Characteristics of Pain Changes in Rats with Nerve Injury Within 24 hours After One-Time Tuina Intervention
Summary
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.
Abstract
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-α 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-α, the analgesic efficacy of tuina within 24 h after intervention is significantly different at different time points.
Introduction
Peripheral neuropathic pain (pNP) refers to pain caused by a lesion or disease of the peripheral somatosensory nervous system, manifested as a series of symptoms and signs, with hyperalgesia as one of the main symptoms1,2. Hyperalgesia is a heightened experience of pain caused by a noxious stimulus, including pinprick, cold, and heat3. Large epidemiological studies have been conducted, which show pNP is the most common, with a prevalence rate of 6.9%-10% in neuropathic pain4. pNP can be caused by multiple diseases, including injury of the nerve, postherpetic neuralgia, painful diabetic polyneuropathy, multiple sclerosis, stroke, cancer, etc.5. Nowadays, the main method for treating pNP is medication, but the effect is not ideal; and the side effects are significant, which is the main reason for the high economic burden on individuals and society6.
Tuina is a green, economical, safe, and effective external treatment method of traditional Chinese medicine7. Many clinical studies have proven the analgesic effect of tuina on pNP, and basic studies have verified the immediate and cumulative analgesic effects of tuina8,9. The main cumulative analgesic mechanism of tuina is to reduce the levels of inflammatory factors and inhibit the activation of glial cells10,11. The previous research confirmed the cumulative analgesic effect of tuina and found differentially expressed genes (DEGs) in the dorsal root ganglia (DRG) and spinal dorsal horn (SDH) of rats with sciatic nerve injury after 20 times of tuina treatment, mainly related to protein binding, pressure response, and neuronal projection12. In recent studies, it has been confirmed that tuina has an immediate analgesic effect and that 1-time tuina intervention could alleviate the hyperalgesia of minor CCI rats and especially ease thermal hyperalgesia more effectively13. However, the optimal time point for the analgesic effect of tuina may vary depending on the injury sensation (cold, heat, mechanical), affecting the exploration of the initiation mechanism of tuina analgesia.
Inflammatory mediators can sensitize and activate pain receptors, leading to decreased discharge thresholds and ectopic discharges, thereby contributing to peripheral sensitization14,15. After peripheral nerve injury, TNF-α is an initiator of the inflammatory response, which can promote the synthesis of inflammatory factors such as IL-10 and IL-1β, causing direct tissue inflammatory injury, stimulating local nerve endings, and causing pain16,17,18. Tuina can achieve analgesic effects by reducing the expression of inflammatory factors such as TNF-α, IL-10, IL-6, and IL-1β19,20,21. The study selected minor CCI model rats to simulate clinical pNP and selected different time points for behavioral testing of cold, thermal, and mechanical stimulation pain after a 1-time tuina intervention by cold sensitivity threshold (CST), mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), and choose IL-10 and TNF-α in the serum by enzyme-linked immunosorbent assay (ELISA), in order to select the time point at which the analgesic effect of tuina is significant, providing a basis for the study of the initiation mechanism of tuina analgesia in the later stage.
Protocol
Representative Results
Discussion
The study used the minor CCI model to simulate pNP caused by clinical sciatic nerve injury. The minor CCI model involves the continuous, chronic compression and restraint of the nerve trunk through ligation, accompanied by the gradual swelling of the ligature, resulting in edema within the sciatic nerve and forming stable chronic pain in 3-5 days27,28. In the preliminary study, the research group found that the minor CCI model group was more stable than the class…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors have received funding for research, writing, and publication of this paper from the National Natural Science Foundation of China (Nos. 82074573 and 82274675) and the Beijing Natural Science Foundation (No. 7232278).
Materials
| Anesthesia machine | Ruiwode Life Technology Co., Ltd., Shenzhen, China | R500 | Animal respiratory anesthesia related equipment |
| Chromic intestinal suture | Shandong Boda Medical Products Co., Ltd., China | BD210903 | An absorbable surgical suture mainly made from collagen protein processed from the intestines of healthy young goats |
| Electronic Von Frey instrument | Bioseb, USA | BIO-EVF5 | An instrument for detecting mechanical withdrawal threshold |
| Intelligent cold and hot plate pain detector | Anhui Zhenghua Biological Instrument Equipment Co., Ltd,China. | ZH-6C | An instrument for detecting cold sensitivity threshold |
| Isoflurane | Ruiwode Life Technology Co., Ltd., Shenzhen, China | R510-22-10 | An anesthetic |
| Multi-function full-wavelength microplate reader | Molecular Devices (Shanghai) Co., Ltd. | SpectraMax M2 | An instrument for detecting optical density (OD) |
| Thermal analgesia device | Chengdu Techman Software Co., Ltd., China | PL-200 | An instrument for detecting thermal withdrawal latency |
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