Name: Author Spotlight: Exploring the Initiation Mechanism of &#039;Three-Method and Three-Acupoint&#039; Tuina Analgesia in Rats with Nerve Injury
Uploaded: 2024-01-26T13:15:00
Description: Read this Scientific Article Protocol about Author Spotlight: Exploring the Initiation Mechanism of 'Three-Method and Three-Acupoint' Tuina Analgesia in Rats with Nerve Injury at JoVE.com

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).

The Committee on Animal Protection and Use of Beijing University of Chinese Medicine (BUCM-4-2022082605-3043) approved all procedures used in this study.
1. Animals and study design
<ol>
	<li>Obtain 49 male Sprague-Dawley (SD) rats of 8-week-old, weighing 200 &#177; 10 g.</li>
	<li>Using a random number table method, divide the rats into a sham operation group (n = 7), a model group (n = 7), and immediately after tuina group (n = 7), 6 h after tuina group (n = 7), 12 h after...

Assessing Tuina Therapy&#039;s Pain Relief in Chronic Constriction Rats

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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silent+cold-sensing+neurons+contribute+to+cold+allodynia+in+neuropathic.">Silent cold-sensing neurons contribute to cold allodynia in neuropathic.</a> Brain. 144 (6), 1711-1726 (2021).</li><li>Pan, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chinese+tuina+downregulates+the+elevated+levels+of+tissue+plasminogen+activator+in+sciatic+nerve+injured+Sprague-Dawley+rats.">Chinese tuina downregulates the elevated levels of tissue plasminogen activator in sciatic nerve injured Sprague-Dawley rats.</a> Chinese J Integr Med. 23 (8), 617-624 (2017).</li><li>Lv, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+into+the+rehabilitative+mechanism+of+tuina+in+the+treatment+of+sciatic+nerve+injury.">An investigation into the rehabilitative mechanism of tuina in the treatment of sciatic nerve injury.</a> Evid Based Complement Alternat Med. 2020, 5859298 (2020).</li><li>Lv, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+RNA+sequencing+technology+to+explore+repair+mechanism+of+Tuina+on+gastrocnemius+muscle+in+sciatic+nerve+injury+rats.">Applying RNA sequencing technology to explore repair mechanism of Tuina on gastrocnemius muscle in sciatic nerve injury rats.</a> Chin Med J. 135 (19), 2378-2379 (2022).</li><li>Ruan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+effective+and+concise+device+for+detecting+cold+allodynia+in+mice.">An effective and concise device for detecting cold allodynia in mice.</a> Sci Rep. 8 (1), 14002 (2018).</li><li>Kim, S. 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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...

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-&#945; is an initiator of the inflammatory response, which can promote the synthesis of inflammatory factors such as IL-10 and IL-1&#946;, 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-&#945;, IL-10, IL-6, and IL-1&#946;19,20,21. 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-&#945; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthesia machine</td>
			<td>Ruiwode Life Technology Co., Ltd., Shenzhen, China</td>
			<td>R500</td>
			<td>Animal respiratory anesthesia related equipment</td>
		</tr>
		<tr>
			<td>Chromic intestinal suture</td>
			<td>Shandong Boda Medical Products Co., Ltd., China</td>
			<td>BD210903</td>
			<td>An absorbable surgical suture mainly made from collagen protein processed from the intestines of healthy young goats</td>
		</tr>
		<tr>
			<td>Electronic Von Frey instrument</td>
			<td>Bioseb, USA</td>
			<td>BIO-EVF5</td>
			<td>An instrument for detecting mechanical withdrawal threshold</td>
		</tr>
		<tr>
			<td>Intelligent cold and hot plate pain detector</td>
			<td>Anhui Zhenghua Biological Instrument Equipment Co., Ltd,China.</td>
			<td>ZH-6C</td>
			<td>An instrument for detecting cold sensitivity threshold</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Ruiwode Life Technology Co., Ltd., Shenzhen, China</td>
			<td>R510-22-10</td>
			<td>An anesthetic</td>
		</tr>
		<tr>
			<td>Multi-function full-wavelength microplate reader</td>
			<td>Molecular Devices (Shanghai) Co., Ltd.</td>
			<td>SpectraMax M2</td>
			<td>An instrument for detecting optical density (OD)</td>
		</tr>
		<tr>
			<td>Thermal analgesia device</td>
			<td>Chengdu Techman Software Co., Ltd., China</td>
			<td>PL-200</td>
			<td>An instrument for detecting thermal withdrawal latency</td>
		</tr>
	</tbody>
</table>

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 &#039;Three-Method and Three-Acupoint&#039; Tuina Analgesia in Rats with Nerve Injury

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

Our research scope belonged to the efficacy verification and the mechanism exploration of tuina therapy for neuropathic pain. We want to answer the effectiveness of tuina through animal experiments, providing scientific evidence for the analgesic efficacy of tuina in clinical practice. At present, RNA sequencing, calcium imaging, and the molecular biology technologies are used to promote further research in this field.The analgesic efficacy of tuina, within 24 hours after intervention, is significantly different at different time points. This provides a scientific basis for selecting appropriate behavioral time points and sampling for further molecular mechanism exploration in the later study of the initiation mechanism of the Three-Method and Three-Acupoint"tuina analgesia. The research protocol further validates the immediate and sustained analgesic effects of Three-Method and Three-Acupoint"tuina technique and provide a more accurate time point for exploring the initiation mechanism of tuina analgesia in the later stage.In subsequent research, we will continue to focus on exploring the initiation mechanism of analgesia by tuina in order to provide scientific evidence for clinical tuina treatment of neuropathic pain-related diseases.

CST: Compared with the model group, the number of foot lifts in the group 6 h after tuina was significantly reduced, and the difference was statistically significant (P &#60; 0.05). Compared with the sham group, the number of foot lifts in the model group was significantly increased, and the difference was statistically significant (P &#60; 0.05) (Table 3, Figure 3). 
MWT: Compar...

Read this Scientific Article Protocol about Author Spotlight: Exploring the Initiation Mechanism of 'Three-Method and Three-Acupoint' Tuina Analgesia in Rats with Nerve Injury at JoVE.com

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) ...

characteristics-pain-changes-rats-with-nerve-injury-within-24-hours

Research

JoVE Journal

Neuroscience

Establishing a Rat Model of Minor Chronic Constriction Injury and Intervention

After anesthesia, fix the rat in a prone position on a table. Shave the fur of the right hip joint area, and disinfect the area with iodine. Make a skin incision of approximately 0.5 to one centimeter along the walking direction of the sciatic nerve.And separate the muscle layer bluntly to expose the sciatic nerve trunk. For the tuina group rat, loosely tie an absorbable chrome intestinal suture around the sciatic nerve without interrupting the blood circulation of extraneural vessels. On the computer operation interface, set the instrument to a stimulation force of four Newtons, a frequency of 60 times per minute, and a temperature of 36 degrees Celsius.Place the rat in the fixator, and expose the hind limb acupoints. On the computer screen, click on the point pressing, plucking, and kneading in the sequence on the

Behavioral Measurement in Tuina-Intervened Rats

To begin, place the TuiNa intervened rat on an intelligent cold and hot plate pain detector with a surface temperature of four degrees Celsius. Cover the rat with a transparent plastic cage. Once the rat is acclimatized to the plastic cage, record the number of foot lifts in the operative side of the hind limb for the next five minutes.Place the rat on a grid surface in a test box for 15 to 30 minutes before testing. Move the probe of the pain to the center of the right posterior plantar region of the rat, and linearly increase the pressure by hand. When the rat lifts or licks its feet, record the threshold displayed on the instrument screen.Adapt the rat in a glass-bottom test box for 15 minutes before starting the test. On a thermal stimulation pain instrument, click on set and direction buttons to set the cutoff time to 30 seconds, and intensity to 50%Then click on start. Position the infrared probe to the center of the right posterior plantar region of the rat.When the rat lifts or licks its feet, record the latency of the foot retraction reflex. In cold sensitivity threshold analysis, compared to the model group, the Tuina group showed a significant reduction in foot lifts six hours post-treatment. The model group exhibited a significant increase in foot lifts compared to the sham group, highlighting the effect of the model intervention.The mechanical withdrawal threshold of each subgroup of Tuina was significantly higher than that of the model group. Except for 18 hours after Tuina, the model group had a significantly lower mechanical withdrawal threshold than the sham operation group. Thermal withdrawal latency analysis revealed a significant increase immediately, six hours, and 18 hours after Tuina in comparison to the model group.The model group showed a significant decrease compared to the sham operation group.