This work was supported by ShanghaiCritical Clinical Specialties ConstructionProject(Grant Number: Shslczdzk04001); the Sailing program of Shanghai Science andTechnology Commission (Grant Number:22YF1444300); Projects within the budget ofShanghai University of Traditional ChineseMedicine(Grant Number: 2021LK091).

This work was carried out at the Pain Lab of the Neurobiology Institute at Fudan University. The experiments were approved and strictly adhered to the guidelines for the protection of laboratory animals established by the International Association for the Study of Pain (LASP) for all surgical procedures and animal handling. Clean-grade Sprague-Dawley (SD) rats, consisting of 32 males between 40-50 days old, with an average weight of 220 &#177; 1.38 g, were used for the present study. These rats were obtained from the Experimental Animal Center of the Shanghai Academy of Life Sciences, Chinese Academy of Sciences. The animals were properly cared for and housed in a dedicated room with independent ventilation, regulated temperature (22 &#177; 1 &#176;C), and humidity (40%-50%). The rats had access to adequate food and water in their cages. The laboratory animal room followed a 12 h light-dark cycle to maintain the regularity of the rats&#39; circadian rhythms, and designated personnel regularly replaced the padding. The X-ray was performed at the Radiology Department of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, affiliated with the Shanghai University of Traditional Chinese Medicine.
1. Study participants and grouping
<ol>
	<li>Assign 32 rats to four groups: naive (control), sham (sham operation), CCD (chronic dorsal root ganglion compression), and CCD + Tuina (8 rats/group). Rats had free access to food and water in animal facilities. 
	&#8203;NOTE: Naive rats had no intervention, while the sham group underwent the same surgical procedure as the CCD group rats, but without leaving an &#34;L&#34;-shaped stainless steel rod in the L4 and L5 intervertebral foramen. Rats in the CCD group underwent complete chronic dorsal root ganglion compression model surgery. Rats in the CCD + Tuina group received tuina therapy starting on the fourth day after the CCD surgery.</li>
</ol>
2. Establishing animal model
<ol>
	<li>Administer isofluorane&#160;to anesthetize rats. Once the rats lose consciousness (no tail flick reflex or leg flexion reflex), shave the hair in the surgical area using a razor. 
	NOTE:&#160;Analgesic drugs were applied 15 min before surgery and rats were injected subcutaneously with tramadol 20 mg/kg.</li>
	<li>Secure the rat on a foam board (see Table of Materials) and use rubber bands to secure its limbs and incisors. Wipe the prepared area with a sterile prep of alcohol and iodine alternation for a minimum of 3 cycles.</li>
	<li>Insert the &#34;L&#34;-shaped probe (see Table of Materials).
	<ol>
		<li>Use scissors to make a 2-3 cm incision through the skin, superficial fascia, and deep fascia layer by layer. First, locate the anterior superior iliac spine, which corresponds to the fifth lumbar spine. Then, sequentially locate the third and fourth spinous processes.</li>
		<li>Clamp the spinous process with toothed forceps and lift it to allow the scissors to be close to the right side of the spinous process and cut the muscle attached to the right side of the spinous process.</li>
		<li>Then, bluntly dissect the muscle attached to the outer surface of the vertebral plate until there is resistance to the right side. The protrusion is the zygapophysial joint. Similarly, bluntly dissect the muscle and fascia on the zygapophysial joint. 
		NOTE: The transverse process pointing to the rat&#39;s head direction will be touched first in the lower outer front of the zygapophysial joint. Below the transverse process is the intervertebral foramen, which is filled with nerve roots and surrounding soft tissues and is not usually easy to find.</li>
		<li>First, use an &#34;L&#34;-shaped probe to determine the position of the intervertebral foramen, and then use an &#34;L&#34;-shaped stainless steel rod (which needs to be made with a diameter of 0.4 mm and a length of 4 mm) to insert it into the intervertebral foramen.</li>
		<li>If the dorsal root ganglion is compressed successfully, the rat will exhibit a tail flick and leg flexion reflex. Insert the stainless steel rod into the fourth and fifth lumbar intervertebral foramen. Then, suture (3-0, see Table of Materials) the muscle, fascia, and skin layer by layer.</li>
	</ol>
	</li>
	<li>Place the rat in a thermostatic box until it wakes up. After waking up, observe whether the function of the right hind limb of the rat is normal. If there is dragging, it means that the operation has injured the motor nerves, and the rat should be discarded. If the function of the right hind limb is normal, the rat can be used and placed in a cage for feeding.</li>
</ol>
3. Tuina therapy
<ol>
	<li>Establish a comfortable environment: before initiating Tuina therapy, acclimate the rat in the restrainer for 30 min to allow it to adapt (Figure 1). This device can fully expose the rat&#39;s thigh and immobilize it, facilitating Tuina maneuvers (see Table of Materials). 
	NOTE: The temperature in the treatment room should be maintained between 22-26 &#176;C, and the humidity should be between 40%-50%.</li>
	<li>Standardization of Tuina: ensure that the therapists wear wireless finger sleeves that can monitor the pressure and frequency of Tuina and provide real-time feedback data. First, practice Tuina with the feedback data of the finger sleeves, adjusting the force to 5 N and the frequency to 2 Hz. Then, perform the same maneuvers on the rats, maintaining a consistent force and frequency throughout the procedure (Figure 2). 
	NOTE: Based on our previous work, the calculated optimal pressing force is 5 N (see Discussion section for details).</li>
	<li>Identify the acupoint: select the gastrocnemius muscle of the right hind limb as the Tuina area of the rat, at the junction of the two heads of the gastrocnemius muscle, approximately at the location of BL5714.</li>
	<li>Perform the Tuina: ensure that the therapist faces the posterior aspect of the rat&#39;s thigh and holds the rat&#39;s right hind limb with their right upper limb. Position the thumb vertically on the BL57 acupoint, and ensure the forearm and fingers exert force to perform rhythmic small-range rotary motion while applying a 5 N pressure (Figure 3).</li>
	<li>During treatment, ensure the manipulation feedback&#39;s force and frequency correspond to the preset values. Initiate the intervention from the fourth day after surgery, with Tuina performed once a day for 15 min, continuously for 18 days.</li>
</ol>
4. Behavioral testing for pain
NOTE: Behavioral tests were conducted before modeling, after modeling, on intervention day 1, intervention day 3, intervention day 7, intervention day 14, intervention day 17, and intervention day 21.
<ol>
	<li>Perform Mechanical Stimulation Response Threshold (Paw Withdrawal Threshold, PWT) following the steps below (Figure 4).
	<ol>
		<li>Use the von Frey method to test the response threshold of mechanical stimulation in the rats&#39; feet. Place the rats in a transparent tempered glass compartment measuring 20 cm &#215; 10 cm &#215; 20 cm, which was placed on a metal wire grid stand with 10 mm &#215; 10 mm apertures at a height of 40 cm. Keep the room temperature at 23 &#177; 2 &#176;C, and the surrounding environment quiet.</li>
		<li>Measure the mechanical withdrawal threshold with electronic Von Frey fibers (see Table of Materials). Stimulate the rat&#39;s foot center until it moves noticeably, such as raising the leg or avoiding it. The machine records the maximum pressure value (N) automatically.</li>
		<li>Wait for 15 s or more before stimulating the same rat again. Keep each stimulation under 5 s to prevent tactile sensitization in the rat&#39;s paws. Repeat the test five times until the three consecutive measurements differ by less than 10 N.</li>
	</ol>
	</li>
	<li>Perform Paw Withdrawal Latency (PWL) in response to thermal stimulation (Figure 5).
	<ol>
		<li>Assess the PWL using the Hargreaves method15,16. Place the rats in a small chamber made of transparent tempered glass, measuring 20 cm x 10 cm x 20 cm, with a transparent glass lid with a vent hole. Heat the central area of the glass lid to 45 &#176;C using a heating plate until it reaches a stable temperature.</li>
		<li>During the behavioral testing phase, acclimate the rats to the behavioral lab for at least 2 h each day to minimize the impact of environmental factors on test results.</li>
		<li>Before the formal testing, place the rats in the behavioral lab for 30 min to allow them to adapt to the environment and reduce interference.</li>
		<li>Heat the heating plate to 45 &#176;C, and place the rat&#39;s hind limbs on the heating plate.</li>
		<li>The paw withdrawal latency (PWL) was defined as the time from the start of heating to the appearance of the paw withdrawal reflex in response to thermal stimulation. In each test, test the same hind limb three times in a row, and the average value to obtain the response latency of that hind limb. After testing, return the rats to their cages for feeding.</li>
	</ol>
	</li>
</ol>
5. Perfusion
<ol>
	<li>Preparation: prepare a 0.9% saline solution and a 4% paraformaldehyde solution in advance. Place the saline solution in an oven set to a constant temperature of 37 &#176;C, and store the paraformaldehyde solution in a refrigerator at 4 &#176;C for later use.</li>
	<li>Perform anesthesia and establish access.
	<ol>
		<li>Start by injecting 25% Urethane (0.6 mL/100 g, see Table of Materials) into the rat&#39;s abdominal cavity to induce deep anesthesia. Wait until no toe, corneal, or turning reflex is observed. Fix the rat on a foam board.</li>
		<li>Cut the chest bone with scissors and open the skin and fascia layer by layer. Cut the diaphragm and cut off the ribs on both sides to fully expose the heart. Carefully separate the pericardium. Separate the lungs from the heart.</li>
		<li>Use forceps to pull and expose the aorta by pulling the heart toward oneself. Align the needle, left ventricle, and aorta in a straight line and on the same horizontal plane. Then insert the needle from the left ventricle into the aorta until the needle is visible inside the aorta.</li>
		<li>Use forceps to clamp the aorta and the needle inside the aorta, and then cut open the left atrium with scissors. At this time, a large amount of blood will spurt out of the left atrium. Open the valve for the saline solution and connect the saline injection to perfuse 37 &#176;C saline solution, for a total of about 150-200 mL.</li>
	</ol>
	</li>
	<li>After the saline perfusion is completed, switch to the 4% paraformaldehyde solution and perfuse with 4% paraformaldehyde solution for a total of about 400 mL at 4 &#176;C. When starting the paraformaldehyde perfusion, hold the rat&#39;s front teeth with one pair of forceps and pull them forward, while holding the tail with one hand and pulling it backward, which is advantageous for fully extending the spine and increasing the intervertebral foramen to facilitate DRG sampling.</li>
	<li>During the perfusion, the rat&#39;s liver, mesentery, and greater omentum gradually turn pale until the liver becomes stiff. Then, slow down the flow rate and adjust it to about 2 drops per second until all the paraformaldehyde is perfused.</li>
</ol>
6. Dorsal root ganglion collection
NOTE: After perfusion, quickly cut off the lumbar section of the rat spine. Locate the L5 and L4 intervertebral foramen by connecting the highest points of the iliac crest on both sides to the L5 lumbar spinous process using this positioning method, and remove the dorsal root ganglion from the intervertebral foramen. The specific collection method is as follows:
<ol>
	<li>From the entrance of the spinal canal (thoracic spinal canal), insert the scissors into the spinal canal and cut off the lamina on both sides until all the laminae can be completely removed, exposing the entire spinal canal. 
	NOTE: Be careful not to damage the spinal cord and nerve roots outside the spinal canal when cutting the laminae.</li>
	<li>Carefully remove the spinal cord and posterior longitudinal ligament. Separate the dura mater attached to the inner hole of the intervertebral foramen.</li>
	<li>Use ophthalmic forceps to clamp and pull out the dorsal root ganglion, which is shaped like a pearl and slightly yellowish. 
	NOTE: Due to the fragility and poor toughness of nervous tissue, it is necessary to grasp the strength and pulling direction when pulling out the dorsal root ganglion and not use brute force.</li>
	<li>When pulling out the dorsal root ganglion, ensure to clean the surrounding soft tissues, including the dura mater and arachnoid, in advance to facilitate the smooth pulling of the dorsal root ganglion.</li>
	<li>Place the dorsal root ganglion on absorbent paper, cut off the axon with a blade, and clean the blood vessels on the surface of the dorsal root ganglion.</li>
	<li>After trimming, weigh the dorsal root ganglion and immerse it in a 4% paraformaldehyde solution with a concentration of 4% and a temperature of 4 &#176;C for sufficient time, usually about 2-4 h, then transfer the dorsal root ganglion to 10%, 20%, and 30% PB sucrose solution (4 &#176;C) prepared in advance for stepwise dehydration.</li>
</ol>
7. Cryosectioning
<ol>
	<li>Start by placing the dorsal root ganglion in a 0.01 M PBS solution. Shake them for 10 min and then wash away the sucrose solution. Carefully trim the axon fibers of both segments of the dorsal root ganglia. Clean the cryosectioning machine&#39;s freezing head and add the optimal cutting temperature (OCT) compound to it (see Table of Materials).</li>
	<li>Place the freezing head on the surface of a metal shell containing liquid nitrogen, wait until the tissue is frozen, remove it, and trim it flat. After that, place the freezing head on the base of the slicer. The thickness of the dorsal root ganglia slices should be 15 mm. Arrange the sliced thin sections in a wooden box in order and store them in a -20 &#176;C refrigerator, away from light.</li>
</ol>
8. Hematoxylin and Eosin staining
<ol>
	<li>Place the sections 2 min in xylene, and dehydrate them in a series of alcohols, including 100%, 95%, 80%, and 70%, for 2 min each. Then place the sections 2 min in distilled water, 1 min in hematoxylin, perform 5 min of tap water rinse, and perform differentiation in 1% saline alcohol solution for 30 s, followed by 30 s in saturated lithium carbonate.</li>
	<li>Next, place the slices 2 min each in distilled water and tap water, 5 min in eosin solution (0.5%), a 1 min quick rinse in distilled water, two rounds of 2 min each in 95% and 100% alcohol, 30 s in 100% xylene with added sodium bicarbonate, three rounds of 3 min each in xylene, and finally, seal with neutral balsam (see Table of Materials).</li>
</ol>

Standardized Tuina Protocol for Rat DRG Compression Model

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The authors have nothing to disclose.

Our research group has conducted relevant studies on the parameters of Tuina manipulation in early stage. First, it is important to set the force intensity for Tuina manipulation. In clinical Tuina, practitioners adjust the force intensity according to their experience and patients&#39; subjective feelings, achieving the best Tuina effect through communication. However, this is not feasible in animal experiments. In animal experiments, a &#34;response threshold&#34; is used to define the intensity of Tuina manipulation. These judges whether the applied Tuina force is appropriate based on the animal&#39;s instinctive response. Randall17 used this method to study pain behavior in rat inflammation models. The difference is that Randall operated in the local inflammation area, while this experiment operated in the non-injury area. Heavy Tuina manipulation will make rats produce screaming and escape reflexes. The setting of this heavy force intensity determines that the formal Tuina force intensity is less than this heavy force intensity. Based on the distribution of maximum scream and escape reflex pressing values of 150 rats between 5-25 N, with a mean and standard deviation of 9.93 and 3.018 (Figure 11), our previous work calculated the optimal pressing force value to be 5 N. Second, the operating frequency of the Tuina method is described in the Science of Tuina as 120-160 times/min18. Therefore, the Tuina frequency in this experiment was set at 2 Hz.
The CCD model is a surgical model that can be traumatic, as it requires exposure to the articular process and intervertebral foramen. Therefore, rats need 1-3 days to recover after surgery. According to both the literature and preliminary experimental data, the pain behavior of rats tends to be stable on the fourth day after surgery. This provides a favorable time to observe the analgesic effect of Tuina intervention. However, prolonged Tuina may not be beneficial to the analgesic effect of rats and could lead to tissue damage. This study compared the analgesic effects of Tuina and kneading for 5, 15, and 30 min, and ultimately chose 15 min as the optimal duration of the intervention.
This method has limitations, as only a portion of the clinical operations was used to select manipulations and body parts. BL57 (Chengshan) is situated in the triceps surae of rats, and it is innervated by the dorsal root ganglia of L4 and L519. Because the triceps surae muscles are thick and suitable for Tuina, massaging this area can have a direct impact on the dorsal root ganglia of L4 and L5 in the rat lumbar vertebrae that are used for experimental sampling.
During the molding process, one needs to ensure accurate insertion of the &#34;L&#34; shaped stainless steel rod into the intervertebral foramina of L4 and L5 in the CCD pain model by performing X-ray verification. Before the X-ray was taken, we administered intraperitoneal 25% duration anesthesia (0.6 mL/100 g) to anesthetize the rats. The X-ray confirmed the successful insertion of the &#34;L&#34; shaped stainless steel rod into the intervertebral foramina of L4 and L5 (Figure 12).
Overall, this study focused on observing Tuina treatment operations and evaluating the therapeutic effects of Tuina on CCD rats. The team conducted an animal experiment to explore how to set Tuina parameters, select appropriate parts, and determine suitable treatment times. This provides a standardized and reproducible animal model intervention demonstration for future research on the analgesic effect of Tuina.

In clinical settings, it is common to observe neurological pathological pain caused by nerve root compression due to various reasons. The most typical form of this neuropathic pain is lumbar disc herniation (LDH), which is often persistent, recurrent, and difficult to cure. Approximately 9% of the global population is affected by LDH, leading to significant social and economic burdens1. The incidence of this type of neuropathic pain is increasing yearly, with a trend toward younger patients, due to changes in human production and lifestyle2. Despite the use of non-steroidal painkillers, patients&#39; symptoms cannot be completely alleviated. As a result, alternative therapies, such as Tuina, for treating pain caused by LDH have gained increasing attention.
Tuina therapy, a form of conservative treatment for LDH, is widely recommended in various clinical practice guidelines worldwide for preventing and treating lower back pain3,4. Research has shown that Tuina can significantly lower inflammatory factors such as serum IL-6 and tumor necrosis factor-alpha (TNF-&#945;) levels in LDH patients while improving patients&#39; pain and lumbar function impairment5. However, the specific mechanism behind Tuina therapy&#39;s pain-relieving effects remains unclear.
Animal models are a valuable tool for studying neuropathic pain caused by LDH6. They allow for behavioral measurements to evaluate the effectiveness of Tuina therapy and provide samples of the pathological physiology of LDH. For example, samples from the dorsal root ganglia in the thigh can be taken to verify changes in dorsal root ganglion cells. The chronic compression of the Dorsal Root Ganglion (CCD) model is commonly used to evaluate the pathological physiology of LDH, as it causes damage to the morphology of dorsal root ganglion cells that are consistent with the pathological changes seen in clinical cases of nerve compression caused by disc herniation7.
Many scholars have conducted several animal experiments on acupressure analgesia8,9,10. However, when implementing acupressure operations on animal models, they often imitate human acupressure. The therapeutic effect of acupressure is affected by factors such as the size, frequency, and the direction of the applied force11,12,13. If the experiment lacks a unified acupressure standard, such as the force, frequency, and duration of the operation, this may cause some deviation in the experimental results. This article introduces a set of acupressure treatment plans based on the characteristics of CCD rats, and promotes the development of standardized acupressure operations in animal models.

<table><tbody><tr><td>&quot;L&quot; stainless steel rod (4 mm long and 0.4 mm in diameter)</td><td>hand-made</td><td>/</td><td>For CCD models making</td></tr><tr><td>ALMEMO admeasuring apparatus</td><td>ahlborn</td><td>2450-1</td><td>Mechanical Withdrawal Threshold test</td></tr><tr><td>Constant temperature slicer CM-1900</td><td>Leica</td><td>1491950C1US</td><td>For specimen production</td></tr><tr><td>Disinfectant (iodine) 100 mL/bottle</td><td>LIRCON/Shandong Lilkang</td><td>/</td><td>For disinfection</td></tr><tr><td>Disposable sterile syringe 5 mL</td><td>Shanghai Misha Wa Medical Industry</td><td>/</td><td>For injection</td></tr><tr><td>Electron microscope CX-31</td><td>Olympus, Japan</td><td>BJ002318</td><td>For specimen observation</td></tr><tr><td>Finger pressure recordings</td><td>Suzhou Changxian Optoelectronic Technology</td><td>CX1003w</td><td>For Tuina manipulation</td></tr><tr><td>Foam board (35 cm x 20 cm)</td><td>hand-made</td><td>/</td><td>It is our homemade apparatus for fixing rats</td></tr><tr><td>MERSILK W2512</td><td>Johnson &amp;amp; Johnson</td><td>/</td><td>For tissue suture</td></tr><tr><td>Neutral balsam</td><td>Sinopharm Chemical Reagent</td><td>10004160</td><td>For specimen production</td></tr><tr><td>paraformaldehyde</td><td>China National Chemical Reagent</td><td>/</td><td>For specimen production</td></tr><tr><td>Pentobarbital sodium</td><td>Sigma-Aldrich</td><td>P3761</td><td>For anesthesia of rat</td></tr><tr><td>Plantar Test Apparatus (Hargreaves Method) for Mice and Rats</td><td>IITC Life Science</td><td>/</td><td>Paw Withdrawal Latency</td></tr><tr><td>Precision electronic scale for experiment JY3002</td><td>Shanghai Precision Scientific Instrument</td><td>/</td><td>Weighing of rat</td></tr><tr><td>Rat hair clipper</td><td>Philips</td><td>HP6341/00</td><td>Shaving of rat fur</td></tr><tr><td>Restrainer for rats</td><td>Tongji University (self-made)</td><td>/</td><td>It is a homemade apparatus made by Tongji University, which can effectively immobilize the rats and fully expose their hind limbs.</td></tr><tr><td>Tissue-Tek&amp;nbsp;O.C.T. Compound</td><td>SAKURA</td><td>4583</td><td>For specimen production</td></tr><tr><td>Uratan</td><td>China National Chemical Reagent</td><td>/</td><td>For anesthesia of rat</td></tr><tr><td>X-ray detector XR-600</td><td>Dongguan Kaso Electronic Technology</td><td>/</td><td>Examination of CCD models</td></tr><tr><td>xylene</td><td>Shanghai Sinopharm Group</td><td>100092</td><td>For specimen production</td></tr></tbody></table>

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.

Tuina therapy can help decrease rats&#39; mechanical and thermal stimulation thresholds caused by CCD modeling 
After 17 days of Tuina therapy, a significant difference was observed in PWT thresholds between the CCD rats receiving Tuina therapy and the untreated CCD group (P = 0.021, &#60;0.05) (Figure 6 and Table 1).
The rats in the CCD group receiving Tuina therapy showed improvement in pain threshold from the beginning of treatment, and a significant difference in thermal pain threshold was found between the CCD group and the group receiving Tuina therapy from day 14 after modeling (P = 0.0047, 0.0056, 0.0049, &#60; 0.01) (Figure 7 and Table 2).
The application of Tuina therapy did not improve the cell necrosis caused by the CCD model 
Based on the HE staining of the dorsal root ganglia, the CCD group of rats, which were subjected to physical compression with an &#34;L&#34;-shaped stainless steel rod, had incurred cell membrane damage and apoptosis (Figure 8). In contrast, the control group of rats had neatly defined edges, intact neuron contours, and full cell bodies (Figure 9). In the CCD + Tuina group, some neuron contours in the dorsal root ganglia of rats were incomplete (Figure 10).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65535/65535fig01.jpg" /> 
Figure 1: Restrainer for rats. It is a homemade apparatus fabricated by Tongji University, which can effectively immobilize the rats and fully expose their hind limbs. The silver screw controls the baffle that holds the rat&#39;s tail and restrains the rat. The black screw adjusts the device to the length of the rat. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65535/65535fig01large.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/65535/65535fig02.jpg" /> 
Figure 2: Wearing wireless tactile force measurement finger sleeves. A device that measures and displays the force and frequency of finger pressure provides real-time feedback on the intensity and frequency during Tuina manipulation. (a) Pressure sensor and transmission equipment. (b) Finger pressure measurements. (c) The force measured during Tuina manipulation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65535/65535fig02large.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/65535/65535fig03.jpg" /> 
Figure 3: Body position and acupoint location for rat Tuina. The rat restrainer can completely expose the position of BL57. The limbs, together with the thumb, grasp the lower limbs of the rat to fix it in place so that the rat can cooperate quietly during treatment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65535/65535fig03large.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/65535/65535fig04.jpg" /> 
Figure 4. Paw withdrawal threshold test. This is a test to measure pain behavior in rats. It involves mechanically stimulating their feet and measuring their paw withdrawal latency. (a) shows the apparatus, and (b) shows how it was manipulated on the mouse during the experiment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65535/65535fig04large.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/65535/65535fig05.jpg" /> 
Figure 5: Paw withdrawal latency test. This setup measures the rats&#39; pain threshold by their sensitivity to the heat generated by a spotlight. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65535/65535fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65535/65535fig06.jpg" /> 
Figure 6: Results of the Paw withdrawal threshold test. Measurements are represented using the mean plus or minus standard error (<img alt="Equation 1" src="/files/ftp_upload/65535/65535eq01.jpg" style="margin: auto;" />). Results of the mechanical stimulus-induced hindlimb withdrawal reflex threshold test at different stages for each group of rats. Significant differences (P &#60; 0.05) were observed between the CCD + Tuina and the CCD model groups from day 17 onwards. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65535/65535fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65535/65535fig07.jpg" /> 
Figure 7: Results of the paw withdrawal latency test. Measurements are represented using the mean plus or minus standard error (<img alt="Equation 1" src="/files/ftp_upload/65535/65535eq01.jpg" style="margin: auto;" />). Results of threshold testing of heat stimulation-induced leg withdrawal reflex in different stages of rats in each group. Significant differences were observed between the CCD + Tuina group and the CCD model group starting from the seventh day (P &#60; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65535/65535fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65535/65535fig08.jpg" /> 
Figure 8: Microscopic observation of dorsal root ganglion neurons in the CCD group (longitudinal section). This image was obtained by scanning the HE-stained sample of the dorsal root ganglion. The red ellipse in the figure indicates that some neurons are undergoing necrosis. This indicates that the CCD model has caused damage to the neurons in the dorsal root ganglion.&#160;Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65535/65535fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/65535/65535fig09.jpg" /> 
Figure 9. Microscopic observation of dorsal root ganglion neurons in the naive group (longitudinal section and cross section). As shown in the above figure, (a) is a longitudinal section, and (b) is a transverse section. There is no CCD group vacancy phenomenon in the area where the blank group of rats&#39; dorsal root ganglia is gathered. The neurons are continuous and compact, and the cell bodies are filled. The satellite glial cells are located between the neurons.Scale bar: (a),50 &#181;m; (b), 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65535/65535fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/65535/65535fig10.jpg" /> 
Figure 10: Microscopic observation of dorsal root ganglion neurons in the CCD + Tuina group (cross-section). The neurons are continuous, and there is a cell dissolution phenomenon. This indicates that in the short term, massage therapy was unable to improve the cell necrosis (red ellipse) caused by the CCD model.Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65535/65535fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/65535/65535fig11.jpg" /> 
Figure 11: Tuina pressure test. Rats with minimum hissing-escape reflex compression values vary in size, ranging from 5 N to 25 N. The abscissa represents the degree of pressure, while the ordinate represents the number of rats. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65535/65535fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/65535/65535fig12.jpg" /> 
Figure 12: X-ray images of CCD rats. Validation of CCD fabrication using X-ray imaging of rat models. (a) shows the X-ray taken in the vertical plane, and (b)shows the X-ray taken in the sagittal plane. The two different cross-sectional X-rays allow a clearer view of the position of the &#34;L&#34;-shaped stainless steel rod. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65535/65535fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>N</td>
			<td>Before Modeling</td>
			<td>D1</td>
			<td>D3</td>
			<td>D7</td>
			<td>D14</td>
			<td>D17</td>
			<td>D21</td>
		</tr>
		<tr>
			<td>Na&#239;ve</td>
			<td>8</td>
			<td>18.1&#177;1.4</td>
			<td>16.8+1.5</td>
			<td>16.8&#177;1.5</td>
			<td>18.6&#177;1.7</td>
			<td>16.8&#177;1.5</td>
			<td>15.6&#177;1.8</td>
			<td>16.8&#177;1.5</td>
		</tr>
		<tr>
			<td>Sham</td>
			<td>8</td>
			<td>18.0&#177;1.7</td>
			<td>18.6&#177;1.7</td>
			<td>16.0&#177;1.3</td>
			<td>17.6&#177;1.7</td>
			<td>16.8&#177;1.5</td>
			<td>18.9&#177;1.7</td>
			<td>16.8&#177;1.5</td>
		</tr>
		<tr>
			<td>CCD</td>
			<td>8</td>
			<td>17.7&#177;1.1</td>
			<td>10.7&#177;2.8#</td>
			<td>10.0&#177;1.5</td>
			<td>4.4&#177;2.2</td>
			<td>3.8&#177;1.7</td>
			<td>4.1&#177;2.4</td>
			<td>3.8&#177;2.5</td>
		</tr>
		<tr>
			<td>CCD + Tuina</td>
			<td>8</td>
			<td>18.9&#177;1.7</td>
			<td>9.8&#177;2.3#</td>
			<td>8.3&#177;1.4</td>
			<td>4.8&#177;1.2</td>
			<td>5.8&#177;2.0</td>
			<td>7.2&#177;1.8*</td>
			<td>7.5&#177;1.8*</td>
		</tr>
		<tr>
			<td colspan="9">#Comparison before and D1 modeling, p&#60;0.05.</td>
		</tr>
		<tr>
			<td colspan="9">*Comparison between CCD group and CCD+Tuina group, p&#60;0.05.</td>
		</tr>
	</tbody>
</table>
Table 1: Paw Withdrawal Threshold, PWT (<img alt="Equation 1" src="/files/ftp_upload/65535/65535eq01.jpg" style="margin: auto;" />, s). Comparison before and D1 modeling, #p &#60; 0.05. Comparison between the CCD group and CCD + Tuina group, *p &#60; 0.05.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>N</td>
			<td>Before Modeling</td>
			<td>D1</td>
			<td>D3</td>
			<td>D7</td>
			<td>D14</td>
			<td>D17</td>
			<td>D21</td>
		</tr>
		<tr>
			<td>Na&#239;ve</td>
			<td>8</td>
			<td>11.9&#177;1.2</td>
			<td>12.0&#177;1.6</td>
			<td>12.2&#177;1.9</td>
			<td>12.4&#177;1.1</td>
			<td>12.2&#177;1.9</td>
			<td>12.0&#177;1.4</td>
			<td>12.0&#177;1.4</td>
		</tr>
		<tr>
			<td>Sham</td>
			<td>8</td>
			<td>11.9&#177;1.2</td>
			<td>11.6&#177;1.5</td>
			<td>12.2&#177;1.9</td>
			<td>11.6&#177;1.5</td>
			<td>12.2&#177;0.9</td>
			<td>11.6&#177;1.5</td>
			<td>11.6&#177;1.5</td>
		</tr>
		<tr>
			<td>CCD</td>
			<td>8</td>
			<td>10.8&#177;1.1</td>
			<td>8.9&#177;0.7#</td>
			<td>7.9&#177;0.8</td>
			<td>7.7&#177;0.5</td>
			<td>7.8&#177;1.0</td>
			<td>7.7&#177;0.8</td>
			<td>7.7&#177;0.8</td>
		</tr>
		<tr>
			<td>CCD + Tuina</td>
			<td>8</td>
			<td>11.3&#177;1.5</td>
			<td>9.1&#177;0.6#</td>
			<td>8.0&#177;0.7</td>
			<td>8.3&#177;0.7*</td>
			<td>8.9&#177;0.6*</td>
			<td>9.1&#177;0.7*</td>
			<td>9.2&#177;0.9*</td>
		</tr>
		<tr>
			<td colspan="9">#Comparison before and D1 modeling, p &#60; 0.05.</td>
		</tr>
		<tr>
			<td colspan="9">Comparison between CCD group and CCD+Tuina group,*p &#60; 0.05.</td>
		</tr>
	</tbody>
</table>
Table 2: Paw Withdrawal Latency, PWL (<img alt="Equation 1" src="/files/ftp_upload/65535/65535eq01.jpg" style="margin: auto;" />, s).&#160;Comparison before and D1 modeling,&#160;#p &#60; 0.05. Comparison between the CCD group and CCD + Tuina group,*p &#60; 0.05.

Watch this Scientific Journal Video about Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain at JoVE.com

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.

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 ...

analgesic-effect-tuina-on-rat-models-with-compression-dorsal-root

Nanyang Technological University

Research

JoVE Journal

Medicine

1.3K Views.  Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Shanghai University of Traditional  Chinese Medicine. Tuina-analgesie wordt veel gebruikt in de klinische praktijk. Ons onderzoek richt zich op het werkingsmechanisme van Tuina analgesie, en onderzoekt hoe Tuina zijn pijnstillende werking bereikt. Het bereiken van standaardisatie in Tuina is op dit moment het belangrijkste obstakel.De werkzaamheid van Tuina wordt beïnvloed door verschillende factoren, zoals behandeling, gebiedstype, intensiteit en frequentie. Het huidige protocol is op basis van eerder onderzoek in twee opzichten geoptim...

Video: Auteur in de schijnwerpers: Pijnstillend effect van Tuina op rattenmodellen met compressie van de dorsale wortel Ganglionpijn  

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This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

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Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

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A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

Spontaneous and Evoked Measures of Pain in Murine Models of Monoarticular Knee Pain

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are necessary and useful for studying the mechanisms of pain and for evaluating efficacy of arthritis therapies. Measuring pain in animal models of arthritis is challenging. We have developed methods for measuring evoked and spontaneous pain in three models of murine arthritis. We quantitate the evoked pain responses of mice subjected to firm palpation of a painful knee. We also evaluate spontaneous pain by the proportion of weight and the amount of time placed on each of their 4 limbs after induction of arthritis pain in one knee. Joint pain in these mouse models produces a significant increase in evoked pain responses and an alteration in weight bearing. Since mice are quadrupeds, they offload the painful limb to the contralateral limb, to the forelimbs, or some combination. These methods are simple, require minimal equipment, and are reproducible and sensitive for detecting pain. They are useful for studying both disease-modifying arthritis treatments and analgesics in mice.

We have developed an evoked measure of arthritis pain and coupled it with a standardized method for measuring spontaneous pain in different murine models of chemically induced arthritis. These measures are sensitive and reproducible for different types of joint pain.

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are ...

Three-Dimensional Finger Motion Tracking during Needling: A Solution for the Kinematic Analysis of Acupuncture Manipulation

Three-dimensional (3D) motion tracking has been used in many fields, such as the researches of sport and medical skills. This experiment aimed to use 3D motion tracking technology to measure the kinematic parameters of the joints of fingers during acupuncture manipulation (AM) and establish three technical indicators "amplitude, velocity and time". This method can reflect the operation characteristics of AM and provide quantitative parameters along three axes of multiple finger joints. The current evidence shows that the method has great potential for future applications such as the study of the dose-effect relationship of acupuncture, teaching, and learning of AM, and the measurement and preservation of famous acupuncturists' AM.

This experimental method describes a solution for the kinematic analysis of acupuncture manipulation with three-dimensional finger motion tracking technology.

Three-dimensional (3D) motion tracking has been used in many fields, such as the researches of sport and medical skills. This experiment aimed to use 3D ...

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

Standardized Tuina Manipulation for Knee Osteoarthritis in Rats

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Author Spotlight: Understanding the Mechanism of Tuina Manipulation in Rats 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...

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 ...

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

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