Mimicking Ding's Roll Method on Notexin-Induced Muscle Injury in Rats
August 25th, 2023
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.
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 Ding's rolling manipulation with rats by creating a messenger that can simulate Ding's rolling manipulation for rats and verifying the effectiveness of the messenger on rats with muscle injuries.
We are focused on the questions of how efficacious the rolling massager is on the skeletal muscle injury in rat on the different parameters in future standardized research. 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. 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 iodophor 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. 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.
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 3, 000G 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.
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Overview
This study presents a device that mimics Ding's roll method to establish a rat model of skeletal muscle injury. The protocol includes hematoxylin-eosin staining for pathology observation and enzyme-linked immunosorbent assay for serum damage marker detection.
Key Study Components
Area of Science
- Neuroscience
- Musculoskeletal Disorders
- Animal Models
Background
- Tuina is a traditional Chinese therapeutic technique.
- Standardization of Tuina treatments is essential for laboratory research.
- Existing methods for muscle injury treatment in rats are limited.
- Ding's rolling manipulation is difficult to perform on rats.
Purpose of Study
- To create a device that simulates Ding's rolling manipulation.
- To evaluate the efficacy of the rolling massager on skeletal muscle injuries in rats.
- To establish a standardized method for future research.
Methods Used
- Development of a self-made Tuina device.
- Use of a rubber roller, fork holder, and spring in the device.
- Application of hematoxylin-eosin staining for tissue analysis.
- Enzyme-linked immunosorbent assay for serum marker detection.
Main Results
- Successful simulation of Ding's rolling manipulation in rats.
- Observation of muscle injury pathology through staining.
- Detection of serum damage markers post-treatment.
- Establishment of a reliable model for future studies.
Conclusions
- The rolling massager is effective for treating muscle injuries.
- This method can enhance the standardization of Tuina treatments.
- Further research is needed to explore different parameters of efficacy.
Frequently Asked Questions
Executive Industry Relevance
Standardizing manual therapeutic interventions in preclinical models is critical for generating reproducible, quantitative data on musculoskeletal injury and repair. The development of a device that mimics Ding's roll method enables controlled, scalable evaluation of mechanotherapeutic effects in rat models, directly supporting early-stage target validation and mechanistic de-risking for myopathy research. This approach enhances predictive confidence in translational studies by minimizing operator variability and enabling robust biomarker assessment.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables quantitative interrogation of mechanotherapeutic hypotheses in disease-relevant animal models.
- Supports functional target validation by linking intervention parameters to plasma injury biomarkers.
- Facilitates mechanistic de-risking by standardizing force, frequency, and duration of intervention.
Screening & Assay Development
- Prepares validated animal models for downstream pharmacological or mechanistic screening workflows.
- Improves assay reproducibility by minimizing subjective operator effects in intervention delivery.
- Generates quantitative outputs (CK, FABP3) suitable for comparative compound evaluation.
Translational & Preclinical Research
- Aligns preclinical intervention parameters with clinical mechanotherapy concepts for translational continuity.
- Enables risk-adjusted advancement decisions by providing standardized injury and recovery metrics.
- Supports biomarker-driven assessment of therapeutic efficacy in musculoskeletal disease models.
Pipeline & Workflow Integration
This device-based method integrates into the discovery-to-preclinical continuum by enabling standardized mechanistic studies, quantitative biomarker analysis, and reproducible intervention delivery in animal models of muscle injury.
- Discovery Biology: Supports hypothesis testing on mechanotherapy effects and biological pathway clarification in muscle injury.
- Screening: Provides reproducible, quantitative injury models for compound or intervention screening.
- Analytics: Delivers measurable readouts (CK, FABP3) for statistical comparison across experimental groups.
- Translational Research: Bridges preclinical findings to clinical mechanotherapy by aligning intervention parameters and outcomes.
- Enterprise Reuse: Establishes a reusable, standardized platform for future mechanistic and therapeutic studies in musculoskeletal research.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in myopathy research.
- Operational Value: Enhances standardization, reproducibility, and scalability of manual intervention studies.
- Strategic Value: Improves go/no-go decision quality and capital efficiency by enabling robust, quantitative preclinical data.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of mechanotherapeutic and pharmacological candidates.
Implementation Considerations
- Requires expertise in animal handling and standardized device operation.
- Needs access to analytical infrastructure for biomarker quantification (e.g., ELISA for CK, FABP3).
- Demands cross-team agreement on intervention parameters for reproducibility.
- May require adaptation for different animal models or injury types.
- Device force and frequency calibration are essential to ensure translational relevance and minimize variability.
