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.

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
<ol>
	<li>Purchase 48 maleSprague-Dawley (SD) rats (age: 8-10 weeks, weight: 250-300 g).</li>
	<li>House rats individually in ventilated cages at 24 &#177; 2 &#176;C and a 12-h dark and light cycle.</li>
	<li>Feed rats a standard pellet diet with sterile drinking water ready to use, and provide soft bedding.</li>
</ol>
2. Grouping of animals
<ol>
	<li>Randomly divide 48 SD rats into six groups (n = 8 per group): the sham group, CCI group, CCI+FSN group, CCI+TENS group, FSN alone group, and TENS alone group as in the previous study conducted by Chan et al.24. 
	&#8203;NOTE: The details of six groups: (1) Sham group: no surgery and no treatment; (2) CCI group: prepared for surgery with no treatment; (3) CCI+FSN treatment group: FSN treatment after successful CCI modeling; (4) CCI+TENS treatment group: TENS treatment after successful CCI modeling; (5) FSN treatment alone group: only FSN treatment without surgery; (6) TENS treatment alone group: only TENS treatment without surgery.</li>
</ol>
3. Establishment of a CCI rat model
NOTE: The CCI surgery model in rats was modified according to Bennett and Xie conducted in 198825.
<ol>
	<li>Ensure the operator wears a surgical mask, disposable operating cap, and sterile gloves.</li>
	<li>Disinfect the surface of the surgical table with 70% ethanol. Sterilize instruments (e.g., scissors, forceps, and retractors), gauze, staples, and cotton swabs by autoclaving. 
	NOTE: Aseptic techniques are used throughout the surgical procedure.</li>
	<li>Anesthetize the rats with 4% isoflurane after standard skin preparation (shaving) and maintain with 2% isoflurane (Figure 2A).
	<ol>
		<li>Confirm the appropriate depth of anesthesia by observing the lack of response after pinching the hind paw and monitoring the anesthetized rats throughout the procedure.</li>
		<li>Apply enough veterinary ophthalmic ointment over the eyes for protection against drying.</li>
	</ol>
	</li>
	<li>Place the rat in the prone position on the operating table and shave the hair on the side of the right hind leg, then disinfect the skin with povidone-iodine solution and 75% ethanol three times. Provide thermal support throughout the procedure and use sterile drapes to cover the surgical site.
	<ol>
		<li>Make a parallel incision in the skin 3-4 mm below the femur of about 20-50 mm.</li>
		<li>Prioritize the identification of the positions of gluteus maximus and biceps femoris. Separate the subcutaneous fat and superficial fascia layer by layer using surgical scissors, cutting through the surrounding connective tissue to expose the muscle (Figure 2B). 
		NOTE: To distinguish the subcutaneous fat and superficial fascia layers, observe the texture and color. The subcutaneous fat layer should appear soft and pliable with a yellowish or whitish appearance. The superficial fascia is a thin fibrous layer situated directly beneath the subcutaneous fat. Differentiate between the layers by gently palpating or probing with a blunt instrument, noting that the subcutaneous fat offers more resistance to pressure compared to the superficial fascia.</li>
	</ol>
	</li>
	<li>Using blunt scissors, cut the connective tissue between the superficial gluteus and biceps femoris muscles.
	<ol>
		<li>Widen the gap between these two muscles using a retractor to expose the sciatic nerve (Figure 2C). 
		NOTE: To visually identify the sciatic nerve in a rat, focus on the thigh region. Locate the midpoint of the thigh region to visualize the sciatic nerve. Typically, the nerve runs along the posterior aspect of the thigh, starting from the hip region and extending toward the knee.</li>
		<li>Without altering the nerve morphology, pick the sciatic nerve with a microneedle through a good light source. Ligate the sciatic nerve twice using 3-0 chromic gut ligatures, positioning the ligature points approximately 1 mm apart between the two sutures.</li>
		<li>Starting with a loose loop for each ligature, grasp the ends of the ligature close to the loop and tighten until the loop is just snug, ensuring that the ligature does not slip along the nerve. Stop when slight twitching of the limb is observed during ligation (Figure 2D).</li>
	</ol>
	</li>
	<li>Close the muscle and skin layer by layer with 4-0 suture lines. Finally, disinfect the wound with iodine (Figure 2E).</li>
	<li>Closely monitor the rats&#39; vital signs&#160;during anesthesia and place them in individual recovery cages until they are awake before placing them back in their cages. Line the cages with flat paper bedding to prevent asphyxiation in unconscious animals. A brief twitch in the post-operative limb indicates a successful operation (Figure 2F).</li>
	<li>Perform pain hypersensitivity testing several times before CCI (baseline) and at different time points after CCI.</li>
	<li>Observe for spontaneous pain and behavioral changes on days 1, 3, 5, and 7 following model construction. 
	NOTE: Observe the gait and posture of the right hind limb and the presence of licking and biting on the limb.
	<ol>
		<li>Identify the presence of neuropathic pain to determine the successful establishment of the model and exclude unsuccessful rats. 
		&#8203;NOTE: Assess the success of the model by observing signs such as weakness in lower limb walking, toes of the right limb held together with mild valgus, frequent dangling, and reluctance to land. Observe the rat standing with the left hind limb supporting the weight, while the right hind limb is raised and close to the abdomen.</li>
	</ol>
	</li>
</ol>
4. Administration of FSN manipulation
<ol>
	<li>Fix the rat of the FSN treatment group (including CCI+FSN and FSN alone group) in the rodent restraint with the affected limb exposed laterally. Provide thermal support throughout the procedure. Both groups were treated with FSN disposable needles (Figure 3A).</li>
	<li>Without anesthesia, extend the rat&#39;s hind limbs gradually and slowly until they were stretched tight (Figure 3B). 
	NOTE: The rat head is covered with a surgical drape to keep the animal calm and stable. Do not overextend the leg to cause injury to the rat. Observe the rat&#39;s response closely for any signs of distress or discomfort. If the rat shows signs of pain or discomfort, stop the extension and provide a break before attempting again.</li>
	<li>Remove the protecting sheath of the FSN needle.
	<ol>
		<li>Insert the tip of the FSN needle toward the TMs (muscles with MTrPs), approximately close to the gluteus maximus muscle, located on the lower back and rear.</li>
	</ol>
	</li>
	<li>Place the FSN needle flat and enter the skin at an angle of approximately 15&#176;.
	<ol>
		<li>Push it carefully and quickly through the skin and into the subcutaneous space to prevent stress in the rat until fully inserted. Ensure that the needle is inserted sufficiently to completely bury the soft tube under the skin.</li>
		<li>When pushing forward, raise the needle tip slightly to observe if the skin bulge moves along the needle tip (Figure 3C).</li>
	</ol>
	</li>
	<li>Perform the swaying movement by smoothly and softly fanning the FSN needle tip with the thumb as the fulcrum while keeping the index finger, middle finger, and ring finger aligned in a straight line.
	<ol>
		<li>Hold the FSN needle between the middle finger and thumb in a face-to-face position, and alternate the movement back and forth using the index and ring fingers.</li>
		<li>Set the frequency to 100 strokes per minute and perform the operation for approximately 1 min (Figure 3D).</li>
	</ol>
	</li>
	<li>After completing the manipulation, quickly withdraw the FSN needle. 
	&#8203;NOTE: The operation was performed every 2 days for a total of four sessions (days 1, 3, 5, and 7 after the CCI model was created). Disposable FSN needles must be used once. Repeated use will blunt the needle and cause increased pain in rats.</li>
</ol>
5. Administration of TENS manipulation
<ol>
	<li>Fix the rat of the TENS treatment group (including CCI+TENS and TENS alone group) in the rodent restraint with the affected limb exposed laterally. Provide thermal support throughout the procedure. Ensure that the fur is shaved before being treated. 
	NOTE: Electrodes were cut to 45 mm (length) by 5 mm (width) (Figure 4A).</li>
	<li>Choose Zusanli point (ST36) and Sanyinjiao point (SP6) as the locations for TENS. This is based on the theory for treating neuropathic pain26,27.
	<ol>
		<li>Locate the Zusanli point (ST36) approximately 5 mm lateral to the anterior tubercle of the tibia between the tibia and fibula just below the knee28.</li>
		<li>Locate the Sanyinjiao point (SP6) at the posterior border of the tibia, 3 mm proximal to the medial malleolus28. 
		NOTE: Both of these two acupuncture points are located by manual inspection as described by Stux and Pomeranz and in the animal acupuncture atlas28,29 (Figure 4B).</li>
	</ol>
	</li>
	<li>Deliver a low-frequency electrical stimulation (2 Hz continuous sine wave, 3 mA) for 10 min using the TENS device with the electrode applied on the leg around the nerve. Cover the head of the rat head with a surgical drape to keep it calm and stable. 
	&#8203;NOTE: This procedure is performed every 2 days for a total of four sessions (days 1, 3, 5, and 7 after the CCI model was created).</li>
</ol>
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.
<ol>
	<li>Design rat walkways with clear plexiglass and tilt mirrors to capture the footprints and body orientation of rats during the walk. 
	NOTE: The walkway is a platform 10 cm long, 50 cm wide, and 15 cm high with a white paper lining at the bottom (Figure 5A).</li>
	<li>Gently and freely place the rats in the box and allow them to acclimatize to their new environment for at least 5 min before recording. 
	NOTE: Special care is taken to minimize unnecessary stress on the animal to avoid its possible effect on postural muscle tension.</li>
	<li>Dip the rat&#39;s paws in red ink and allow the rat to walk along the walkway strip, leaving traces on the backing paper. Record at least 2 s of continuous walking for each test. Make the rat walk at least 3 times in one direction (Figure 5B). 
	NOTE: Apply quick-drying, nontoxic, water-soluble red ink to both hind feet to make the hind footprints clearly visible.</li>
	<li>At the end of the experiment, dry the walkway strips to measure the parameters. Measure their footprints with a ruler and round to the nearest 0.5 mm. 
	NOTE: Three clear footprints from each rat were selected from several footprints, and three different parameters were measured. Factors for SFI include print length (PL), toe spread (TS), and intermediate toe spread (ITS). 
	SFI values are calculated using the following formula31: 
	<img alt="Equation 1" src="/files/ftp_upload/65406/65406eq01.jpg" style="margin: auto;" /> 
	(EPL, experimental print length; NPL, normal print length; ETS, experimental toe spread; NTS, normal toe spread; EIT, experimental intermediate toe spread; NIT, intermediate toe spread.) 
	SFI = 0 and - 100 indicates normal and complete dysfunction. Rats that dragged their toes were arbitrarily assigned a value of -100. For normal neurological function, the SFI oscillates around 0, while around -100 SFI represents complete dysfunction32.</li>
</ol>
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.
<ol>
	<li>Administer Zoletil 50 (40 mg/kg, ip) to anesthetize the rats. Prepare the skin according to standard protocols (shaving).</li>
	<li>Place disposable adhesive surface electrodes (20 mm outer diameter) on the designated areas. Fix the recording electrodes to the lateral and dorsal surfaces of the gastrocnemius muscle (Figure 6A).</li>
	<li>Apply electrical stimulation (intensity 1.2 mA) to the right proximal sciatic nerve stem. Record a compound muscle action potential (CMAP) on the belly of the gastrocnemius muscle (Figure 6B). 
	NOTE: Be careful when inserting the electrodes to avoid the muscle tissue.</li>
	<li>Record the effect of three repeated measurements for each rat. 
	NOTE: CMAP is expressed as the mean &#177; SD of each group. The signal was amplified by an amplifier, filtered (0.3-3 kHz). After integration (time constant = 0.05 s), both the original signal and the integrated signal are input. The original signal and the integrated signal are then digitized in the PowerLab system and stored on the computer hard disk.</li>
	<li>After completing the electrophysiology procedures, move the rat to a different cage and monitor it until it regains enough consciousness to maintain a sternal recumbent position. Once the rat has fully recovered from the anesthetic, transfer it back to its original cage.</li>
</ol>
8. Statistics:
<ol>
	<li>Evaluate differences in SFI and CMAPs between groups using repeated measures analysis of variance (ANOVA).</li>
	<li>Quantify the data by assistants who are blind to the experimental conditions. Express the data as mean &#177; standard deviation.</li>
	<li>Compare the data, when appropriate, using Student&#39;s two-tailed paired and unpaired t-test. Establish statistical significance as p &#60; 0.05.</li>
</ol>

Improving FSN Therapy in the Rat Model&#58; Minimizing Discomfort and Maximizing Efficiency

The authors declare that no competing conflicts of interest exist.

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 t...

Neuropathic pain, defined as pain caused by nerve damage, is estimated to affect 6.9%-10% of the world&#39;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&#39;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.

<table><tbody><tr><td>Forceps</td><td>World Precision Instruments</td><td>14098</td><td></td></tr><tr><td>Fu&amp;rsquo;s subcutaneous needling</td><td>Nanjing Paifu Medical Science and Technology Co.&amp;nbsp;</td><td></td><td>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).</td></tr><tr><td>Medelec Synergy electromyography</td><td>Oxford Instrument Medical Ltd.</td><td>034W003&amp;nbsp;</td><td>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.</td></tr><tr><td>Normal saline (0.9%) 20 mL</td><td>Taiwan Biotech Co.,Ltd.</td><td>4711916010323</td><td>Lot: 1TKB2022</td></tr><tr><td>POLYSORB 4-0 VIOLET 30&quot; CV-25</td><td>UNITED STATES SURGICAL, A DIVISION OF TYCO HEALTHC</td><td>GL-181</td><td></td></tr><tr><td>Retractor</td><td>COOPERSURGICAL, INC.(USA)</td><td>3311-8G</td><td></td></tr><tr><td>Rompun</td><td>Elanco Animal Health Korea Co. Ltd.</td><td>27668</td><td></td></tr><tr><td>SCISSORS CVD 90MM</td><td>BBRUAN</td><td>XG-LBB-BC101R</td><td></td></tr><tr><td>Transcutaneous Electrical Nerve Stimulation</td><td>Well-Life Healthcare Co.</td><td>Model Number 2205A</td><td>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&amp;ndash;30 s. Interval OFF time 1&amp;ndash;30 s.</td></tr><tr><td>Zoletil&amp;nbsp;</td><td>VIRRBAC</td><td>8V8HA</td><td></td></tr></tbody></table>

efficacy of fu's subcutaneous needling on sciatic nerve pain: behavioral and electrophysiological changes in a chronic constriction injury rat model

Fu&#39;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&#39;s body, including needle insertion and direction, needle retention, and swaying movement. Minimizing the rat&#39;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.

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 &#177; 3.46) and CCI+TENS (-29.58 &#177; 9.19) groups improved significantly compared to the CCI alone group (-87.40 &#177; 14.22). The improvement was significant in the CCI+FSN group compared to the CCI+TENS group&#160;(<strong class="x...

Watch this Scientific Journal Video about Efficacy of Fu's Subcutaneous Needling on Sciatic Nerve Pain: Behavioral and Electrophysiological Changes in a Chronic Constriction Injury Rat Model at JoVE.c

Author Spotlight: Unveiling the Therapeutic Effects of FSN Treatment &#8211; Bridging Research and Clinical Applications in Neuropathic Pain

We present a protocol for using Fu's subcutaneous needling in a chronic constriction injury model to induce sciatic nerve pain in rats.

Efficacy of Fu's Subcutaneous Needling on Sciatic Nerve Pain: Behavioral and Electrophysiological Changes in a Chronic Constriction Injury Rat Model

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the ...

efficacy-fu-s-subcutaneous-needling-on-sciatic-nerve-pain-behavioral

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1.8K Views.  Chang Bing Show Chwan Memorial Hospital. 1 Dit onderzoek onderzoekt het pijnstillende effect2 van Fu's subcutane needling-therapie 3 en laagfrequente elektrotherapie 4 met chronisch vernauwingsletsel en neuropathische pijn. 5 Het onderzoekt niet-farmacologische behandelingsopties 6 voor neuropathische pijn 7 en onderzoekt het potentieel van FSN-therapie 8 als een effectieve interventie. 9 De huidige experimentele uitdagingen omvatten10 het ontwikkelen van geschikte naaldtechnieken 11 om ongema...

Video: Auteur in de schijnwerpers: onthulling van de therapeutische effecten van FSN-behandeling - een brug slaan tussen onderzoek en klinische toepassingen bij neuropathische pijn

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor vehicle accidents, and falls, a reliable animal model of mTBI had previously not been developed for this important aspect of development. The modified weight-drop technique employs a glancing impact to the head of a freely moving rodent transmitting acceleration, deceleration, and rotational forces upon the brain. When applied to juvenile rats, this modified weight-drop technique induced clinically relevant behavioural outcomes that were representative of post-concussion symptomology. The technique is a rapidly applied procedure with an extremely low mortality rate, rendering it ideal for high-throughput studies of therapeutics. In addition, because the procedure involves a mild injury to a closed head, it can easily be used for studies of repetitive brain injury. Owing to the simplistic nature of this technique, and the clinically relevant biomechanics of the injury pathophysiology, the modified weight-drop technique provides researchers with a reliable model of mTBI that can be used in a wide variety of behavioural, molecular, and genetic studies.

The modified weight-drop technique is an easy, cost-effective procedure used for the induction of mild traumatic brain injury in juvenile rats. This novel technique produces clinically relevant symptomology that will advance the study of mild traumatic brain injury (mTBI) and concussion.

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor ...

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

Assessing Tuina Therapy's Pain Relief in Chronic Constriction Rats

Characteristics of Pain Changes in Rats with Nerve Injury Within 24 hours After One-Time Tuina Intervention

Tuina, as an external treatment method of traditional Chinese medicine, has been proven to have an analgesic effect on peripheral neuropathic pain (pNP) in clinical and basic research. However, the optimal time point for the analgesic effect of tuina may vary according to different injury sensations, affecting the exploration of the initiation mechanism of tuina analgesia. 
The research used minor chronic constriction injury (minor CCI) model rats to simulate pNP and used the intelligent tuina manipulation simulator to simulate the three methods (point-pressing, plucking, and kneading) and three acupoints (Yinmen BL37, Chengshan BL57, and Yanglingquan GB34) for performing tuina therapy. The study evaluated the changes in pain within 24 h and the optimal time point for the efficacy of tuina analgesia in rats with minor CCI models by testing cold sensitivity threshold (CST), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL). Furthermore, the study evaluated IL-10 and TNF-&#945; expression changes through Elisa detection. The results show that tuina has both immediate and sustained analgesic effects. For the three different injury sensitivity thresholds of CST, MWT, TWL, and two cytokines of IL-10 and TNF-&#945;, the analgesic efficacy of tuina within 24 h after intervention is significantly different at different time points.

Author Spotlight: Exploring the Initiation Mechanism of 'Three-Method and Three-Acupoint' Tuina Analgesia in Rats with Nerve Injury

We present a protocol using the tuina manipulation simulator to perform the "Three-Manipulation and Three-Acupoint" tuina therapy for minor chronic constriction injury rats and evaluate the effective analgesic time points of tuina within 24 h by testing pain changes through behavioral analysis and changes in inflammatory factor expression using enzyme-linked immunosorbent assay.

Tuina, as an external treatment method of traditional Chinese medicine, has been proven to have an analgesic effect on peripheral neuropathic pain (pNP) ...

Neuroscience

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...

FSN Therapy for Knee Osteoarthritis Pain Management

Fu's Subcutaneous Needling for Knee Osteoarthritis Pain

Fu's subcutaneous needling (FSN) is a new acupuncture and dry needling technique based on traditional Chinese medicine. It rapidly produces long-lasting effects in soft tissue injuries, particularly in painful musculoskeletal conditions, by providing stimulation primarily in the subcutaneous area. Osteoarthritis (OA) is the most common joint disease in adults worldwide and is often accompanied by a painful syndrome of structural changes in the peripheral joints of the knee. However, the etiology of OA pain is not fully understood, though myofascial trigger points (MTrPs) are commonly found in the lower limb muscles (so-called "tightened muscles") of patients with knee OA. 
FSN has been used in many fields for the treatment of acute pain problems and can relieve muscle contraction from MTrPs, thereby improving the local circulation. This study recruited patients with pain from knee OA into an FSN group or a transcutaneous electrical nerve stimulation (TENS) group with three treatment sessions and a follow-up over the course of 2 weeks. The results showed that FSN was effective in treating soft tissue pain around the knee with OA. This study aimed to establish and visualize three key technical indicators during FSN therapy, including the FSN needle insertion point and layer; the frequency and duration of the swaying movement; and the manipulation of the reperfusion approach. These findings have great potential for future applications in myofascial pain treatment, especially for pain management. Following this protocol could enhance FSN skills.

Author Spotlight: Fu's Subcutaneous Needling for Knee Osteoarthritis Pain  

We present a protocol for using Fu's subcutaneous needling for knee osteoarthritis pain, which combines swaying movement and reperfusion approach techniques. This protocol has great potential for future applications in myofascial pain treatment and could enhance Fu's subcutaneous needling (FSN) manipulation skills.

Fu's subcutaneous needling (FSN) is a new acupuncture and dry needling technique based on traditional Chinese medicine. It rapidly produces long-lasting ...

Standardized Tuina Protocol for Rat DRG Compression Model

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

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar disc herniation, spinal canal stenosis, and intervertebral foramen stenosis. Although Tuina, a traditional Chinese manual therapy, has shown analgesic effects in clinical practice for the treatment of neuropathic pain, its underlying neurobiological mechanisms remain unclear. Animal models are essential for elucidating the basic principles of Tuina. In this study, we propose a standardized Tuina protocol for rats with compression of the dorsal root ganglion (DRG), which involves inducing DRG compression by inserting a stainless steel rod into the intervertebral foramen, performing Tuina manipulation with specific parameters of location, intensity, and frequency in a controlled environment, and assessing the behavioral and histopathological outcomes of Tuina treatment. This article also discusses the potential clinical implications and limitations of the study and suggests directions for future research on Tuina.

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

This article presents a manipulation for treating chronic compression of the dorsal root ganglion in rats using Tuina therapy, along with a method for evaluating its effectiveness based on pain behavior and histopathological results.

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 ...

Efficacy of Fu's Subcutaneous Needling on Sciatic Nerve Pain: Behavioral and Electrophysiological Changes in a Chronic Constriction Injury Rat Model

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