This project was sponsored by National Natural Science Foundation of China (81873365 and 81603676), Zhejiang Provincial Natural Science Funds for Distinguished Young Scholars (LR17H270001) and research funds from Zhejiang Chinese Medical University (Q2019J01, 2018ZY37, 2018ZY19).

The animal protocols were approved by Zhejiang Chinese Medical University Animal Ethics Committee.
1. Animals
<ol>
	<li>Obtain male Sprague-Dawley (SD) rats (280&#8211;320 g, 8-10 weeks of age) from Shanghai Laboratory Animal Center. House the animals in Zhejiang Chinese Medical University Laboratory Animal Center. Note that the breeding conditions should include 12 h/ 2h light/dark cycles and keep temperature constant at 24 &#176;C. Provide water and food ad libitum. Note that a total of 48 rats are used in this study. One note, in this model, we need to observe the pain response during the whole process. If pain medication is applied, then we cannot successfully establish the pain model. After the experiments, the rats are sacrificed.</li>
</ol>
2. CPIP model establishment
<ol>
	<li>Anesthetize all rats (including sham and CPIP model groups) with sodium phenobarbital (50 mg/kg, intraperitoneal injection [i.p.]). Maintain anesthesia with up to 20 mg/kg/h phenobarbital [i.p.], if necessary. Check the reflexes of each animal by pinching its hind paw or tail tip using forceps. Make sure that the rats are not responsive before model establishment. Place vet ointment on eyes to avoid dryness during the procedure. Place the anesthetized rats on a heated pad maintained at 37 &#176;C for the following procedure.</li>
	<li>Ischemia and reperfusion of the hind paw
	<ol>
		<li>Lubricate the right hind paw and ankle with glycerol once the rat is anesthetized.</li>
		<li>Slide a Nitrile 70 Durometer O-ring with a 7/32&#34; (5.5 mm) internal diameter into the larger side of a 1.5 mL Eppendorf tube (with the snap-cap cut off before use). Carefully insert the hind paw into the hollow Eppendorf tube until reaching the bottom.</li>
		<li>Gradually slide the O-ring from the tube to the right hind limb near the ankle joint and place for 3 h. Apply the same treatment to a sham group of rats, except that a broken O-ring, which is cut off and should not induce ischemia, should be placed around the ankle.</li>
		<li>Cut off the O-ring 3 h after the ischemia step. Carefully watch the rat until it recovers enough consciousness to maintain sternal recumbency. Note that the rat that received anesthesia should not be placed back to the company of other rats until it fully recovered.</li>
	</ol>
	</li>
</ol>
3. Nocifensive behavioral tests
<ol>
	<li>Place the rat in a transparent Plexiglas chamber that is sitting on a mesh floor. Habituate the rat for 0.5 h before any behavioral testing.</li>
	<li>Mechanical allodynia
	<ol>
		<li>Use von Frey filaments (0.4, 0.6, 1.0, 2.0, 4.0, 6.0, 8.0, 15.0, and 26.0 g filaments) for the test. Begin the test from the middle filament (4.0 g). Vertically apply the filaments to the middle plantar surface of the hind paw. Slightly apply suitable force to bend the filament for up to 5 s. A sudden retraction of the hind paw in response to the stimuli is considered a nocifensive behavior. Conduct the mechanical allodynia test on days -3, -2, -1, and every other day until day 13.</li>
		<li>Apply the up-down testing method to test the threshold. Apply the Dixon method for calculating 50% paw withdrawal threshold (PWT)13,14,15.</li>
	</ol>
	</li>
	<li>Thermal hyperalgesia
	<ol>
		<li>Use Hargreaves&#39; method to examine thermal hyperalgesia. Directly aim the light beam emitted from a bulb (50 W) to the hind paw to measure the paw withdrawal latency (PWL). Set 20 s as the cut-off threshold to avoid excessive injury from the heating.</li>
		<li>Repeat each test 3x in 5 min intervals for each hind paw. Take the average of these three tests as the PWL of each rat16. Conduct the thermal hyperalgesia test on days -3, -2, -1, and every other day until day 13.</li>
	</ol>
	</li>
	<li>Capsaicin-induced acute nocifensive behavior
	<ol>
		<li>Prepare capsaicin stock solution (200 mM) using dimethyl sulfoxide (DMSO) and further dilute to 1:1000 in sterile phosphate-buffered saline (PBS) for hind paw injection. The final DMSO concentration in PBS is 0.1% (vehicle contains 0.1% DMSO in PBS only). Inject capsaicin or vehicle into the hind paw (intraplantar injection) at a volume of 50 &#956;L using a 30 G needle attached to 1 mL syringe.</li>
		<li>Record the nocifensive behavior (i.e., licking, biting, or flinching of the injected paw) using a video camera for 10 min right after the injection and quantified thereafter as previously described17,18,19.</li>
	</ol>
	</li>
	<li>Hind paw edema evaluation: Evaluate the hind paw edema by measuring the increase in paw diameter. Measure with a digital caliper and calculate the difference between the basal value and the test value observed at different time points. Assess the changes in paw thickness at 15 min, 24 h, 48 h, and 72 h after model establishment.</li>
</ol>

<ol><li>Goh, E. L., Chidambaram, S., Ma, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complex+regional+pain+syndrome%3A+a+recent+update%5BTitle%5D)+AND+%22Burns%2C+Trauma%22%5BJournal%5D)">Complex regional pain syndrome: a recent update.</a> Burns, Trauma. 5 (1), 2(2017).</li>
<li>Birklein, F., Ajit, S. K., Goebel, A., Rsgm, P., Sommer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complex+regional+pain+syndrome+-+phenotypic+characteristics+and+potential+biomarkers%5BTitle%5D)+AND+%22Nature+Reviews+Neurology%22%5BJournal%5D)">Complex regional pain syndrome - phenotypic characteristics and potential biomarkers.</a> Nature Reviews Neurology. 14 (5), (2018).</li>
<li>Shim, H., Rose, J., Halle, S., Shekane, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complex+regional+pain+syndrome%3A+a+narrative+review+for+the+practising+clinician%5BTitle%5D)+AND+%22British+Journal+of+Anaesthesia%22%5BJournal%5D)">Complex regional pain syndrome: a narrative review for the practising clinician.</a> British Journal of Anaesthesia. , (2019).</li>
<li>Urits, I., Shen, A. H., Jones, M. R., Viswanath, O., Kaye, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complex+Regional+Pain+Syndrome%2C+Current+Concepts+and+Treatment+Options%5BTitle%5D)+AND+%22Current+Pain%2C+Headache+Reports%22%5BJournal%5D)">Complex Regional Pain Syndrome, Current Concepts and Treatment Options.</a> Current Pain, Headache Reports. 22 (2), 10(2018).</li>
<li>Helyes, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transfer+of+complex+regional+pain+syndrome+to+mice+via+human+autoantibodies+is+mediated+by+interleukin-1-induced+mechanisms%5BTitle%5D)+AND+%22Proceedings+of+National+Academy+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Transfer of complex regional pain syndrome to mice via human autoantibodies is mediated by interleukin-1-induced mechanisms.</a> Proceedings of National Academy Sciences of the United States of America. 116 (26), 13067-13076 (2019).</li>
<li>Coderre, T. J., Xanthos, D. N., Francis, L., Bennett, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+post-ischemia+pain+%28CPIP%29%3A+a+novel+animal+model+of+complex+regional+pain+syndrome-Type+I+%28CRPS-I%3B+reflex+sympathetic+dystrophy%29+produced+by+prolonged+hindpaw+ischemia+and+reperfusion+in+the+rat%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Chronic post-ischemia pain (CPIP): a novel animal model of complex regional pain syndrome-Type I (CRPS-I; reflex sympathetic dystrophy) produced by prolonged hindpaw ischemia and reperfusion in the rat.</a> Pain. 112 (1), 94-105 (2004).</li>
<li>Coderre, T. J., Bennett, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+hypothesis+for+the+cause+of+complex+regional+pain+syndrome-type+I+%28reflex+sympathetic+dystrophy%29%3A+pain+due+to+deep-tissue+microvascular+pathology%5BTitle%5D)+AND+%22Pain+Medicine%22%5BJournal%5D)">A hypothesis for the cause of complex regional pain syndrome-type I (reflex sympathetic dystrophy): pain due to deep-tissue microvascular pathology.</a> Pain Medicine. 11 (8), 1224-1238 (2010).</li>
<li>Klafke, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acute+and+chronic+nociceptive+phases+observed+in+a+rat+hind+paw+ischemia%2Freperfusion+model+depend+on+different+mechanisms%5BTitle%5D)+AND+%22Pflugers+Archiv+European+Journal+of+Physiology%22%5BJournal%5D)">Acute and chronic nociceptive phases observed in a rat hind paw ischemia/reperfusion model depend on different mechanisms.</a> Pflugers Archiv European Journal of Physiology. 468 (2), 229-241 (2015).</li>
<li>Tang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+between+astrocytic+colony+stimulating+factor+and+its+receptor+on+microglia+mediates+central+sensitization+and+behavioral+hypersensitivity+in+chronic+post+ischemic+pain+model%5BTitle%5D)+AND+%22Brain+Behavioral+Immunology%22%5BJournal%5D)">Interaction between astrocytic colony stimulating factor and its receptor on microglia mediates central sensitization and behavioral hypersensitivity in chronic post ischemic pain model.</a> Brain Behavioral Immunology. 68, 248-260 (2018).</li>
<li>Kim, J. H., Kim, Y. C., Nahm, F. S., Lee, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Therapeutic+Effect+of+Vitamin+C+in+an+Animal+Model+of+Complex+Regional+Pain+Syndrome+Produced+by+Prolonged+Hindpaw+Ischemia-Reperfusion+in+Rats%5BTitle%5D)+AND+%22International+Journal+of+Medical+Sciences%22%5BJournal%5D)">The Therapeutic Effect of Vitamin C in an Animal Model of Complex Regional Pain Syndrome Produced by Prolonged Hindpaw Ischemia-Reperfusion in Rats.</a> International Journal of Medical Sciences. 14 (1), 97-101 (2017).</li>
<li>Yin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptome+profiling+of+dorsal+root+ganglia+in+a+rat+model+of+complex+regional+pain+syndrome+type-I+reveals+potential+mechanisms+involved+in+pain%5BTitle%5D)+AND+%22Journal+of+Pain+Research%22%5BJournal%5D)">Transcriptome profiling of dorsal root ganglia in a rat model of complex regional pain syndrome type-I reveals potential mechanisms involved in pain.</a> Journal of Pain Research. 12, 1201-1216 (2019).</li>
<li>Hu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TRPV1+Channel+Contributes+to+the+Behavioral+Hypersensitivity+in+a+Rat+Model+of+Complex+Regional+Pain+Syndrome+Type+1%5BTitle%5D)+AND+%22Frontiers+in+Pharmacology%22%5BJournal%5D)">TRPV1 Channel Contributes to the Behavioral Hypersensitivity in a Rat Model of Complex Regional Pain Syndrome Type 1.</a> Frontiers in Pharmacology. 10, 453(2019).</li>
<li>Dixon, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+analysis+of+experimental+observations%5BTitle%5D)+AND+%22Annual+Review+of+Pharmacology+and+Toxicology%22%5BJournal%5D)">Efficient analysis of experimental observations.</a> Annual Review of Pharmacology and Toxicology. 20, 441-462 (1980).</li>
<li>Chai, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electroacupuncture+Alleviates+Pain+Responses+and+Inflammation+in+a+Rat+Model+of+Acute+Gout+Arthritis%5BTitle%5D)+AND+%22Evidence-Based+Complementary+and+Alternative+Medicine%22%5BJournal%5D)">Electroacupuncture Alleviates Pain Responses and Inflammation in a Rat Model of Acute Gout Arthritis.</a> Evidence-Based Complementary and Alternative Medicine. 2018, 2598975(2018).</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/pubmed?term=((Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">Quantitative assessment of tactile allodynia in the rat paw.</a> Journal of Neuroscience Methods. 53 (1), 55-63 (1994).</li>
<li>Fang, J. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parameter-specific+analgesic+effects+of+electroacupuncture+mediated+by+degree+of+regulation+TRPV1+and+P2X3+in+inflammatory+pain+in+rats%5BTitle%5D)+AND+%22Life+Sciences%22%5BJournal%5D)">Parameter-specific analgesic effects of electroacupuncture mediated by degree of regulation TRPV1 and P2X3 in inflammatory pain in rats.</a> Life Sciences. 200, 69-80 (2018).</li>
<li>Tai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Involvement+of+Transient+Receptor+Potential+Cation+Channel+Member+A1+activation+in+the+irritation+and+pain+response+elicited+by+skin-lightening+reagent+hydroquinone%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Involvement of Transient Receptor Potential Cation Channel Member A1 activation in the irritation and pain response elicited by skin-lightening reagent hydroquinone.</a> Scientific Reports. 7 (1), 7532(2017).</li>
<li>Liu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TRPM8+is+the+principal+mediator+of+menthol-induced+analgesia+of+acute+and+inflammatory+pain%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">TRPM8 is the principal mediator of menthol-induced analgesia of acute and inflammatory pain.</a> Pain. 154 (10), 2169-2177 (2013).</li>
<li>Liu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxidized+Phospholipid+OxPAPC+Activates+TRPA1+and+Contributes+to+Chronic+Inflammatory+Pain+in+Mice%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Oxidized Phospholipid OxPAPC Activates TRPA1 and Contributes to Chronic Inflammatory Pain in Mice.</a> PLoS One. 11 (11), 0165200(2016).</li>
<li>Terkelsen, A. J., Gierthmuhlen, J., Finnerup, N. B., Hojlund, A. P., Jensen, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bilateral+hypersensitivity+to+capsaicin%2C+thermal%2C+and+mechanical+stimuli+in+unilateral+complex+regional+pain+syndrome%5BTitle%5D)+AND+%22Anesthesiology%22%5BJournal%5D)">Bilateral hypersensitivity to capsaicin, thermal, and mechanical stimuli in unilateral complex regional pain syndrome.</a> Anesthesiology. 120 (5), 1225-1236 (2014).</li>
<li>Drummond, P. D., Morellini, N., Finch, P. M., Birklein, F., Knudsen, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complex+regional+pain+syndrome%3A+intradermal+injection+of+phenylephrine+evokes+pain+and+hyperalgesia+in+a+subgroup+of+patients+with+upregulated+alpha1-adrenoceptors+on+dermal+nerves%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Complex regional pain syndrome: intradermal injection of phenylephrine evokes pain and hyperalgesia in a subgroup of patients with upregulated alpha1-adrenoceptors on dermal nerves.</a> Pain. 159 (11), 2296-2305 (2018).</li>
<li>Minert, A., Gabay, E., Dominguez, C., Wiesenfeld-Hallin, Z., Devor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spontaneous+pain+following+spinal+nerve+injury+in+mice%5BTitle%5D)+AND+%22Experimental+Neurology%22%5BJournal%5D)">Spontaneous pain following spinal nerve injury in mice.</a> Experimental Neurology. 206 (2), 220-230 (2007).</li>
<li>Kingery, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Capsaicin+sensitive+afferents+mediate+the+development+of+heat+hyperalgesia+and+hindpaw+edema+after+sciatic+section+in+rats%5BTitle%5D)+AND+%22Neuroscience+Letters%22%5BJournal%5D)">Capsaicin sensitive afferents mediate the development of heat hyperalgesia and hindpaw edema after sciatic section in rats.</a> Neuroscience Letters. 318 (1), 39-43 (2002).</li>
<li>Xu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+cannabinoid+receptor+2+attenuates+mechanical+allodynia+and+neuroinflammatory+responses+in+a+chronic+post-ischemic+pain+model+of+complex+regional+pain+syndrome+type+I+in+rats%5BTitle%5D)+AND+%22European+Journal+of+Neuroscience%22%5BJournal%5D)">Activation of cannabinoid receptor 2 attenuates mechanical allodynia and neuroinflammatory responses in a chronic post-ischemic pain model of complex regional pain syndrome type I in rats.</a> European Journal of Neuroscience. 44 (12), 3046-3055 (2016).</li>
<li>Coderre, T. J., Xanthos, D. N., Francis, L., Bennett, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+post-ischemia+pain+%28CPIP%29%3A+a+novel+animal+model+of+complex+regional+pain+syndrome-type+I+%28CRPS-I%3B+reflex+sympathetic+dystrophy%29+produced+by+prolonged+hindpaw+ischemia+and+reperfusion+in+the+rat%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Chronic post-ischemia pain (CPIP): a novel animal model of complex regional pain syndrome-type I (CRPS-I; reflex sympathetic dystrophy) produced by prolonged hindpaw ischemia and reperfusion in the rat.</a> Pain. 112 (1-2), 94-105 (2004).</li>
<li>Weissmann, R., Uziel, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pediatric+complex+regional+pain+syndrome%3A+a+review%5BTitle%5D)+AND+%22Pediatric+Rheumatology+Online+Journal%22%5BJournal%5D)">Pediatric complex regional pain syndrome: a review.</a> Pediatric Rheumatology Online Journal. 14 (1), 29(2016).</li>
<li>Kim, H., Lee, C. H., Kim, S. H., Kim, Y. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidemiology+of+complex+regional+pain+syndrome+in+Korea%3A+An+electronic+population+health+data+study%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Epidemiology of complex regional pain syndrome in Korea: An electronic population health data study.</a> PLoS One. 13 (6), 0198147(2018).</li>
<li>Tang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sex+differences+in+complex+regional+pain+syndrome+type+I+%28CRPS-I%29+in+mice%5BTitle%5D)+AND+%22Journal+of+Pain+Research%22%5BJournal%5D)">Sex differences in complex regional pain syndrome type I (CRPS-I) in mice.</a> Journal of Pain Research. 10, 1811-1819 (2017).</li>
</ol>

The authors declare no conflicts of interest in this work.

This protocol describes the detailed methods for establishing a rat CPIP model by applying ischemia/reperfusion to hind limbs of the rats. It involves the evaluation of hind limb appearance, edema, mechanical/thermal hypersensitivities, and acute nocifensive behaviors in response to capsaicin injection.
Limb ischemia/reperfusion is a common factor contributing to CRPS-I in human patients12. This protocol describes how to establish the rat CPIP model, which is a commonly used animal model to recapitulate human CRPS-I6. Ischemia was induced in the rat&#39;s hind limb with a tight O-ring applied to the ankle for 3 h under anesthesia. The O-ring was removed, and reperfusion occurred. After the model establishment, the CPIP model rat developed early hyperemia and edema in the hind limb. The model also displays neuropathic pain behaviors, including persistent mechanical/thermal hypersensitivities. These symptoms all mimic the typical characteristics of human CRPS-I 21.
CRPS patients exhibit obvious increased responses to capsaicin-induced pain in the affected limbs20. However, it remains unclear whether CPIP model animals exhibit similar responses. Thus, in this study, nocifensive behaviors of CPIP rats were examined in response to intraplantar capsaicin injection into the ipsilateral hind paw. It was found that CPIP model rats showed significantly higher nocifensive responses to capsaicin injection compared to sham rats. To our knowledge, this is the first report of this phenomenon in CPIP model rats.
In a previous study, TRPV1 channel expression was shown to be upregulated in ipsilateral hind paw tissues and DRG neurons that innervated the hind paw12. Furthermore, it has been found that spinal glial cells are activated in CPIP model rats, resulting in central pain sensitization and facilitation of pain perception and transmission9,12. Therefore, peripheral and central pain sensitization may both be involved in the increased nocifensive behaviors in response to capsaicin injection in CPIP model rats. Future studies are needed to test whether capsaicin injection results in enhanced responses in contralateral hind paws of CPIP model rats, as well. In the tests performed here, we did not observe autotomy in the CPIP model rats. This contrasts with rats that have undergone sciatic sectioning, who usually develop autotomy in addition to chronic pain hypersensitivities22,23. In addition, no referred pain response has been reported in CRPS-I patients or CPIP mode animals.
One of the critical steps in the model is to maintain anesthesia throughout the procedure. In this study, the anesthesia was initiated by intraperitoneal injection of sodium phenobarbital (50 mg/kg). This dosage is frequently used in many studies involving rat anesthesia, including studies with CPIP rat model establishment9,12,24. Besides, anesthesia should be maintained via 20 mg/kg/h (i.p.) sodium phenobarbital when necessary to guarantee successful implement of the procedure12,24.
In the initial CPIP model established by Coderre et al., the O-ring was actually slid off from the outside of a 3 cm3 syringe (cut in half) when the hind paw was placed into the barrel of the syringe as far as possible25. Here, in our study, we made minor modifications to this method. We used a 1.5 mL Eppendorf tube (with the snap-cap cut off beforehand) instead of the syringe, since this material was more accessible and fit just well with the diameter of the rat&#39;s hind paw. The O-ring can be easily placed on the ankle part of the rats in this modified method without injuries to allow for model establishment.
At present, the CPIP rat model was exclusively established in male rats based upon previous studies, including our present study25. However, it should be noted that the incidence of CRPS-I is more frequent in female than in male patients according to epidemiological analysis26,27. This is further supported by the findings that female animals exhibited more pain responses than male animals when CPIP was established28. Therefore, it will be necessary to establish the CPIP model in both male and female animals in the future. This will be important for studying the translational significance of this model.
In conclusion, described here are methods for establishing a rat CPIP model. This CPIP rat model recapitulates many clinical characters of human CRPS-I, and its properties may render it a suitable animal model to study CRPS-I.

Complex regional pain syndrome (CRPS) reprents complex and chronic pain symptoms resulting from fractures, trauma, surgery, ischemia or nerve injury1,2,3. CRPS is classified into 2 subcategories: CRPS type-I and type-II (CRPS-I and CRPS-II)4. Epidemiological studies revealed that the prevalence of CRPS was approximately 1:20005. CRPS-I, which shows no obvious nerve damage, can result in chronic pain and dramatically affects the life quality of the patients. Current available treatments show inadequate therapeutic effects. Therefore, CRPS-I still remains an important and challenging clinical problem that needs to be addressed.
Establishing a preclinical animal model mimicking CRPS-I is crucial for exploring the mechanisms underlying CRPS-I. In order to address this issue, Coderre et al. designed a rat model by applying prolonged ischemia and reperfusion to the hind limb to recapitulate CRPS-I6. It is known that ischemia/reperfusion injury is among one of the major causes of CRPS-I7. The rat CPIP model exhibits many CRPS-I-like symptoms, which include hind limb edema and hyperemia in the early stage after model establishment, followed with persistent thermal and mechanical hypersensitivities6. With the aid from this model, it is proposed that central pain sensitization, peripheral TRPA1 channel activation and reactive oxygen species generation, etc. contribute to CRPS-I8,9,10. We recently successfully established the CPIP rat model and performed RNA-sequencing of the dorsal root ganglia (DRGs) that innervate the affected hind paw11. We discovered some potential mechanisms that are possibly involved in mediating the pain hypersensitivities of CRPS-I11. We further identified transient receptor potential vanilloid 1 (TRPV1) channel in DRG neurons as an important contributor to the mechanical and thermal hypersensitivities of CRPS-I12.
In this study, we described the detailed procedures involved in the establishment of the rat model of CPIP. We further evaluated the rat CPIP model by measuring the mechanical and thermal hypersensitivities as well as its responsiveness to acute capsaicin challenge. We propose that the rat CPIP model can be a reliable animal model for further investigation of the mechanisms involved in CRPS-I.

<table><tbody><tr><td>1.5 ml Eppendorf tube</td><td>Eppendorf</td><td>22431021</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D1435</td><td></td></tr><tr><td>Capsaicin</td><td>APEXBIO</td><td>A3278</td><td></td></tr><tr><td>Digital caliper</td><td>Meinaite</td><td>NA</td><td></td></tr><tr><td>O-ring</td><td>O-Rings West</td><td>Nitrile 70 Durometer</td><td>7/32 in.&lt;br/&gt; internal diameter</td></tr><tr><td>Plantar Test Apparatus</td><td>UGO Basile, Italy</td><td>37370</td><td></td></tr><tr><td>von Frey filaments</td><td>UGO Basile, Italy</td><td>NC12775</td><td></td></tr></tbody></table>

chronic post-ischemia pain model for complex regional pain syndrome type-i in rats

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical condition. However, the underlying mechanisms of CRPS-I have yet to be revealed. It is known that ischemia/reperfusion is one of the leading factors that causes CRPS-I. By means of prolonged ischemia and reperfusion of the hind limb, the rat chronic post-ischemia pain (CPIP) model has been established to mimic CRPS-I. The CPIP model has become a well-recognized animal model for studying the mechanisms of CRPS-I. This protocol describes the detailed procedures involved in the establishment of the rat model of CPIP, including anesthesia, followed by ischemia/reperfusion of the hind limb. Characteristics of the rat CPIP model are further evaluated by measuring the mechanical and thermal hypersensitivities of the hind limb as well as the nocifensive responses to acute capsaicin injection. The rat CPIP model exhibits several CRPS-I-like manifestations, including hind limb edema and hyperemia in the early stage after establishment, persistent thermal and mechanical hypersensitivities, and increased nocifensive responses to acute capsaicin injection. These characteristics render it a suitable animal model for further investigation of the mechanisms involved in CRPS-I.

After placing the O-ring on the ankle, the ipsilateral hind paw skin showed cyanosis, an indication of tissue hypoxia (Figure 1A). After cutting the O-ring, the ipsilateral hind paw began to fill with blood and showed robust swelling, which demonstrated an intense sign of hyperemia (Figure 1A). The paw swelling gradually diminished and returned to normal 48 h after the ischemic/reperfusion procedure (two-way ANOVA with Sidak post-hoc test, Figure 1B). All of these signs are consistent with previous studies6,12.
Then, mechanical allodynia was measured using a von Frey hair test. The ipsilateral hind paw of the CPIP group exhibited obvious mechanical allodynia 1 day after model establishment compared to the sham group. The mechanical allodynia of the ipsilateral hind paw persisted until 13 days of the observation timeframe (two-way ANOVA with Sidak post-hoc test, Figure 1C). The contralateral hind paw of the CPIP group also displayed mechanical hyperalgesia similar to the ipsilateral hind paw, lasting for 13 days (two-way ANOVA with Sidak post-hoc test, Figure 1D).
Thermal hyperalgesia was then measured using a Hargreaves&#39; test. Bilateral hind paws of CPIP rats exhibited significantly reduced withdrawal latency in response to noxious thermal stimuli, a sign of thermal hyperalgesia, compared to the sham group rats (two-way ANOVA with Sidak post hoc test, Figure 1E,F). This observation is consistent with previous studies11,12. The thermal hyperalgesia of the ipsilateral hind paw persisted until the end of the observation timeframe, whereas the thermal hyperalgesia of the contralateral hind paw lasted for 7 days (two-way ANOVA with Sidak post-hoc test, Figure 1E,F). The above results suggest that CPIP rats develop robust and persistent mechanical and thermal hypersensitivities, consistent with previous observations11,12.
CRPS patients exhibited an obvious increased response to capsaicin-induced pain in affected areas20. It was then examined whether the CPIP rat model can recapitulate this phenomenon. Nocifensive behavior in CPIP rats was observed in response to intraplantar capsaicin (a TRPV1 agonist) injection into the ipsilateral hind paw. First, the nocifensive responses of the rats when a vehicle was injected were tested. The sham group showed a slight nocifensive response to vehicle injection, whereas the CPIP group showed a significantly higher response compared to the sham group (one-way ANOVA with Sidak post-hoc test, Figure 2). Furthermore, capsaicin injection resulted in robust nocifensive response in the sham group (Figure 2). More importantly, CPIP rats showed significantly higher responses to capsaicin injection than the sham group (Figure 2). These data suggest that CPIP rats exhibited enhanced nocifensive responses to capsaicin, a phenomenon mimicking human patients with CRPS-I.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60562/60562fig1b.jpg" /> 
Figure 1: The CPIP rat model showed thermal and mechanical pain hypersensitivities in bilateral hind limbs. (A) Typical images taken during different time points (during ischemia, 10 min after reperfusion, and 7 days later). (B) Ipsilateral hind paw thickness measurements of both CPIP and sham groups. (C,D) 50% paw withdraw threshold (50% PWT, index of mechanical hyperalgesia) of ipsilateral (C) and contralateral (D) hind paws of rats. (E,F) Paw withdrawal latency (PWL, index of thermal hyperalgesia) of ipsilateral (E) and contralateral (F) hind paws of rats (n = 8 rats per group, **p &#60; 0.01). Results are expressed as mean &#177; SEM (two-way ANOVA followed by Sidak post-hoc test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60562/60562fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60562/60562fig2b.jpg" /> 
Figure 2: CPIP rats exhibited more nocifensive behaviors compared to sham rats in response to intraplantar capsaicin injection into ipsilateral hind paws. The cumulated time the rats spent licking, biting, or flinching the hind paws was calculated over 10 min after vehicle (0.1% DMSO in PBS, intraplantar) or capsaicin (10 nmol in 50 &#956;L volume, intraplantar) injection (n = 8 rats per group, **p &#60; 0.01). Results are expressed as mean &#177; SEM (one-way ANOVA followed by Sidak post-hoc test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60562/60562fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Chronic Post-Ischemia Pain Model for Complex Regional Pain Syndrome Type-I in Rats

Provided here is a protocol that details steps to establish an animal model of chronic post-ischemia pain (CPIP). This is a well-recognized model mimicking human complex regional pain syndrome type-I. Mechanical and thermal hypersensitivities are further evaluated, as well as capsaicin-induced nocifensive behaviors observed in the CPIP rat model.

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical ...

chronic-post-ischemia-pain-model-for-complex-regional-pain-syndrome

Nanyang Technological University

Research

JoVE Journal

Behavior

7.6K Views.  Key Laboratory of Acupuncture and Neurology of Zhejiang Province. Het mechanisme dat ten grondslag ligt aan CRPS I nog steeds onduidelijk. Het opzetten van een preklinisch diermodel nabootsen CRPS Ik zal cruciaal zijn voor het verkennen van de mechanismen die ten grondslag liggen CRPS I.The rat CPIP model vertoont veel CRPS Ik hou van simitance, waaronder hindlimb oedeem en hyperemie in het vroege stadium. Na model vestiging, gevolgd door aanhoudende thermische en mechanische overgevoeligheid.Begin met het verdoven van alle ratten met natriumfenobarb...

Video: Chronic Post-Ischemia Pain Model for Complex Regional Pain Syndrome Type-I in Rats

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

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

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

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

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

Medicine

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 ...

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 ...

Urokinase-type Plasminogen Activator-induced Mouse Back Pain Model

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid induction of a persisting back pain model in mice are provided here using an injection of urokinase-type plasminogen activator (urokinase), a serine protease present in humans and other animals. The methodology for induction of persisting lower back pain in mice involves a simple injection of urokinase along the ligamentous insertion region of the lumbar spine. The urokinase inflammatory agent activates plasminogen to plasmin. Typically, the model can be induced within 10 min and hypersensitivity persists for at least 8 weeks.
Hypersensitivity, gait disturbance, and other standard anxiety- and depression-like measures can be tested in the persisting model.&#160;Back pain is the most prevalent type of pain. To improve awareness of back pain, the International Association for the Study of Pain (IASP) named 2021 the &#34;Global Year about Back Pain&#34; and 2022 the &#34;Global Year for Translating Pain Knowledge to Practice.&#34; One limitation of the therapeutic advancement of pain therapeutics is the lack of suitable models for testing persistent and chronic pain. The&#160;features of this model&#160;are suitable for testing potential therapeutics aimed at the reduction of back pain and its ancillary characteristics, contributing to IASP&#39;s naming 2022 as the Global Year for Translating Pain Knowledge to Practice.

Methods for simple, rapid induction of a back pain model in mice are provided here using an intraligament injection of urinary plasminogen activator.

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid ...

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 ...

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

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

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

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

Neuroscience

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...

Creating a Preterm Rat Model for Early Postnatal Pain Research

A Preterm Rat Model for Pain Studies

This research delves into the consequences of consistent pinprick stimulation on preterm offspring to ascertain its long-term implications for pain sensitivity. The primary objective of this protocol was to investigate the impact of neonatal pinprick stimuli on the pain threshold in the later stages of life using a preterm rat model. By establishing this model, we aim to advance the research on understanding and managing early postnatal pain associated with prematurity. The findings of this study indicate that while the baseline thresholds to mechanical stimuli remained unaffected, there was a notable increase in mechanical hypersensitivity following complete Freund's adjuvant (CFA) injection in adult rats. Interestingly, compared with male rats, female rats demonstrated heightened inflammatory hypersensitivity. Notably, maternal behavior, the weight of the litters, and the growth trajectory of the offspring remained unchanged by the stimulation. The manifestation of altered nociceptive responses in adulthood after neonatal painful stimuli could be indicative of changes in sensory processing and the functioning of glucocorticoid receptors. However, further research is needed to understand the underlying mechanisms involved and to develop interventions for the consequences of prematurity and neonatal pain in adults.

Here, we present a concise protocol for creating a preterm rat model, facilitating research on early postnatal pain management. The method involves performing a cesarean section three days before the expected birth, extracting preterm rat pups via hysterectomy, and integrating them with the biological offspring of a surrogate dam.

This research delves into the consequences of consistent pinprick stimulation on preterm offspring to ascertain its long-term implications for pain ...

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.

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. ...

Establishing the Mouse Model for Trigeminal Neuropathic Pain

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

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

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

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

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

Chronic Post-Ischemia Pain Model for Complex Regional Pain Syndrome Type-I in Rats

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