Differentiating Thermal Allodynia and Hyperalgesia Using Dynamic Hot and Cold Plate in Rodents

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1 The Journal of Pain, Vol 10, No 7 (July), 2009: pp Available online at Differentiating Thermal Allodynia and Hyperalgesia Using Dynamic Hot and Cold Plate in Rodents Ipek Yalcin,* Alexandre Charlet,* Marie-José Freund-Mercier,* y Michel Barrot,* and Pierrick Poisbeau* y * Nociception and Pain Department, Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique. y Université de Strasbourg, Strasbourg, France. Abstract: In animal studies, thermal sensitivity is mostly evaluated on the basis of nociceptive reaction latencies in response to a given thermal aversive stimulus. However, these techniques may be inappropriate to differentiate allodynia from hyperalgesia or to provide information differentiating 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 working on animal models. As shown after stimulation of capsaicin-sensitive fibers or during inflammatory 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. Received October 17, 2008; Revised January 7, 2009; Accepted January 13, Supported by Centre National de la Recherche Scientifique, Université de Strasbourg, Région Alsace, Institut Universitaire de France, and Institut UPSA de la douleur. Drs Yalcin and Charlet equally contributed to this work. Address reprint requests to Prof Pierrick Poisbeau; Département Nociception et Douleur, UPR 3212 CNRS, 21 rue René Descartes, Strasbourg cedex, France. poisbeau@neurochem.u-strasbg.fr /$36.00 ª 2009 by the American Pain Society doi: /j.jpain Thermonociception in rodents is usually evaluated by measuring escape latency in response to a nociceptive stimulus at fixed temperature. Tail-flick 8 and the hot plate 28 are widely used to assess responses to heat stimuli. 17 These tests have been used by researchers for over half a century; they are well standardized and considered as references for testing analgesic drugs. 17 In the late 1980s, the development of the Hargreave method improved research on local analgesics and pain models by individualizing the response of both hind paws. 10 Other methods, such as contact heating systems 21,30 or infrared diode laser-based stimulators 25 were also used to allow the selective stimulation of A- or C-thermonociceptors in anesthetized rats. Despite the presence of altered sensitivity to cold in patients with nerve injury, 11,12 cold stimuli are more rarely used in animal models. 13 Some approaches were inspired by those using contact heat. The paw or tail withdrawal was thus measured after immersion in cold water 22 or after placing the animal on a cold plate. 3 Direct applications of acetone, 6 allowing the measure of cold allodynia, or contact with a Peltier thermode 24 have also been used to differentiate both hind paw responses. Although thermal tests have been used for over half a century in rodents, the measured parameters mainly remain the responses (latency or intensity) to a stimulus 767

2 768 Dynamic Hot and Cold Plate known to be nociceptive, that is, hyperalgesia. Despite its relevance to clinical conditions, 4,9,27 thermal allodynia is more rarely considered in animal studies. The objective of our study was to establish a reliable method to score mouse and rat nociceptive behaviors using computer-controlled ramps of temperature and to validate this method in animals with pain symptoms. Using a dynamic hot and cold plate analgesia meter, we identified the most relevant behavioral parameters in C57Bl/6J mice and in Sprague-Dawley rats and we evaluated the responses to the sensitization of capsaicin-sensitive C-fibers or to carrageenan-induced hind paw inflammation. Our results reveal that the dynamic hot and cold plate allows the measure of both thermal allodynia and thermal hyperalgesia within the same experimental procedure. Materials and Methods Animals Male C57Bl/6J mice (weight, 25 to 30 g; Charles River, L Arbresle, France) and Sprague-Dawley rats (weight, 250 to 350 g; Janvier, Le Genest St Isle, France) were used for the experiments. Mice and rats were housed 4 per cage in independent animal rooms. They were maintained under a 12-hours light/dark cycle in standard animal housing conditions, with food and water ad libitum. All animals were habituated to the apparatus before the onset of the experiments. All procedures were performed in accordance with European directives (86/609/EEC) and were approved by the regional ethics committee and French ministry of agriculture (license No , to P.P.). ethanol in saline) or 45 C for rats (n = 8 for the characterization of DHP, n = 4 for DMSO, capsaicin and control rats: 0.9% NaCl or 50% ethanol in saline), with 1 C.min -1 speed (r 2 = 1). During each degree interval, we scored hind paw lickings, escape behavior (jumps), and rearing for mice; hind paw lickings, paw withdrawals, and rearing for rats. Dynamic Cold Plate Test Animals were placed on the dynamic cold plate (DCP) at C, and the plate temperature was decreased down to 1 C for mice (n = 10 for the characterization of DHP, n = 6 for DMSO, n = 7 for capsaicin, n = 8 for the respective control rats with 0.9% NaCl and 50% ethanol in saline) or 5 C for rats (n = 8 for the characterization of DHP, n = 4 for DMSO, for capsaicin and control rats: 0.9% NaCl or 50 % ethanol in saline), with a 1 C.min -1 speed (r 2 = 0.99). Scored behaviors were determined as described with DHP. Hargreaves Test Mice (n = 5/group) and rats (n = 6 for DMSO, capsaicin or control rats with 50% ethanol in saline; n = 8 for control with 0.9% NaCl) were placed in clear Plexiglas boxes (mice: 7 cm 9cm7cm; rats: 22 cm 19 cm 14 cm) on a glass surface, and testing began after exploration and grooming behaviors ended (15 minutes). 2,5 The infrared beam of the radiant heat source (7370 Plantar Test; Ugo Basile, Comerio, Italy) was applied to the plantar surface of each hind paw. The experimental cutoff to prevent damage to the skin was set at 15 seconds. Three measures of the paw withdrawal latency were taken and averaged for each hind paw. Tests For the hot or cold plate tests, we used a computercontrolled hot and cold plate analgesia meter (Bioseb, Chaville, France). With no animal on it, the plate can be heated up to 65 C or cooled down to 3 C Cin an ambient room temperature (20 to 25 C). The animal was placed in a Plexiglas cylinder (mice: 10-cm diameter, 15-cm height; rats: 15.5-cm diameter, 25-cm height) with a drilled cover. Conventional Hot Plate Test Mice or rats were placed on the hot plate with the temperature adjusted to C (n = 5/group for mice; n = 6/group for rats) or to C (n = 5/group for mice; n = 4/group for rats). The latency to the first hind paw licking or withdrawal was taken as an index of nociceptive threshold. The cutoff time was set at 15 seconds and 60 seconds, respectively, to avoid damage to the paw. Dynamic Hot Plate Test Animals were placed on the dynamic hot plate (DHP) at C, and the plate temperature increased up to 43 C for mice (n = 10 for the characterization of DHP, for dimethylsulfoxyde (DMSO) and for capsaicin, n = 5 for the respective control rats with 0.9% NaCl or 50% Peripheral Sensitization We studied the effect of local application of capsaicin or DMSO in mice and rats. Capsaicin (10 mm, dissolved in 50% ethanol with 0.9% NaCl) or DMSO (100%) was applied topically on the plantar surface of the mice or rat hind paws with cotton-tipped applicators. 25 They were tested 15 minutes after the application of the drugs. As a control, we also tested mice or rats at fixed temperature (30 C) for 15 minutes after a topical application of saline, capsaicin, or DMSO. In rats, we determined the consequence of carrageenaninduced inflammation using the dynamic hot and cold plate test. The l-carrageenan (3% in 0.9% NaCl, 150 ml) was injected in the right hind paw of the rats. 19,20,23 We only evaluated the effects of carrageenan-induced inflammation in rats because this model was well characterized in rats, and few studies exist in mice. Injections and applications (all drugs from Sigma- Aldrich, St. Quentin Fallavier, France) were done under light 3% halothane anesthesia. Control groups received the vehicle of the administered drug. Statistics The data are expressed as mean 6 SEM. Because data did not fit the conditions of parametric testing (homogeneity of variances and normality of samples), nonparametric tests

3 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 measured on the DCP from 20 to 1 C. B, The same parameters were measured on the DHP from 30 to 43 C. The tests were done on C57Bl/ 6J mice (n = 10/group). The cooling and heating rate for the dynamic cold and hot plate was 1 C 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- TICA 7.1 (Stat-soft, Tulsa, OK). The independent variables were analyzed by Kruskal-Wallis H tests followed by Mann-Whitney U test when significant differences were detected. The application of capsaicin or carrageenan was taken as independent variable (capsaicin or carrageenan vs control). The Wilcoxon test for 2-group comparisons or multiple-factor analysis of variance (ANOVA), Friedman & Concordance of Kendal, was used when the variables were dependent. The behavioral changes due to temperature were taken as dependent variables because the repeated measures were done on the same animal. The significance level was set at P <.05. Results Nociceptive Parameters in C57Bl/6J Mice During DCP testing in mice (Fig 1A), we observed no 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 52 Corat 42 C) 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 52 Corat42 C) or in the Hargreaves test (n = 5/group). The cooling and heating rate for the dynamic cold and hot plate was 1 C min -1. Statistical significance against the respective control animals is indicated (*P<.05, 1P <.01). y-axes give the number (nb) of responses.

4 770 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 52 C and 42 C, and in the Hargreaves test (Fig 2C). Topical application of capsaicin significantly augmented the number of jumps from 19 C in the DCP test (P <.05; Fig 2D). It also decreased temperature threshold for jumps (from 39 to 35 C) in the DHP (P <.05 for 35 C and above; Fig 2E). The number of jumps was significantly increased for temperatures above 39 C(P <.05). No jump was observed after capsaicin application when mice were tested for 15 minutes on the plate setup at 30 C (data not shown). Capsaicin did not alter the conventional hot plate response at 52 C, but it decreased licking latency at 42 C (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 = , P <.001) was seen at 9 C. In the DHP tests (Fig 3B) and for temperature above 39 C, we found significantly elevated scores for rearing behavior (H = 51.84, P <.001), hind paw licking (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 = , P <.001; DCP: H = , P <.001) (Fig 3, C and D). The temperature inducing the first significant withdrawal reaction was of C with DCP (Fig 3E) and of C with DHP (Fig 3F). This value was not affected 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 30 Cor20 C (data not shown). Figure 3. Dynamic cold (DCP) and hot plate (DHP) test in rats. A, Numbers (nb) of rearing, hind paw licking, and paw withdrawal were measured on the DCP from 20 to 5 C. B, The same parameters were measured on the DHP from 30 to 45 C. Tests were done in Sprague-Dawley rats (n = 8/group). C, Sum of nociceptive responses (paw licking and withdrawal) for the DCP test. D, Sum of nociceptive responses (paw licking and withdrawal) for the DHP test. E, Time course showing the stability of the temperature inducing the first paw withdrawal reaction during the DCP test. F, Time course showing the stability of the temperature inducing the first paw withdrawal reaction during the DHP test. The cooling and heating rate for the DCP and DHP was 1 C.min -1. Statistical significance is indicated (*P <.05, 1 P <.01 when compared with the response at starting temperatures). y-axes give the number (nb) of responses. significant escape behavior (jumps) appeared at 2 C (H = 48.45, P <.001). During DHP testing (Fig 1B), the mice displayed jumps (temperature effect; H = , P <.001) but no significant licking behavior. First jumps were observed at 39 C (6 of 10 mice; P <.05) and were seen in all mice (10 of 10) from 40 to 43 C(P <.05 for all temperatures). The mice displayed constant rearing behavior from 30 to 39 C, and it decreased from 39 to 43 C(H = 24.77, P <.05). Peripheral Sensitization in Sprague- Dawley Rats DMSO application did not modify nociceptive responses in the DCP (Fig 4A), in the DHP (Fig 4B), in the conventional HP at 52 C and 42 C, and in the Hargreaves test (Fig 4C) in rats. Topical application of capsaicin significantly augmented the number of jumps between 11 C and 9 C(P <.05; Fig 4D) in the DCP test. It also decreased temperature threshold for jumps (from 40 Cto 34 C) in the DHP (P <.05 for 34 C and above; Fig 4E). Capsaicin did not alter hot plate response at 52 C or in the Hargreaves test but it decreased licking latency in the conventional hot plate at 42 C; P <.05) (Fig 4F). Carrageenan-Induced Inflammation in Sprague-Dawley Rats Carrageenan-injected rats displayed significant nociceptive response in DCP (Fig 5A) at 13 C instead of 9 C for control rats (P <.05) and in DHP (Fig 5B) at 37 C instead of 39 C(P <.05). In addition to cold and heat allodynia, thermal hyperalgesia in carrageenan-injected

5 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 52 C: n = 6/group; at 42 C: 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 52 C: n = 6/group; at 42 C: n = 4/group) or in the Hargreaves test (n = 6/group). The cooling and heating rate for the DCP and DHP was 1 C.min -1. Statistical significance against the respective controls is indicated (*P <.05). y-axes give the number (nb) of responses. rats was indicated by increased nociceptive responses at 9 Cor45 C(P <.05). 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 species, 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 nociceptors 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 allodynia and hyperalgesia within a single procedure in awake and freely moving animals. C57Bl/6J mice and Sprague-Dawley rats exhibit different responses in the DCP/DHP test. In both species, rearing behavior that reflects spontaneous activity and exploration did not appear suitable to measure nociceptive response. Jumps, which can be interpreted as an escape behavior, were only present in mice when hot- or cold-nociceptive temperatures were reached and the number of jumps increased with the intensity of nociceptive stimulus. It is thus the most relevant parameter to score during DCP/DHP tests in the C57Bl/6J mice. Similarly, hind paw lickings and withdrawals appeared as the most relevant parameters in Sprague-Dawley rats. Cumulating both parameters in rats gave a clear-cut detection of the nociceptive response and threshold. When we tested mice or rats during 15 minutes on the plate setup at 30 Cor20 C, we did not observe any nociceptive Figure 5. Carrageenan-induced paw inflammation in rats. A, Total number of nociceptive responses (sum of hind paw licking and withdrawal) in DCP, 7 hours after the intraplantar injection of saline (control) or carrageenan. B, Total number of nociceptive responses in DHP, 7 hours after the intraplantar injection of saline (control) or carrageenan. The saline solution or the l-carrageenan (3% in 0.9% NaCl, 150 ml) was injected in the right hind paw of Sprague-Dawley rats (n = 8/group). The cooling and heating rate for the dynamic cold and hot plate was 1 C.min -1. Statistical significance against the respective control animals is indicated (*P <.05, 1 P <.01, # P <.001). y-axes give the number (nb) of responses.

6 772 Dynamic Hot and Cold Plate behavior. Moreover, the procedure offers reproducible measures as shown by repeated testing on a 9-day period. The nociceptive threshold to heat in the DCP/DHP test was between 39 C and 40 C. This is in agreement with thermonociceptor thresholds as measured by in vivo electrophysiology. 31 However, there is no clear agreement in the literature as to when a cold stimulus begins to be nociceptive. 13 The results that we obtained with the cold ramp revealed a behavioral response starting at 4 C for mice and at 9 C for rats. These temperatures could be considered as nociceptive because the animals did not display escape behavior (mice) or paw licking/withdrawal (rats) before reaching them. Thermal nociception is thought to be mediated by C- and Ad-fibers, 14 which have distinct electrophysiological and pharmacological properties. 16 Based on in vivo electrophysiological approaches, it has been proposed that slow ramps (speed) of temperature heating preferentially activate C-fibers, whereas fast ramps (speed) of temperature heating preferentially activate Ad-fibers. 29 This idea is also supported by experiments using contact heating 21 or infrared diode laser systems 25 to distinguish thermonociceptors. Thus, the 1 C.min -1 heating rate that we used in this study can be considered as very slow when compared with other studies, 18,29,32 and we suggest that this slow rate heating preferentially activates C-fibers. Unfortunately, we have no direct evidence such as peripheral nerve recordings. Moreover, for doses similar to the ones we used, capsaicin and DMSO were proposed to act respectively on C- or Ad-thermonociceptors. 1,7,15,26,29 Accordingly, we found significant heat hypersensitivity with topical application of capsaicin in mice and rats with DHP testing. However, we observed no effect of topical DMSO application either in DHP or DCP testing in both mice and rats. Our data indicating that C- or Ad-thermonociceptors could be differentially activated depending of the rate of skin heating are in good agreement with the results of the groups of Lumb 21 and Yeomans, 25 and a main interest of the DCP/ DHP test was to provide a procedure to study this sensitization of thermonociceptors in freely moving animals. Interestingly, we observed that capsaicin-treated mice and rats showed nociceptive behavior at innocuous temperatures, which revealed the presence of a thermal allodynia. On the other hand, the conventional HP setup at 52 C did not allow the detection of an effect of either capsaicin or DMSO application in mice and rats. The Hargreaves test and the conventional hot plate at 42 C with a long cutoff allowed detection of the capsaicin effect in mice but did not discriminate between thermal allodynia and thermal hyperalgesia. On the contrary, in rats we observed the effects of capsaicin with the conventional hot plate at 42 C but not with the Hargreaves test. The DCP/DHP is thus of interest to provide both within the same testing procedure. Similarly, we investigated whether this method is able to measure the changes in hypersensitivity evoked by a carrageenan-induced inflammatory pain model. 23 Because this model is first and well characterized in rats, we decided to evaluate the effects of carrageenaninduced inflammatory pain in DCP/DHP testing in rats. We observed that carrageenan-injected rats display a nociceptive reaction at innocuous temperatures (ie, thermal allodynia) as well as an exaggerated number of responses at noxious temperatures (ie, thermal hyperalgesia) in comparison to saline-injected rats. These data revealed that inflammatory pain is inducing a strong thermal allodynia and confirmed in a widely used pain model the interest of the DCP/DHP procedure to observe thermal allodynia as well as hyperalgesia in the same procedure. We observed that capsaicin-treated mice and rats or carrageenan-injected rats display hyperalgesia and/or allodynia as compared with their control animals. Although the DCP/DHP requires longer testing time than other thermal tests, it allows measuring the nociceptive temperature thresholds and behavioral score for nonnoxious and noxious stimuli in a single experiment with a freely moving animal. References 1. 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