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.
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.
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 pain3,4. 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 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.
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
2. Establishing animal model
3. Tuina therapy
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.
5. Perfusion
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:
7. Cryosectioning
8. Hematoxylin and Eosin staining
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 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, < 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 "L"-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).
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's tail and restrains the rat. The black screw adjusts the device to the length of the rat. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 5: Paw withdrawal latency test. This setup measures the rats' pain threshold by their sensitivity to the heat generated by a spotlight. Please click here to view a larger version of this figure.
Figure 6: Results of the Paw withdrawal threshold test. Measurements are represented using the mean plus or minus standard error (). Results of the mechanical stimulus-induced hindlimb withdrawal reflex threshold test at different stages for each group of rats. Significant differences (P < 0.05) were observed between the CCD + Tuina and the CCD model groups from day 17 onwards. Please click here to view a larger version of this figure.
Figure 7: Results of the paw withdrawal latency test. Measurements are represented using the mean plus or minus standard error (). 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 < 0.05). Please click here to view a larger version of this figure.
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. Scale bar = 50 µm. Please click here to view a larger version of this figure.
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' 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 µm; (b), 100 µm. Please click here to view a larger version of this figure.
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 µm. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 "L"-shaped stainless steel rod. Please click here to view a larger version of this figure.
N | Before Modeling | D1 | D3 | D7 | D14 | D17 | D21 | |
Naïve | 8 | 18.1±1.4 | 16.8+1.5 | 16.8±1.5 | 18.6±1.7 | 16.8±1.5 | 15.6±1.8 | 16.8±1.5 |
Sham | 8 | 18.0±1.7 | 18.6±1.7 | 16.0±1.3 | 17.6±1.7 | 16.8±1.5 | 18.9±1.7 | 16.8±1.5 |
CCD | 8 | 17.7±1.1 | 10.7±2.8# | 10.0±1.5 | 4.4±2.2 | 3.8±1.7 | 4.1±2.4 | 3.8±2.5 |
CCD + Tuina | 8 | 18.9±1.7 | 9.8±2.3# | 8.3±1.4 | 4.8±1.2 | 5.8±2.0 | 7.2±1.8* | 7.5±1.8* |
#Comparison before and D1 modeling, p<0.05. | ||||||||
*Comparison between CCD group and CCD+Tuina group, p<0.05. |
Table 1: Paw Withdrawal Threshold, PWT (, s). Comparison before and D1 modeling, #p < 0.05. Comparison between the CCD group and CCD + Tuina group, *p < 0.05.
N | Before Modeling | D1 | D3 | D7 | D14 | D17 | D21 | |
Naïve | 8 | 11.9±1.2 | 12.0±1.6 | 12.2±1.9 | 12.4±1.1 | 12.2±1.9 | 12.0±1.4 | 12.0±1.4 |
Sham | 8 | 11.9±1.2 | 11.6±1.5 | 12.2±1.9 | 11.6±1.5 | 12.2±0.9 | 11.6±1.5 | 11.6±1.5 |
CCD | 8 | 10.8±1.1 | 8.9±0.7# | 7.9±0.8 | 7.7±0.5 | 7.8±1.0 | 7.7±0.8 | 7.7±0.8 |
CCD + Tuina | 8 | 11.3±1.5 | 9.1±0.6# | 8.0±0.7 | 8.3±0.7* | 8.9±0.6* | 9.1±0.7* | 9.2±0.9* |
#Comparison before and D1 modeling, p < 0.05. | ||||||||
Comparison between CCD group and CCD+Tuina group,*p < 0.05. |
Table 2: Paw Withdrawal Latency, PWL (, s). Comparison before and D1 modeling, #p < 0.05. Comparison between the CCD group and CCD + Tuina group,*p < 0.05.
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. These judges whether the applied Tuina force is appropriate based on the animal'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 "L" 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 "L" 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.
The authors have nothing to disclose.
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).
"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 |