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

Summary

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.

Abstract

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.

Introduction

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' 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 pain^3,4. Research has shown that Tuina can significantly lower inflammatory factors such as serum IL-6 and tumor necrosis factor-alpha (TNF-α) levels in LDH patients while improving patients' pain and lumbar function impairment⁵. However, the specific mechanism behind Tuina therapy'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 analgesia^8,9,10. 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 force^11,12,13. 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.

Protocol

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 ± 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 ± 1 °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' 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

1. 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.
   ​NOTE: Naive rats had no intervention, while the sham group underwent the same surgical procedure as the CCD group rats, but without leaving an "L"-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.

2. Establishing animal model

1. Administer isofluorane 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: Analgesic drugs were applied 15 min before surgery and rats were injected subcutaneously with tramadol 20 mg/kg.
2. 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.
3. Insert the "L"-shaped probe (see Table of Materials).
   1. 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.
   2. 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.
   3. 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'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.
   4. First, use an "L"-shaped probe to determine the position of the intervertebral foramen, and then use an "L"-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.
   5. 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.
4. 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.

3. Tuina therapy

1. 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'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 °C, and the humidity should be between 40%-50%.
2. 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).
3. 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.
4. Perform the Tuina: ensure that the therapist faces the posterior aspect of the rat's thigh and holds the rat'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).
5. During treatment, ensure the manipulation feedback'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.

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.

1. Perform Mechanical Stimulation Response Threshold (Paw Withdrawal Threshold, PWT) following the steps below (Figure 4).
   1. Use the von Frey method to test the response threshold of mechanical stimulation in the rats' feet. Place the rats in a transparent tempered glass compartment measuring 20 cm × 10 cm × 20 cm, which was placed on a metal wire grid stand with 10 mm × 10 mm apertures at a height of 40 cm. Keep the room temperature at 23 ± 2 °C, and the surrounding environment quiet.
   2. Measure the mechanical withdrawal threshold with electronic Von Frey fibers (see Table of Materials). Stimulate the rat's foot center until it moves noticeably, such as raising the leg or avoiding it. The machine records the maximum pressure value (N) automatically.
   3. 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's paws. Repeat the test five times until the three consecutive measurements differ by less than 10 N.
2. Perform Paw Withdrawal Latency (PWL) in response to thermal stimulation (Figure 5).
   1. Assess the PWL using the Hargreaves method^15,16. 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 °C using a heating plate until it reaches a stable temperature.
   2. 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.
   3. 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.
   4. Heat the heating plate to 45 °C, and place the rat's hind limbs on the heating plate.
   5. 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.

5. Perfusion

1. 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 °C, and store the paraformaldehyde solution in a refrigerator at 4 °C for later use.
2. Perform anesthesia and establish access.
   1. Start by injecting 25% Urethane (0.6 mL/100 g, see Table of Materials) into the rat's abdominal cavity to induce deep anesthesia. Wait until no toe, corneal, or turning reflex is observed. Fix the rat on a foam board.
   2. 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.
   3. 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.
   4. 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 °C saline solution, for a total of about 150-200 mL.
3. 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 °C. When starting the paraformaldehyde perfusion, hold the rat'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.
4. During the perfusion, the rat'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.

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:

1. 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.
2. Carefully remove the spinal cord and posterior longitudinal ligament. Separate the dura mater attached to the inner hole of the intervertebral foramen.
3. 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.
4. 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.
5. 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.
6. 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 °C for sufficient time, usually about 2-4 h, then transfer the dorsal root ganglion to 10%, 20%, and 30% PB sucrose solution (4 °C) prepared in advance for stepwise dehydration.

7. Cryosectioning

1. 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's freezing head and add the optimal cutting temperature (OCT) compound to it (see Table of Materials).
2. 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 °C refrigerator, away from light.

8. Hematoxylin and Eosin staining

1. 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.
2. 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).

Results

Tuina therapy can help decrease rats' 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, <0.05) (Figure 6 and Table 1).

The rats in the CCD group receiving Tuina therapy showed improvement in pain threshold from the begi...

Discussion

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' subjective feelings, achieving the best Tuina effect through communication. However, this is not feasible in animal experiments. In animal experiments, a "response threshold" is used to define the intensity of Tuina manipulation. ...

Materials

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