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

Summary

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.

Abstract

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.

Introduction

Chronic pain is a significant healthcare issue across the world and is one of the costliest health problems in the United States. Chronic pain is better managed when both pharmacological and non-pharmacological modalities are utilized in a multidisciplinary fashion¹. Management of chronic pain is challenging and, in some cases, does not adequately treat the pain². Therefore, new and complementary methods are needed to improve chronic pain management, and animal models are crucial to investigate innovative therapies.

Chronic neuropathic pain results from lesions or diseases in the somatosensory system, including diabetes, infections, nerve compressions, or autoimmune diseases³. Neuropathic pain relies both on peripheral and central sensitization mechanisms and originates from a lesion of the nerves. This pain can be characterized by both touch- and thermal-evoked hyperalgesia and allodynia, ongoing pain, and changes in the temperature of the affected limb⁴. To better understand the mechanisms and advance new treatments, several models have been developed in rodents to mimic the symptoms and causes of neuropathic pain⁵. For example, neuropathic pain can be induced with chemotherapeutic agent injections, spinal nerve ligation (SNL), chronic constriction injury (CCI) of the sciatic nerve, pSNL, spared nerve injury, sciatic nerve transection, and sciatic nerve trisection⁶. Notably, ligation of the sciatic nerve reproduces multiple features of neuropathic pain observed in humans, such as mechanical and thermal hypersensitivity, or changes in temperature of the affected limb, characteristic of complex regional pain syndrome (CRPS)⁷. Thus, this model is well-suited for the study of CRPS or any other nerve injury affections that induce chronic neuropathic pain. The model was first developed by Seltzer in 1990⁸, and is widely used in pain studies to investigate novel analgesic compounds or evaluate the cognitive effects of chronic pain^{9,10,11,12,13}. The model presents high reproducibility, and the partial ligation preserves behavioral responses to peripheral stimuli⁶.

Many of the currently used models have shortcomings not observed in pSNL. The CCI model has a much higher variability of injury between each animal depending on the snugness of the constrictor, and autotomy alters the hind paw digits rendering the model unsuitable for behavioral analysis⁶. The SNL model is a far more complicated and longer surgery that not only requires advanced technical skills but also carries a high risk of severe motor deficits³. These shortcomings are not seen in the pSNL model. The ease of reproducibility, short duration of the surgery, and the reduced risk of motor deficits seen postoperatively make this model valuable for studying peripheral neuropathic pain^8,14. Nevertheless, the partial ligation procedure itself can have variability between experimenters, resulting in less consistency in the number of ligated nerve fibers. Thus, presenting the details of the surgery is crucial to increase reproducibility among studies.

To induce chronic neuropathy, a 9-0 non-absorbable nylon suture is used to ligate a third of the width of the sciatic nerve. Following surgery, responses to thermal and mechanical stimuli are exaggerated, starting at day 1 postoperatively and lasting more than 50 days⁸. Here, both thermal and mechanical sensitivities were evaluated over 28 days using Hargreaves', hot plate, and von Frey filament tests. All of the behavioral assays demonstrated the consistency of the long-lasting hypersensitivity. This model has been shown to have dose-dependent effects of both morphine and ibuprofen, confirming it is well-suited for preclinical pain studies. Notably, this article describes the instructions for a unique handmade glass tool, referred to as "nerve glass hook." This tool is used in place of forceps to manipulate the nerve and prevent unintended additional nerve injury during surgery.

Protocol

All procedures were approved by the Institutional Animal Care and Use Committee of the University of Arizona and conform to the guidelines for the use of laboratory animals of the National Institutes of Health (NIH publication no. 80-23, 1966). Pathogen-free, adult C57Bl6/J mice (weight at testing: 22-28 g) were housed in standard vivarium mouse cages (five mice per cage) in climate-controlled rooms on a 12 h light/dark cycle and were allowed access to food and water ad libitum. All behavioral experiments were conducted by experimenters blinded to the treatment conditions.

1. Baseline: the measure of mechanical sensitivity

1. Upon arrival of the mice, allow them to habituate to the animal facility for 1 week. Then, habituate the animals to experimenter handling for ≥7 days thereafter.
2. Habituate the mice to the von Frey testing apparatus for 1 h prior to testing by placing them in clear Plexiglas boxes, on a wire mesh, in the same room as the testing room-preferably with the experimenter present in the room during habituation.
3. Establish the baseline paw withdrawal threshold via the "up-and-down" method using von Frey filaments described in Supplementary Table S1, starting with the 3.61 (3.9 mN) filament.
   1. Measure the withdrawal response to probing the mid-plantar hind paw with a series of calibrated fine (von Frey) monofilaments. Apply each filament perpendicularly once to the plantar surface of the pSNL ipsilateral hind paw of the animals held in suspended wire mesh cages. Evaluate mechanical sensitivity using the "up-and-down" method¹⁵: determine the withdrawal threshold by sequentially increasing or decreasing the stimulus strength, corresponding to the size of the filament. Sequentially apply each filament once.
      NOTE: The experimenter must avoid stimulating any of the footpads to obtain consistent results between the animals.
   2. For example, if the animal does not respond to the 3.61 filament, use the thicker 4.08 filament (9.8 mN) (a response is noted visually as withdrawal, shake, or licking of the affected paw); if the animal responded the first time, use the thinner 3.22 (1.6 mN) filament. Continue to use either decreasingly or increasingly thick filaments depending on whether the animal had positive or negative subsequent responses, respectively. Report negative and positive responses in the datasheet presented in Supplementary Table S1. Test the same paw 4x with different filaments following the first positive response.

2. Baseline: the measure of thermal sensitivity using the Hargreaves test

1. Upon arrival of the mice, allow them to habituate to the animal facility for 1 week. Then, habituate the animals to experimenter handling for ≥7 days thereafter.
2. Habituate the mice to the Hargreaves testing apparatus for 1 h prior to testing by placing them in clear Plexiglas boxes, in the same room as the testing room-preferably with the experimenter present in the room during habituation.
   NOTE: The Hargreaves test requires that the animal stand still for a few seconds. With mice, habituation is key to a successful experiment. Thus, if the mice remain very active after 1 h of habituation, allow them to acclimate for longer as needed.
   1. Determine paw withdrawal latencies as described by Hargreaves et al.¹⁶. Acclimate the mice within Plexiglas enclosures on a clear Plexiglass plate.
   2. Focus a radiant heat source (high-intensity projector lamp) onto the plantar surface of the hind paw ipsilateral to the pSNL. Adjust the intensity of the heat source to obtain a baseline of paw withdrawal latency of approximately 10 s. Then, keep the intensity constant for the remainder of the experiment.
   3. Wait for a motion detector to automatically halt the stimulus and timer when the paw is withdrawn. Use a maximal cutoff of 33.5 s to prevent tissue damage.
      NOTE: The cut-off is determined based on previous experiments and articles to avoid any additional skin damage^11,17,18. With the intensity used in this study, 33.5 is the cut-off, corresponding to a stimulus intensity of 30 (50 W) using the Hargreaves apparatus. The observed behavior is a reflex behavior, not a voluntary one.
   4. Establish the baseline paw withdrawal latencies using the Hargreaves apparatus and aiming at the plantar surface of the pSNL ipsilateral hind paw. Start thermal stimulation and record withdrawal latency. To avoid affecting the temperature of the heat stimulus, clean up any urine during the trials.

3. Baseline: the measure of thermal sensitivity using the hot plate test

1. Habituate the animals to the testing room for 1 h prior to testing.
   NOTE: As room temperature is important and can affect responses to the hot plate test, ensure the temperature of the room is consistently around 22 °C during the habituation period and through the testing period.
2. Set the hot plate to 52 °C, as this temperature has been shown to ideally elicit an aversive thermal response¹⁹.
3. Place the animal in the testing chamber and start a chronometer.
4. Observe for nocifensive behaviors (i.e., paw withdrawal, licking, shaking). As the pSNL surgery affects the hindlimb, disregard any behaviors observed in the forelimbs (especially forelimb licking).
5. Stop the chronometer as soon as nocifensive behavior is observed.
6. Remove the animal from the chamber and record the latency to this behavior.
   NOTE: Remove the animals from the chamber after a maximum of 30 s to prevent tissue damage. Additionally, it is important to note that the observed behavior is a reflex behavior, not a voluntary one.
7. Clean the testing chamber with 70% ethanol between animals to reduce the behavioral impact of odors. To avoid affecting the temperature of the heat stimulus, clean the apparatus of any urine between each animal tested.
8. To confirm the results, record videos of the animals in the hot plate chamber during testing for review after the animals have been tested.
   ​NOTE: By using video review to quantify latencies, the experimenter can repeatedly observe the test and closely analyze nocifensive behaviors that may have been missed during real-time observation.

4. Preoperative preparation

NOTE: Ensure clean cages are available for recovering the mice after surgery. Clean the surgical area with 70% ethanol, disinfect hands with 70% ethanol, use sterile gloves, wear proper personal protective equipment (PPE) (lab coat, hair net, shoe covers), and practice sterile techniques throughout the surgery.

1. Prepare the tools (Supplementary Figure S1) and additional resources (gauze) to be used in surgery by autoclaving them beforehand.
2. Induce anesthesia using volatile isoflurane and adjust as needed to maintain a surgical plane. Ensure oxygen is at an appropriate flow rate.
3. To ensure the animal is anesthetized, pinch the toes on a hind paw with tweezers to ensure the absence of paw reflex and check the corneal blink reflex before applying lubricating ophthalmic ointment.
   ​NOTE: Analgesics cannot be offered in this study as they may alter the pain pathway intended to be analyzed or even neutralize and invalidate the behavior being measured in accordance with pain research goals^20,21,22.
4. Upon choosing which side to perform the surgery on (left is demonstrated here), shave the animal's hind leg around the thigh region, inferiorly toward the patella, superiorly toward the hip, and above the femur. Wipe 3x with chlorhexidine in one direction with three separate gauzes, alternated with warm sterile saline.
   ​NOTE: Going forward, ensure every animal has the surgery performed on the same side to maintain consistency.
5. Slip the leg through a slit made in a 10 cm x 10 cm sterile drape to create a sterile field around the leg of choice.

5. Surgical procedure

1. Using fine surgical scissors (Supplementary Figure S1F), make a small 2 mm cut of the skin in the midline of the lateral aspect of the thigh. Slide the scissors under the skin in a circular motion to break through the fascia and create a clearance, enlarging the incision space.
2. Using tying forceps (Supplementary Figure S1H), create a sharp incision vertically at a 90° angle in the thigh muscles, 1 cm deep.
3. Insert the fine small scissors (Supplementary Figure S1G) into the same incision, also at a 90° angle, and spread them open gently to separate the muscles. Continue to do this until the sciatic nerve is visualized.
4. Locate the sciatic nerve, which can appear glossy and thin, running parallel to the vertical thigh, in the direction of the hip to the knee. Remove the scissors and the tying forceps from the body before proceeding.
5. Use the extra fine forceps (Supplementary Figure S1D) and the nerve glass hook (Supplementary Figure S1E) to isolate the nerve from underneath. Carefully free the nerve from surrounding connective tissues at a site near the trochanter of the femur, which is closest to the hip and furthest from the knee.
6. Allow the nerve to rest on the glass rod and ensure that the end of the rod prevents the nerve from rolling off.
7. Place a surgical knot to tie 1/3 of the width of the sciatic nerve using a 9-0 nylon suture, prior to it dividing into the common peroneal, tibial, and sural nerve branches³.
   NOTE: The branching occurs as the sciatic nerve courses down the knee, away from the hip. Since these three branches of the nerve have three different innervations, it is imperative to place the surgical knot prior to the branching to ensure the same nerve deficits across all animal surgeries.
8. Take care to hold the threads close to the knot when pulling the threads tight, so as not to tug on the nerve with excessive force to avoid sliding the nerve off the glass rod and avoid further stretch injury.
9. Carefully slip the nerve off the glass rod once the knot is complete and tuck it back into the original location at the level below the separated muscles.
10. Suture the muscle incision using an absorbable polyglycolic 5-0 suture. Separately, suture the skin using a non-absorbable polypropylene 6-0 suture.
11. Record the surgery and anesthesia stop time. Allow the mouse to wake up, alone in a recovery cage, before returning it to a new clean cage.
    ​NOTE: Throughout the surgery, pinch the animal's toes to confirm adequate maintenance of anesthesia and monitor its breathing and bodily perfusion (red, pink, pale). If the breathing is significantly reduced or the animal appears pale, consider reducing the anesthesia flow or increasing the oxygen flow and have a syringe filled with saline ready to inject subcutaneously to rehydrate the animal. At all times, the animal should have a heat source placed below it to maintain body warmth.

6. Sham surgery procedure for control animals

1. Follow steps 5.1-5.11 of the surgical procedure; exclude steps 5.4-5.9.

7. Postsurgical behavioral tests

NOTE: Ensure that the experimenter is blinded to any treatment. Chronic neuropathic pain will develop over 2 weeks post-surgery, after which behavioral tests can be conducted following administration of compounds of interest.

1. Use the von Frey, Hargreaves, or hot plate test to evaluate both thermal and mechanical hypersensitivity and its potential reversal.
2. Remove any animal from the study if it meets endpoint criteria as described by the institutional animal care and use committee.
3. Euthanize the animals following procedures described by the institutional animal care and use committee at the end of the behavioral testing.

8. Data analysis

1. von Frey:
   1. Analyze the data using the nonparametric method of Dixon, as described by Chaplan and colleagues²³, and express the data as the mean withdrawal threshold.
      1. On the main page of the referenced software (see Table of Materials), select all the filaments that were used for the study (2.44, 2.83, 3.22, 3.61, 4.08, 4.31, and 4.56). In the group panel, select the filament corresponding to the last simulation. In the blank box, report the positive (X) and negative (o) responses. Write down the thresholds reported in the box on the left of the observed pattern of responses.
         NOTE: An example of pattern and quantification is presented in Supplementary Figure S2.
2. Hargreaves and hot plate:
   1. Report the latencies in a spreadsheet for further statistical analysis.
   2. Plot the results as the mean of the sensitivities (thresholds or latencies) as a function of time.

9. Instructions on how to make the nerve glass hook

NOTE: Practice fire safety throughout this process. Wear proper protection, such as heat-resistant gloves or eyewear as necessary.

1. Turn the Bunsen burner on.
2. Hold one end of the glass rod (A) to the fire in one hand. As this glass rod melts, use another glass rod (B) in the other hand to guide and pull at the melting glass on rod A. Remove glass rod A from the fire and allow the end of the melted portion to naturally roll inward to form a small ball shape. Use the glass rod B to guide this shape.

Results

Chronic neuropathic pain was induced through partial ligation of the sciatic nerve of C57Bl6/J male mice (Figure 1A). Mechanical sensitivity was evaluated using von Frey filaments and the "up-and-down" method. Thermal sensitivity to heat was evaluated using the Hargreaves and hot plate tests. All data were analyzed with a repeated measures two-way ANOVA with Geisser-Greenhouse correction, to compare the effect of pSNL surgery to sham animals over time or the effects of different dose...

Discussion

Chronic pain treatment often requires long-term medication, rendering pain management challenging. Thus, preclinical models are an essential tool to evaluate the potential benefits of innovative therapies relying on pharmacological or non-pharmacological approaches. The numerous models of chronic neuropathic pain bring challenges due to increased variability in the surgical techniques among different researchers, leading to reduced reproducibility. Thus, it is essential to characterize the potential antinociceptive effec...

Materials

Name	Company	Catalog Number	Comments
5/0, FS-2, 30" Undyed PGA Braided Polyglycolic Acid Synthetic Absorbable Suture	CP Medical	421A	https://cpmedical.com/suturesearch/product/421a-visorb-50-fs-2-30/
6/0, P-1, 18" Blue Polypropylene Monofilament Non-Absorbable Suture	CP Medical	8697P	https://cpmedical.com/suturesearch/product/8697p-polypro-60-p-1-18/
9/0 (0.3 metric) Nylon Black Monofilament Suture	Crestpoint Ophthalmics	MANI 1407	https://crestpointophthalmics.com/mani-1407-suture-trape-spatula-nylon-black-mono-box-of-12.html
Allodynia Software	National Instruments, LabView 2015		Quantification of mean withdrawal thresholds (Von Frey data)
C57Bl6/J mice	The Jackson Laboratory, Bar Harbor, ME	000664	https://www.jax.org/strain/000664
Castroviejo needle holder	Fine Science Tools	12565-14	https://www.finescience.com/en-US/Products/Wound-Closure/Needle-Holders/Castroviejo-Needle-Holder/12565-14
Cold Hot Plate Test	Bioseb	BIO-CHP	https://www.bioseb.com/en/pain-thermal-allodynia-hyperalgesia/563-cold-hot-plate-test.html
Elevated metal mesh stand for Von Frey	Bioseb	BIO-STD2-EVF	https://www.bioseb.com/en/pain-mechanical-allodynia-hyperalgesia/1689-elevated-metal-mesh-stand-30-cm-height-to-fit-up-to-2-pvf-cages.html
Extra fine Graefe forceps	Fine Science Tools	11152-10	https://www.fishersci.com/shop/products/fisherbrand-curved-medium-point-general-purpose-forceps/16100110
Fine Castroviejo needle holder	Simovision/Geuder	17565	https://simovision.com/assets/Uploads/Brochure-Geuder-Ophthalmic-Surgical-Instruments-EN2.pdf
Fine scissors (11.5 cm)	Fine Science Tools	14558-11	https://www.finescience.com/en-US/Products/Scissors/Standard-Scissors/Fine-Scissors-Tungsten-Carbide-ToughCut%C2%AE/14558-11
Fine scissors (9 cm)	Fine Science Tools	14558-09	https://www.finescience.com/en-US/Products/Scissors/Standard-Scissors/Fine-Scissors-Tungsten-Carbide-ToughCut%C2%AE/14558-09
Iris forceps	Fine Science Tools	11064-07	https://www.finescience.com/en-US/Products/Forceps-Hemostats/Fine-Forceps/Iris-Forceps/11064-07
Micro Adson forceps	Fine Science Tools	392487	https://www.fishersci.com/shop/products/micro-adson-tissue-forceps-1x2-teeth-german-steel/13820072#?keyword=adson%20forceps
Modular holder cages for rats and mice	Bioseb	BIO-PVF	https://www.bioseb.com/en/pain-mechanical-allodynia-hyperalgesia/1206-modular-holder-cages-for-rats-and-mice.html
Moretti/Effetre #240 Light Cobalt Blue glass rods 4 mm	Ebay	N/A	https://www.ebay.com/itm/402389491328?hash=item5db0485e80:g:agYAAOS w9CtfnIVJ&amdata=enc %3AAQAHAAAAwCoqvgWRo NTe5Vq8PWOgfE4ygWeW4tL k81J1AFu%2Fkcbsk6pxYtJi6 digE5TL9SzlgMzYUMNDr%2B dku2%2B%2FEvB1qXqFmebE 020SGs9LPDXLL5w21un7jrM0 9xfWYvIzBYQYh6FRWyUJngC uuA9Bkjb9lxtZoYlg5y6PyFR2P 34xFk5xaNC5ib65M1%2Fr%2F 4w2Iw45QqsSyXH2cuUKRom0 AGBoBaIr%2BbJw1VnlMjGuc9dtx 4fbPbqoBNSWjj3RbZPOPTYS8Q %3D%3D%7Ctkp%3ABk9SR4q6- 6LfYA
Plantar Test for Thermal Stimulation - Hargreaves Apparatus	Ugo Basile	37570	https://ugobasile.com/products/categories/pain-and-inflammation/plantar-test-for-thermal-stimulation
Touch-Test Sensory Evaluators, Set of 20 Monofilaments	North Coast Medical	NC12775-99	https://www.ncmedical.com/products/touch-test-sensory-evaluators_1278.html
Tying forceps	Duckworth & Kent	2-504ER8	https://duckworth-and-kent.com/product/tying-forceps-9/