This research was supported by the National Center for Complementary and Integrative Health [R01AT009716, 2017] (M.M.I.), the Comprehensive Chronic Pain and Addiction Center-University of Arizona (M.M.I.), and the Medical Scientist Training Program (MSTP) at the University of Arizona, College of Medicine, Tucson.

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
<ol>
	<li>Upon arrival of the mice, allow them to habituate to the animal facility for 1 week. Then, habituate the animals to experimenter handling for &#8805;7 days thereafter.</li>
	<li>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.</li>
	<li>Establish the baseline paw withdrawal threshold via the &#34;up-and-down&#34; method using von Frey filaments described in Supplementary Table S1, starting with the 3.61 (3.9 mN) filament.
	<ol>
		<li>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 &#34;up-and-down&#34; 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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Baseline: the measure of thermal sensitivity using the Hargreaves test
<ol>
	<li>Upon arrival of the mice, allow them to habituate to the animal facility for 1 week. Then, habituate the animals to experimenter handling for &#8805;7 days thereafter.</li>
	<li>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.
	<ol>
		<li>Determine paw withdrawal latencies as described by Hargreaves et al.16. Acclimate the mice within Plexiglas enclosures on a clear Plexiglass plate.</li>
		<li>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.</li>
		<li>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 damage11,17,18. 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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Baseline: the measure of thermal sensitivity using the hot plate test
<ol>
	<li>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 &#176;C during the habituation period and through the testing period.</li>
	<li>Set the hot plate to 52 &#176;C, as this temperature has been shown to ideally elicit an aversive thermal response19.</li>
	<li>Place the animal in the testing chamber and start a chronometer.</li>
	<li>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).</li>
	<li>Stop the chronometer as soon as nocifensive behavior is observed.</li>
	<li>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.</li>
	<li>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.</li>
	<li>To confirm the results, record videos of the animals in the hot plate chamber during testing for review after the animals have been tested. 
	&#8203;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.</li>
</ol>
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.
<ol>
	<li>Prepare the tools (Supplementary Figure S1) and additional resources (gauze) to be used in surgery by autoclaving them beforehand.</li>
	<li>Induce anesthesia using volatile isoflurane and adjust as needed to maintain a surgical plane. Ensure oxygen is at an appropriate flow rate.</li>
	<li>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. 
	&#8203;NOTE:&#160;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 goals20,21,22.</li>
	<li>Upon choosing which side to perform the surgery on (left is demonstrated here), shave the animal&#39;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&#160;warm sterile saline. 
	&#8203;NOTE: Going forward, ensure every animal has the surgery performed on the same side to maintain consistency.</li>
	<li>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.</li>
</ol>
5. Surgical procedure
<ol>
	<li>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.</li>
	<li>Using tying forceps (Supplementary Figure S1H), create a sharp incision vertically at a 90&#176; angle in the thigh muscles, 1 cm deep.</li>
	<li>Insert the fine small scissors (Supplementary Figure S1G) into the same incision, also at a 90&#176; angle, and spread them open gently to separate the muscles. Continue to do this until the sciatic nerve is visualized.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Allow the nerve to rest on the glass rod and ensure that the end of the rod prevents the nerve from rolling off.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Suture the muscle incision using an absorbable polyglycolic 5-0 suture. Separately, suture the skin using a non-absorbable polypropylene 6-0 suture.</li>
	<li>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. 
	&#8203;NOTE: Throughout the surgery, pinch the animal&#39;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.</li>
</ol>
6. Sham surgery procedure for control animals
<ol>
	<li>Follow steps 5.1-5.11 of the surgical procedure; exclude steps 5.4-5.9.</li>
</ol>
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.
<ol>
	<li>Use the von Frey, Hargreaves, or hot plate test to evaluate both thermal and mechanical hypersensitivity and its potential reversal.</li>
	<li>Remove any animal from the study if it meets endpoint criteria as described by the institutional animal care and use committee.</li>
	<li>Euthanize the animals following procedures described by the institutional animal care and use committee at the end of the behavioral testing.</li>
</ol>
8. Data analysis
<ol>
	<li>von Frey:
	<ol>
		<li>Analyze the data using the nonparametric method of Dixon, as described by Chaplan and colleagues23, and express the data as the mean withdrawal threshold.
		<ol>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Hargreaves and hot plate:
	<ol>
		<li>Report the latencies in a spreadsheet for further statistical analysis.</li>
		<li>Plot the results as the mean of the sensitivities (thresholds or latencies) as a function of time.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Turn the Bunsen burner on.</li>
	<li>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.</li>
</ol>

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Powerful+antinociceptive+effects+of+the+cone+snail+venom-derived+subtype-selective+NMDA+receptor+antagonists+conantokins+G+and+T.">Powerful antinociceptive effects of the cone snail venom-derived subtype-selective NMDA receptor antagonists conantokins G and T.</a> Pain. 101 (1-2), 109-116 (2003).</li><li>Nakamura, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathic+pain+in+rats+with+a+partial+sciatic+nerve+ligation+is+alleviated+by+intravenous+injection+of+monoclonal+antibody+to+high+mobility+group+box-1.">Neuropathic pain in rats with a partial sciatic nerve ligation is alleviated by intravenous injection of monoclonal antibody to high mobility group box-1.</a> PLoS One. 8 (8), 73640 (2013).</li><li>Sherman, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+patterns+of+pain+development+after+nerve+injury+in+rats+and+the+influence+of+sex.">Heterogeneity in patterns of pain development after nerve injury in rats and the influence of sex.</a> Neurobiology of Pain. 10, 100069 (2021).</li><li>Ba, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cinobufacini+protects+against+paclitaxel-induced+peripheral+neuropathic+pain+and+suppresses+TRPV1+up-regulation+and+spinal+astrocyte+activation+in+rats.">Cinobufacini protects against paclitaxel-induced peripheral neuropathic pain and suppresses TRPV1 up-regulation and spinal astrocyte activation in rats.</a> Biomedicine Pharmacotherapy. 108, 76-84 (2018).</li><li>Hao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huachansu+suppresses+TRPV1+up-regulation+and+spinal+astrocyte+activation+to+prevent+oxaliplatin-induced+peripheral+neuropathic+pain+in+rats.">Huachansu suppresses TRPV1 up-regulation and spinal astrocyte activation to prevent oxaliplatin-induced peripheral neuropathic pain in rats.</a> Gene. 680, 43-50 (2019).</li><li>Guo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+resveratrol+in+the+signaling+of+neuropathic+pain+involving+P2X3+in+the+dorsal+root+ganglion+of+rats.">Effects of resveratrol in the signaling of neuropathic pain involving P2X3 in the dorsal root ganglion of rats.</a> Acta Neurologica Belgica. 121 (2), 365-372 (2021).</li><li>Ni, W., Zheng, X., Hu, L., Kong, C., Xu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventing+oxaliplatin-induced+neuropathic+pain:+Using+berberine+to+inhibit+the+activation+of+NF-kappaB+and+release+of+pro-inflammatory+cytokines+in+dorsal+root+ganglions+in+rats.">Preventing oxaliplatin-induced neuropathic pain: Using berberine to inhibit the activation of NF-kappaB and release of pro-inflammatory cytokines in dorsal root ganglions in rats.</a> Experimental and Therapeutic Medicine. 21 (2), 135 (2021).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+activation+of+metabotropic+glutamate+receptor+7+blocks+paclitaxel-induced+acute+neuropathic+pain+and+suppresses+spinal+glial+reactivity+in+rats.">Selective activation of metabotropic glutamate receptor 7 blocks paclitaxel-induced acute neuropathic pain and suppresses spinal glial reactivity in rats.</a> Psychopharmacology. 238 (1), 107-119 (2021).</li><li>Sun, C., Wu, G., Zhang, Z., Cao, R., Cui, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+tyrosine+phosphatase+receptor+type+D+regulates+neuropathic+pain+after+nerve+injury+via+the+STING-IFN-I+pathway.">Protein tyrosine phosphatase receptor type D regulates neuropathic pain after nerve injury via the STING-IFN-I pathway.</a> Frontiers in Molecular Neuroscience. 15, 859166 (2022).</li><li>Coyle, D. E., Sehlhorst, C. S., Mascari, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Female+rats+are+more+susceptible+to+the+development+of+neuropathic+pain+using+the+partial+sciatic+nerve+ligation+(PSNL)+model.">Female rats are more susceptible to the development of neuropathic pain using the partial sciatic nerve ligation (PSNL) model.</a> Neuroscience Letters. 186 (2-3), 135-138 (1995).</li></ol>

The authors have no conflict of interest to report. None of the authors of the manuscript received any remuneration or any reimbursement or honorarium in any other manner. The authors are not affiliated with any vendor or pharmaceutical company associated with this study.

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...

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 pain9,10,11,12,13. 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 pain8,14. 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&#39;, 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 &#34;nerve glass hook.&#34; This tool is used in place of forceps to manipulate the nerve and prevent unintended additional nerve injury during surgery.

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

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.

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 &#34;up-and-down&#34; 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...

Watch this Scientific Journal Video about Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies at JoVE.com

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

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. ...

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6.0K Views.  The University of Arizona, Tucson. Omdat een overvloed aan innovatieve therapieën nu wordt onderzocht om chronische pijn te behandelen, biedt ons protocol cruciale concepten voor de standaardisatie van operaties die nodig zijn om neuropathische pijn te induceren. Partiële heupzenuwligatie, of PSNL, is een voordelig model om neuropathische pijn te bestuderen, omdat het veel symptomen reproduceert die worden waargenomen bij mensen met neuropathische pijn. Omdat PSNL veel symptomen reproduceert die worden waargenomen bij mensen...

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

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared nerve injury (SNI) model induces symptoms of neuropathic pain such as mechanical allodynia i.e. pain due to tactile stimuli that do not normally provoke a painful response [1]. 
The SNI mouse model involves ligation of two of the three branches of the sciatic nerve (the tibial nerve and the common peroneal nerve), while the sural nerve is left intact [2]. The lesion results in marked hypersensitivity in the lateral area of the paw, which is innervated by the spared sural nerve. The non-operated side of the mouse can be used as a control. The advantages of the SNI model are the robustness of the response and that it doesn&#x2019;t require expert microsurgical skills.
The threshold for mechanical pain response is determined by testing with von Frey filaments of increasing bending force, which are repetitively pressed against the lateral area of the paw [3], [4]. A positive pain reaction is defined as sudden paw withdrawal, flinching and/or paw licking induced by the filament. A positive response in three out of five repetitive stimuli is defined as the pain threshold. 
As demonstrated in the video protocol, C57BL/6 mice experience profound allodynia as early as the day following surgery and maintain this for several weeks.

The Spared Nerve Injury SNI Model of Induced Mechanical Allodynia in Mice

The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared ...

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 ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

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 ...

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

Neuroscience

Establishing the Mouse Model for Trigeminal Neuropathic Pain

Chronic Constriction Injury of the Distal Infraorbital Nerve (DIoN-CCI) in Mice to Study Trigeminal Neuropathic Pain

Animal models remain necessary tools to study neuropathic pain. This manuscript describes the distal infraorbital nerve chronic constriction injury (DIoN-CCI) model to study trigeminal neuropathic pain in mice. This includes the surgical procedures to perform the chronic constriction injury and the postoperative behavioral tests to evaluate the changes in spontaneous and evoked behavior that are signs of ongoing pain and mechanical allodynia. The methods and behavioral readouts are similar to the infraorbital nerve chronic constriction injury (IoN-CCI) model in rats. However, important changes are necessary for the adaptation of the IoN-CCI model to mice. First, the intra-orbital approach is replaced by a more rostral approach with an incision between the eye and the whisker pad. The IoN is thus ligated distally outside the orbital cavity. Secondly, due to the higher locomotor activity in mice, allowing rats to move freely in small cages is replaced by placing mice in custom-designed and constructed restraining devices. After DIoN ligation, mice exhibit changes in spontaneous behavior and in response to von Frey hair stimulation that are similar to those in IoN-CCI rats, i.e., increased directed face grooming and hyperresponsiveness to von Frey hair stimulation of the IoN territory.

Author Spotlight: Utilizing Infraorbital Nerve Ligation in Mice for Investigating Trigeminal Neuropathic Pain and Treatment Strategies

Chronic constriction injury of the distal infraorbital nerve in mice induces changes in spontaneous behavior (increased face grooming activity) and nocifensive behavior in response to tactile stimulation (hyperresponsiveness to von Frey hair stimulation) that are signs of ongoing pain and allodynia and serves as a model for trigeminal neuropathic pain.

Animal models remain necessary tools to study neuropathic pain. This manuscript describes the distal infraorbital nerve chronic constriction injury ...

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

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.

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.

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

Targeting Mouse Sympathetic Neurons: A Lumbar Sympathectomy Approach

Surgical Lumbar Sympathectomy in Mice

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays many critical roles in maintaining bodily homeostasis, but it has rarely been studied in the context of peripheral nerve injury. The extent of postganglionic sympathetic neuronal functions in distal targets in the periphery is currently unclear. To better explore the role of sympathetic innervation of peripheral targets, a surgical &#34;knock-out&#34; model provides an alternative approach. Although this can be achieved chemically, chemical destruction of postganglionic sympathetic neurons can be nonspecific and dose-dependent. The use of a surgical lumbar sympathectomy in mice, once thought to be &#34;virtually not practicable&#34; in small animals, allows for specific targeting of postganglionic sympathetic neurons that innervate the hind limbs. This manuscript describes how to surgically remove the L2-L5 lumbar sympathetic ganglia from a mouse as a survival surgery, which reliably decreases the hind paw sweat response and the number of sympathetic axons in the sciatic nerve.

Author Spotlight: Exploring Plasticity of Sympathetic Neurons

This manuscript presents a protocol for surgically removing the postganglionic lumbar sympathetic neurons from a mouse. This procedure will facilitate a multitude of studies aimed at investigating the role of sympathetic innervation in distal tissue targets.

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays ...

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

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