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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		<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>
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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.

partial-sciatic-nerve-ligation-mouse-model-chronic-neuropathic-pain

Research

JoVE Journal

Medicine

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 ...

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 ...

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

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

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

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

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

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

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

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

Chronic Constriction Injury of the Rat's Infraorbital Nerve (IoN-CCI) to Study Trigeminal Neuropathic Pain

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral procedures to study trigeminal neuropathic pain in rats. To meet the specificity of trigeminal neuropathic pain syndromes, the infraorbital nerve (IoN) is subjected to a chronic constriction injury (CCI) by loosely ligating the nerve. An intra-orbital approach is presented here to expose and ligate the IoN in the orbital cavity. After IoN ligation, rats exhibit changes in spontaneous behavior and in response to von Frey hair stimulation that are indicative of persistent pain and mechanical allodynia. Two phases can be defined in the development of the behavioral changes. During the first week following IoN-CCI (phase 1), rats show an increased and asymmetric face grooming activity, i.e., with face wash strokes primarily directed to the nerve-injured IoN territory. A distinction is made between face grooming behavior that is part of a more general body grooming behavior, which remains largely unaffected by IoN-CCI, and face grooming that is neither preceded nor followed by body grooming, which is significantly increased after IoN-CCI. During this period, responsiveness to mechanical stimulation of the IoN territory is reduced. This hyporesponsiveness is abruptly replaced by an extreme hyperresponsiveness whereby even very weak stimulus intensities provoke nocifensive behavior (phase 2). The phenomenological similarities between these behavioral alterations and reported signs of facial pain (i.e., responses to noxious stimulation of the face) suggest the presence of dysesthesia/paresthesia and mechanical allodynia in the ligated IoN territory.

Chronic Constriction Injury of the Rat's Infraorbital Nerve IoN-CCI to Study Trigeminal Neuropathic Pain

This manuscript describes a rodent model to study trigeminal neuropathic pain. The methods described include surgical procedures to perform a chronic constriction injury of the rat&#8217;s infraorbital nerve and the post-surgical behavioral tests to measure the changes in spontaneous and evoked behavior that are indicative of persistent pain and allodynia.

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...