Name: partial sciatic nerve ligation: a mouse model of chronic neuropathic pain to study the antinociceptive effect of novel therapies
Uploaded: 2022-10-06T14:30:00
Description: 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

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 c...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peripheral+mononeuropathy+in+rat+that+produces+disorders+of+pain+sensation+like+those+seen+in+man.">A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man.</a> Pain. 33 (1), 87-107 (1988).</li><li>Seltzer, Z., Dubner, R., Shir, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+behavioral+model+of+neuropathic+pain+disorders+produced+in+rats+by+partial+sciatic+nerve+injury.">A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury.</a> Pain. 43 (2), 205-218 (1990).</li><li>Hasnie, F. S., Wallace, V. C., Hefner, K., Holmes, A., Rice, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+cold+hypersensitivity+in+nerve-injured+C57BL/6J+mice+is+not+associated+with+fear-avoidance-and+depression-related+behaviour.">Mechanical and cold hypersensitivity in nerve-injured C57BL/6J mice is not associated with fear-avoidance-and depression-related behaviour.</a> British Journal of Anaesthia. 98 (6), 816-822 (2007).</li><li>Ito, H., 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=Suvorexant+and+mirtazapine+improve+chronic+pain-related+changes+in+parameters+of+sleep+and+voluntary+physical+performance+in+mice+with+sciatic+nerve+ligation.">Suvorexant and mirtazapine improve chronic pain-related changes in parameters of sleep and voluntary physical performance in mice with sciatic nerve ligation.</a> PLoS One. 17 (2), 0264386 (2022).</li><li>Martin, L., 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=Conotoxin+contulakin-G+engages+a+neurotensin+receptor+2/R-type+calcium+channel+(Cav2.3)+pathway+to+mediate+spinal+antinociception.">Conotoxin contulakin-G engages a neurotensin receptor 2/R-type calcium channel (Cav2.3) pathway to mediate spinal antinociception.</a> Pain. 163 (9), 1751-1762 (2021).</li><li>Peiser-Oliver, J. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Pain. , 1299-1300 (2007).</li><li>Zahn, P. K., Brennan, T. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+foot+withdrawal+response+to+noxious+radiant+heat+in+the+rat.">Characterization of the foot withdrawal response to noxious radiant heat in the rat.</a> Pain. 59 (1), 85-94 (1994).</li><li>Cheah, M., Fawcett, J. W., Andrews, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+thermal+pain+sensation+in+rats+and+mice+using+the+Hargreaves+test.">Assessment of thermal pain sensation in rats and mice using the Hargreaves test.</a> Bio-Protocol. 7 (16), 2506 (2017).</li><li>Hook, M. A., 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=The+impact+of+morphine+after+a+spinal+cord+injury.">The impact of morphine after a spinal cord injury.</a> Behavioural brain research. 179 (2), 281-293 (2007).</li><li>Loram, L. C., 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=Prior+exposure+to+repeated+morphine+potentiates+mechanical+allodynia+induced+by+peripheral+inflammation+and+neuropathy.">Prior exposure to repeated morphine potentiates mechanical allodynia induced by peripheral inflammation and neuropathy.</a> Brain, behavior, and immunity. 26 (8), 1256-1264 (2007).</li><li>Green-Fulgham, S. M., 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=Oxycodone,+fentanyl,+and+morphine+amplify+established+neuropathic+pain+in+male+rats.">Oxycodone, fentanyl, and morphine amplify established neuropathic pain in male rats.</a> Pain. 160 (11), 2634-2640 (2019).</li><li>Deuis, J. R., Dvorakova, L. S., Vetter, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+used+to+evaluate+pain+behaviors+in+rodents.">Methods used to evaluate pain behaviors in rodents.</a> Frontiers in Molecular Neuroscience. 10, 284 (2017).</li><li>Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., Yaksh, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw.">Quantitative assessment of tactile allodynia in the rat paw.</a> Journal of Neuroscience Methods. 53 (1), 55-63 (1994).</li><li>Jensen, T. S., Finnerup, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allodynia+and+hyperalgesia+in+neuropathic+pain:+clinical+manifestations+and+mechanisms.">Allodynia and hyperalgesia in neuropathic pain: clinical manifestations and mechanisms.</a> Lancet Neurology. 13 (9), 924-935 (2014).</li><li>Malmberg, A. B., Gilbert, H., McCabe, R. T., Basbaum, A. 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>

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.

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	<tbody>
		<tr>
			<td>5/0, FS-2, 30&#34; 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&#34; 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&#160;</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&#160;</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 
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			digE5TL9SzlgMzYUMNDr%2B 
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			%3D%3D%7Ctkp%3ABk9SR4q6- 
			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 &#38; Kent</td>
			<td>2-504ER8</td>
			<td>https://duckworth-and-kent.com/product/tying-forceps-9/</td>
		</tr>
	</tbody>
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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. ...

As a plethora of innovative therapies are now being investigated to treat chronic pain, our protocol provides crucial concepts for the standardization of surgeries required to induce neuropathic pain. Partial sciatic nerve ligation, or PSNL, is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. As PSNL reproduces many symptoms observed in humans with neuropathic pain, this protocol provides crucial concepts to study and develop new pain management therapies.Demonstrating the procedure will be Kevin Cheng, an MD PhD candidate in my laboratory. To begin, habituate the mice to the Von Frey testing apparatus for one hour prior to testing by placing them in clear plexiglass boxes on a wire mesh in the same room as the testing room. Apply a series of calibrated fine mono filaments perpendicularly to the planter surface of the PSNL ipsilateral hind paw of the animals held in suspended wire mesh cages.Determine the withdrawal threshold by sequentially increasing or decreasing the stimulus strength corresponding to the size of the filament. Increase the size of the filament when the response is negative and decrease the size of the filament when the animal withdraws its paw. Report negative and positive responses in the data sheet and test the same paw four times with different filaments following the first positive response.Habituate the mice to the Hargreaves testing apparatus for one hour prior to testing by placing them in clear plexiglass boxes in the same room as the testing room. Establish the baseline paw withdrawal latencies using the Hargreaves apparatus. Focus a radiant heat source onto the planter surface of the hind paw ipsilateral to the PSNL and start stimulation.When the animal withdraws its paw, wait for a motion detector to halt the stimulus and stop the timer. Adjust the intensity of the heat source to obtain a baseline of paw withdrawal latency of approximately 10 seconds. Next, record the withdrawal time in a table.Set the hot plate to 52 degrees Celsius. Place the animal in the testing chamber and start a chronometer. Observe for nocifensive behaviors and stop the chronometer as soon as nocifensive behavior is observed.Then remove the animal from the chamber and record the latency to this behavior. Make sure to clean the apparatus of any urine between each animal tested to avoid affecting the temperature of the heat stimulus. Clean the testing chamber with 70%ethanol between animals to reduce the behavioral impact of odors.To make a nerve glass hook, turn on the bunsen burner. Hold one end of the glass rod to the fire in one hand. As this glass rod melts, use another glass rod in the other hand to guide and pull at the melting glass on the first rod.Remove the first glass rod from the fire and allow the end of the melted portion to naturally roll inward to form a small ball shape using the second glass rod. 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. Upon choosing which side to perform the surgery on, shape the animal's hind leg around the thigh region, inferiorly toward the patella, superiorly toward the hip and above the femur.Next, wipe three times with chlorhexidine in one direction with three separate gauzes alternated with warm sterile saline. Then slip the leg through a slit made in a 10 by 10 centimeters sterile drape to create a sterile field around the leg of choice. Using fine surgical scissors make a small two millimeter 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. Next, using tying forceps create a sharp one centimeter deep incision vertically at a 90 degree angle in the thigh muscles. Then insert the fine small scissors into the same incision also at a 90 degree angle and spread them open gently to separate the muscles until the sciatic nerve is visualized.Locate the sciatic nerve running parallel to the vertical thigh in the direction of the hip to the knee. Remove the scissors in the tying forceps from the body before proceeding. Use the extra fine forceps in the nerve glass hook to isolate the nerve from underneath.Carefully free the nerves from surrounding connective tissues at a site near the trochanter of the femur which is closest to the hip and furthest from the knee. Allow the nerve to rest on the glass rod and ensure that the end of the rod prevents the nerve from rolling off. Place a surgical knot to tie one third of the width of the sciatic nerve using a nine zero nylon suture prior to it dividing into the common peroneal, tibial and sural nerve branches.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. Next, suture the muscle incision using an absorbable polyglycolic five zero suture. Separately, suture the skin using a non-absorbable polypropylene six zero suture.Then 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. Use the Von Frey, Hargreaves or hot plate test to evaluate both thermal and mechanical hypersensitivity and its potential reversal.Mice that underwent PSNL surgery demonstrated lower thresholds to mechanical stimuli compared to sham animals over 28 days. Similar results were obtained with thermal hypersensitivity evaluation. Paw withdrawal latencies after exposure to radiant heat stimulus were increased in PSNL animals as well as the withdrawal latencies when animals were placed on a 52 degree Celsius plate.Groups injected with two different doses of morphine displayed a reversal of hypersensitivity which lasted from one to two hours. Mechanical hypersensitivity returned to baseline four hours after the injection of morphine. When two different doses of ibuprofen were administered intraperitoneally to the mice, the results demonstrated decreased mechanical hypersensitivity compared to saline injected mice.Limiting additional damage to the nerve is essential as it can affect pain sensory thresholds. The experimenter should pay particular attention and use the glass hook to reduce unintentional damage Following this procedure, this model of chronic neuropathic pain can be used to identify and characterize the antinociceptive effect of drugs leading to the development of new pain management therapies.

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