This research was supported by grants from the National Natural Science Foundation of China (Grant Nos.82174521), Innovation Project for Graduate Students of Hunan University of Chinese Medicine(2022CX109)

Procedures involving animals have been approved by the Institutional Care and Use Committee at Hunan University of Chinese Medicine.
1. Assembly of the rolling massager
<ol>
	<li>Select a massager that consists of a rubber roller, fork holder, spring, limit baffle, adjustment splint, screw, and acrylic handle (Figure 1). Ensure the rubber roller measures 3 cm long and 1.6 cm in diameter, the spring measures 3 cm long and 0.9 cm in diameter, the limit baffle is 3 cm long and 2 cm wide, and the handle measures 12 cm long and 0.9 cm in diameter.</li>
	<li>Force control: According to the literature results23, Ding&#39;s roll method downward pressure was found to be about 10% of the body weight, so the pressure applied during the design of forward rolling is about 10% of the rat&#39;s body weight (0.2-0.3 N). Test the maximum pressure of the massager on the weighing controller to be about 0.3 N by adjusting the angle of the limit baffle. This pressure requirement meets the needs of the rat.</li>
	<li>Ensure the minimum pressure is about 0.08 N when rolling back (Figure 2). Ensure the pressure is precisely in conformity with the requirement of Ding&#39;s roll method that the ratio of the forward and backward forces is 3:1.</li>
	<li>Before treatment, ask the operator to work with the metronome software to control the rolling frequency to 140 rolls/min and practice this more than 3x in the pre-experiment to ensure the operation is standardized.</li>
</ol>
2. Establishment of a rat model of skeletal muscle injury
<ol>
	<li>Randomly divide 24 male, Sprague-Dawley rats (weighing 200-250 g) into three groups of eight rats each, including control (C), notexin (NTX), and notexin with Tuina (NTX + Tuina), and feed on a standard diet. Maintain on a 12 h light/12 h dark cycle, house at 20-25 &#176;C and 50%-70% humidity.</li>
	<li>Anesthetize with 1% pentobarbital sodium (40 mg/kg) by intraperitoneal injection and then remove hair from the right lower limb with hair removal cream. After removing the fur, wipe the residual cream using saline. Confirm adequate anesthesia by toe-pinch response. Apply ophthalmic ointment to moisturize the eyes while the animal is under anesthesia. Provide thermal support throughout the procedure.</li>
	<li>Alternate skin disinfection for the right lower limb with iodophor disinfecting solution and 75% alcohol before injection. Touch a cotton swab soaked in iodophor disinfecting solution to the center of the skin of the lower extremity and apply in a circular motion outward. Repeat with an ethanol-soaked cotton swab.</li>
	<li>Establish skeletal muscle injury models according to the reference method24. Inject notexin in only one leg (to prevent double notexin injection). Draw 200 &#181;L of notexin solution (10 &#181;g/mL notexin solution prepared by adding 100 &#181;g of notexin to 10 mL of normal saline in a 15 mL centrifuge tube) into a 1 mL syringe with a 30G needle and inject the notexin solution intramuscularly into the gastrocnemius muscle to produce muscle injury.</li>
	<li>Inject the notexin slowly and wait for 3 s before pulling out the needle (to be fully injected). 
	CAUTION: Notexin is a toxic chemical that requires immediate flushing with plenty of water upon contact with an open wound and prompt medical attention if necessary.</li>
	<li>Inject the rats in the control group with 200 &#181;L of saline solution. Move anesthetized rats to empty cages with clean bedding. Take care to remove the padding around the rats&#39; nose and mouth to keep their breathing clear. Visually observe the tissue color and respiratory rate at the end of the injection until the rats regain sufficient consciousness.</li>
	<li>Return rats to the home cage and typically rear them for 24 h.</li>
</ol>
3. Tuina therapy
<ol>
	<li>Place an SD rat in a prone position with its head covered with a black cloth on the experimental platform disinfected with 75% alcohol to expose the gastrocnemius muscle. Do not cover too tightly.</li>
	<li>Using the Tuina massager for the NTX+Tuina group: Hold the massager and place the roller on the gastrocnemius muscle of the rat and roll forward until the spring contacts the limit baffle. Then retract the force and return to its original position, thereby reciprocating movement (Figure 3).</li>
	<li>Roll the massager at a speed of 140 rolls per min, and perform each operation for 3 min. Perform the massages once in the morning and once in the afternoon for 3 consecutive days.</li>
	<li>Return rats to the home cage after each treatment and fast for 8 h after the last treatment.</li>
</ol>
4. Collecting blood and tissues from rats after the experiment
<ol>
	<li>According to the requirements of the relevant animal experiment ethics committee, anesthetize rats by intraperitoneal injection of 1% pentobarbital sodium (40 mg/kg, intraperitoneal injection). Confirm adequate anesthesia by toe-pinch response. Euthanize rats by abdominal aorta bloodletting after blood collection.</li>
	<li>Alternate skin disinfection with iodophor disinfecting solution and 75% alcohol before injection. Touch a cotton swab soaked in povidone-iodine to the center of the abdominal skin and apply in a circular motion outward. Repeat with an ethanol-soaked cotton swab. Repeat disinfection 3x.</li>
	<li>Ask the assistant to use two hemostats to lift the skin in the middle of the abdomen. As the operator, use a scalpel to cut through the abdominal skin and muscles from the raphe to the pubic symphysis.</li>
	<li>After opening the abdominal cavity, separate the bowel with sterile cotton balls to expose the abdominal aorta in the posterior abdominal wall.</li>
	<li>Locate the abdominal aorta, take 5 mL of rat blood into blood collection tubes, and obtain the plasma into 1.5 microtubes by centrifuging at 3000 x g for 10 min after standing blood for 1 h. Store plasma at -80 &#176;C.</li>
	<li>Cut open the skin with surgical scissors along the lower abdominal opening toward the lateral aspect of the right lower limb, exposing the lower limb muscles, and after carefully separating the fascia with forceps, cut the scalpel to remove the intact gastrocnemius muscle.</li>
	<li>Wash the gastrocnemius muscle in sterile saline to remove adhering hair and blood.</li>
	<li>Place the removed gastrocnemius muscle into a 15 mL centrifuge tube containing 4% paraformaldehyde.</li>
</ol>
5. Detection of plasma CK and FABP 3 levels by ELISA
<ol>
	<li>Calculate and determine the number of pre-wrapped plates required for one experiment. Remove the required plates, place them in the 96-well frame, put the remaining microplates back into the aluminum foil bag for sealing, and store them at 4 &#176;C.</li>
	<li>Equilibrate the kits and samples at room temperature (25-28 &#176;C) for 120 min, fully equilibrate to room temperature. 
	NOTE: Equilibration of the kit and the sample is critical and must be equilibrated to sufficient time.</li>
	<li>Set standard, sample, and blank wells. Add 50 &#181;L of CK or FABP3 standard at different concentrations (100, 50, 25, 12.5, 6.25, 0 ng/mL) to the standard wells. Repeat each standard once, occupying a total of 12 wells.</li>
	<li>Fill sample wells with 40 &#181;L of sample diluent (0.8 g NaCl, 0.02 g KH2PO4, 0.29 g Na2HPO412H2O, 0.02 g KCl, 0.01 g NaN3in 100 mL of double distilled water, pH 7.4), followed by 10 &#181;L of the sample to be tested. Repeat each sample once, occupying 48 wells in total.</li>
	<li>Except for the blank wells located two wells behind the last sample well, add 100 &#181;L of HRP-labeled anti-human CK or FABP3 antibody (enzyme-labeled antibody) to each standard and sample well.</li>
	<li>Seal the wells with sealing film and incubate in a 37 &#176;C water bath or thermostat for 60 min.</li>
	<li>Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid, leave for 20 s, shake off the washing liquid, pat dry on blotting paper, and repeat washing the plate 5x (or use a plate washer).</li>
	<li>Add 50 &#181;L of chromogen solution A (20 mg tetramethylbenzidine 10 mL ethanol in 100 mL of double distilled water) and 50 &#181;L of chromogen solution B (0.1 M/L citric acid, 0.2 M/L sodium dihydrogen phosphate buffer, pH 5.0-5.4) to each well. Keep away from light for 15 min at 37 &#176;C.</li>
	<li>For standard wells, sample wells, and blank wells, add 50 &#181;L of termination solution to each well, and measure the optical density value of each well at 450 nm within 15 min.</li>
</ol>
6. Histological analysis of notexin-induced gastrocnemius muscle injury in rats
<ol>
	<li>Prepare 5 &#181;m thick paraffin sections stained with hematoxylin and eosin for light microscopic examination as described in25.</li>
</ol>
7. Image processing and data analysis
<ol>
	<li>Read and analyze the images captured by the imaging system with analysis software. Move the selected image field of view to the center of the screen with the mouse, click 40x, and then click Take Snapshot.</li>
	<li>Record the OD values from the ELISA in a spreadsheet and calculate the rat CK and FABP3 levels in the samples using the standard curve.</li>
	<li>Use statistical analysis software for the statistical analyses. Express measurements as mean &#177; standard deviation (<img alt="Equation 1" src="/files/ftp_upload/65820/65820eq01.jpg" style="margin: auto;" />), and analyze the comparisons between groups by one-way ANOVA, with the LSD test when the variance was uniform, and the Tamhane T2 method when the variance was not uniform. The difference was considered statistically significant at a p-value less than 0.05.</li>
</ol>

A Massager Based on Ding's Roll Manipulation to Treat Muscle Injury in a Rat Model

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</ol>

The authors have nothing to disclose.

Here, we described a protocol for Tuina treatment of skeletal muscle injury in rats and then analyzed the degree of skeletal muscle injury after treatment to verify the effectiveness of the method. Notably, rat skeletal muscle injury models, including but not limited to drug induction (notexin, bupivacaine)16, blunt contusion26, crush27, and ischemia-reperfusion28, can be intervened with Tuina. Through HE staining to observe the histopathological changes and ELISA detection to determine the markers of skeletal muscle damage, it is intuitively shown that the notexin causes severe damage to the gastrocnemius muscle of rats, while the massager simulating the Ding's roll method can reduce the muscle damage. These provide convincing evidence for the effectiveness of Tuina in treating skeletal muscle injuries.
There are several necessary procedures that should be considered when performing Tuina on rats mimicking Ding's roll method. Before the treatment, the Tuina operator should be trained in advance to use the rolling massager at least 3x, to adjust the angle of the baffle with the assistance of the pressure sensor for proper force control, and to control the frequency of Tuina at 140 rolls/min. Meanwhile, keep the rats quiet by covering their heads with a black bandana without being unduly tight to ensure they can breathe freely. If the rat suddenly struggles to be active in the Tuina process, wait until it is quiet before continuing the Tuina.
The limitation of this experiment is that this massager is suitable for rats rather than smaller animals, such as mice. For animals other than rats, some instrument configurations can be changed. Such as the size of the rollers, the angle of the baffle, the strength of the spring, and naturally, the pressure sensor needs to be used to test the rolling force. We have only made a Tuina massager simulating Ding's roll method here. However, as a non-pharmacological therapy of traditional Chinese medicine, there are various Tuina manipulations, such as pushing, moistening, pressing, and kneading. All these manipulations can be made according to the operating parameters of relevant famous Tuina masters to produce an instrument more suitable for animal research to standardize the experimental operation and allow the experimental results to be more accurate and credible.
In conclusion, we provide a detailed description of the manufacturing method of the rolling massager that effectively simulates Ding's roll method during animal experiments. The massager has significant therapeutic efficacy on skeletal muscle injury in rats and lays the foundation for further research on the standardization of Tuina.

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.

<table><tbody><tr><td>1 mL syringe</td><td>JIANGXI FENGLIN</td><td>20220521</td><td></td></tr><tr><td>1.5 microtubes&amp;nbsp;</td><td>Servicebio</td><td>EP-150X-J</td><td></td></tr><tr><td>15 mL centrifuge tube</td><td>Servicebio</td><td>EP-1501-J</td><td></td></tr><tr><td>30G needle</td><td>CONPUVON</td><td>220318</td><td></td></tr><tr><td>5 mL blood collection tube</td><td>Servicebio</td><td>QX0023</td><td></td></tr><tr><td>Acrylic handle</td><td>Guangdong Guangxingwang Plastic Materials Co., Ltd</td><td>65643645</td><td></td></tr><tr><td>Adjustment splint</td><td>CREROMEM</td><td>20220729</td><td></td></tr><tr><td>Cotton Swab</td><td>INOHV</td><td>22080215</td><td></td></tr><tr><td>Enzyme-labeled Instrument</td><td>Rayto</td><td>RT-6100&amp;nbsp;</td><td></td></tr><tr><td>Ethanol</td><td>INOHV</td><td>211106</td><td></td></tr><tr><td>Fork holder</td><td>Yongkang Kangzhe Health Technology Co., Ltd</td><td>JL001</td><td></td></tr><tr><td>Hair removal cream</td><td>Veet, France</td><td>LOTC190922002</td><td></td></tr><tr><td>Hematoxylin dyeing solution set</td><td>Wuhan Google Biotech</td><td>G1005</td><td></td></tr><tr><td>Imaging system&amp;nbsp;</td><td>Nikon, Japan</td><td>Nikon DS-U3</td><td></td></tr><tr><td>IODOPHOR disfecting solution</td><td>Hale&amp;amp;Hearty</td><td>20221205</td><td></td></tr><tr><td>Light microscope</td><td>Nikon, Japan</td><td>Nikon Eclipse E100</td><td></td></tr><tr><td>Limit baffle</td><td>CREROMEM</td><td>20220724</td><td></td></tr><tr><td>Notexin</td><td>Latoxan S.A.S.</td><td>L8104-100UG</td><td></td></tr><tr><td>Pentobarbital sodium</td><td>Merck KGaA</td><td>P3761</td><td></td></tr><tr><td>Rat creatine kinase (CK) ELISA kit</td><td>LunChangShuoBiotech</td><td>YD-35237</td><td></td></tr><tr><td>Rat fatty acid-binding protein 3 (FABP3) ELISA kit</td><td>LunChangShuoBiotech</td><td>YD-35730</td><td></td></tr><tr><td>Rubber roller</td><td>Hebei Mgkui Chemical Technology Co.,Ltd</td><td>202207</td><td></td></tr><tr><td>Screw</td><td>Weiyan Hardware</td><td>B05Z122</td><td></td></tr><tr><td>Sprague Dawley rats</td><td>Hunan Slake Kingda Laboratory Animal Co.</td><td>SYXK2019-0009</td><td></td></tr><tr><td>Spring</td><td>Bingzhang Hardware</td><td>TH001</td><td></td></tr><tr><td>Surgical blade</td><td>Covetrus</td><td>#23</td><td></td></tr><tr><td>Weigh controller</td><td>Iyoys</td><td>HY-XSQ</td><td></td></tr></tbody></table>

mimicking ding's roll method on notexin-induced muscle injury in rats

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.

In order to observe the morphological properties of rat skeletal muscle after injury, the gastrocnemius muscle was stained with hematoxylin and eosin, and the stained images were read with an analysis software as described in the protocol for 8 rats per group. In rats with gastrocnemius muscle injury induced by notexin (NTX group), many muscle cells were ruptured, atrophic, necrotic, and irregularly arranged. There was also a high infiltration of neutrophils and lymphocytes around the affected area (Figure 4B). However, after Tuina treatment with the rolling massager, the pathological condition of muscle cells in the NTX+Tuina group improved, with fewer ruptured, atrophic, and necrotic cells, and only a small number of inflammatory cells infiltrating as compared to the NTX group (Figure 4C). In the control group, the muscle cells of rats were evenly sized, closely arranged, and without inflammatory cell infiltration (Figure 4A).
To further confirm the therapeutic effect of Tuina on gastrocnemius injury in rats utilizing Ding&#39;s rolling massager, we used ELISA to detect the levels of skeletal muscle injury markers CK and FABP3 for 8 rats per group. Compared with the control group, CK and FABP3 levels were significantly increased in the NTX group, and both CK and FABP3 levels were markedly decreased in the NTX+Tuina group compared with the NTX group (Figure 5). These results suggest that injection of notexin caused severe damage to the gastrocnemius muscle in rats, while Tuina can reduce this damage.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65820/65820fig01.jpg" /> 
Figure 1: Rolling massager physical map. It mainly comprises a rubber roller, fork holder, spring, limit baffle, adjustment splint, screw, and acrylic handle. Scale bar = 1 cm. <a href="https://www.jove.com/files/ftp_upload/65820/65820fig01large.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/65820/65820fig02.jpg" /> 
Figure 2: Rolling massager force measurement. The maximum pressure of forward rolling is 0.3 N, and the minimum pressure is about 0.08 N when rolling back. <a href="https://www.jove.com/files/ftp_upload/65820/65820fig02large.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/65820/65820fig03.jpg" /> 
Figure 3: Tuina therapy using rolling massager. Place the roller on the gastrocnemius muscle of the rat and roll forward until the spring contacts the limit baffle, then retract the force and return to its original position, thereby reciprocating the movement. <a href="https://www.jove.com/files/ftp_upload/65820/65820fig03large.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/65820/65820fig04.jpg" /> 
Figure 4: Representative images of HE-stained cross-section of rat gastrocnemius muscle. (A) The gastrocnemius muscle of the control (C) group rats was not injured significantly after saline injection. (B) Notexin injection into the gastrocnemius muscle of rats in the notexin (NTX) group caused severe muscle injury, resulting in myocyte atrophy, necrosis, and varying sizes, accompanied by a massive infiltration of neutrophils and lymphocytes. (C) The gastrocnemius muscle injury was attenuated in the notexin and Tuina (NTX+Tuina) group of rats, and the number of atrophic and necrotic myocytes, neutrophils, and lymphocytes were reduced. Scale bar = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65820/65820fig04large.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/65820/65820fig05.jpg" /> 
Figure 5: CK and FABP3 expression. Plasma was harvested after the completion of Tuina, and the concentrations of CK and FABP3 in the plasma of rats in control (C), notexin (NTX), and notexin and Tuina (NTX+Tuina) were detected by ELISA. *P&#160;&#60;&#160;0.05, **P &#60; 0.01 using one-way ANOVA with post-hoc LSD test. Values are mean &#177; SEM, n=8 in all groups. <a href="https://www.jove.com/files/ftp_upload/65820/65820fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Performing Tuina Massage in Rats Using a Self-Made Massager at JoVE.com

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

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.

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

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

mimicking-ding-s-roll-method-on-notexin-induced-muscle-injury-in-rats

Nanyang Technological University

Research

JoVE Journal

Medicine

1.0K Views.  Hunan University of Chinese Medicine. 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.

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

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite cells. Satellite cells are characterized by the expression of the transcription factor Pax7 and their location underneath the basal lamina in the resting skeletal muscle. Upon injury, satellite cells get activated, undergo self-renewal or differentiation to either form new myofibers or to fuse with damaged ones. The functionality of satellite cells in vivo can be investigated using a cardiotoxin based injury model of skeletal muscle. To study the function of one gene during the regeneration of skeletal muscle, transgenic mouse models are mostly used. Here, we present an alternative method to transgenic mice, to investigate the gene function in satellite cells during regeneration, e.g., in cases where transgenic mice are not available. We combine the cardiotoxin mediated injury of a specific skeletal muscle with the injection of a self-delivering siRNA into the regenerating muscle which is then taken up by satellite cells among other cells. Thereby, we provide a method to analyze gene function in satellite cells during regeneration under physiological conditions without the need for transgenic mice.

We describe an in vivo method to investigate the functional loss of a specific gene in satellite cells by using a combination of cardiotoxin mediated injury of skeletal muscle and the injection of a self-delivering siRNA.

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite ...

Genetics

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Developmental Biology

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Assessing Tuina Therapy's Pain Relief in Chronic Constriction Rats

Characteristics of Pain Changes in Rats with Nerve Injury Within 24 hours After One-Time Tuina Intervention

Tuina, as an external treatment method of traditional Chinese medicine, has been proven to have an analgesic effect on peripheral neuropathic pain (pNP) in clinical and basic research. However, the optimal time point for the analgesic effect of tuina may vary according to different injury sensations, affecting the exploration of the initiation mechanism of tuina analgesia. 
The research used minor chronic constriction injury (minor CCI) model rats to simulate pNP and used the intelligent tuina manipulation simulator to simulate the three methods (point-pressing, plucking, and kneading) and three acupoints (Yinmen BL37, Chengshan BL57, and Yanglingquan GB34) for performing tuina therapy. The study evaluated the changes in pain within 24 h and the optimal time point for the efficacy of tuina analgesia in rats with minor CCI models by testing cold sensitivity threshold (CST), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL). Furthermore, the study evaluated IL-10 and TNF-&#945; expression changes through Elisa detection. The results show that tuina has both immediate and sustained analgesic effects. For the three different injury sensitivity thresholds of CST, MWT, TWL, and two cytokines of IL-10 and TNF-&#945;, the analgesic efficacy of tuina within 24 h after intervention is significantly different at different time points.

Author Spotlight: Exploring the Initiation Mechanism of 'Three-Method and Three-Acupoint' Tuina Analgesia in Rats with Nerve Injury

We present a protocol using the tuina manipulation simulator to perform the "Three-Manipulation and Three-Acupoint" tuina therapy for minor chronic constriction injury rats and evaluate the effective analgesic time points of tuina within 24 h by testing pain changes through behavioral analysis and changes in inflammatory factor expression using enzyme-linked immunosorbent assay.

Tuina, as an external treatment method of traditional Chinese medicine, has been proven to have an analgesic effect on peripheral neuropathic pain (pNP) ...

Neuroscience

Improving FSN Therapy in the Rat Model&#58; Minimizing Discomfort and Maximizing Efficiency

Efficacy of Fu's Subcutaneous Needling on Sciatic Nerve Pain: Behavioral and Electrophysiological Changes in a Chronic Constriction Injury Rat Model

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the mechanisms of action are still not fully understood. During FSN treatment, the FSN needle is inserted and retained in the subcutaneous tissues for a long duration with a swaying movement. However, challenges arise from maintaining a posture while manipulating FSN in animal models (e.g., rats) for researchers. Uncomfortable treatment can lead to fear and resistance to FSN needles, increasing the risk of injury and may even affect research data. Anesthesia may also affect the study results too. Hence, there is a need for strategies in FSN therapy on animals that minimize injury during the intervention. This study employs a chronic constriction injury model in Sprague-Dawley rats to induce neuropathic pain. This model replicates the pain induced by nerve injury observed in humans through surgical constriction of a peripheral nerve, mimicking the compression or entrapment seen in conditions such as nerve compression syndromes and peripheral neuropathies. We introduce an appropriate manipulation for easily inserting an FSN needle into the subcutaneous layer of the animal&#39;s body, including needle insertion and direction, needle retention, and swaying movement. Minimizing the rat&#39;s discomfort prevents the rat from being tense, which causes the muscle to contract and hinder the entry of the needle and improves the study efficiency.

Author Spotlight: Unveiling the Therapeutic Effects of FSN Treatment &#8211; Bridging Research and Clinical Applications in Neuropathic Pain

We present a protocol for using Fu's subcutaneous needling in a chronic constriction injury model to induce sciatic nerve pain in rats.

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the ...

Standardized Tuina Protocol for Rat DRG Compression Model

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar disc herniation, spinal canal stenosis, and intervertebral foramen stenosis. Although Tuina, a traditional Chinese manual therapy, has shown analgesic effects in clinical practice for the treatment of neuropathic pain, its underlying neurobiological mechanisms remain unclear. Animal models are essential for elucidating the basic principles of Tuina. In this study, we propose a standardized Tuina protocol for rats with compression of the dorsal root ganglion (DRG), which involves inducing DRG compression by inserting a stainless steel rod into the intervertebral foramen, performing Tuina manipulation with specific parameters of location, intensity, and frequency in a controlled environment, and assessing the behavioral and histopathological outcomes of Tuina treatment. This article also discusses the potential clinical implications and limitations of the study and suggests directions for future research on Tuina.

Author Spotlight: Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain  

This article presents a manipulation for treating chronic compression of the dorsal root ganglion in rats using Tuina therapy, along with a method for evaluating its effectiveness based on pain behavior and histopathological results.

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar ...

Tuina Therapy for Frozen Shoulders in a Rat Model

Tuina in a Frozen Shoulder Rat Model: An Efficient and Reproducible Protocol

Frozen shoulder (FS) is a common condition with no defined optimal therapy. Tuina therapy, a traditional Chinese medicine (TCM) technique used to treat FS patients in Chinese hospitals, has demonstrated excellent results, but its mechanisms are not fully understood. Building on a previous study, this work aimed to develop a Tuina protocol for an FS rat model. We randomly divided 20 SD rats into control (C; n = 5), FS model (M; n = 5), FS model Tuina treatment (MT; n = 5), and FS model oral treatment (MO; n = 5) groups. This study used the cast immobilization method to establish the FS rat model. The effect of Tuina and oral dexamethasone on the glenohumeral range of motion (ROM) was evaluated, and the histological findings were assessed. Our study showed that Tuina and oral dexamethasone were able to improve shoulder active ROM and preserve the structure of the capsule, with Tuina therapy proving to be more effective than oral dexamethasone. In conclusion, the Tuina protocol established in this study was highly effective for FS.

Author Spotlight: Investigating the Effectiveness of Tuina Therapy for Frozen Shoulder 

This study develops an efficient and reproducible Tuina protocol for treating frozen shoulder established in a rat model. This approach will help to study the Tuina therapy treatment method for frozen shoulders.

Frozen shoulder (FS) is a common condition with no defined optimal therapy. Tuina therapy, a traditional Chinese medicine (TCM) technique used to treat FS ...

Electroporation and Nerve Injury in Mouse Levator Auris Longus Muscle

Combined In Vivo Electroporation and Short&#45;Term Reinnervation of the Cranial Levator Auris Longus Skeletal Muscle

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many organisms. At the NMJ, a presynaptic motor axon terminal contacts a muscle postsynaptic domain and is covered by terminal Schwann cells. The integrity and function of the NMJ is compromised under several conditions, including aging, neuromuscular diseases, and traumatic injuries. To analyze the potential contribution of muscle-derived proteins to NMJ maintenance and regeneration, an in vivo gene transfer strategy has been combined with the denervation of the cranial levator auris longus (LAL) muscle after mechanical nerve injury. Previous findings showed that the forced expression of control fluorescent proteins does not alter NMJ organization or neurotransmission. This procedure aims to describe a detailed method of in vivo electroporation of the LAL muscle followed by transection or crushing of the specific branch of the facial nerve innervating cranial muscles, leading to NMJ denervation and reinnervation, respectively. The combination of these experimental strategies in the convenient LAL muscle constitutes an efficient method to study the potential contribution of muscle protein overexpression or silencing in the context of short-term NMJ reinnervation.

Author Spotlight: Regenerative Roles of Muscle Proteins at Neuromuscular Junction Post-Nerve Injury

Here, we present a protocol that combines in vivo electroporation and denervation of the cranial levator auris longus (LAL) muscle. This procedure enables the study of the potential role of muscle-derived proteins in the regeneration of the neuromuscular synapse.

The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many ...

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

Summary

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Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Characteristics of Pain Changes in Rats with Nerve Injury Within 24 hours After One-Time Tuina Intervention

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