This study elaborately describes a modeling method for cervical spondylosis in rats and an acupotomy intervention strategy, providing a visualized operational protocol for relevant research.
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Method Article
* These authors contributed equally
This study elaborately describes a modeling method for cervical spondylosis in rats and an acupotomy intervention strategy, providing a visualized operational protocol for relevant research.
Cervical spondylosis (CS) is a chronic degenerative disease. Degenerative changes in the intervertebral discs of the cervical spine, along with subsequent pathological alterations, affect surrounding tissues and structures, leading to clinical manifestations such as neck pain, stiffness, limited mobility, and occasionally numbness and radiating symptoms in the upper limbs. Among these, nerve root compression is the most common. Therefore, this study established a rat model of CS induced by cervical nerve root compression. Acupotomy therapy has shown potential in clinical CS treatment by releasing local soft tissue adhesions and alleviating nerve compression. However, there is currently a lack of visualized procedural protocols in animal experiments. This study provides a detailed description of a reliable method for establishing a rat model of CS and the corresponding acupotomy intervention protocol. In this procedure, we elaborate on the model induction process, the steps of acupotomy manipulation, therapeutic localization, rat restraint techniques, and critical precautions. Additionally, this study used electromyography (EMG) to confirm the successful establishment of the CS model and evaluated the intervention through mechanical withdrawal thresholds, gait analysis, and nerve root histopathology. This research not only provides a reference for the establishment of the CS rat model and acupotomy intervention but also offers a systematic and feasible experimental framework for future studies investigating the mechanisms of action.
Cervical spondylosis (CS) is a common degenerative spinal disease. Its core pathological process originates from the degeneration of intervertebral discs, which further triggers a series of secondary pathological changes, including intervertebral space stenosis, osteophyte formation, and ligament hypertrophy1,2. These pathological alterations result in a variety of clinical symptoms, such as soreness and pain in the neck and shoulders, and radiating numbness in the upper extremities. In severe cases, neurological impairments may occur, including motor dysfunction and even tetraplegia2,3. Notably, the incidence of cervical spondylosis has gradually increased in recent years, with a progressively younger onset age, imposing a substantial socioeconomic burden4. Degeneration of the cervical intervertebral discs and osteophytic proliferation may involve the adjacent bony and soft-tissue structures, resulting in stenosis of the cervical spinal canal or intervertebral foramina and consequent compression of nearby structures, including cervical nerve roots, the spinal cord, and the vertebral arteries1,2,3,4. Among these, compression of the cervical nerve roots is the most frequent, accounting for approximately 60%–70% of all cases of CS5,6,7. Although multiple therapeutic approaches are available in clinical practice, including physical therapy, pharmacological intervention, and surgery, non-surgical treatments have become the preferred option for most patients due to the high cost, substantial trauma, and uncertain postoperative outcomes of surgical procedures4. In contemporary medical practice, nonsteroidal anti-inflammatory drugs and neurotrophic agents are commonly employed. Although these medications can relieve pain and delay disease progression to a certain extent, they are accompanied by adverse reactions such as digestive system injury and renal damage8,9.
Acupotomy therapy is a characteristic therapeutic modality developed on the basis of traditional Chinese acupuncture theories combined with modern minimally invasive surgical techniques. It exerts effects by dissecting and releasing soft tissue adhesions, regulating local inflammatory factors, and improving regional microcirculation10,11,12. In recent years, the clinical efficacy of acupotomy for cervical spondylosis has been widely recognized, showing promising potential in alleviating nerve compression, relieving pain, and improving cervical motor function13,14. Nevertheless, when applied in animal experiments, acupotomy interventions face challenges such as imprecise localization and difficulty in controlling the depth of manipulation. Meanwhile, to translate these clinical observations into robust evidence-based conclusions, the establishment of stable and reproducible animal models is an essential prerequisite. The intravertebral nylon suture method induces direct mechanical compression by inserting nylon sutures into the intervertebral foramen, thereby precisely mimicking the pathophysiological process of clinical nerve root compression15. This modeling approach also offers advantages, such as a short modeling cycle and controllable compression segments15. However, its surgical procedure largely relies on personal experience, and detailed, visualized operational descriptions are currently lacking.
This study presents a complete, detailed, and visualized experimental protocol covering the establishment of a rat model of cervical spondylosis and the entire procedure of acupotomy intervention. Since anesthetic agents may affect the measurement of mechanical withdrawal threshold and consequently interfere with experimental results16,17,18,19, a dedicated rat restraint device was adopted in this study. This article provides a detailed description of the rat model induction process, the stepwise acupotomy manipulation, the precise localization of treatment sites, the methods for rat restraint and fixation, and other key experimental precautions. Multiple evaluation dimensions were adopted, including mechanical withdrawal threshold measurement, electrophysiological recordings, gait analysis, and nerve root histopathological observation. This study aims to provide a visualized protocol for model induction and acupotomy intervention in a rat model of cervical spondylosis, thereby furnishing methodological support for mechanistic investigations and the evaluation of the efficacy of acupotomy therapy in the treatment of CS.
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All experimental procedures were reviewed, approved, and stringently monitored by the Laboratory Animal Ethics Committee of Beijing MDKN Biotech Company (Approval ID: MDKN-2025-096). All protocols were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health (NIH). The reagents and equipment employed in this study are listed in the Table of Materials.
1. Experimental animals and groups
2. Establishment of the CS model
NOTE: Withhold food and water from all rats for 12 h prior to modeling. Weigh and record body weight. Sterilize all surgical instruments by high-pressure steam autoclaving (Figure 2A).
3. Restraint procedure
4. Acupotomy treatment
5. Mechanical withdrawal threshold (MWT) test
NOTE: Conduct the measurement on the day before model establishment surgery, the 3rd day, and the 14th day after surgery, using a plantar mechanical pain stimulator for rats and mice.
6. Electromyography (EMG) recording
NOTE: Perform resting-state EMG on Day 3 after model establishment using a biological signal acquisition and analysis system.
7. Gait analysis in rats
NOTE: Perform gait analysis on the first day after completion of acupotomy intervention (postoperative Day 15 after model surgery). The gait analysis system consists of a treadmill equipped with a transparent belt, a high-speed digital camera positioned beneath the belt, and a computer workstation.
8. HE staining of the nerve root tissues
NOTE: After the behavioral tests on postoperative Day 15 after model surgery, collect nerve root tissue samples for HE staining.
9. Statistical analysis
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Mechanical withdrawal threshold (MWT) assessments demonstrated comparable baseline values across all groups prior to model induction. On the 3rd day after modeling and sham operation, no significant difference was observed between the sham group and the control group (p > 0.05). In contrast, both the model and acupotomy groups exhibited a marked reduction in MWT relative to the sham group (p < 0.01, Figure 4). Upon completion of treatment, the acupotomy group showed a...
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The selection of an appropriate animal model is a prerequisite for elucidating the pathogenesis of cervical spondylosis (CS) and for rigorously evaluating therapeutic interventions. Currently, the intravertebral nylon suture method is widely adopted due to its advantages of relative simplicity, short modeling cycle, and high reproducibility and reliability15,20. In this approach, a compressive material is implanted into the rat spinal canal to exert continuous co...
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All authors have declared no potential conflicts of interest.
This study was supported by the National Key Research and Development Program of China (Grant No. 2023YFC3502703) and the Key Project of the Hubei Provincial Natural Science Foundation Innovation and Development Joint Fund (Grant No. 2024AFD271).
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 0.5 mL syringe | Hebei Yunsheng Biotechnology Co., Ltd | SN-04442 | |
| 1 mL syringe | Shanghai Zhiyu Medical Equipment Co., LTD | ZSQ-1 | |
| 4-0 Suture with Needle, Single-armed (4×12) | GLASS | JZZSX193 | |
| 75% alcohol | Shandong Anjie Gaoke Disinfection Technology Co. LTD | 75-500ml | |
| Acupotomy Needle | Beijing Huaxia Blue Chip Biotechnology Co., Ltd. | HY0440 | |
| Adherent slide | CITOTEST | 188105 | |
| Biological Signal Acquisition and Analysis System | Techman | BL-420N | |
| Biological Signal Acquisition and Analysis System (Software) | Techman | BL-420N Version 3.0.0.237 | |
| Cover glass | Citotest Labware Manufacturing Co.,Ltd | 10212432C | |
| Dehydrator | DIAPATH | Donatello | |
| Electronic balance | Mettler-Toledo | ME203E/02 | |
| Electrothermal blast drying oven | Labotery | GEL-70 | |
| Embedding machine | Wuhan Junjie Electronics Co., Ltd | JB-P5 | |
| Environmental Friendly Dewaxing Transparent Liquid | Servicebio | G1128-1L | |
| ESD-Safe Black Stainless Steel Tweezers (Curved Tip) | solomen | ESD-15 | |
| Ethanol | SCRC | 100092683 | |
| Fat-free cotton ball | Caoxian Hualu Sanitary Material Co. LTD | TZMQ-500 | |
| Hematoxylin-eosin (HE) HD constant dye kit | Servicebio | G1076 | |
| Hemostatic Forceps | solomen | B0557 | |
| IBM SPSS Statistics | IBM Corporation | Version 22.0 | |
| Imaging system | Nikon | NIKON DS-U3 | |
| Iodophor | Shandong Anjie Gaoke Disinfection Technology Co. LTD | DF-500 | |
| Isoflurane | RWD life science | R510-22-10 | |
| Latex gloves | Beijing Ruijing Latex Products Co. LTD | RJJC-S | |
| Mechanical Paw Poking Test Device | Kew basis | KW-CT-1 | |
| Medical forceps | AIZANCHENG | AZN00008 | |
| Mosquito hemostatic forceps | Servicebio | QX1310D | |
| Neutral gum | SCRC | 10004160 | |
| Normal saline solution | SHIMEN | SLYS-500ml | |
| Ophthalmic scissors | solomen | B0754 | |
| Paraffin liquid | Macklin | 8012-95-1 | |
| Paraformaldehyde Fixative (Neutral) | Servicebio | G1101 | |
| Pathology slicer | Shanghai Leica Instrument Co., Ltd | RM2016 | |
| Penicillin G Sodium for Injection | Jilin Huamu Animal Health Product Co., Ltd. | Veterinary Drug Approval No.070011248 | |
| Poly-L-lysine solution | Shanghai Yuanye Biotechnology Co., Ltd. | R23126-50ml | |
| Rodent Clipper | solomen | B1564 | |
| Rodent Gait Imaging and Analysis System | Clever | TreadScan; BCamCapture | |
| Rodent Gait Imaging and Analysis System (Software) | Clever | TreadScan Version 4.00; BCamCapture Version 3.00 | |
| SD rats | Beijing Sibefei Biotechnology Co., Ltd | A102 | |
| Small Animal Anesthesia Machine | YuYAN | ABM | |
| Steam sterilizer (autoclave) | Yamato | SQ810C | |
| Surgical Blade | solomen | B1177 | |
| Surgical mask | winner | WJKZ-1 | |
| Surgical needle holder | solomen | B0772 | |
| Surgical scissors | GLASS | JZZSX-ZC544R | |
| Tissue spreader | Zhejiang Kehua Instrument Co., Ltd | KD-P | |
| Ultrapure Water Polishing System | Aiken water El | AK-RO-C2 | |
| Universal tissue fixative (neutral) | Servicebio | G1101-15ML | |
| Upright optical microscope | Nikon | NIKON ECLIPSE E100 | |
| Xylene | SCRC | 10023418 |
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