Insert a needle (22G, 0.7 mm x 30 mm) from Waixiyan. Inject 4% papain solution (0.1 mL/kg, 0.275 mL 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's lateral recess, and Neixiyan (EX-LE4) is located in the medial recess of the patellar ligament^20,21,22.
6. Press the pinhole for 2 min to avoid solution spillage.
7. Place your hands above and below the rabbit's left knee joint. Gently and passively flex the rabbit'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.
8. At week 7, observe the rabbit'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.

4. Tuina manipulation

1. 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.
   1. 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).
   2. Analyze the force in three directions of X, Y, and Z axes by the Tuina manipulation parameter processing software and check the force's magnitude, frequency, and time of action displayed on the screen (Figure 3B, D).
   3. 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,D^25,26,27.
2. 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.
3. Perform the rotatory kneading method with the thumb on the rabbit'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.
4. 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.
5. Perform the rotary correction method on the rabbit knee joint and perform this 3x separately for each animal in the group.
   1. 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.
   2. Fix the femur with one hand. Fix the patella'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.
   3. 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.

5. Measurement of the knee joint ROM

NOTE: Before measurement, calm the rabbit. The measurement statistician and operator are different from one another.

1. 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.
2. Position the rabbit on its right side in the rabbit fixation box and fix its left femur with one hand.
3. Align the center of the circle of the medical arthroscope with the lateral center of the rabbit's left knee joint. Extend the fixation arm such that it is parallel to the line extension connecting the circle's center to the greater trochanter. Extend the mobile arm such that it is parallel to the longitudinal axis of the tibia.
4. 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°/s²⁸.
5. Perform this till the rabbit'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's surface.
6. Measure ROM for each knee 3x and take the average value²⁸.

6. Hematoxylin-eosin (HE) staining

1. Sample collection
   1. 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's ear marginal vein for humane euthanasia^29,30.
   2. 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.
   3. 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.
2. Fixation and decalcification
   1. Place the cartilage in 4% paraformaldehyde solution and fix it for 72 h.
   2. 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.
3. Dehydration of embedded sections
   1. Place the specimen in an automatic dehydrator for dehydration.
   2. 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 µm.
   3. 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.
4. De-waxing and hydration
   1. Bake the sections at 65 °C for 60 min.
   2. Soak the sections in xylene for 7 min, followed by 2 more rounds of soaking in fresh xylene for 7 min each.
   3. Soak the slice in anhydrous ethanol for 5 min, followed by soaking for 2 min each in 95% ethanol, 85% ethanol, and 75% ethanol.
   4. Soak sections in distilled water for 2 min.
5. 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.
6. Eosin re-staining: Stain sections with eosin for 30 s. Rinse sections with tap water.
7. Dehydration for transparency of the sample
   1. Place sections in 95% ethanol twice for 3 s each, followed by placing in anhydrous ethanol for 3 s.
   2. Again, place the slices in anhydrous ethanol for 1 min, followed by 2 rounds of xylene wash for 1 min each.
8. 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.
9. Photographing the sample: Observe and photograph under the field of view of a light microscope at 100x.
10. Evaluation: Evaluate the cartilage tissue by Mankin's score for each group³².

7. Data analysis

1. 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.
2. Express results as mean ± standard deviation (SD). Represent results as statistical plots using commercial software. Differences were statistically significant at p < 0.05.

Results

The degree of restricted knee motion and cartilage tissue damage reflects the severity of KOA. The knee joint'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, ...

Discussion

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 model

Materials

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