The Journal of Pain, Vol 10, No 7 (July), 2009: pp 767-773 Available online at www.sciencedirect.com Differentiating Thermal Allodynia and Hyperalgesia Using Dynamic Hot and Cold Plate in Rodents Ipek Yalcin,* Alexandre Charlet,* Marie-Jose´ Freund-Mercier,*y Michel Barrot,* and Pierrick Poisbeau*y *Nociception and Pain Department, Institut des Neurosciences Cellulaires et Inte´ gratives, Centre National de la Recherche Scientifique. y Universite´ de Strasbourg, Strasbourg, France. Abstract: In animal studies, thermal sensitivity is mostly evaluated on the basis of nociceptive re- action latencies in response to a given thermal aversive stimulus. However, these techniques may be inappropriate to differentiate allodynia from hyperalgesia or to provide information differentiat- ing the activation of nociceptor subtypes. The recent development of dynamic hot and cold plates, allowing computer-controlled ramps of temperature, may be useful for such measures. In this study, we characterized their interest for studying thermal nociception in freely moving mice and rats. We showed that escape behavior (jumps) was the most appropriate parameter in C57Bl/6J mice, whereas nociceptive response was estimated by using the sum of paw lickings and withdrawals in Sprague- Dawley rats. We then demonstrated that this procedure allows the detection of both thermal allodynia and hyperalgesia after peripheral pain sensitization with capsaicin in mice and in rats. In a condition of carrageenan-induced paw inflammation, we observed the previously described thermal hyperalgesia, but we also revealed that rats exhibit a clear thermal allodynia to a cold or a hot stimulus. These results demonstrate the interest of the dynamic hot and cold plate to study thermal nociception, and more particularly to study both thermal allodynia and hyperalgesia within a single paradigm in awake and freely moving rodents. Perspective: Despite its clinical relevance, thermal allodynia is rarely studied by researchers work- ing on animal models. As shown after stimulation of capsaicin-sensitive fibers or during inflamma- tory pain, the dynamic hot and cold plate validated in the present study provides a useful tool to distinguish between thermal allodynia and thermal hyperalgesia in rodents. ª 2009 by the American Pain Society Key words: Pain, dynamic hot/cold plate, hyperalgesia, allodynia, capsaicin, carrageenan. hermonociception in rodents is usually evaluated greave method improved research on local analgesics by measuring escape latency in response to a noci- and pain models by individualizing the response of ceptive stimulus at fixed temperature. Tail-flick8 both hind paws.10 Other methods, such as contact heat- T 28 21,30 and the hot plate are widely used to assess responses ing systems or infrared diode laser-based stimula- to heat stimuli.17 These tests have been used by re- tors25 were also used to allow the selective stimulation searchers for over half a century; they are well standard- of A- or C-thermonociceptors in anesthetized rats. ized and considered as references for testing analgesic Despite the presence of altered sensitivity to cold in pa- drugs.17 In the late 1980s, the development of the Har- tients with nerve injury,11,12 cold stimuli are more rarely used in animal models.13 Some approaches were inspired Received October 17, 2008; Revised January 7, 2009; Accepted January 13, by those using contact heat. The paw or tail withdrawal 2009. 22 Supported by Centre National de la Recherche Scientifique, Universite´ de was thus measured after immersion in cold water or af- Strasbourg, Re´ gion Alsace, Institut Universitaire de France, and Institut ter placing the animal on a cold plate.3 Direct applica- UPSA de la douleur. 6 Drs Yalcin and Charlet equally contributed to this work. tions of acetone, allowing the measure of cold 24 Address reprint requests to Prof Pierrick Poisbeau; De´ partement Nocicep- allodynia, or contact with a Peltier thermode have tion et Douleur, UPR 3212 CNRS, 21 rue Rene´ Descartes, 67084 Strasbourg also been used to differentiate both hind paw responses. cedex, France. E-mail: [email protected] 1526-5900/$36.00 Although thermal tests have been used for over half ª 2009 by the American Pain Society a century in rodents, the measured parameters mainly re- doi:10.1016/j.jpain.2009.01.325 main the responses (latency or intensity) to a stimulus 767 768 Dynamic Hot and Cold Plate known to be nociceptive, that is, hyperalgesia. Despite its ethanol in saline) or 45C for rats (n = 8 for the character- relevance to clinical conditions,4,9,27 thermal allodynia is ization of DHP, n = 4 for DMSO, capsaicin and control more rarely considered in animal studies. rats: 0.9% NaCl or 50% ethanol in saline), with 1C.min-1 The objective of our study was to establish a reliable speed (r2 = 1). During each degree interval, we scored method to score mouse and rat nociceptive behaviors hind paw lickings, escape behavior (jumps), and rearing using computer-controlled ramps of temperature and for mice; hind paw lickings, paw withdrawals, and rear- to validate this method in animals with pain symptoms. ing for rats. Using a dynamic hot and cold plate analgesia meter, we identified the most relevant behavioral parameters Dynamic Cold Plate Test in C57Bl/6J mice and in Sprague-Dawley rats and we eval- Animals were placed on the dynamic cold plate (DCP) uated the responses to the sensitization of capsaicin-sen- at 20 6 0.1C, and the plate temperature was decreased sitive C-fibers or to carrageenan-induced hind paw down to 1C for mice (n = 10 for the characterization of inflammation. Our results reveal that the dynamic hot DHP, n = 6 for DMSO, n = 7 for capsaicin, n = 8 for the re- and cold plate allows the measure of both thermal allo- spective control rats with 0.9% NaCl and 50% ethanol in dynia and thermal hyperalgesia within the same experi- saline) or 5C for rats (n = 8 for the characterization of mental procedure. DHP, n = 4 for DMSO, for capsaicin and control rats: 0.9% NaCl or 50 % ethanol in saline), with a 1C.min-1 2 Materials and Methods speed (r = 0.99). Scored behaviors were determined as described with DHP. Animals Hargreaves Test Male C57Bl/6J mice (weight, 25 to 30 g; Charles River, L’Arbresle, France) and Sprague-Dawley rats (weight, Mice (n = 5/group) and rats (n = 6 for DMSO, capsaicin 250 to 350 g; Janvier, Le Genest St Isle, France) were or control rats with 50% ethanol in saline; n = 8 for con- used for the experiments. Mice and rats were housed 4 trol with 0.9% NaCl) were placed in clear Plexiglas boxes per cage in independent animal rooms. They were main- (mice: 7 cm  9cm 7 cm; rats: 22 cm  19 cm  14 cm) on tained under a 12-hours light/dark cycle in standard a glass surface, and testing began after exploration and 2,5 animal housing conditions, with food and water ad libi- grooming behaviors ended (15 minutes). The infrared tum. All animals were habituated to the apparatus beam of the radiant heat source (7370 Plantar Test; Ugo before the onset of the experiments. All procedures Basile, Comerio, Italy) was applied to the plantar surface were performed in accordance with European directives of each hind paw. The experimental cutoff to prevent (86/609/EEC) and were approved by the regional ethics damage to the skin was set at 15 seconds. Three measures committee and French ministry of agriculture (license of the paw withdrawal latency were taken and averaged No. 67-116, to P.P.). for each hind paw. Tests Peripheral Sensitization For the hot or cold plate tests, we used a computer- We studied the effect of local application of capsaicin controlled hot and cold plate analgesia meter (Bioseb, or DMSO in mice and rats. Capsaicin (10 mM, dissolved in Chaville, France). With no animal on it, the plate can be 50% ethanol with 0.9% NaCl) or DMSO (100%) was ap- heated up to 65C or cooled down to À3C 6 0.1Cin plied topically on the plantar surface of the mice or rat 25 an ambient room temperature (20 to 25C). The animal hind paws with cotton-tipped applicators. They were was placed in a Plexiglas cylinder (mice: 10-cm diameter, tested 15 minutes after the application of the drugs. As 15-cm height; rats: 15.5-cm diameter, 25-cm height) with a control, we also tested mice or rats at fixed tempera- a drilled cover. ture (30C) for 15 minutes after a topical application of saline, capsaicin, or DMSO. Conventional Hot Plate Test In rats, we determined the consequence of carrageenan- Mice or rats were placed on the hot plate with the induced inflammation using the dynamic hot and cold temperature adjusted to 52 6 0.1C (n = 5/group for plate test. The l-carrageenan (3% in 0.9% NaCl, 150 mL) 19,20,23 mice; n = 6/group for rats) or to 42 6 0.1C (n = 5/group was injected in the right hind paw of the rats. We for mice; n = 4/group for rats). The latency to the first only evaluated the effects of carrageenan-induced inflam- hind paw licking or withdrawal was taken as an index mation in rats because this model was well characterized in of nociceptive threshold. The cutoff time was set at 15 rats, and few studies exist in mice. seconds and 60 seconds, respectively, to avoid damage Injections and applications (all drugs from Sigma- to the paw. Aldrich, St. Quentin Fallavier, France) were done under light 3% halothane anesthesia. Control groups received Dynamic Hot Plate Test the vehicle of the administered drug. Animals were placed on the dynamic hot plate (DHP) at 30 6 0.1C, and the plate temperature increased up to Statistics 43C for mice (n = 10 for the characterization of DHP, The data are expressed as mean 6 SEM.