We present a protocol for using Fu’s subcutaneous needling in a chronic constriction injury model to induce sciatic nerve pain in rats.
Fu's subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the mechanisms of action are still not fully understood. During FSN treatment, the FSN needle is inserted and retained in the subcutaneous tissues for a long duration with a swaying movement. However, challenges arise from maintaining a posture while manipulating FSN in animal models (e.g., rats) for researchers. Uncomfortable treatment can lead to fear and resistance to FSN needles, increasing the risk of injury and may even affect research data. Anesthesia may also affect the study results too. Hence, there is a need for strategies in FSN therapy on animals that minimize injury during the intervention. This study employs a chronic constriction injury model in Sprague-Dawley rats to induce neuropathic pain. This model replicates the pain induced by nerve injury observed in humans through surgical constriction of a peripheral nerve, mimicking the compression or entrapment seen in conditions such as nerve compression syndromes and peripheral neuropathies. We introduce an appropriate manipulation for easily inserting an FSN needle into the subcutaneous layer of the animal's body, including needle insertion and direction, needle retention, and swaying movement. Minimizing the rat's discomfort prevents the rat from being tense, which causes the muscle to contract and hinder the entry of the needle and improves the study efficiency.
Neuropathic pain, defined as pain caused by nerve damage, is estimated to affect 6.9%-10% of the world's population, and the reported lifetime prevalence is 49%-70%1,2. It is also considered to be one of the most difficult pain syndromes to manage. The use of pharmacological agents to manage neuropathic pain has yielded limited success as commonly prescribed pain medications like non-steroidal anti-inflammatory drugs and opioids have shown little efficacy in relieving this type of pain3,4. There is therefore a great need to explore new treatment options, especially non-pharmacological treatments. Acupuncture, as a non-pharmacological intervention, potentially alleviate neuropathic pain by exerting analgesic effects on the somatosensory system. Both clinical and preclinical studies have indicated that acupuncture is effective in relieving neuropathic pain symptoms without significant side effects5,6,7. However, the central mechanism of acupuncture treatment for pain relief in neuropathic pain remains to be further investigated.
In recent years, Fu's subcutaneous needling (FSN) has gained popularity for treating pain-related neurological disorders8. FSN originated from traditional Chinese acupuncture and was first described by traditional Chinese physician Zhonghua Fu in 19969,10. While originating from traditional acupuncture, FSN differs significantly in its techniques and theories from meridian-based acupuncture, yin and yang principles, and acupuncture point concepts. FSN places greater emphasis on neurophysiological and anatomical approaches to effectively address myofascial pain11. FSN therapy is applied in clinical practice to address various painful muscular disorders, targeting the connective tissues closely associated with the muscles, particularly focusing on the treatment of tightened muscles (TMs)12. As a complementary therapy for pain relief, there is also clinical evidence that FSN is effective in treating soft tissue injuries in addition to providing rapid pain management and significant improvement in soft tissue spasms13,14. FSN therapy involves specific techniques tailored to address the underlying myofascial trigger points (MTrPs) associated with the condition. The FSN needle insertion position is carefully chosen based on the location of these trigger points, allowing precise targeting of affected areas. During the procedure, the FSN needle is inserted into the subcutaneous layer, where it is intentionally stopped to optimize therapeutic effects. A distinctive technique known as the swaying movement is then employed, involving a gentle oscillating motion of the needle to stimulate the tissues and promote the therapeutic responses10. The development of MTrPs is associated with the energy crisis theory, which explains that factors such as chronic muscle overload, excessive exercise, improper exertional postures, muscle atrophy, and degeneration can contribute to the onset of muscle tissue ischemia and hypoxia. This oxygen and energy deficiency within the muscle tissue is believed to play a key role in the formation of MTrPs15,16. Previous animal studies have found that FSN treatment for chronic pain in rats improves the morphological structure and function of mitochondria in TMs to some extent, validating the potential of FSN therapy to promote the recovery of damaged nerves and muscles17.
Sciatica has been classified as neuropathic pain18. The origin of neuropathic pain is thought to lie anywhere between the motor endplate and the outer fibrous layer of the muscle, involving the microvascular system and neurotransmitters at the cellular level. Loss of muscle innervation and apoptosis of innervated nerve cells occurs when nerve damage occurs19, leading to pain-related gait in the affected limb. Additionally, chronic compression or irritation of the nerve can lead to a variety of changes in the way of nerve functions, which can further exacerbate the symptoms of sciatica20. However, the complexity of the nervous system makes it difficult to replicate it in vitro, thus necessitating the use of animal models for such studies. In the investigation of neuropathic pain disorders, model organisms are commonly employed, involving various methods of direct peripheral nerve injury, such as sciatic nerve ligature, transection, or compression21,22. The chronic constriction injury (CCI) model in Sprague-Dawley rats has been used to induce neuropathic pain. This model replicates the pain induced by nerve injury observed in humans through surgical constriction of a peripheral nerve, mimicking the compression or entrapment seen in conditions such as nerve compression syndromes and peripheral neuropathies.
In this study, we evaluated the analgesic effects of FSN therapy and low-frequency electrotherapy (transcutaneous electrical nerve stimulator, TENS) in rats with chronic constriction injury and neuropathic pain. As anesthesia slows or blocks nerve impulses and affects synaptic transmission and neuronal function23, animals cannot be anesthetized under all needling procedures and swaying movements. Therefore, an appropriate needle technique is required to reduce discomfort in rats. The steps for establishing a rat CCI model, the way the rats were treated with FSN combined swaying movement without anesthesia, feasible animal behavioral pattern tests, and electrophysiological investigations are described in detail.
All procedures involving animal subjects were approved by the Institutional Animal Care and Use Committee (IACUC) of the Chang Bing Show Chwan Memorial Hospital, Changhua, Taiwan (111031) on October 2022 (Figure 1).
1. Preparation of animals
2. Grouping of animals
3. Establishment of a CCI rat model
NOTE: The CCI surgery model in rats was modified according to Bennett and Xie conducted in 198825.
4. Administration of FSN manipulation
5. Administration of TENS manipulation
6. Physiological measurements performing the animal behavioral test
NOTE: Sciatic function index (SFI)30 is a widely used index by researchers studying pathology and potential treatment of nerve injuries, determined by comparing the geometry of the affected hind paw in injured rats with that of the contralateral paw and comparing it with the opposite paw.
7. Neurophysiological assessment by electrophysiological measurement33
NOTE: Electromyography was used to record the electrophysiological activity in this study. The compound muscle action potential (CMAP) is caused by the activation of muscle fibers in the target muscle supplied by the nerve. CMAP amplitude and latency are investigated. The CMAP amplitude is measured from baseline to negative peak.The latency of CMAPs is determined by measuring the time between the application of the stimulus and the onset of the compound action potential, which is influenced by the distance between the stimulation site and the recording site. Electrophysiology provides an objective assessment of peripheral nerve function in rats.
8. Statistics:
Footprints and determination of the SFI
We examined the development of SFI in the CCI alone, CCI+FSN, and CCI+TENS groups (Figure 7). After 4 sessions of FSN and TENS treatments on day 7 for CCI surgery, the SFI in the CCI+FSN (-15.85 ± 3.46) and CCI+TENS (-29.58 ± 9.19) groups improved significantly compared to the CCI alone group (-87.40 ± 14.22). The improvement was significant in the CCI+FSN group compared to the CCI+TENS group (Figure 7A).
We also investigated the SFI in the sham (0.02 ± 0.52), FSN alone (0.06 ± 1.75), and TENS alone (-2.36 ± 1.22) groups. The results showed that none of these last two groups and the sham group had any significant difference from each other (Figure 7B). This indicates that FSN and TENS are safe treatments that do not cause any harm to rats in their healthy state (Table 1).
Electrophysiological response
We examined the development of the amplitude of CMAP in the CCI alone, CCI+FSN, and CCI+TENS treatment groups (Figure 8). The amplitude of CMAP in the CCI+FSN (5.01 mV ± 0.67 mV) and CCI+TENS (4.64 mV ± 1.96 mV) groups improved significantly compared to the CCI alone group (1.80 mV ± 0.34 mV) (Table 2). CCI+FSN and CCI+TENS groups showed no significant difference (Figure 8A).
We also examined the development of latency peaks of CMAP in the CCI alone, CCI+FSN, and CCI+TENS groups. The latency peaks of CMAP in the CCI+FSN (2.46 ms ± 0.72 ms) and CCI+TENS (2.26 ms ± 0.97 ms) groups improved significantly compared to the CCI alone group (1.23 ms ± 0.22 ms) (Table 3). CCI+FSN and CCI+TENS groups showed no significant difference (Figure 8C).
The CMAP amplitude and latency were investigated in the sham (5.80 mV ±0.53 mV; 2.35 ms ± 0.37 ms), FSN alone (5.70 mV ± 0.45 mV; 2.64 ms ± 0.41 ms), and TENS alone (5.54 mV ± 0.92 mV; 2.61 ms ± 0.20 ms) groups, no significant difference between any of them were noted. This indicates that FSN and TENS are both safe treatments and do not cause harm to rats in their healthy state (Figure 8B,D).
Figure 1: Schematic view of the timeline for establishing the CCI rat model. Pain thresholds are measured from the first day after modeling (-7 days) and then every 2 days thereafter (-5, -3, -1 days). Pain thresholds measured on day 1 indicate successful modeling. After modeling, intervention and electrophysiological measurements are started on day 1. CCI+FSN, CCI+TENS, FSN alone, and TENS alone groups were treated with FSN or TENS at fixed time points on days 1, 3, 5, and 7, respectively. The rats were sacrificed on day 7 after electrophysiological and physiological measurements. Abbreviations: FSN: Fu's subcutaneous needling; TENS: transcutaneous electrical nerve stimulation; CCI: chronic constriction injury; SFI: sciatic function index. Please click here to view a larger version of this figure.
Figure 2: Chronic constriction injury (CCI) for inducing neuropathic pain of the sciatic nerve of rats. (A) After positioning and disinfection, the right hind leg of the rat is shaved. A parallel incision is made in the skin 3-4 mm above the femur. (B) The site is entered carefully while straining the muscle, separating the muscle fibers, and then removing them in layers without closing them completely. The connective tissue between the superficial gluteus and biceps femoris muscles is incised and the fascia is separated layer by layer. (C) The incision remains open, exposing the right lateral sciatic nerve. (D) The ligature is tied around the sciatic nerve using a 3-0 chromium suture, ensuring that the ligature is secured in place and does not slip along the nerve and restrict blood flow to the outer membrane of the nerve. (E) The muscle layer and skin are closed using sutures. (F) A brief twitch in the postoperative limb (The red circles indicate the terminal branch of the sciatic nerve) indicates a successful operation. Please click here to view a larger version of this figure.
Figure 3: Manipulating Fu's subcutaneous needling (FSN). (A) Fix the rat in the rodent restraint with the hind limbs exposed, avoiding straining (B) FSN needle is inserted toward the tightened muscle, approximately close to the gluteus maximus muscle. (C) The needle is inserted into the skin with the needle tip placed at approximately 15° to the skin. (D) Swaying movement (black fan) of Fu subcutaneous needling. Please click here to view a larger version of this figure.
Figure 4: Position and fixation of the electrodes on the rat's skin surface for applying transcutaneous electrical nerve stimulation (TENS). (A) Electrodes, cut to 45 mm (length) by 5 mm (width), placed on the rat. (B) Location of ST 36 and SP6 acupoints. Please click here to view a larger version of this figure.
Figure 5: Recording footprints on the walkway. (A) The walkway for physiological measurements by sciatic functional index (SFI) evaluation. (B) Recorded footprints on the 7th day postoperatively. Differences in several animal paw measurements can differentiate between the paws of the sham, CCI, CCI+FSN, CCI+TENS, FSN alone, and TENS alone groups. Measurements such as inter-toe (IT, the transverse distance between the second toe to the fourth toe), toe spread (TS, the transverse distance between the first to the fifth toe), and paw length (PL) are used to calculate values such as the SFI. Abbreviations: FSN: Fu's subcutaneous needling; TENS: transcutaneous electrical nerve stimulation; CCI: chronic constriction injury. Please click here to view a larger version of this figure.
Figure 6: Variation in digital nerve activity as a function of stimulus intensity and distal recording site measurements of rats via electrophysiology. (A) Electrophysiological measurements for recording compound muscle action potentials (CMAP). The recording and reference electrodes (blue patch) are placed on the lateral and medial gastrocnemius muscles, respectively, and electrical stimulation is applied to the proximal end of the right sciatic nerve trunk under the anesthesia induced and maintained with Zoletil. (B) Representative CMAPs tracings sham, CCI, CCI+FSN, CCI+TENS, FSN alone, and TENS alone groups after 4 treatments (prior to euthanasia of the animal). To calculate the baseline-to-peak (B-P) and peak-to-peak (P-P) compound muscle action potential (CMAP) amplitudes, the waveform is measured from the baseline (I) to the negative peak (II) and from the negative peak (II) to the positive peak (III), respectively. X-axis represents time (ms), and Y-axis represents voltage (mV). Sensitivity: 1 mV; Duration: 2 ms, 1 ms per frame. Please click here to view a larger version of this figure.
Figure 7: Sciatic functional index (SFI) for each group. (A) Comparison of SFI between the CCI, CCI+FSN, and CCI+TENS groups (* p < 0.05). (B) Comparison of SFI between the sham, FSN alone, and TENS alone groups. Abbreviations: FSN: Fu's subcutaneous needling; TENS: transcutaneous electrical nerve stimulation; CCI: chronic constriction injury; SFI: sciatic function index. Please click here to view a larger version of this figure.
Figure 8: Electrophysiological findings for each group. (A) Amplitude of CMAP, comparison between CCI, CCI+FSN, and CCI+TENS groups (* p < 0.05). (B) Amplitude of CMAP, comparison between Sham, FSN alone, and TENS alone groups. (C) Latency peaks of CMAP, comparison between CCI, CCI+FSN, and CCI+TENS groups (* p < 0.05). (D) Latency peaks of CMAP, comparison between Sham, FSN alone, and TENS alone groups. Abbreviations: FSN: Fu's subcutaneous needling; TENS: transcutaneous electrical nerve stimulation; CCI: chronic constriction injury; CMAP: compound muscle action potential. Please click here to view a larger version of this figure.
N | Mean | SD | |
Sham | 8 | 0.02 | 0.52 |
CCI | 8 | -87.40 | 14.22 |
CCI+FSN | 8 | -15.85 | 3.46 |
CCI+TEN | 8 | -29.58 | 9.19 |
FSN | 8 | 0.06 | 1.75 |
TEN | 8 | -2.36 | 1.22 |
FSN: Fu’s subcutaneous needling; TENS: transcutaneous electrical nerve stimulation; CCI: chronic constriction injury. |
Table 1: Summary of values of sciatic functional index in rats.
N | Mean (mV) | SD | |
Sham | 8 | 5.80 | 0.53 |
CCI | 8 | 1.80 | 0.34 |
CCI+FSN | 8 | 5.01 | 0.67 |
CCI+TEN | 8 | 4.64 | 1.96 |
FSN | 8 | 5.70 | 0.45 |
TEN | 8 | 5.54 | 0.92 |
FSN: Fu’s subcutaneous needling; TENS: transcutaneous electrical nerve stimulation; CCI: chronic constriction injury; CMAP: compound muscle action potential. |
Table 2: Summary of values of the electrophysiological response on CMAP amplitude in rats.
N | Mean (ms) | SD | |
Sham | 8 | 2.35 | 0.37 |
CCI | 8 | 1.23 | 0.22 |
CCI+FSN | 8 | 2.46 | 0.72 |
CCI+TEN | 8 | 2.26 | 0.97 |
FSN | 8 | 2.64 | 0.41 |
TEN | 8 | 2.61 | 0.20 |
FSN: Fu’s subcutaneous needling; TENS: transcutaneous electrical nerve stimulation; CCI: chronic constriction injury. |
Table 3: Summary of values of the electrophysiological response on latency peaks in rats.
This study observes the effect of FSN treatment on neuropathic pain in rat CCI models. This study presents a protocol for SFI and electrophysiological testing to evaluate the therapeutic effects after FSN or TENS treatment. Additionally, it illustrates how to evaluate the functional recovery of the injured nerve using noninvasive behavioral tests and physiological measurements. Results showed that the FSN treatment after CCI-induced sciatic nerve pain showed significantly better improvement in all prognostic indicators than the TENS treatment. This research has great potential for future applications in animal studies focused on FSN therapy to bridge the gap between basic research and clinical application. This study will improve patient outcomes by better understanding disease mechanisms
Acupuncture has been used in China for more than 3000 years and is often considered as a safe and effective method for relieving pain in humans and experimental animals34,35. Previous clinical studies have confirmed that acupuncture could alleviate pain behavior in CCI models36. FSN, as an acupuncture technique developed from traditional Chinese acupuncture37, is widely used for treating many pain-related musculoskeletal disorders38,39. Despite its satisfactory efficacy in painful musculoskeletal disorders, the underlying mechanism of FSN treatment remains unclear. The difficulties of performing acupuncture experiments on animals, including the complexity and difficulty of quantifying traditional acupuncture techniques and the uncomfortable posture of the animal, can lead to fear and resistance to acupuncture, making proper acupuncture technique and point positioning more difficult, increasing the risk of injury and affecting the experimental data40. Recent research in animal studies has shed light on the mechanisms underlying acupuncture analgesia; acupuncture-induced analgesia is associated with the release of endogenous opioid peptides41. However, pain perception is not solely modulated during the transmission of pain signals from the periphery to the cortex, indicating that other factors and mechanisms may also influence the experience of pain.
Based on the hypothesis presented by Simons et al.42,43, MTrP formation plays a key role in pain sensation. The pathogenesis of trigger point formation is thought to be related to abnormal motor endplate within the muscle. Excessive acetylcholine release leads to abnormal endplate potentials and formation of bandages which can lead to persistent muscle spasms resulting in local ischemia and hypoxia leading to hyperalgesia and abnormal pain44,45. An animal model for the MTrP study on rabbits using dry needles established by Hong46 demonstrates that dry needling can modulate proximal MTrPs in the muscle and spinal cord. However, only the relationship between acupuncture and muscle was discussed in this experiment, and fewer animal experiments were performed in relation to nerve injury. FSN is not the same as traditional acupuncture or dry needling in terms of technique and theoretical basis. FSN treatment does not work directly on the injured nerve but is clinically effective47,48. It was shown that in many clinical neurological disorders, the main problem may be on the muscle rather than the nerve itself. In this study, by incorporating a nerve injury model with CCI and treating the muscles around the sciatic nerve with FSN, FSN therapy showed a significant reduction in neuropathic pain and promoted recovery of the injured nerve and muscle.
An additional focus of this experiment is the benefits of FSN treatment without anesthesia. A previous study has shown that performing acupuncture experiments on rats without anesthesia can change physiological parameters such as heart rate, blood pressure, and hormone levels due to immobilization stress49. However, some researchers have argued that the benefits of conducting acupuncture experiments without anesthesia outweigh the potential effects of immobilization stress, even counteract the effects of immobilization stress50. Because of the small size of rodents and their differential sensitivity to anesthetics and analgesics, in addition to the loss of consciousness caused by general anesthesia, animals cannot perceive pain. However, in unconscious animals, painful stimuli are still transmitted and processed through the central nervous system51. In addition to the small size of rodent animals and their differential sensitivity to anesthetics and analgesics, in unconscious animals, painful stimuli are still transmitted and processed through the central nervous system. The use of µ- and δ-opioid receptor antagonists in animals treated under anesthesia may even cause reversal of the efficacy of acupuncture40,52.
We selected TENS as the control group for this study. TENS is a pain-relieving treatment that utilizes low-frequency pulsed electrical currents transmitted through the skin via electrodes without the use of medication53. It was observed that low frequency TENS was more effective than high frequency TENS in increasing the vascular response and may be a potential treatment for neuropathic pain caused by CCI54. Acupuncture is effective for conditions including neuropathic pain. However, the results can vary considerably depending on the acupuncture point chosen55, whereas TENS reduces nociceptive hypersensitivity by activating central inhibitory pathways with low variability in the selection of different locations56.
Although the results of this study are encouraging, some study limitations should be noted. According to Dr. Fu's guidance, a complete FSN treatment method should include swaying movement (passive treatment) and the reperfusion technique (active treatment). However, this experiment only has a swaying movement; designing a better animal experiment in the future is necessary. Previous standardized trials have established that the assessment of animal models of nerve compression requires the integration of multiple indicators including behavioral analysis, electromyography, immunohistochemistry and morphological evaluation57. In our study, we primarily used behavioral analysis and electromyography to evaluate the efficacy of FSN therapy. However, due to the potential importance of immunohistochemistry and morphological evaluation in this context, we recognize the need to prioritize these components in future follow-up trials to better validate the effectiveness of FSN therapy in improving this type of disease.
The authors have nothing to disclose.
This study was supported by a grant from the animal center of Chang Bing Show Chwan Memorial Hospital, Changhua, Taiwan. The authors would like to thank Show Chwan Memorial Hospital IRCAD TAIWAN for their invaluable support and assistance throughout this research project.
Forceps | World Precision Instruments | 14098 | |
Fu’s subcutaneous needling | Nanjing Paifu Medical Science and Technology Co. | FSN needles are designed for single use. The FSN needle is made up of three parts: a solid steel needle core (bottom), a soft casing pipe (middle), and a protecting sheath (top). | |
Medelec Synergy electromyography | Oxford Instrument Medical Ltd. | 034W003 | Electromyogram (EMG) are used to help in the diagnosis and management of disorders such as neuropathies. Contains a portable two-channel electromyography/nerve conduction velocity system. |
Normal saline (0.9%) 20 mL | Taiwan Biotech Co.,Ltd. | 4711916010323 | Lot: 1TKB2022 |
POLYSORB 4-0 VIOLET 30" CV-25 | UNITED STATES SURGICAL, A DIVISION OF TYCO HEALTHC | GL-181 | |
Retractor | COOPERSURGICAL, INC.(USA) | 3311-8G | |
Rompun | Elanco Animal Health Korea Co. Ltd. | 27668 | |
SCISSORS CVD 90MM | BBRUAN | XG-LBB-BC101R | |
Transcutaneous Electrical Nerve Stimulation | Well-Life Healthcare Co. | Model Number 2205A | Digital unit which offers TENS. Supplied complete with patient leads, self-adhesive electrodes, 3 AAA batteries and instructions in a soft carry bag. Interval ON time 1–30 s. Interval OFF time 1–30 s. |
Zoletil | VIRRBAC | 8V8HA |