establishment of a rat model of skeletal muscle injury

A Massager Based on Ding&#039;s Roll Manipulation to Treat Muscle Injury in a Rat Model

Muscle injuries are common traumatic injuries in clinical and daily life, caused by external blows (contusions) or chronic overstrain of muscle fibers (strains), etc., resulting in muscle dysfunction and pain, even seriously affecting the patient&#39;s quality of life1. Starting rehabilitation as early as possible after an acute strain injury is the key to reducing the time to return to sports2&#160;and in reducing pain3,4. In modern Western medicine, clinical first aid for muscle injuries follows the principles of rest, ice, compression, and elevation (RICE) to stop injurious bleeding into the muscle tissue5&#160;and non-steroidal anti-inflammatory drugs to relieve pain6. The discovery of novel therapies such as exosomes7&#160;and tissue engineering8&#160;became potential treatment strategies for skeletal muscle diseases, compensating for previous pharmacological treatments&#39; shortcomings. However, it can also increase the cost of treatment for patients, putting them under tremendous financial pressure9. Therefore, alternative and complementary therapies are recommended for treating musculoskeletal problems10. Tuina is widely used clinically in China as a traditional medical method and is popular among patients for its efficacy and fewer side effects. Tuina therapy for musculoskeletal disorders can alleviate pain and improve function11,12,13. Mr. Ding Jifeng, a famous Shanghai Tuina practitioner, founded Ding&#39;s roll method14. It is a unique rolling and crushing technique with a large force area, uniform and gentle force, and intense penetration.
Different animal models are based on different etiologies. They have advantages and disadvantages, and the selection of correct and appropriate animal models is of great significance to basic experiments, which helps understand the cellular and molecular signaling pathways of regeneration and repair after skeletal muscle injury to develop new therapies for treating the treatment of skeletal muscle diseases. Chemically induced models of muscle injury are widely used, with injections of skeletal muscle causing myofiber necrosis and producing regenerated areas that can effectively regenerate within 2 weeks15. Both notexin and bupivacaine can cause muscle damage. However, notexin can cause more severe myotoxic damage to skeletal muscle than bupivacaine, and natural functional recovery is relatively slower16. Drug intramuscular injection molding not only takes less time but also has controlled effects and extent of skeletal muscle damage. This quantifiable control makes successful molding less difficult15,17.
Inflammatory response is an essential biological response that has been extensively studied in the context of myopathy18,19. In the early stages of skeletal muscle injury, myofiber necrosis disrupts local muscle homeostasis, and many inflammatory cells infiltrate the injury site, secreting many pro-inflammatory cytokines19. Creatine kinase (CK) is a traditional serum biomarker for assessing skeletal muscle injury. However, it lacks tissue specificity20&#160;and sensitivity21, which limits its ability to assess the extent of drug-induced muscle damage and indirectly report the extent of muscle recovery after injury. Novel biomarkers, including fatty acid binding protein 3 (FABP3), have recently shown relatively high tissue specificity and sensitivity in rodent models of skeletal muscle injury. FABP3 is a family of binding proteins expressed primarily in cardiac and skeletal muscle cells and implicated in fatty acid metabolism, transport, and signaling22. Therefore, we chose a combination of two biomarkers, CK and FABP3, to assess the extent of notexin-induced skeletal muscle damage and recovery after treatment.
In rodents, the muscles are shallow, and the skin area is small, which also determines that the various parameters of massage in rodents will not be the same as in humans, such as in animal therapy, the massage therapist should treat them with less force using Ding&#39;s roll method, and may not be conducive to the operation of this technique due to the small size of the injured area, which can ultimately lead to a reduction in the effectiveness of the massage. Therefore, the experiment utilized the rolling massager made in-house, which conforms to the characteristics of Ding&#39;s roll method, to intervene and evaluate the therapeutic effect of the notexin-induced skeletal muscle injury model in rats, which helps to standardize the parameters of Tuina in experimental animal studies in order to profoundly investigate the molecular mechanism of action of Tuina, a traditional Chinese medicine treatment method, on musculoskeletal diseases.

Author Spotlight: Performing Tuina Massage in Rats Using a Self-Made Massager 

Mimicking Ding's Roll Method on Notexin-Induced Muscle Injury in Rats

The scope of our research is the biological mechanism of tuina in the treatment of musculoskeletal diseases. Here we are trying to make the self-made tuina device to standardize and effectively perform tuina treatments on laboratory animals. We are addressing the inability to perform this rolling manipulation with pairs by creating a massager that can simulate this rolling manipulation on rats, and verifying the effectiveness of the massager on rats with muscle injuries.We will focus on the questions of how efficacious the rolling massager is on the skeletal muscle injury in rats under different parameters in future standardized research.

Watch this Scientific Journal Video about Establishment of a Rat Model of Skeletal Muscle Injury at JoVE.com

Establishment of a Rat Model of Skeletal Muscle Injury  

Begin by randomly dividing the rats into three groups of eight rats each, including control, notexin, and notexin with tuina groups. Remove the hair from the right lower limb with hair removal cream after anesthetizing the rat. Then disinfect the skin with three alternating rounds of iota four disinfecting solution and 75%alcohol in a circular outward motion.To establish the skeletal muscle injury model, use a one milliliter syringe with a 30 gauge needle to inject the notexin solution intramuscularly into the gastrocnemius muscle in only one leg. Wait for three seconds before pulling out the needle. Inject the rats in the control group with 200 microliters of saline solution.Move anesthetized rats to empty cages with clean bedding. Visually observe the tissue color and respiratory rate until the rats regain sufficient consciousness. Return the rats to the home cage and typically rear them for 24 hours.The morphological properties of rat skeletal muscle after injury were observed by hematoxylin and eosin staining. The gastrocnemius muscle injured by notexin injection showed the rupture of many muscle cells compared to controls. The cells were atrophic, necrotic, and irregularly arranged, with a high infiltration of neutrophils and lymphocytes around the affected area.

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153 Aufrufe.  Hunan University of Chinese Medicine. Beginnen Sie damit, die Ratten nach dem Zufallsprinzip in drei Gruppen zu je acht Ratten einzuteilen, einschließlich Kontroll-, Notexin- und Notexin- mit Tuina-Gruppen. Entfernen Sie die Haare von der rechten unteren Extremität mit Haarentfernungscreme, nachdem Sie die Ratte betäubt haben. Desinfizieren Sie dann die Haut mit drei abwechselnden Runden Iota vier Desinfektionslösung und 75%igem Alkohol in kreisenden Bewegungen nach außen.Um das Skelettmuskelverletzungsmodell zu erste...

Serie: Establishment of a Rat Model of Skeletal Muscle Injury  

Ding's roll method is one of the most commonly used manipulations in traditional Chinese massage (Tuina) clinics and one of the most influential contemporary Tuina manipulations in China. It is based on the traditional rolling method commonly used in the one finger Zen genre and named Ding's roll method. Due to its anti-inflammatory and blood circulation-promoting effects, Ding's rolling method has sound therapeutic effects on myopathy. Because of the large area of force applied to human skin, Ding's roll method is challenging to perform on experimental animals with small skin areas, such as rats and rabbits. Additionally, the strength of Tuina applied to the human body differs from that applied to experimental animals, so it may happen that the strength is too high or too low to achieve the therapeutic effect of Tuina during the experiment. This experiment aims to create a simple massager suitable for rats based on Ding's rolling manipulation parameters (strength, frequency, Tuina duration). The device can standardize manipulation in animal experiments and reduce the variation in Tuina force applied to different animals due to subjective factors. A rat model of notexin-induced skeletal muscle injury was established, and plasma injury markers creatine kinase (CK) and fatty acid binding protein 3 (FABP3) were used to assess the therapeutic effect of Tuina on skeletal muscle injury. The results showed that this Tuina massager could reduce the levels of CK and FABP3 expression and slow down the degree of skeletal muscle injury. Therefore, the Tuina massager described here, mimicking Ding's roll method, contributes to standardizing Tuina manipulation in experimental research and is of great help for subsequent research on the molecular mechanism of Tuina for myopathy.

This protocol describes a simple device that mimics Ding's roll method, establishes a rat model of skeletal muscle injury, and uses hematoxylin-eosin staining to observe the pathology of damaged tissue and enzyme-linked immunosorbent assay to detect changes in serum damage markers.

Ding's roll method is one of the most commonly used manipulations in traditional Chinese massage (Tuina) clinics and one of the most influential ...

Assembly of a Rolling Massager for Tuina Therapy

To begin, select a massager that consists of a rubber roller of length three centimeters and diameter 1.6 centimeters, a fork holder, and a three-centimeter long spring of diameter 0.9 centimeters. The roller should also consist of a limit baffle three centimeters long and two centimeters wide, an adjustment splint, a screw, and an acrylic handle measuring 12 centimeters in length and 0.9 centimeters in diameter. To control the force in the Ding's roll method, test the maximum pressure of the massager on the weighing controller.Ensure the force is about 0.3 newton by adjusting the angle of the limit baffle. Next, ensure the minimum pressure is about 0.08 newton when rolling back. Before treatment, familiarize yourself with the metronome application to control the rolling frequency to 140 rolls per minute, and practice this more than three times to standardize the operation.

Tuina Therapy in Rat Model of Skeletal Muscle Injury

First, induce skeletal muscle injury in rats using notexin. To perform therapy, place the rat on the experimental platform in a prone position. Cover its head with a black cloth to expose the gastrocnemius muscle.Next, hold the massager and place the roller on the gastrocnemius muscle of the rat. Roll forward until the spring contacts the limit baffle. Then retract the force and return to its original position, thereby reciprocating movement.Roll the massager at a speed of 140 rolls per minute. Perform each operation for three minutes for three consecutive days. Return the rats to the home cage after each treatment and fast for eight hours after the last treatment.After Tuina treatment with the rolling massager, the morphology of muscle cells improved in the gastrocnemius muscle injured by notexin injection. As observed by hematoxylin and eosin staining, fewer ruptured atrophic and necrotic cells and only a small number of inflammatory cells were observed as compared to the injured muscle. Also, the levels of skeletal muscle injury markers CK and FABP3 were analyzed using Eliza.These markers were markedly decreased in the Tuina group compared to the notexin group, suggesting that Tuina can reduce damage to skeletal muscle.

Blood and Tissue Collection from Rats After Tuina Therapy

Begin by placing the anesthetized animal on the operation table. Disinfect the skin with three alternate swabs of Iodophor disinfecting solution and 75%alcohol. Use two hemostats to lift the skin in the middle of the abdomen.Use a scalpel to cut through the abdominal skin and muscles from the raphe to the pubic synthesis. After opening the abdominal cavity, separate the bowel with sterile cotton balls. To expose the abdominal aorta in the posterior abdominal wall, locate the abdominal aorta and take five milliliters of rat blood into blood collection tubes.Keep the tubes containing the blood standing for one hour and centrifuge at 3000 G for 10 minutes to obtain the plasma. To collect the gastrocnemius muscle, cut open the skin with surgical scissors along the lower abdominal opening toward the lateral aspect of the right lower limb, and expose the lower limb muscles. After carefully separating the fascia with forceps, cut using a scalpel to remove the intact gastrocnemius muscle.Wash the gastrocnemius muscle in sterile saline to remove adhering hair and blood, and place the removed gastrocnemius muscle into a 15 milliliter centrifuge tube containing 4%Paraformaldehyde.