Manual Therapy for a Chronic Non-Specific Low Back Pain Rat Model

This study proposes a standardized procedure for manual therapy on chronic low back pain model rats, which will be of reference value for future experimental research on manual therapy.

Chronic low back pain (CLBP) is a highly prevalent condition worldwide and a major cause of disability. The majority of patients with CLBP are diagnosed with chronic non-specific low back pain (CNLBP) due to an unknown pathological cause. Manual therapy (MT) is an integral aspect of traditional Chinese medicine and is recognized as Tuina in China. It involves techniques like bone-setting and muscle relaxation manipulation. Despite its clinical efficacy in treating CNLBP, the underlying mechanisms of MT remain unclear. In animal experiments aimed at investigating these mechanisms, one of the main challenges is achieving normative MT on CNLBP model rats. Improving the stability of finger strength is a key issue in MT. To address this technical limitation, a standardized procedure for MT on CNLBP model rats is presented in this study. This procedure significantly enhances the stability of MT with the hands and alleviates common problems associated with immobilizing rats during MT. The findings of this study are of reference value for future experimental investigations of MT.

Chronic low back pain (CLBP) is characterized by persistent low back pain lasting more than 3 months, typically between the rib cage and transverse hip line, with or without lower limb pain^1,2. It is a highly prevalent disease, with an estimated global annual prevalence of 38% and a lifetime prevalence of 39%, making it a common public health issue^3,4. The majority of patients with CLBP, more than 90-95%, cannot be given a definitive pathological and anatomical diagnosis (such as the tumor, fracture, and infection), leading to the classification of Chronic non-specific low back pain (CNLBP)^5,6. Due to the non-specific nature of the pathological mechanism, opioid analgesics and nonsteroidal anti-inflammatory drugs (NSAIDs) are the main treatment options in Western medicine but are associated with safety concerns^7,8. Therefore, there is an increasing demand for complementary and alternative medicines that are both safe and effective.

Manual therapy (MT), commonly referred to as Tui Na in China, is a significant aspect of traditional Chinese medicine (TCM) and encompasses techniques such as bone-setting and muscle relaxation manipulation. Its widespread use in China is attributed to its clinical effectiveness, high safety profile, and non-invasiveness⁹. In the treatment of CNLBP, MT has been found to be an effective complementary and alternative medicine with proven efficacy demonstrated in previous studies^10,11,12. TCM believes that Qi stagnation and blood stasis are the core causes of chronic pain, and Tui Na relieves pain by promoting blood circulation and promoting Qi to muscles and soft tissues. However, despite its efficacy, the precise mechanism underlying its therapeutic effect remains elusive.

In the domain of experimental research on MT, discrepancies exist in the implementation of interventions on animal models. Presently, the majority of researchers opt for machine-based MT, whereas a minority perform MT using human hands^13,14,15,16. While machine-based MT is more standardized, MT is more clinically relevant and produces more representative data. However, MT on experimental animals is hindered by the unstable strength of fingers, leading to a crucial challenge in animal experiments and hampering the comparability of experimental results. To overcome this technical challenge, this study proposes an appropriate solution, which enhances the stability of finger strength during MT on CNLBP model rats. The feasibility of this solution is verified, providing valuable guidance for future experimental studies of MT.

This experimental protocol was approved by the Animal Care and Use Committee of Zhejiang University of Traditional Chinese Medicine, and all procedures adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study used adult male SD rats (see Table of Materials) with a body weight ranging from 330 g to 350 g. All rats were housed in a standard animal facility with a light-dark cycle of 12 h, temperature of 24 ± 2 °C, and humidity of 50 ± 5%. The rats were provided with adequate food and water supply. The following protocol provides a detailed description of the CNLBP model establishment and manual therapy Procedure.

1. Establish the low back pain model according to the following steps (Figure 1)

1. Feed the rats until they weigh 330-350 g, weigh them, and then inject 3% sodium pentobarbital (1 mL/kg) intraperitoneally to anesthetize them (see Table of Materials). Wait for 2 min, then gently pinch the four toes of the hind limbs with forceps, and touch the corneas of the rats with forceps to check for the absence of retraction or blinking response, indicating that the anesthesia is successful.
   NOTE: Autoclave all surgical instruments used in rat modeling in advance and sterilize other non-autoclavable instruments such as shavers, ELFS devices, and thermostatic tables with alcohol. The operator should wear a clean lab coat, disposable sterile gloves, mask, surgical cap, and facemask.
2. Apply an appropriate amount of veterinary ointment to the eyes of rats to prevent drying due to prolonged inability to close eyes after anesthesia.
3. Place the rat in the sternal recumbent position on the constant temperature table, locate its L4-6 segment, shave the hair of the segment and disinfect it with Iodophor 3x before spreading the disposable surgical cavity drape.
4. Cut the skin and fascia of the L4-6 segment with surgical scissors, then separate the muscles on both sides of the spinous process of the lumbar vertebrae of this segment with a scalpel.
   NOTE: Ensure that the muscle is entirely separated from the spinous process and that no muscle fibers remain attached to the spinous process. Furthermore, the depth of separation should extend to the transverse process of the lumbar spine.
5. Drill a hole in the middle of the spinous process of L4-6 with a 5 mL syringe needle, then load the External link fixation system (ELFS) (see Table of Materials) onto the spinous process of L4-6.
6. Disinfect with iodophor, then suture after intramuscular injection of 100,000 units of penicillin (see Table of Materials) on each side of the wound.
   NOTE: Push the ELFS device forward to lower the height of the ELFS device before suturing, then suture under the skin to prevent infection.
7. Take an X-ray before the rat is awake to determine whether the ELFS device is successfully attached to the L4-6 spinous processes (Figure 2).
   NOTE: Place the rat in the sternal recumbent position on the constant temperature table (see Table of Materials) after taking the radiographs, wait until it is awake, then return it to the cage, and do not leave it unattended until it is awake.
8. As previously reported¹⁷, feed each model rat in a single cage for 2 weeks, and if the ELFS device does not fall off at the end of the 2 weeks, the CNLBP modeling is considered successful.
   NOTE: During the postoperative recovery period, a few rats will develop abscesses in the lumbar region due to infection; in this case, use a 2.5 mL syringe to aspirate the pus, then flush the wound with saline, and finally press the wound with a cotton ball for a few seconds.
9. To prevent the rats from damaging each other's ELFS devices, continue to keep each model rat in a single cage after the 2-week recovery period.
   ​NOTE: To prevent the psychological depression of rats caused by single-cage feeding, place a plastic ball toy (see Table of Materials) in the cage.

2. Procedure for manual therapy on CNLBP model rats (Figure 3)

1. Before MT, conduct finger strength stability exercises on a weighing table (see Table of Materials) for 2 weeks. During the exercise, maintain a controlled finger kneading force at approximately 600 g with a frequency of two times per second. Conduct the exercise once a day for a duration of 10 min each time.
2. Before MT, acclimate the model rats to the rat pouch for 1 week. Place the upper body of the rat into the pouch, with the lumbar region and lower limbs exposed. Simultaneously, softly stroke the lumbar region of the rats to encourage them to accept the contact between the fingers and the lumbar region.
   NOTE: Cut the shape of a piece of fabric into a fan shape with a top angle of 90° and a radius of 12 cm. Then, sew the two sides of the scalloped fabric together to make a rat pouch (see Supplementary Figure S1).
3. Place the rat in a rat pouch. At this time, the upper half of the rat's body is covered by the rat pouch, and only the waist and lower limbs are exposed. Hold the rat's upper body in a "C" shape using the non-dominant hand, while performing MT on the rat's lumbar region with the dominant hand.
   NOTE: Do not hold the rat's torso too tightly when holding it with the non-dominant hand. The rat's thorax will be compressed and breathing will be impeded if the rat is held too tightly. It will also cause the rat to become very resistant.
4. Find the exact location of the Jiaji (EX-B2) acupoints. First, determine the location of the rat's L4-6 spinous process. The Jiaji acupoints are located on both sides of L4-6, 0.5 cm from the vertebral spinous process. There are six Jiaji acupoints in total.
   NOTE: In rats, the spines of L6 are on the line of the two anterior superior iliac spines, and the positions of L4 and L5 can be found by counting up along the spines of L6.
5. Place the thumb on the Jiaji acupoint of the L4-6 segment of the rat and gradually increase the kneading intensity. When the model rats show resistance behaviors such as limb writhing, loud screaming, and strong struggling in the quiet state, the kneading force at that time is the maximum pressure value. Ensure that the most appropriate pressure is slightly less than the maximum pressure value, which is the maximum pressure value that the rats can tolerate without showing resistance behaviors.
6. Start with the Jiaji acupoint on the left side of L6. After finding the most appropriate pressure level for the rat's Jiaji acupoint, hold the thumb at that pressure level as long as possible while performing a kneading action at a frequency of two times per second for a duration of one minute. Repeat for the remaining five Jiaji acupoints.
7. If resistant behavior is observed in rats during the kneading procedure, stop the thumb pressure until the rat calms down, and then resume the procedure. Ensure that the kneading process lasts for a duration of 1 min for each acupoint, even if there is an interruption during the process.

3. Paw withdrawal threshold (PWT)

1. Place the rats in a clear Plexiglass box on wire mesh for 30 min before the official test to acclimate them to the environment.
2. Use different sizes of von Frey filaments (0.6, 1, 2, 4, 6, 8, 15, 26 g) (see Table of Materials), after the rats have stopped exploring and grooming, vertically stimulate the middle of the left hind paw, slowly increase the pressure until the filaments are slightly bent, then stop the pressure and maintain the pressure value for 5 s.
   NOTE: Avoid the thicker area of skin in the middle of the hind paw when stimulating the hind paws with the filament.
3. Record positive behavior as X if the rat shows a leg retraction or foot-licking response and give a lower level of stimulus filament. Record the absence of positive behavior as O and give a higher level of stimulus filament.
   NOTE: The interval between two adjacent stimuli should be greater than 30 s.
4. Detect 4x in a row when X and then a sequence of consecutive X O is obtained, and then convert the sequence to the corresponding PWT value according to the method of Chaplan¹⁸.

4. Paw withdrawal latency (PWL)

1. Place the rats in the Hargreaves Apparatus (see Table of Materials) for 30 min before the formal test to allow them to acclimate to the environment.
2. Set the instrument parameters as follows: maximum light exposure time 20 s; light exposure intensity 50% (see Supplementary Figure S2).
   NOTE: The maximum light exposure time should not exceed 20 s to avoid burning the rat's paws, which would affect the accuracy of subsequent test values.
3. Align the + on the heat stimulator with the center of the rat's left hind paw after the rat has stopped exploring and grooming. Click Start, and when the rat exhibits foot lifting behavior and the instrument automatically stops timing, record the resulting time value. Perform a total of five tests for each rat.
   NOTE: The interval between each test for the same rat is at least 5 min to avoid burning the rat's hind paw skin.
4. Remove the maximum and minimum values from the five values measured for each rat, and take the average of the remaining three values as the PWL value of the rat.

5. Hematoxylin and eosin (H&E) staining

1. Anesthetize rats with sodium pentobarbital at the end of 2 weeks of MT and euthanize all rats by cardiac perfusion after they are completely anesthetized.
2. Remove the left psoas muscle from rat L5 and immerse in 4% paraformaldehyde (see Table of Materials) for 48 h fixation.
3. Trim the tissue to the appropriate size and place it in an embedding box (see Table of Materials), and then use the Automatic Dehydrator (see Table of Materials) for dehydration.
4. Use paraffin wax to embed the tissue, and then cut the tissue into 5 µm thick sections.
5. Mount the sections on slides and bake for 4 h.
6. Place tissues in the Automatic Stainer (see Table of Materials) for subsequent dewaxing, hematoxylin staining, differentiation, bluing, eosin staining, dehydration, and transparency¹⁹.
7. Seal the sections with Neutral Resin (see Table of Materials) and leave in a ventilated place for 24 h.
8. Scan the slides using a Digital Slide Scanner (see Table of Materials).

In this study, the aim was to investigate the analgesic effect of MT on CNLBP model rats. For this purpose, 24 rats were randomly assigned to four groups, namely the blank, sham-operated, model, and MT groups, each containing six rats. The blank group did not receive any intervention, while the sham-operated group underwent only a surgical procedure in which the lumbar muscles on both sides of the L4-6 spinous process were separated and sutured without any subsequent intervention. The CNLBP model rats were established in the model group according to the described method without any further intervention. In the MT group, the CNLBP model rats were treated with MT for 2 weeks after successful modeling. All the rats were fed individually in cages with a plastic ball toy. The rats were assessed for paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) on days 15, 17, 21, 24, and 28 after modeling (Figure 4). The statistical analysis of the data was conducted to express all data as mean ± standard deviation. One-way ANOVA was used for comparing multiple groups, while the LSD test was used for comparing two groups when the variance was equal, and the Dunnett T3 test was used when it was not equal. A significance level of p < 0.05 was considered statistically significant. Figure 5 shows the changes in PWT and PWL in each group of rats. The baseline values of PWT and PWL were similar among the groups. No significant fluctuations in PWT and PWL were observed between rats in the blank and sham-operated groups during the 15-28 days after the end of modeling. However, the PWT and PWL of the rats in the model group decreased significantly. In contrast, the PWT and PWL of the MT group showed a trend of decrease followed by an increase and started to increase from day 15 after the end of modeling, exhibiting a statistical difference with the model group on day 17.

In addition, the histological examination was conducted to evaluate the inflammatory status of the lumbar muscle in the CNLBP model rats with the anti-inflammatory effect of the MT. The left lumbar muscle of L5 in each group of rats was subjected to HE staining, and the results are presented in Figure 6. No significant inflammatory cell infiltration was observed in the lumbar muscle of rats in the blank and sham-operated groups. In contrast, the lumbar muscle of rats in the model group showed significant inflammatory cell infiltration. Notably, inflammatory cell infiltration in the lumbar muscle of rats in the MT group was significantly reduced. Our previous study also revealed that the levels of inflammatory mediators such as substance P, CGRP, and NGF were significantly elevated in the lumbar muscles of model rats, whereas MT significantly reduced the levels of these inflammatory mediators, resulting in an improvement of the inflammatory microenvironment in the lumbar muscle of the model rats^20,21. In summary, this study provides detailed guidance on how to properly establish the CNLBP model and perform MT on rats.

Figure 1: CNLBP model construction. (A) Components of the ELFS. (B) Shave and sterilize. (C) Separate the muscles on either side of the spinous process. (D) Drill a hole in the middle of the spinous process with a 5 ml syringe needle. (E) Load the ELFS. (F) Suture the ELFS into the subcutis. (G) Lateral and oblique views of the rat model²². Abbreviations: CNLBP = chronic non-specific low back pain; ELFS = External link fixation system. Please click here to view a larger version of this figure.

Figure 2: The X-ray of the model rats. After modeling, the X-ray was taken before the rats were awake to observe whether the ELFS device was successfully attached to the L4-6 spinous process. The red rectangle indicates that the ELFS was successfully attached to the L4-6 spinous process. Abbreviation: ELFS = External link fixation system. Please click here to view a larger version of this figure.

Figure 3: Pictorial representation of MT process on CNLBP model rats. (A) Place the model rat in the rat pouch. (B) Fix the model rat in the rat pouch with both hands. (C) MT on CNLBP model rats. The three red dots on the rat's body represent the Jiaji acupoint. The red dot on the right thumb is where the dominant hand (here right) performs MT on the Jiaji acupoint. Abbreviations: MT = manual therapy; CNLBP = chronic non-specific low back pain. Please click here to view a larger version of this figure.

Figure 4: Flow chart of animal experiments. The first day after the end of modeling was considered the first day of the whole experiment. PWT and PWL were measured on days 15, 17, 21, 24, and 28 after the start of the experiment for each group of rats. Abbreviations: PWT = paw withdrawal threshold; PWL = paw withdrawal latency. Please click here to view a larger version of this figure.

Figure 5: The changes in PWT and PWL of rats in each group. The values of PWT and PWL of rats in the blank group, sham-operated group, model group, and MT group on days 15, 17, 21, 24, and 28. (A) The PWT values of rats in the model group decreased significantly, and the PWT values of rats in the MT group increased gradually. (B) The PWL values of rats in the model group fluctuated but showed an overall decreasing trend, while the PWL values of rats in the MT group gradually increased. n = 6 per group. ^*P < 0.05, ^**P < 0.01, compared with blank group; ^#P < 0.05, ^##P < 0.01, compared with model group. Abbreviations: PWT = paw withdrawal threshold; PWL = paw withdrawal latency; MT = manual therapy. Please click here to view a larger version of this figure.

Figure 6: HE staining of lumbar muscles from each group of rats. Single examples from each group of rats. (A, B) Normal lumbar muscles of rats in the blank and sham-operated groups after 28 days. (C) After 28 days, the lumbar muscles of rats in the model group showed significant inflammatory cell infiltration. (D) After 28 days, the inflammatory cell infiltration in the lumbar muscles of rats in the MT group was significantly reduced. The black arrows indicate the infiltration of inflammatory cells. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Supplementary Figure S1: Preparation of the rat pouch. (A) The size of the scalloped cloth. (B) The shape of the rat pouch. Please click here to download this File.

Supplementary Figure S2: Setting the parameters of the Hargreaves apparatus. IR Intensity (light exposure intensity): 50%; Cutoff Time (maximum light exposure time): 20 s. Please click here to download this File.

Currently, a consensus on an animal model that accurately replicates chronic non-specific low back pain (CNLBP) is lacking. Multiple animal models of CNLBP exist, such as disc-derived, neurogenic, osteoarticular-derived, and muscle-derived models²³. However, these models have limitations due to the heterogeneous nature of CNLBP in clinical practice. The CNLBP model used in this study was modified from Henderson's "external link" model²⁴, which is an osteoarticular-derived CNLBP model. In a previous study, the X-ray showed that long-term fixation of the lumbar spine resulted in osteophytes in the vertebral joint of the fixed segment, i.e., Slight misalignment of the vertebrae and joints²⁵. According to TCM theory, maintaining the biomechanical balance between the soft tissue structure (Jin) and the bony structure (Gu) of the lower back is crucial for lower back health. Slight misalignment of the vertebrae and joints can lead to an imbalance in the Gu biomechanics of the lower back, which, if left untreated, may eventually result in CNLBP due to an imbalance in Jin-Gu biomechanics. The conventional method of inducing CNLBP in an osteoarticular-derived model involves the injection of chemicals that induce inflammation associated with osteoarthritis^26,27. In contrast, the approach utilized in this study presents a distinct advantage wherein CNLBP is physically induced by immobilizing the lumbar spine, resulting in slight misalignment of the vertebrae and joints. This physical induction method excludes the potential influence of inflammation or nociceptive sensitivity triggered by the chemicals themselves. Furthermore, we verified the correlation between CNLBP and slight misalignment of lumbar vertebrae and joints from multiple perspectives, including lumbar range of motion, spinal stiffness, gait changes, and nerve conduction velocity^28,29,30,31, demonstrating the high research value of our proposed model.

In Western pharmacologic treatment of CNLBP, the prolonged utilization of opioid analgesics can lead to respiratory depression and overdose, as well as dependency, tolerance, exacerbation of pain, dermatitis, constipation, and cognitive confusion in patients³². Similarly, nonsteroidal anti-inflammatory drugs (NSAIDs) commonly give rise to gastrointestinal complications, cardiovascular risks, and adverse effects on renal function³³. MT is being increasingly investigated for its safety and efficacy in the management of various chronic musculoskeletal disorders, including neck pain, shoulder pain, low back pain, and knee osteoarthritis^11,34,35,36. MT is usually considered a low-risk intervention. Adverse effects, if any, are usually mild and transient, such as temporary soreness or bruising at the site of manipulation. Serious complications of massage therapy are rare but can occur in certain circumstances such as improper application of the technique or failure to address contraindications (e.g., open wounds or infected skin conditions). To investigate the molecular mechanisms underlying the efficacy of MT, animal studies have been conducted. However, the application of MT to animals in research studies presents a challenge. While many researchers opt for machine-based simulations to standardize and quantify the MT, this method is often cost-prohibitive and does not account for individual variations in pressure tolerance. Moreover, given that MT is typically performed by clinicians, data obtained from machine simulations may not accurately reflect the clinical efficacy of MT. As a result, developing an effective method for applying MT to animals in research remains an area of active investigation.

This study presents a detailed protocol for performing MT on rats, including necessary precautions. Prior to the MT, it is recommended that the experimenter completes 2 weeks of finger strength stability exercises to improve intervention stability. If the experimenter lacks clinical experience in MT, the duration of the stability exercise can be extended. Determining the most appropriate pressure values for each rat is also important, and this is achieved by referencing the clinician's method of determining appropriate pressure levels in patients. In the absence of verbal communication, resistance behaviors exhibited by the rat can indicate that the MT pressure level is too high. By applying MT at the most appropriate pressure level for each rat, the resulting experimental data can be compared. To maintain a quiet state during MT, anesthesia or forced immobilization is not recommended, as these methods may interfere with neurotransmission or increase anxiety in the rats³⁷. Instead, a rat pouch made of cloth is used, which is readily available and low-cost. If the rat pouch does not hold well, it can be optimized by creating an opening above the pouch and extending its length to limit the rat's range of motion. Additionally, rats can be placed on a 38 °C thermostat during MT, as we have found that they are quieter at this temperature than at room temperature. We hypothesize that this might be due to the "psychological suggestion" effect of placing the rats on the 38 °C constant-temperature table. The rats are normally housed at 24 °C and are only placed at 38 °C when they undergo MT. Over time, the rat will know when it touches the 38 °C constant temperature table that it is going to undergo MT and be quieter. However, if the rat does not know what happens next, it will resist out of fear, and the experimental data obtained in this state will lack accuracy.

Several limitations are associated with the procedure of MT described in this study that warrant attention. First and foremost, it necessitates the experimenter to have clinical experience in MT to ensure the desired force and pressure levels during the intervention are achieved. Consequently, inexperienced experimenters may find it challenging to control the force of MT on rats, which can impede the accuracy of the experimental results. Second, due to the ELFS device's substantial size and incision, ~40% of the model rats experienced abscess formation in the lumbar region, necessitating focused attention and appropriate management during the recovery phase. Notably, the incidence of infection in the model rats demonstrated a positive correlation with the incision size. However, by mastering the modeling technique, it is possible to minimize the incision size, resulting in a significant decrease in the infection rate among the model rats to approximately 20%. Third, the rats may display resistance behaviors during the first three days of MT because they have not yet acclimatized to the intervention. Thus, it is crucial to consider this when designing the experiment and interpreting the results. Finally, while the rat pouch and the 38 °C constant temperature table are used to keep the rats quiet during MT, it is important to monitor the rats for any indications of discomfort or distress and adopt appropriate measures as necessary.

In summary, the proposed standardized procedure for MT on CNLBP model rats, as described in this paper, offers several notable advantages. First, it is characterized by its cost-effectiveness, making it a financially accessible approach for conducting experiments. Second, the procedure is user-friendly and easy to operate, allowing for straightforward implementation. Importantly, the chosen methodology demonstrates enhanced clinical relevance, thereby increasing its applicability to future animal studies aimed at investigating the mechanisms underlying the therapeutic effects of MT.

The authors declare no competing interests or relationships that might constitute a conflict of interest.

This work was supported by the General Program of the National Natural Science Foundation of China (81774442, 82274672), the Zhejiang Provincial Natural Science Foundation Project (Q23H270025), the Zhejiang Province Lv Lijiang Famous Old Chinese Medicine Expert Inheritance Studio Construction Project Fund (GZS2021026), and the School-Level Scientific Research Project of Zhejiang Chinese Medicine University (2021RCZXZK03, 2022FSYYZQ13, 2022GJYY045).