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

Podsumowanie

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.

Streszczenie

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 µL of sodium monoiodoacetate (MIA) solution (4 mg MIA diluted in 25 µL 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.

Wprowadzenie

Knee osteoarthritis (KOA) is a common degenerative osteoarthritis, characterized by chronic pain and disability, and relatively severe KOA will lead to irreversible structural damage to the joint¹. The high global prevalence of KOA has become a major global public health challenge^2,3, which seriously impairs the quality of life of patients^4,5. Studies have shown that KOA affects more than 260 million people worldwide⁶. With the aging of the population, the total prevalence rate of KOA in China is as high as 17.0% over the age of 40, which puts a heavy burden on the families of those affected^7,8.

According to Chinese medicine, KOA belongs to the category of "paralysis"⁹, and the Yellow Emperor's Classic of Internal Medicine associates tendons, bones, and flesh with paralysis. Hence, we need to pay attention to the connection between the cartilage, muscle, synovium, as well as other tissues around the knee joint in KOA. In modern medicine, both muscle and bone inflammation, which are major components of the immune homeostasis of arthritis, drive the progression of KOA pain. However, most studies focus on cartilage inflammation and synovial inflammation in bone joints, and there is a paucity of articles examining inflammatory phenomena in skeletal muscles. Therefore, more research on the role of skeletal muscle in inflammation is needed to gain a more comprehensive understanding of KOA and provide ideas for more effective treatment modalities. In a review of the literature, we found that inhibition of the immune-related factor programmed death ligand 1 (PD-L1) exacerbated the progression of KOA^10,11 and that there was differential expression of IL-15 and FOXO1 in the skeletal muscle of patients with KOA¹², as well as significant skeletal muscle inflammation in patients with end-stage KOA, which was also associated with changes in gait. It has been shown that the skeletal muscle can also secrete factors closely related to inflammatory immunity such as IL-6, and that TNF-α is also closely related to the PD-L1 pathway and KOA^13,14,15.

Currently, available treatments for KOA include weight control, exercise, medication, and surgery, which usually include intra-articular injections, analgesic medication, arthroscopic surgery, and periprosthetic osteotomy of the knee¹⁶. Currently, KOA disease progression cannot be completely stopped or reversed, and without a clear understanding of the complex mechanisms underlying KOA pain, KOA patients are at risk for overuse of analgesic medications and a high incidence of side effects⁸. Most treatment strategies are effective only for short periods and focus only on the temporary relief of symptoms rather than on the pathogenesis of the disease. Therefore, KOA pain remains a priority and a challenge for treatment, posing a serious clinical challenge. However, Tuina can directly stimulate the skeletal muscles around the affected joint in KOA treatment and can provide some benefits. Combined with the previous description, it is the ideal therapy to study the role of skeletal muscle tissue in KOA, and clarifying the role of skeletal muscle tissue in inflammation can also provide better theoretical support for Tuina of KOA.

Studies have shown that massage therapy, osteopathic manipulative therapy, and spinal manipulation can help relieve pain and restore body function^17,18. These manipulations can act on local inflammation and relieve spasms and analgesia through external mechanical action. They can relieve pain after total knee arthroplasty, reduce the use of analgesic drugs, promote postoperative recovery, and improve biomechanical balance¹⁹. Swedish massage is feasible in the treatment of osteoarthritis and can reduce stress and improve quality of life²⁰. The therapeutic effect of Tuina on KOA has been preceded by certain studies.

In this experiment, our team will explore whether the expression of PD-L1 and related inflammatory factors in skeletal muscle can induce KOA and promote its development based on the MIA-induced KOA model in rats. Tuina will also be implemented to see whether it can alleviate KOA symptoms caused by increased inflammatory factor expression. Tuina will be combined with PD-L1 inhibition to demonstrate the association between Tuina-mediated PD-1 pathway in skeletal muscle inflammation affecting KOA development and pain, laying the foundation for further multi-level studies on the therapeutic mechanisms of Tuina intervention in KOA.

In summary, this paper describes the investigation and elaboration of the mechanisms of skeletal muscle inflammation in the development of KOA and its pain, as well as the therapeutic effects of Tuina on KOA, from the perspective of immune-inflammatory factor expression, combined with behavioral related indicators, to provide modern research evidence for the "imbalance of tendon and bone" theory in traditional Chinese medicine.

Protokół

All experiments were approved and supervised by the Animal Care and Use Committee of the Shanghai University of Traditional Chinese Medicine (approval number: SYXK2018-0040), which conforms to the stipulations in the World Medical Association Helsinki Declaration.

1. Animal preparation

1. House 32 Sprague Dawley (SD) male rats aged 8 weeks and weighing 180-220 g in a specific pathogen-free housing apparatus under 12 h light/12 h dark cycles at 24 ± 2 °C and 60% humidity with a standard rodent pellet diet. All experimental operations on animals comply with the welfare ethics of experimental animals and animal experimental safety regulations.
2. Randomize and cage rats into four groups of eight animals each after 1 week of adaptive feeding: antiPD-L1+Tuina (group A), model (group B), Tuina (group C), and sham surgery (group D).

2. Intra-articular injection of monosodium iodoacetate (MIA) in the knee

NOTE: Except for group D, the KOA rat model will be prepared by the knee joint cavity injection of MIA in all the groups. For group D, inject 25 µL of sterile physiological saline into the right knee joint cavity.

1. Anesthetize the rat by placing it in an anesthetic cage and then introducing 2.5% isoflurane until it is fully anesthetized. Confirm the animal is fully anesthetized by waiting for the point when the young rat is lying on its back, its limbs stop moving, and there is no response in toe pinch (pedal reflex), palpebral reflex, and muscle relaxation.
2. Add pet eye ointment and eye drops to the anesthetized young rats to prevent eye dryness.
3. Shave the right hind limb of the rats and disinfect the knee joint with three alternating rounds of iodophor and 75% ethanol.
4. To maintain a standardized experimental condition, carefully fix the knee flexion at a precise angle of 90°, ensuring that the patellar ligament is oriented upwards. Subsequently, inject 25 µL of MIA solution, consisting of 4 mg of MIA diluted in 25 µL of sterile saline solution into the right knee joint cavity of the rats.
   NOTE: This injection was carried out with a specialized microinjector, ensuring accuracy and consistency in the procedure^21,22,23. Put the rats back into the cages until they wake up. The animal that has undergone surgery is not returned to the company of other animals until fully recovered.
5. At the end of the study, euthanize the rats by injecting 1% pentobarbital sodium as a 100 mg/kg dose.

3. Implementation of Tuina

NOTE: The implementation ofTuina will be guided by the theory that "for patients with tendon and bone imbalance, tendons need to be treated first". Both groups A and B started the intervention on the first day after the successful evaluation of the model, and both groups were operated once a day for 14 days. The remaining two groups were only observed for 14 days without any intervention. The operators of the manipulation must be strictly trained before the experiment to ensure the consistency of force, frequency, and rhythm.

1. Accurately locate the rat acupuncture point, including EX-LE4, ST35, SP10, ST34, SP9, and GB34 (Figure 1 and Table 1), where finger kneading methods will be carried out by using Experimental Acupuncture²⁴.
2. Fix the rat in the prone position on the rat fixation equipment.
3. After it is quiet and the lower limbs are relaxed, knead the EX-LE4 and ST35 of the affected limb with the thumb and the index finger simultaneously for 4 min at 120-140 times/min.
4. Knead the anterior and medial-lateral muscles of the affected limb with the thumb and the index finger for 3 min at 120-140 times/min, focusing on the SP10, ST34, and the muscles near the knee joint. Emphasize the manipulation of those stiff muscles with an intensity so that the rat does not struggle.
5. Knead the posterior calf muscles of the affected limb from top to bottom for 3 min at 120-140 times/min by using the thumb, the index finger, and the middle finger. To fully expose the treatment area, use the ring finger and the little finger to hold the ankle joint of the affected limb and gently tug on the affected limb. Focus on SP9, GB34, and the muscles near the knee joint with emphasis on the rigid muscles and intensity so that the rat does not struggle.

4. Measurement of behavioral index

1. Measurement of plantar mechanical pain threshold
   NOTE: Measure plantar mechanical pain thresholds 1 day before MIA injection as well as 2, 9, and 16 days after injection.
   1. Bring the rats to a plantar test room with a stainless-steel grid bottom and a plastic cage body in a quiet environment at room temperature (22 ± 2) °C for 30 min of adaptation.
   2. Use an electronic mechanical pain tester with a plastic test needle of 0.8 mm in diameter to stimulate the central area of the right hindfoot of the rats vertically. Evenly and gradually increase the stimulation intensity until the rats show paw retraction and avoidance reactions.
   3. Record the reading of the electronic screen as the mechanical pain threshold of the right plantar foot of the rat.
   4. Measure each rat 5x at 5 min intervals.
   5. Remove the maximum and minimum values from the five values. Take the average value of the middle three values through the trimmed mean method as the result (Table 2).
2. Measurement of paw mechanical withdrawal threshold
   NOTE: Measure paw mechanical withdrawal threshold 1 day before MIA injection, 2, 9, and 16 days after injection.
   1. Bring the rats to a heated plantar test room with a 30 °C glass bottom and a plastic cage body in a quiet environment at room temperature (22 ± 2) °C for 30 min of adaption.
   2. Stimulate the right hind plantar center area with a light source heating device at a stimulation temperature of 65 °C until the rat shows a paw retraction and avoidance response.
   3. Record the time reading of the electronic screen as the right plantar thermal pain threshold of the rat.
   4. Measure each rat 5x continuously at 5 min intervals.
   5. Remove the maximum and minimum values from the five values. Take the average value of the middle three values through the trimmed mean method as the result (Table 3).

5. Measurement of swelling of the right knee joint

NOTE: Measure the swelling of the right knee joint of rats 1 day before MIA injection, 2, 9, and 16 days after the injection.

1. Anesthetize the rat by placing it in an anesthetic cage and exposing it to 2.5% isoflurane until it is fully anesthetized.
2. Place the rat in a flat position on the operating table.
3. Measure the width of the right knee joint with vernier calipers (Figure 2 and Table 4).

6. Measurement of the Lequesne MG score

NOTE: Measure the Lequesne MG score in rats at 2, 9, and 16 days after MIA injection. Lequesne et al developed an index of severity for osteoarthritis for the hip (ISH), which can be used to assess the effectiveness of therapeutic interventions. We consider four parameters: pain stimulation, gait change, joint mobility, and joint swelling.

1. Bring the rats to a manipulation platform in a quiet environment at room temperature (22 ± 2) °C.
2. Have two operators, blinded to each other, measure the local pain stimulation response, gait change, joint movement, and joint swelling.
   1. Stimulate the lateral side of the right knee joint with a plastic test needle and score on a scale of 0-3 according to the response. 0 no response; 3 a good response; 1 contraction of the affected limb; 2 contraction and spasm of the affected limb, accompanied by mild generalized reactions, such as trembling, licking, and sucking.
   2. Place the rat on the operating table and observe the gait of its right hind limbs. Score on a scale of 0-3 according to the response. 0 no disturbance in the movement of the affected limb, normal running, and strong footwork; 3 the affected limb cannot participate in walking, touch the ground, or stomp on the ground; 1 mild trekking during running with strong stomps; 2 the affected limb participates in walking, but the disturbance in movement of the rats (trekking) is obvious.
   3. Flex and extend the right knee joint of the rat by hand and observe the joint mobility. Score on a scale of 0-3 according to the response. 0 joint mobility angle of more than 90°; 3 joint mobility angle of less than 15°; 1 joint mobility angle or 45°-90°; 2 joint mobility angle of 15°-45°.
   4. Touch the right knee joint of the rat and compare the response with that of a normal rat. Score on a scale of 0-2 according to the response. 0 no obvious swelling and visible bony markings; 2 marked swelling and no bony markings; 1 mild swelling and superficial bony markings.
   5. Add these scores to produce the Lequesne MG score for each rat and calculate the average value through the trimmed method (Table 5).

7. Measurement of skin temperature

NOTE: Measure the skin temperature 1 day before MIA injection and 9 and 16 days after injection.

1. Anesthetize the rat by placing it in an anesthetic cage and then introducing isoflurane until it is fully anesthetized.
2. Place the rat in a lateral position on the operating table, at room temperature (22 ± 2) °C.
3. Straighten the right knee joint by gently grasping the foot with the hand. Photograph to determine the skin temperature by using a Filr infrared camera.
4. Read the skin temperature of the knee joint and around the knee joint of the rat by using supporting editing software. Set control points at the ankle joint and knee joint where the temperature gradually decreases.

8. Statistical analysis

1. Use statistical software to express the experimental data in quartiles.
2. Use the trim mean method to deal with the outliers. The outcomes of behavioral tests are noted as the mean ± standard error of the mean.
3. Perform independent samples t-test for comparison between groups. Analyze the behavioral data using a two-way repeated measure analysis of variance (ANOVA), followed by Bonferroni's multiple comparison tests. Where data do not exhibit normality or homoscedasticity, perform a non-parametric test (Kruskal-Wallis k samples).
   NOTE: P < 0.05 indicates that the differences are statistically significant. All the data meet the assumptions of the statistical tests that are applied.

Wyniki

The described protocol was implemented in a clinical setting at Yueyang Hospital of Integrated Traditional Chinese and Western Medicine. Figure 1 shows the exact location of the acupoints pushed in rats, and Table 1 illustrates the common benefits of stimulating these points. Table 2 and Table 3 offer compelling evidence of notable pain relief achieved through Tuina therapy, even in the absence of PD-1 pathway inhibition. The findings presen...

Dyskusje

This study aims to assess the improvement of KOA after Tuina intervention by using standardized behavioral indicators and investigate the mechanisms of Tuina for KOA and the association between skeletal muscle and KOA. Unlike pharmacological and surgical therapies, Tuina has a positive regulatory effect on the motor, immune, and endocrine systems. Tuina can relieve inflammation and pain produced by disease by acting on different targets. For example, by regulating the TLR4 pathway and miRNA, it can inhibit the activation...

Materiały

Name	Company	Catalog Number	Comments
Anti-PD-L1	Abcam, Cambridge, MA, USA	ab80276
electric von Frey esthesiometer	IITC/Life Science, Woodland Hills, CA, USA	ALMEMO 2450
GraphPad Prism 9.0	GraphPad Software		Software for stastistical analysis
monosodium iodoacetate	Sigma-Aldrich Inc	I9148	Resolved into normal saline for injection
pentasorbital sodium	Sigma-Aldrich Inc	P3761
Sprague Dawley (SD) male rats	Shanghai Jihui Experimental Animal Breeding Co., Ltd	No. SCXK (Hu) 2017-0012
thermal analgesia tester	IITC/Life Science	Model 390