The authors would like to acknowledge the original description of the CCI model by Bennett and Xie (1988). 
This work was supported partially by grants from the National Health and Medical Research Council of Australia (ID # 568637) and the NSW Office for Science and Medical Research to GMT.

1. Sciatic Nerve Chronic Constriction
Aseptic techniques should be used for the surgical procedure. Disinfect the surgical work surface with 70% ethanol and prepare in advance sterile instruments (e.g., fine scissors, forceps, and retractor), gauzes, staples and swabs by autoclaving. For multiple surgeries, clean and resterilise instruments with 70% ethanol or a dry bead steriliser between rats. A surgical mask, hair bonnet and sterile gloves should be worn.
For constriction of the nerve, prepare the chromic gut suture by cutting it into small pieces of about 3 cm length immersed in sterile saline, to prevent drying. For closing the wound, use commercially available sterile silk sutures.
<ol>
	<li>Anaesthetise the rat in an induction chamber using 5% isoflurane in O2, then deliver 2% isoflurane in O2 via a custom made facemask, and monitor the anesthetised animal during the procedure. Common alternative anaesthetics are inhaled halothane or intraperitoneal sodium pentobarbital. Ketamine, as an N-methyl-D-aspartate (NMDA) receptor antagonist, should be avoided since these receptors are important in the development of neuropathic pain.</li>
	<li>Shave the left hind leg of the rat. Then, place the animal onto a thermo-regulated heating mat at 37&deg;C and apply lubricating ophthalmic ointment to the eyes.</li>
	<li>Sterilize the shaved area with three alternate applications of 70% isopropyl alcohol and iodine solution.</li>
	<li>With the rat lying on its chest/thorax, elevate left hind leg and hold in position with the femor at 90&deg; to the spine using masking tape on the foot.</li>
	<li>Make an incision in the skin parallel, but 3-4 mm below the femor, and free the skin from the muscle surrounding the incision by cutting through the connective tissue.</li>
	<li>Using blunt scissors, cut through the connective tissue between the gluteus superficialis and the biceps femoris muscles.</li>
	<li>Use a retractor to widen the gap between the two muscles, allowing clear visualisation of the sciatic nerve.</li>
	<li>Using curved blunt-tipped forceps and micro-scissors, gently free approximately 10 mm of the sciatic nerve (proximal to the sciatic trifurcation) from the surrounding connective tissue.</li>
	<li>Under the microscope and a good light source, tie four ligatures (chromic gut 4.0, Ethicon, USA) with a double knot, 1 mm apart, proximal to the trifurcation of the sciatic nerve. For each ligature, start with a single loose loop, then grasp the two ends close to the loop and tighten until the loop is just barely snug and the ligature does not slide along the nerve. To hold the loop in its proper position, place a second loop on top of the first to complete the knot. Finally, cut the loose ends of the ligature to around 1mm. Constriction of the nerve should be minimal, and immediately stopped if a brief twitch is observed, to prevent arresting of the epineural blood flow. Over tightening the ligatures leads to axotomy, and autotomy (self-mutilation), both unwanted side-effects which preclude successful pain hypersensitivity testing.</li>
	<li>Use sutures (Mersilk 5.0, Ethicon) to close the muscle layer and staples (Autoclip, 9 mm) to fasten the skin. Next, use iodine solution (Riodine) to sterilise the wound.</li>
	<li>Observe the rat closely during the anaesthesia recovery period, and allow it to recover in a separate cage with flat paper bedding (not the standard animal husbandry bedding), in order to prevent the unconscious animal choking. 
	Before giving any post-operative analgesia investigators should consider the fact this may interfere with pain hypersensitivity testing. In fact, there is evidence that local application of anaesthetic, lidocaine, at time of injury can alter the development of neuropathic pain, and diminish the degree of hyperalgesia in the preceding weeks7. For further discussion on this matter see review by Bennett et al8.</li>
</ol>
2. Measurements of Pain Behaviours
<ol>
	<li>Daily handling of rats for several days before starting the experiment is recommended in order to accustom the rats to their handler, reduce stress and the risk of biting.</li>
	<li>Prior to behavioural testing, rats need to be habituated to the testing procedure and the animal enclosure. The testing environment should be kept quiet and well controlled, with constant temperature and humidity levels, and carried out at a similar time of day each testing session.</li>
	<li>Pain hypersensitivity testing should be performed several times before CCI (baseline) and at different time points after CCI, as desired.</li>
</ol>
3. Mechanical Withdrawal Threshold
<ol>
	<li>On the day of testing, place rats into the elevated mesh floored testing cage 15-30 minutes before measuring withdrawal thresholds, allowing cage exploration and grooming activities to cease and rats to become relaxed and stationary.</li>
	<li>Using a dynamic plantar von Frey aesthesiometer (Ugo Basile, Italy), position the touch-stimulator unit with the filament directly beneath the mid-plantar surface of the hindpaw, avoiding the metal mesh, and press the start key. This lifts the filament to mechanically stimulate the plantar surface of the hindpaw with an increasing force.</li>
</ol>
Manual von Frey hairs are a series of increasing diameter filaments which are calibrated to induce a specific force when applied to the skin. They can be used as an alternative to an electronic dynamic plantar von Frey aesthesiometer for measurement of paw withdrawal threshold, as previously described4. There are also several alternative manufacturers of electronic von Frey aesthesiometers (e.g. IITC, USA and Somedic, Sweden).
<ol start="3">
	<li>Record the mechanical withdrawal threshold from both injured and uninjured hindpaws, in a counterbalanced order. This device applies pressure to the paw and upon withdrawal reaction the system automatically records and displays the maximum force applied (in grams). If an ambiguous response occurs (i.e. in the case of ambulation or grooming), repeat the stimulus and record the threshold again. Licking or shaking of the hindpaw following stimulation is a sign of behaviour associated with pain.</li>
	<li>Repeat the mechanical stimulation 3 times with an interval of about 5 min between stimuli, record the thresholds, and then calculate the mean of paw withdrawal thresholds.</li>
</ol>
4. Thermal Withdrawal Latency
The infrared (I.R.) heat intensity of the plantar test instrument should be adjusted before starting the experiment. In our laboratory, we adjust the I.R. intensity to give an average paw withdrawal latency of about 10 seconds in na&iuml;ve rats, and we set the cut-off latency at 20 seconds to avoid tissue damage in case of failure to remove a paw.
<ol>
	<li>On the day of testing, place rats into the glass floored testing cage 15-30 minutes before measuring latency, allowing cage exploration and grooming activities to cease and rats to become relaxed and stationary.</li>
	<li>Using a thermal plantar analgesia instrument (Ugo Basile, Italy), position the infrared source directly beneath the mid-plantar surface of the hindpaw, and press the start key. This exposes the plantar surface of the hindpaw to a beam of radiant heat through a transparent glass surface. There are also alternative manufacturers of thermal plantar analgesia instruments (e.g. IITC, USA and Stoelting, USA).</li>
	<li>Record the withdrawal latency from both injured and uninjured hindpaws, in a counterbalanced order. This device automatically records the time taken (in seconds) from the onset of the thermal stimulus to withdrawal of the paw from the heat source. If an ambiguous response occurs (i.e. in the case of ambulation or grooming), repeat the stimulus and record the latency again. Licking or shaking of the hindpaw following stimulation is a sign of behaviour associated with pain.</li>
	<li>Repeat the heat stimulation at least 3 times with an interval of about 5 min between stimuli, record the latencies, and then calculate the mean of paw withdrawal latencies.</li>
</ol>
4. Representative Results
An example of mechanical withdrawal threshold and thermal withdrawal latency in the ipsilateral (injured) and contralateral (uninjured) paws of Wistar rats is shown before (baseline) and 2-12 days after CCI in Figure 3A, and Figure 3B. From these figures it can be seen that CCI causes a significant reduction in both mechanical withdrawal thresholds and thermal withdrawal latencies of the ipsilateral hindpaw, indicating the presence of mechanical and thermal pain hypersensitivity, both features of chronic neuropathic pain.
<img alt="Figure 1" src="/files/ftp_upload/3393/3393fig1.jpg" /> 
Figure 1. A schematic illustrating the positioning of the ligatures in the CCI model. This animal model of neuropathic pain involves placement of four loose chromic-gut ligatures on the sciatic nerve, at mid-thigh level. It results in injuring only a portion of the afferents going to the foot and subsequent pain hypersensitivity of the hindpaw.
<img alt="Figure 2" src="/files/ftp_upload/3393/3393fig2.jpg" /> 
Figure 2. Diagram of the hindpaw area used for pain testing. The mid-plantar surface just posterior to the footpads, as indicated by the red dotted circle, is used for pain testing. This area is innervated mostly by terminal branches of the sciatic nerve. The heel and footpads should be avoided due to differential skin sensitivity.
<img alt="Figure 3" src="/files/ftp_upload/3393/3393fig3.jpg" /> 
Figure 3. Pain hypersensitivity following chronic constriction of the sciatic nerve in rats. After CCI, there is a significant reduction in paw withdrawal threshold to mechanical stimuli (A) and paw withdrawal latency to thermal stimuli (B) in the injured (ipsilateral) hindpaw compared to the uninjured (contralateral) hindpaw (P&lt;0.01 repeated measures 2-way ANOVA). Data show mean &plusmn; s.e.m, n=7 Wistar rats.

<ol>
	<li>Bouhassira, D., Lanteri-Minet, M., Attal, N., Laurent, B., Touboul, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+chronic+pain+with+neuropathic+characteristics+in+the+general+population.">Prevalence of chronic pain with neuropathic characteristics in the general population.</a> Pain. 136, 380-387 (2008).</li><li>Toth, C., Lander, J., Wiebe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+and+impact+of+chronic+pain+with+neuropathic+pain+symptoms+in+the+general+population.">The prevalence and impact of chronic pain with neuropathic pain symptoms in the general population.</a> Pain Med. 10, 918-929 (2009).</li><li>Bennett, G. J., Xie, Y. 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, 87-107 (1988).</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> J. Neurosci. Methods. 53, 55-63 (1994).</li><li>Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+and+sensitive+method+for+measuring+thermal+nociception+in+cutaneous+hyperalgesia.">A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia.</a> Pain. 32, 77-88 (1988).</li><li>Yeomans, D. C., Proudfit, H. 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, 85-94 (1994).</li><li>Dougherty, P. M., Garrison, C. J., Carlton, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+influence+of+local+anesthetic+upon+two+models+of+experimentally+induced+peripheral+mononeuropathy+in+the+rat.">Differential influence of local anesthetic upon two models of experimentally induced peripheral mononeuropathy in the rat.</a> Brain Research. 570, 109-115 (1992).</li><li>Bennett, G. J., Chung, J. M., Honore, M., Seltzer, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+neuropathic+pain+in+the+rat.">Models of neuropathic pain in the rat.</a> Curr. Protoc. Neurosci. Chapter 9, (2003).</li><li>Attal, N., Jazat, F., Kayser, V., Guilbaud, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+evidence+for+&#39;pain-related&#39;+behaviours+in+a+model+of+unilateral+peripheral+mononeuropathy.">Further evidence for &#39;pain-related&#39; behaviours in a model of unilateral peripheral mononeuropathy.</a> Pain. 41, 235-251 (1990).</li><li>Hu, B., Doods, H., Treede, R. -. D., Ceci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depression-like+behaviour+in+rats+with+mononeuropathy+is+reduced+by+the+CB2-selective+agonist+GW405833.">Depression-like behaviour in rats with mononeuropathy is reduced by the CB2-selective agonist GW405833.</a> Pain. 143, 206-212 (2009).</li><li>Jesse, C., Wilhelm, E., Nogueira, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depression-like+behavior+and+mechanical+allodynia+are+reduced+by+bis+selenide+treatment+in+mice+with+chronic+constriction+injury:+a+comparison+with+fluoxetine,+amitriptyline,+and+bupropion.">Depression-like behavior and mechanical allodynia are reduced by bis selenide treatment in mice with chronic constriction injury: a comparison with fluoxetine, amitriptyline, and bupropion.</a> Psychopharmacology. 212, 513-522 (2010).</li><li>Monassi, C. R., Bandler, R., Keay, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subpopulation+of+rats+show+social+and+sleep-waking+changes+typical+of+chronic+neuropathic+pain+following+peripheral+nerve+injury.">A subpopulation of rats show social and sleep-waking changes typical of chronic neuropathic pain following peripheral nerve injury.</a> Eur. J. Neurosci. 17, 1907-1920 (2003).</li><li>Roeska, K., Doods, H., Arndt, K., Treede, R. D., Ceci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anxiety-like+behaviour+in+rats+with+mononeuropathy+is+reduced+by+the+analgesic+drugs+morphine+and+gabapentin.">Anxiety-like behaviour in rats with mononeuropathy is reduced by the analgesic drugs morphine and gabapentin.</a> Pain. 139, 349-357 (2008).</li><li>Kontinen, V. K., Kauppila, T., Paananen, S., Pertovaara, A., Kalso, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+measures+of+depression+and+anxiety+in+rats+with+spinal+nerve+ligation-induced+neuropathy.">Behavioural measures of depression and anxiety in rats with spinal nerve ligation-induced neuropathy.</a> Pain. 80, 341-346 (1999).</li><li>Urban, R., Scherrer, G., Goulding, E. H., Tecott, L. H., 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=Behavioral+indices+of+ongoing+pain+are+largely+unchanged+in+male+mice+with+tissue+or+nerve+injury-induced+mechanical+hypersensitivity.">Behavioral indices of ongoing pain are largely unchanged in male mice with tissue or nerve injury-induced mechanical hypersensitivity.</a> Pain. 152, 990-1000 (2011).</li><li>Myers, R. R., Yamamoto, T., Yaksh, T. L., Powell, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+focal+nerve+ischemia+and+Wallerian+degeneration+in+peripheral+nerve+injury+producing+hyperesthesia.">The role of focal nerve ischemia and Wallerian degeneration in peripheral nerve injury producing hyperesthesia.</a> Anesthesiology. 78, 308-316 (1993).</li><li>Grace, P. M., Hutchinson, M. R., Manavis, J., Somogyi, A. A., Rolan, P. E. <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+animal+model+of+graded+neuropathic+pain:+utility+to+investigate+mechanisms+of+population+heterogeneity.">A novel animal model of graded neuropathic pain: utility to investigate mechanisms of population heterogeneity.</a> J. Neurosci. Methods. 193, 47-53 (2010).</li><li>Kajander, K. C., Pollock, C. H., Berg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+hindpaw+position+in+rats+during+chronic+constriction+injury+&#40;CCI&#41;+produced+with+different+suture+materials.">Evaluation of hindpaw position in rats during chronic constriction injury &#40;CCI&#41; produced with different suture materials.</a> Somatosens Mot. Res. 13, 95-101 (1996).</li><li>Xu, J., Pollock, C. H., Kajander, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromic+gut+suture+reduces+calcitonin-gene-related+peptide+and+substance+P+levels+in+the+spinal+cord+following+chronic+constriction+injury+in+the+rat.">Chromic gut suture reduces calcitonin-gene-related peptide and substance P levels in the spinal cord following chronic constriction injury in the rat.</a> Pain. 64, 503-509 (1996).</li><li>Sommer, C., Schafers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Painful+mononeuropathy+in+C57BL/Wld+mice+with+delayed+wallerian+degeneration:+differential+effects+of+cytokine+production+and+nerve+regeneration+on+thermal+and+mechanical+hypersensitivity.">Painful mononeuropathy in C57BL/Wld mice with delayed wallerian degeneration: differential effects of cytokine production and nerve regeneration on thermal and mechanical hypersensitivity.</a> Brain. Res. 784, 154-162 (1998).</li><li>Walczak, J. S., Beaulieu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+three+models+of+neuropathic+pain+in+mice+using+a+new+method+to+assess+cold+allodynia:+the+double+plate+technique.">Comparison of three models of neuropathic pain in mice using a new method to assess cold allodynia: the double plate technique.</a> Neurosci. Lett. 399, 240-244 (2006).</li><li>Maves, T. J., Pechman, P. S., Gebhart, G. F., Meller, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+chemical+contribution+from+chromic+gut+sutures+produces+disorders+of+pain+sensation+like+those+seen+in+man.">Possible chemical contribution from chromic gut sutures produces disorders of pain sensation like those seen in man.</a> Pain. 54, 57-69 (1993).</li><li>Tall, J. M., Stuesse, S. L., Cruce, W. L., Crisp, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender+and+the+behavioral+manifestations+of+neuropathic+pain.">Gender and the behavioral manifestations of neuropathic pain.</a> Pharmacol Biochem. Behav. 68, 99-104 (2001).</li><li>Chung, J. M., Choi, Y., Yoon, Y. W., Na, H. S. <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+age+on+behavioral+signs+of+neuropathic+pain+in+an+experimental+rat+model.">Effects of age on behavioral signs of neuropathic pain in an experimental rat model.</a> Neurosci. Lett. 183, 54-57 (1995).</li><li>Tanck, E. N., Kroin, J. S., McCarthy, R. J., Penn, R. D., Ivankovich, A. D. <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+age+and+size+on+development+of+allodynia+in+a+chronic+pain+model+produced+by+sciatic+nerve+ligation+in+rats.">Effects of age and size on development of allodynia in a chronic pain model produced by sciatic nerve ligation in rats.</a> Pain. 51, 313-316 (1992).</li><li>Shir, Y., Sheth, R., Campbell, J. N., Raja, S. N., Seltzer, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soy-containing+diet+suppresses+chronic+neuropathic+sensory+disorders+in+rats.">Soy-containing diet suppresses chronic neuropathic sensory disorders in rats.</a> Anesth. Analg. 92, 1029-1034 (2001).</li><li>Yoon, Y. W., Lee, D. H., Lee, B. H., Chung, K., Chung, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+strains+and+substrains+of+rats+show+different+levels+of+neuropathic+pain+behaviors.">Different strains and substrains of rats show different levels of neuropathic pain behaviors.</a> Experimental Brain Research. 129, 167-171 (1999).</li><li>Mogil, J. S., Davis, K. D., Derbyshire, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+necessity+of+animal+models+in+pain+research.">The necessity of animal models in pain research.</a> Pain. 151, 12-17 (2010).</li><li>Mogil, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+pain:+progress+and+challenges.">Animal models of pain: progress and challenges.</a> Nat. Rev. Neurosci. 10, 283-294 (2009).</li><li>Langford, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coding+of+facial+expressions+of+pain+in+the+laboratory+mouse.">Coding of facial expressions of pain in the laboratory mouse.</a> Nat. Methods. 7, 447-449 (2010).</li><li>Austin, P. J., Moalem-Taylor, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuro-immune+balance+in+neuropathic+pain:+involvement+of+inflammatory+immune+cells,+immune-like+glial+cells+and+cytokines.">The neuro-immune balance in neuropathic pain: involvement of inflammatory immune cells, immune-like glial cells and cytokines.</a> J. Neuroimmunol. 229, 26-50 (2010).</li></ol>

Experiments on animals were approved by the Animal Care and Ethics Committee of the University of New South Wales, Australia, and followed guidelines issued by the International Association for the Study of Pain.

CCI is a widely used peripheral nerve injury model of chronic neuropathic pain. It is relatively simple to perform, and produces robust and stable pain hypersensitivity for at least one month after injury. Following CCI, rats exhibit abnormal posture of the injured hindpaw (toes held together and plantar-flexed and paw everted), as well as repeated shaking, guarding and licking of the injured hindpaw suggesting the presence of spontaneous pain9. In addition to sensory dysfunction, several investigators have sh...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Chromic gut</td>
 <td>Ethicon</td>
 <td>G-211</td>
 <td>4-0 thickness</td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td>Delvet Pty. Ltd., Seven Hills, NSW</td>
 <td>n/a</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mersilk</td>
 <td>Ethicon</td>
 <td>W-580</td>
 <td>5-0 thickness</td>
 </tr>
 <tr>
 <td>Autoclip</td>
 <td>Becton Dickinson</td>
 <td>427631 </td>
 <td>9 mm stainless steel</td>
 </tr>
 <tr>
 <td>Riodine</td>
 <td>Orion</td>
 <td>R1000802F</td>
 <td>1% w/v iodine</td>
 </tr>
 <tr>
 <td>Thermal plantar analgesia instrument</td>
 <td>Ugo Basile</td>
 <td>37370</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dynamic plantar aesthesiometer </td>
 <td>Ugo Basile</td>
 <td>37400</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats at JoVE.com

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

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 ...

chronic-constriction-sciatic-nerve-pain-hypersensitivity-testing

Research

JoVE Journal

Medicine

58.6K Views.  University of New South Wales. 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).

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

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50&#8211;60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.

We describe a stepwise procedure for creating pressure overload and left ventricular hypertrophy in Wistar rats by constriction of the ascending aorta using a small metallic clip. This model is extensively used for studying remodeling changes during cardiac hypertrophy and for identifying and evaluating strategies for regression of such changes.

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

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 ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Histological Quantification of Chronic Myocardial Infarct in Rats

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ischemic injury. Following the initial acute phase, chronic remodeling of the left ventricle (LV) modifies the structure and function of the heart. Permanent coronary ligation in small animals has been widely used as a reference model for a chronic model of MI. Thinning of the infarcted wall progressively develops to transmural fibrosis. Histological assessment of infarct size is commonly performed; nevertheless, a standardization of the methods for quantification is missing. Indeed, important methodological aspects, such as the number of sections analyzed and the sampling and quantification methods, are usually not described and therefore preclude comparison across investigations. Too often, quantification is performed on a single section obtained at the level of the papillary muscles. Because novel strategies aimed at reducing infarct expansion and remodeling are under investigation, there is an important need for the standardization of accurate heart sampling protocols. We describe an accurate method to quantify the infarct size using a systematic sampling of harvested rat heart and image analyses of trichromatic stained histological sections obtained from base to apex. We also provide evidence that calculating the expansion index (EI) allowed for infarct size assessment, taking into account changes of the left ventricle throughout the remodeling.

The post-mortem assessment of myocardial infarction (MI) in rodents is based on quantification of the infarct on stained heart sections. We describe an accurate method to quantify the infarct size using systematic sampling of harvested rat hearts from base to apex and image analyses of trichrome-stained histological sections.

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ...

Minimally Invasive Transverse Aortic Constriction in Mice

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.

Minimally invasive transverse aortic constriction (MTAC) conserves the essentials of regular transverse aortic constriction (TAC) while eliminating the use of a ventilator with tracheal intubation. It proves to be a highly desirable method for high-throughput studies on left ventricular overload, particularly in translational studies.

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard ...

Subcutaneous Trigeminal Nerve Field Stimulation for Refractory Facial Pain

Chronic or neuropathic trigeminal facial pain can be challenging to treat. Neurosurgical procedures should be applied when conservative treatment fails. Neuromodulation techniques for chronic facial pain include deep brain stimulation and motor cortex stimulation, which are complex to perform. Subcutaneous nerve field stimulation is certified for chronic back pain and is the least invasive form of neuromodulation. We applied this technique to treat chronic and neuropathic trigeminal pain as an individual therapy concept. First, trial stimulation is performed. Subcutaneous leads are placed in the painful trigeminal dermatome under local anesthesia. The leads are connected to an external neurostimulator that applies constant stimulation. Patients undergo a 12 day outpatient trial to assess the effect of the stimulation. Electrodes are removed after the trial. If the patient reports pain reduction of at least 50% in intensity and/or attack frequency, a reduction in medication or increase in quality of life, permanent implantation is scheduled. New electrodes are implanted under general anesthesia and are subcutaneously tunneled to an infraclavicular internal pulse generator. Patients are able to turn stimulation on and off and to increase or decrease the stimulation amplitude as needed.
This technique represents a minimal invasive alternative to other more invasive means of neuromodulation for trigeminal pain such as motor cortex stimulation or deep brain stimulation.

Treating chronic or neuropathic facial pain can be challenging when medical or standard treatment fails. Subcutaneous nerve field stimulation is the least invasive form of neuromodulation and is used for chronic back pain. We applied this technology to treat chronic and neuropathic trigeminal facial pain.

Chronic or neuropathic trigeminal facial pain can be challenging to treat. Neurosurgical procedures should be applied when conservative treatment fails. ...

Development of Recombinant Proteins to Treat Chronic Pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.

In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain.

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...