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Home , Allodynia, Hot plate test

The Journal of , Vol 10, No 7 (July), 2009: pp 767-773 Available online at www.sciencedirect.com

Differentiating Thermal and Using Dynamic Hot and Cold Plate in Rodents

Ipek Yalcin,* Alexandre Charlet,* Marie-Jose´ Freund-Mercier,*y Michel Barrot,* and Pierrick Poisbeau*y * 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 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 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 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 Despite the presence of altered sensitivity to cold in pa- drugs.17 In the late 1980s, the development of the Har- tients with ,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 3C 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 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 . 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. Because data did for dimethylsulfoxyde (DMSO) and for capsaicin, n = 5 not fit the conditions of parametric testing (homogeneity for the respective control rats with 0.9% NaCl or 50% of variances and normality of samples), nonparametric tests Yalcin et al 769

Figure 1. Dynamic cold (DCP) and hot-plate (DHP) tests in mice. A, Numbers (nb) of rearing, hind paw licking, and jumps were mea- sured on the DCP from 20 to 1C. B, The same parameters were measured on the DHP from 30 to 43C. The tests were done on C57Bl/ 6J mice (n = 10/group). The cooling and heating rate for the dynamic cold and hot plate was 1C min-1. Statistical significance for jumps is indicated (*P < .05, 1P < .01, when compared with the response at starting temperatures). were used. Statistical analyses were performed with STATIS- to temperature were taken as dependent variables because TICA 7.1 (Stat-soft, Tulsa, OK). The independent variables the repeated measures were done on the same animal. The were analyzed by Kruskal-Wallis H tests followed by significance level was set at P <.05. Mann-Whitney U test when significant differences were detected. The application of capsaicin or carrageenan was taken as independent variable (capsaicin or carra- Results geenan vs control). The Wilcoxon test for 2-group compar- isonsormultiple-factoranalysisofvariance(ANOVA), Nociceptive Parameters in C57Bl/6J Mice Friedman & Concordance of Kendal, was used when the During DCP testing in mice (Fig 1A), we observed no variables were dependent. The behavioral changes due major change in paw licking or rearing behaviors, but

Figure 2. Peripheral sensitization in mice. A, Jumps after topical application of DMSO or vehicle (control: 0.9% NaCl) in the DCP test (DMSO, n = 6; control, n = 8). B, Jumps after topical application of DMSO or vehicle (control: 0.9% NaCl) in the DHP test (DMSO, n = 10; control, n = 5). C, Paw licking latency after topical application of DMSO or vehicle in the conventional hot plate test (setup at 52Corat 42C) or in the Hargreaves test (n = 5/group). D, Jumps after topical application of capsaicin or vehicle (50% ethanol in saline) in the DCP test (capsaicin, n = 7; control, n = 8). E, Jumps after topical application of capsaicin or vehicle (50% ethanol in saline) in the DHP test (capsaicin, n = 10; control, n = 5). F, Paw licking latency after topical application of capsaicin or vehicle in the conventional hot plate test (setup at 52Corat42C) or in the Hargreaves test (n = 5/group). The cooling and heating rate for the dynamic cold and hot plate was 1C min-1. Statistical significance against the respective control animals is indicated (*P< .05, 1P < .01). y-axes give the number (nb) of responses. 770 Dynamic Hot and Cold Plate Peripheral Sensitization in C57Bl/6J Mice DMSO application did not alter responses in the DCP (Fig 2A), in the DHP (Fig 2B), or in the conventional hot plate test at 52C and 42C, and in the Hargreaves test (Fig 2C). Topical application of capsaicin significantly augmented the number of jumps from 19C in the DCP test (P < .05; Fig 2D). It also decreased temperature threshold for jumps (from 39 to 35C) in the DHP (P < .05 for 35C and above; Fig 2E). The number of jumps was significantly increased for temperatures above 39C(P < .05). No jump was observed after capsaicin ap- plication when mice were tested for 15 minutes on the plate setup at 30C (data not shown). Capsaicin did not alter the conventional hot plate re- sponse at 52C, but it decreased licking latency at 42C (P < .01) or in the Hargreaves test (P < .05) (Fig 2F).

Nociceptive Parameters in Sprague- Dawley Rats During DCP tests in rats (Fig 3A), a significant increase in rearing behavior (H = 80.71, P < .001), hind paw licking (H = 69.69, P < .001), and withdrawal (H = 105.74, P < .001) was seen at 9C. In the DHP tests (Fig 3B) and for temper- ature above 39C, we found significantly elevated scores for rearing behavior (H = 51.84, P < .001), hind paw lick- ing (H = 58.71, P < .001), and withdrawal (H = 91.46, P < .001). The sum of paw lickings and withdrawals was used as index of nociceptive response (DHP: H = 101.39, P < .001; DCP: H = 112.85, P < .001) (Fig 3, C and D). The temperature inducing the first significant withdrawal re- action was of 9.31 6 0.15C with DCP (Fig 3E) and of 39.58 6 0.21C with DHP (Fig 3F). This value was not af- fected when the test was repeated (Fig 3, E and F) over a period of 10 days. No escape behavior was observed when rats were tested for 15 minutes at 30Cor20C (data not shown).

Figure 3. Dynamic cold (DCP) and hot plate (DHP) test in rats. A, Peripheral Sensitization in Sprague- Numbers (nb) of rearing, hind paw licking, and paw withdrawal Dawley Rats were measured on the DCP from 20 to 5C. B, The same param- eters were measured on the DHP from 30 to 45C. Tests were DMSO application did not modify nociceptive re- done in Sprague-Dawley rats (n = 8/group). C, Sum of nocicep- sponses in the DCP (Fig 4A), in the DHP (Fig 4B), in the tive responses (paw licking and withdrawal) for the DCP test. conventional HP at 52C and 42C, and in the Hargreaves D, Sum of nociceptive responses (paw licking and withdrawal) for the DHP test. E, Time course showing the stability of the tem- test (Fig 4C) in rats. Topical application of capsaicin sig- perature inducing the first paw withdrawal reaction during the nificantly augmented the number of jumps between DCP test. F, Time course showing the stability of the temperature 11C and 9C(P < .05; Fig 4D) in the DCP test. It also de- inducing the first paw withdrawal reaction during the DHP test. The cooling and heating rate for the DCP and DHP was 1C.min-1. creased temperature threshold for jumps (from 40 Cto Statistical significance is indicated (*P < .05, 1 P < .01 when com- 34 C) in the DHP (P < .05 for 34 C and above; Fig 4E). Cap- pared with the response at starting temperatures). y-axes give saicin did not alter hot plate response at 52C or in the the number (nb) of responses. Hargreaves test but it decreased licking latency in the conventional hot plate at 42C; P < .05) (Fig 4F). significant escape behavior (jumps) appeared at 2C (H = 48.45, P < .001). During DHP testing (Fig 1B), the mice displayed jumps (temperature effect; H = 103.36, Carrageenan-Induced Inflammation P < .001) but no significant licking behavior. First jumps in Sprague-Dawley Rats were observed at 39C (6 of 10 mice; P < .05) and were Carrageenan-injected rats displayed significant noci- seen in all mice (10 of 10) from 40 to 43C(P < .05 for ceptive response in DCP (Fig 5A) at 13C instead of 9C all temperatures). The mice displayed constant rearing for control rats (P < .05) and in DHP (Fig 5B) at 37C behavior from 30 to 39C, and it decreased from 39 to instead of 39C(P < .05). In addition to cold and heat 43C(H = 24.77, P < .05). allodynia, thermal hyperalgesia in carrageenan-injected Yalcin et al 771

Figure 4. Peripheral sensitization in rats. A, Sum of nociceptive responses (paw licking and withdrawal) after topical application of DMSO or vehicle (control: 0.9% NaCl) in the DCP test (n = 4/group). B, Sum of nociceptive responses (paw licking and withdrawal) after topical application of DMSO or vehicle (control: 0.9% NaCl) in the DHP test (n = 4/group). C, Paw licking or withdrawal latency after topical application of DMSO or vehicle in the conventional hot plate test (setup at 52C: n = 6/group; at 42C: n = 4/group) or in the Hargreaves test (DMSO, n = 8; control, n = 6). D, Sum of nociceptive responses (paw licking and withdrawal) after topical application of capsaicin or vehicle (50% ethanol in saline) in the DCP test (n = 4/group). E, Sum of nociceptive responses (paw licking and withdrawal) after topical application of capsaicin or vehicle (50% ethanol in saline) in the DHP test (n = 4/group). F, Paw licking latency after topical application of capsaicin or vehicle in the conventional hot plate test (setup at 52C: n = 6/group; at 42C: n = 4/group) or in the Har- greaves test (n = 6/group). The cooling and heating rate for the DCP and DHP was 1C.min-1. Statistical significance against the respec- tive controls is indicated (*P < .05). y-axes give the number (nb) of responses. rats was indicated by increased nociceptive responses at Cumulating both parameters in rats gave a clear-cut de- 9Cor45C(P < .05). tection of the nociceptive response and threshold. When we tested mice or rats during 15 minutes on the plate setup at 30Cor20C, we did not observe any nociceptive Discussion In this study, we characterized the use of the DCP/DHP test to measure nociceptive responses to thermal stimuli in C57Bl/6J mice and in Sprague-Dawley rats. In each spe- cies, we identified the behavioral responses that are the most relevant for nociceptive measure. This method was further validated by using 2 models of pain sensitization including capsaicin-induced sensitization of peripheral in mice and in rats and carrageenan-induced peripheral inflammation in rats. Our results showed that the DCP/DHP test can reveal both thermal cold/hot allo- dynia and hyperalgesia within a single procedure in awake and freely moving animals. C57Bl/6J mice and Sprague-Dawley rats exhibit differ- ent responses in the DCP/DHP test. In both species, rear- ing behavior that reflects spontaneous activity and Figure 5. Carrageenan-induced paw inflammation in rats. A, exploration did not appear suitable to measure nocicep- Total number of nociceptive responses (sum of hind paw licking tive response. Jumps, which can be interpreted as an es- and withdrawal) in DCP, 7 hours after the intraplantar injection cape behavior, were only present in mice when hot- or of saline (control) or carrageenan. B, Total number of nocicep- tive responses in DHP, 7 hours after the intraplantar injection cold-nociceptive temperatures were reached and the of saline (control) or carrageenan. The saline solution or the number of jumps increased with the intensity of nocicep- l-carrageenan (3% in 0.9% NaCl, 150 mL) was injected in the tive stimulus. It is thus the most relevant parameter to right hind paw of Sprague-Dawley rats (n = 8/group). The cool- score during DCP/DHP tests in the C57Bl/6J mice. Simi- ing and heating rate for the dynamic cold and hot plate was 1C.min-1. Statistical significance against the respective control larly, hind paw lickings and withdrawals appeared as animals is indicated (*P < .05, 1 P < .01, # P < .001). y-axes give the most relevant parameters in Sprague-Dawley rats. the number (nb) of responses. 772 Dynamic Hot and Cold Plate behavior. Moreover, the procedure offers reproducible DHP test was to provide a procedure to study this sensiti- measures as shown by repeated testing on a 9-day zation of thermonociceptors in freely moving animals. period. Interestingly, we observed that capsaicin-treated mice The nociceptive threshold to heat in the DCP/DHP and rats showed nociceptive behavior at innocuous test was between 39C and 40C. This is in agreement temperatures, which revealed the presence of a thermal with thermonociceptor thresholds as measured by in allodynia. vivo electrophysiology.31 However, there is no clear On the other hand, the conventional HP setup at 52C agreement in the literature as to when a cold stimulus did not allow the detection of an effect of either capsa- begins to be nociceptive.13 The results that we ob- icin or DMSO application in mice and rats. The Har- tained with the cold ramp revealed a behavioral re- greaves test and the conventional hot plate at 42C sponse starting at 4C for mice and at 9C for rats. with a long cutoff allowed detection of the capsaicin ef- These temperatures could be considered as nociceptive fect in mice but did not discriminate between thermal al- because the animals did not display escape behavior lodynia and thermal hyperalgesia. On the contrary, in (mice) or paw licking/withdrawal (rats) before reaching rats we observed the effects of capsaicin with the con- them. ventional hot plate at 42C but not with the Hargreaves Thermal nociception is thought to be mediated by test. The DCP/DHP is thus of interest to provide both C- and Ad-fibers,14 which have distinct electrophysiolog- within the same testing procedure. ical and pharmacological properties.16 Based on in vivo Similarly, we investigated whether this method is able electrophysiological approaches, it has been proposed to measure the changes in hypersensitivity evoked by that slow ramps (speed) of temperature heating prefer- a carrageenan-induced inflammatory pain model.23 Be- entially activate C-fibers, whereas fast ramps (speed) of cause this model is first and well characterized in rats, temperature heating preferentially activate Ad-fibers.29 we decided to evaluate the effects of carrageenan- This idea is also supported by experiments using contact induced inflammatory pain in DCP/DHP testing in rats. heating21 or infrared diode laser systems25 to distinguish We observed that carrageenan-injected rats display a no- thermonociceptors. Thus, the 1C.min-1 heating rate that ciceptive reaction at innocuous temperatures (ie, ther- we used in this study can be considered as very slow mal allodynia) as well as an exaggerated number of when compared with other studies,18,29,32 and we sug- responses at noxious temperatures (ie, thermal hyperal- gest that this slow rate heating preferentially activates gesia) in comparison to saline-injected rats. These data C-fibers. Unfortunately, we have no direct evidence revealed that inflammatory pain is inducing a strong such as peripheral nerve recordings. Moreover, for doses thermal allodynia and confirmed in a widely used pain similar to the ones we used, capsaicin and DMSO were model the interest of the DCP/DHP procedure to observe proposed to act respectively on C- or Ad-thermonocicep- thermal allodynia as well as hyperalgesia in the same tors.1,7,15,26,29 Accordingly, we found significant heat hy- procedure. persensitivity with topical application of capsaicin in We observed that capsaicin-treated mice and rats or mice and rats with DHP testing. However, we observed carrageenan-injected rats display hyperalgesia and/or al- no effect of topical DMSO application either in DHP or lodynia as compared with their control animals. Al- DCP testing in both mice and rats. Our data indicating though the DCP/DHP requires longer testing time than that C- or Ad-thermonociceptors could be differentially other thermal tests, it allows measuring the nociceptive activated depending of the rate of skin heating are in temperature thresholds and behavioral score for non- good agreement with the results of the groups of noxious and noxious stimuli in a single experiment with Lumb21 and Yeomans,25 and a main interest of the DCP/ a freely moving animal.

References and Rehabilitation Research Injury Measures meeting. J Spinal Cord Med 30:421-440, 2007

1. Baumann TK, Simone DA, Shain CN, LaMotte RH: Neuro- 5. Charlet A, Lasbennes F, Darbon P, Poisbeau P: Fast non-ge- genic hyperalgesia: The search for the primary cutaneous af- nomic effects of progesterone-derived neurosteroids on no- ferent fibers that contribute to capsaicin-induced pain and ciceptive thresholds and pain symptoms. Pain July 7, 2008 hyperalgesia. J Neurophysiol 66:212-227, 1991 [Epub ahead of print].

2. Benbouzid M, Pallage V, Rajalu M, Waltisperger E, 6. Choi Y, Yoon YW, Na HS, Kim SH, Chung JM: Behavioral Doridot S, Poisbeau P, Freund-Mercier MJ, Barrot M: Sciatic signs of ongoing pain and cold allodynia in a rat model of nerve cuffing in mice: A model of sustained neuropathic . Pain 59:369-376, 1994 pain. Eur J Pain 12:591-599, 2008 7. Culp WJ, Ochoa J, Cline M, Dotson R: Heat and mechanical 3. Bennett GJ, Xie YK: A peripheral mononeuropathy in rat hyperalgesia induced by capsaicin: cross modality threshold that produces disorders of pain sensation like those seen in modulation in human C nociceptors. Brain 112:1317-1331, 1989 man. Pain 33:87-107, 1988 8. D’Amour FE, Smith DL: A method for determining loss of 4. Bryce TN, Budh CN, Cardenas DD, Dijkers M, Felix ER, pain sensation. J Pharmacol Exp Ther 72:74-79, 1941 Finnerup NB, Kennedy P, Lundeberg T, Richards JS, Rintala DH, Siddall P, Widerstrom-Noga E: Pain after spinal 9. Forssell H, Tenovuo O, Silvoniemi P, Jaaskelainen SK: Dif- cord injury: An evidence-based review for clinical practice ferences and similarities between and tri- and research: Report of the National Institute on Disability geminal neuropathic pain. 69:1451-1459, 2007 Yalcin et al 773 10. Hargreaves K, Dubner R, Brown F, Flores C, Joris J: A new that distinguishes agonists from mixed agonist-an- and sensitive method for measuring thermal nociception in tagonists. Eur J Pharmacol 119:23-29, 1985 cutaneous hyperalgesia. Pain 32:77-88, 1988 23. Poisbeau P, Patte-Mensah C, Keller AF, Barrot M, 11. Horowitz SH: The diagnostic workup of patients with Breton JD, Luis-Delgado OE, Freund-Mercier MJ, Mensah- neuropathic pain. Med Clin North Am 91:21-30, 2007 Nyagan AG, Schlichter R: Inflammatory pain upregulates spi- nal inhibition via endogenous neurosteroid production. 12. Jaaskelainen SK, Teerijoki-Oksa T, Forssell H: Neurophys- J Neurosci 25:11768-11776, 2005 iologic and quantitative sensory testing in the diagnosis of trigeminal neuropathy and neuropathic pain. Pain 117: 24. Simone DA, Kajander KC: Excitation of rat cutaneous 349-357, 2005 nociceptors by noxious cold. Neurosci Lett 213:53-56, 1996 13. Jasmin L, Kohan L, Franssen M, Janni G, Goff JR: The cold plate as a test of nociceptive behaviors: Description and ap- 25. Tzabazis A, Klyukinov M, Manering N, Nemenov MI, plication to the study of chronic neuropathic and inflamma- Shafer SL, Yeomans DC: Differential activation of tri- tory pain models. Pain 75:367-382, 1998 geminal C or Adelta nociceptors by infrared diode laser in rats: Behavioral evidence. Brain Res 1037:148-156, 14. Julius D, Basbaum AI: Molecular mechanisms of nocicep- 2005 tion. Nature 413:203-210, 2001 26. Wilson SP, Yeomans DC, Bender MA, Lu Y, Goins WF, 15. Kenins P: Responses of single nerve fibres to capsaicin Glorioso JC: Antihyperalgesic effects of infection with a pre- applied to the skin. Neurosci Lett 29:83-88, 1982 proenkephalin-encoding herpes virus. Proc Natl Acad Sci U S A 96:3211-3216, 1999 16. Lawson SN: Phenotype and function of somatic primary afferent nociceptive neurones with C-, Adelta- or Aalpha/ 27. Woolf CJ, Mannion RJ: Neuropathic pain: Aetiology, beta-fibres. Exp Physiol 87:239-244, 2002 symptoms, mechanisms, and management. Lancet 353: 1959-1964, 1999 17. Le Bars D, Gozariu M, Cadden SW: Animal models of no- ciception. Pharmacol Rev 53:597-652, 2001 28. Woolfe G, MacDonald AL: The evaluation of the analge- sic action of pethidine hydrochloride (Demerol). J Pharmacol 18. Leith JL, Wilson AW, Donaldson LF, Lumb BM: Cyclooxy- Exp Ther 80:300-307, 1944 genase-1-derived in the periaqueductal gray differentially control C- versus A-fiber-evoked spinal noci- 29. Yeomans DC, Pirec V, Proudfit HK: Nociceptive responses ception. J Neurosci 27:11296-11305, 2007 to high and low rates of noxious cutaneous heating are mediated by different nociceptors in the rat: Behavioral 19. Luis-Delgado OE, Barrot M, Rodeau JL, Schott G, evidence. Pain 68:133-140, 1996 Benbouzid M, Poisbeau P, Freund-Mercier MJ, Lasbennes F: Calibrated forceps: A sensitive and reliable tool for pain 30. Yeomans DC, Proudfit HK: Characterization of the foot and analgesia studies. J Pain 7:32-39, 2006 withdrawal response to noxious radiant heat in the rat. Pain 59:85-94, 1994 20. Luis-Delgado OE, Barrot M, Rodeau JL, Ulery PG, Freund- Mercier MJ, Lasbennes F: The transcription factor DeltaFosB 31. Yeomans DC, Proudfit HK: Nociceptive responses to high is recruited by inflammatory pain. J Neurochem 98: and low rates of noxious cutaneous heating are mediated 1423-1431, 2006 by different nociceptors in the rat: Electrophysiological 21. McMullan S, Simpson DA, Lumb BM: A reliable method evidence. Pain 68:141-150, 1996 for the preferential activation of C- or A-fibre heat nocicep- tors. J Neurosci Methods 138:133-139, 2004 32. Zachariou V, Goldstein BD, Yeomans DC: Low but not high rate noxious radiant skin heating evokes a capsaicin- 22. Pizziketti RJ, Pressman NS, Geller EB, Cowan A, sensitive increase in spinal cord dorsal horn release of sub- Adler MW: Rat cold water tail-flick: A novel analgesic test stance P. Brain Res 752:143-150, 1997