Validation of a thermal threshold nociceptive model in bearded dragons (Pogona vitticeps)

Accepted Manuscript Validation of a thermal threshold nociceptive model in bearded dragons (Pogona vitticeps) Émilie L. Couture, Beatriz P. Monteiro, Jessica Aymen, Eric Troncy, Paulo V. Steagall PII:

S1467-2987(17)30054-5

DOI:

10.1016/j.vaa.2016.07.005

Reference:

VAA 80

To appear in:

Veterinary Anaesthesia and Analgesia

Received Date: 13 April 2016 Revised Date:

13 July 2016

Accepted Date: 16 July 2016

Please cite this article as: Couture ÉL, Monteiro BP, Aymen J, Troncy E, Steagall PV, Validation of a thermal threshold nociceptive model in bearded dragons (Pogona vitticeps), Veterinary Anaesthesia and Analgesia (2017), doi: 10.1016/j.vaa.2016.07.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

RESEARCH PAPER

2

ÉL Couture et al.

3

Thermal threshold in bearded dragons

4

Validation of a thermal threshold nociceptive model in bearded dragons (Pogona

5

vitticeps)

6

Émilie L Couture*, Beatriz P Monteiro†, Jessica Aymen*, Eric Troncy† & Paulo V

7

Steagall*

8

*Department of Clinical Sciences, Faculty of Veterinary Medicine, Saint-Hyacinthe,

9

Canada

M AN U

SC

RI PT

1

10

†Department of Biomedical Sciences, Faculty of Veterinary Medicine, Saint-Hyacinthe,

11

Canada

12

Correspondence: Paulo Steagall, Département de Sciences cliniques, Faculté de médecine

14

vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, J2S 2M2, Canada.

15 16

EP

TE D

13

Abstract

18

Objectives To validate a thermal threshold (TT) nociceptive model in bearded dragons

19

(Pogona vitticeps) and to document TT changes after administration of morphine.

20

Study design Two part randomized, blinded, controlled, experimental study.

21

Animals Five adult bearded dragons (242–396 g).

22

Methods A TT device delivered a ramped nociceptive stimulus (0.6°C second-1) to the

23

medial thigh until a response (leg kick/escape behavior) was observed, or maximum (cut-

AC C

17

ACCEPTED MANUSCRIPT

off) temperature of 62°C was reached. In Phase I Period 1, six TT readings were

25

determined at 20 minute intervals for evaluation of repeatability. Two of these readings

26

were randomly assigned to be sham to assess specificity of the behavioral response. The

27

same experiment was repeated 2 weeks later (Period 2) to test reproducibility. In Phase II,

28

animals were administered either intramuscular morphine (10 mg kg-1) or saline 0.9%.

29

Thermal thresholds (maximum 68°C) were determined before and 2, 4, 8, 12 and 24 hours

30

after treatment administration. Data were analyzed using one-way ANOVA (temporal

31

changes and repeatability) and paired t-tests (reproducibility and treatment comparisons)

32

using Bonferroni correction (p < 0.05).

33

Results Mean TT was 57.4 ± 3.8°C and 57.3 ± 4.3°C for Periods 1 and 2, respectively.

34

Data were repeatable within each period (p = 0.83 and p = 0.07, respectively).

35

Reproducibility between periods was remarkable (p = 0.86). False-positive responses

36

during sham-testing were 10%. TTs were significantly increased after morphine

37

administration at 2, 4 and 8 hours when compared with baseline, and at 2 and 4 hours when

38

compared with saline 0.9%. Highest TT was 67.7 ± 0.7°C at 4 hours after morphine

39

administration.

40

Conclusions and clinical relevance Testing was repeatable, reproducible, and well

41

tolerated in bearded dragons. TT nociceptive testing detected morphine administration and

42

may be suitable for studying opioid regimens in bearded dragons.

44

SC

M AN U

TE D

EP

AC C

43

RI PT

24

Keywords analgesia, morphine, nociception, opioid, pain, reptile

ACCEPTED MANUSCRIPT

Introduction

46

The class Reptilia includes more than 7200 species that display a wide range of

47

physiological and behavioral adaptations to their environment. These species are housed at

48

zoological parks, rehabilitation centers and veterinary clinics and may require veterinary

49

care. In that sense, pain-induced behaviors can be subtle and difficult to interpret in reptiles

50

which can render the assessment and treatment of pain suboptimal (Fleming & Robertson

51

2012; Sladky & Mans 2012). There are a few published studies describing the

52

pharmacokinetics (PK) of analgesic drugs in reptiles (Kummrow et al. 2008; Norton et al.

53

2015) and their analgesic effects (Sladky & Mans 2012). Nociceptive threshold testing

54

allows objective quantification of the antinociceptive effects of an opioid (Love et al.

55

2011). Studies on antinociception may provide species-specific and evidence-based

56

recommendations for the treatment of clinical pain in reptiles.

M AN U

SC

RI PT

45

The aims of this study were to validate a thermal threshold (TT) nociceptive device

58

in bearded dragons (Pogona vitticeps) and use this device to assess the effect of morphine

59

administration. The hypotheses were that 1) a repeatable, reproducible behavioral response

60

without tissue injury would result from a ramped nociceptive thermal stimulus in

61

unrestrained bearded dragons and 2) administration of morphine would result in thermal

62

antinociception.

64

EP

AC C

63

TE D

57

65

Materials and methods

66

This study was approved by the animal care committee of the Faculty of Veterinary

67

Medicine, Université de Montréal (14-Rech-1736).

ACCEPTED MANUSCRIPT

68 Animals

70

Five adult bearded dragons, three males and two females weighing 242-396 g, were

71

included in this study. They were obtained from different commercial suppliers and the

72

ages were unknown. The animals were considered to be healthy based on physical

73

examination and results of fecal zinc sulfate flotation, complete blood count and serum

74

chemistry profile (Gibbons et al. 2012). They were all administered antiprotozoal treatment

75

(Marquis 15% w/w ponazuril; Bayer Inc., Canada) upon arrival. Bearded dragons were

76

housed individually in a terrarium with 0.80 x 0.46 x 0.51 m dimensions (Atazuki,

77

Mingchang Industrial & Trading Co., LTD, China). A 14-hour daylight period was

78

provided with an Ultraviolet-B (UVB) light (Reptisun T8 10.0 UVB 61 cm; Zoo Med

79

Laboratories Inc., CA, USA). During daytime, animals had access to a basking spot of

80

approximately 40°C (150 W Repticare Ceramic Infrared Heat Emitter; Zoo Med

81

Laboratories Inc.) and room temperature was kept at 27°C. A sloped wood branch and a

82

cardboard box were provided. Three sides of the terrarium were covered with opaque paper

83

so that animals did not have visual contact with each other or the observers. The animals

84

were fed with greens, vegetables, fruits, crickets and worms with appropriate supplements;

85

fresh water was available ad libitum. Animals were allowed to rest for one week before an

86

acclimation period of 10 days when they were conditioned to manipulations and the testing

87

device. Animals were adopted at the end of the study.

AC C

EP

TE D

M AN U

SC

RI PT

69

88 89

Thermal threshold

ACCEPTED MANUSCRIPT

A commercial TT device (Topcat Metrology Ltd., UK) with a 15 x 5 x 10 mm probe

91

containing a heater element and a temperature sensor was used in this study (Fig. 1). The

92

device was calibrated before the study according to the manufacturer. The probe was

93

attached to the medial surface of the thigh of the bearded dragon using bandage material

94

(Fig. 2) and connected by a ribbon cable to a control unit with a digital display of

95

temperature. The ribbon cable exited the terrarium at the top and was long enough to allow

96

free movement of the bearded dragon. This digital display was visualized by one observer

97

(BM, PS or JA) who was behind the terrarium and responsible for connecting and

98

disconnecting the cable, recording temperature values and treatment administration. A

99

video camera (D5100; Nikon Canada Inc, Canada) was directed towards the uncovered side

100

of the terrarium and connected to a computer (Macbook Pro; Apple Canada Inc., Canada)

101

located outside the room. An evaluator (EC) who was blinded to the treatment stayed

102

outside the room to perform TT testing using the wireless temperature remote control. The

103

evaluator could perform live-observation of the animals during testing using an appropriate

104

software (Camera Control Pro 2; Nikon Canada Inc, Canada). The animals were unable to

105

see the observers and evaluator.

EP

TE D

M AN U

SC

RI PT

90

Ten minutes elapsed after probe placement and before thermal stimulation. Skin

107

temperature (ST) was recorded before each test. A wireless remote control increased the

108

temperature of the probe in a ramped thermal stimulus of 0.6°C second-1 until a behavioral

109

response was observed or the cut-off temperature (68°C) was reached. The recorded

110

temperature was considered to be the TT of the animal. In each testing period, thermal

111

stimulation was alternated between pelvic limbs, and bearded dragons were examined for

112

tissue damage daily for up to 72 hours.

AC C

106

ACCEPTED MANUSCRIPT

113 Study design

115

Preliminary study

116

Three sites for probe placement were tested in two bearded dragons: on the thorax, at the

117

base of the tail and at the proximal medial thigh. The band around the thorax securing the

118

probe was not well tolerated. The animals’ behavioral responses were observed when

119

thermal stimuli were applied to the tail or thigh and no response resulted from stimulation

120

of the tail. Stimulation of the thigh resulted in attempts to escape, paddling of the pelvic

121

limbs, kicking of the ipsilateral limb or waking up. Stimulation trials on the pelvic limbs

122

were then performed on the three remaining bearded dragons to confirm behavioral

123

responses.

M AN U

SC

RI PT

114

An initial cut-off temperature of 55°C was chosen based on previous research with

125

green iguanas (Fleming & Robertson 2012). However, three bearded dragons did not

126

respond at this temperature and the cut-off was increased to 62°C after ethical approval.

TE D

124

127 Phase I

129

Phase I was conducted on all five bearded dragons and consisted of TT testing (Period 1)

130

that was repeated 2 weeks later (Period 2). Phase I was completed before Phase II. Six TT

131

tests were performed at 20 minute intervals. Two of these tests were randomly assigned

132

using online software (www.randomization.org) to be sham, where the probe was

133

disconnected from the control unit by one of the observers while the evaluator was not

134

aware of it.

135

AC C

EP

128

ACCEPTED MANUSCRIPT

Phase II

137

In Phase I, the mean TT was close to the cut-off temperature of 62°C which would not

138

allow significant increases in thermal nociception after the administration of an opioid.

139

Thus, the cut-off temperature was increased to 68°C for Phase II after ethical approval.

140

Animals were randomly assigned to be administered either morphine (10 mg kg-1;

141

Morphine 10 mg mL-1; Sandoz Canada Inc., Canada) intramuscularly (IM) into a thoracic

142

limb or an equivalent volume of saline 0.9%. After 14 days, the treatments were reversed.

143

Thermal thresholds were determined before (baseline) and at 2, 4, 8, 12 and 24 hours after

144

treatment administration. Two baseline thresholds were determined 15 minutes apart and

145

averaged.

M AN U

SC

RI PT

136

146 Statistical analysis

148

Data showed normal distribution and passed equal tests of variance using the Shapiro-Wilk

149

test. For Phase I, ST and TT temporal changes within Period 1 or Period 2 (repeatability)

150

were analyzed using one-way ANOVA for repeated measures, excluding sham values.

151

Reproducibility was tested when comparing ST and TT between the Periods 1 and 2 in

152

Phase I, and using a paired t-test. The latter test was also used for treatment comparison in

153

Phase II followed by Bonferroni correction (GraphPad Prism Version 5; GraphPad

154

Software, Inc., CA, USA). Values are reported as mean ± standard deviation (SD) with p <

155

0.05. Specificity is reported as a percentage representing the ratio of false-positive

156

responses and total number of sham stimulations.

AC C

EP

TE D

147

157 158

Results

ACCEPTED MANUSCRIPT

Phase I

160

One bearded dragon did not respond to thermal stimulation at 62°C; only the sham results

161

were considered for this part of the study. Skin temperature was significantly different

162

between Periods 1 (33.8 ± 1°C) and 2 (35.4 ± 1°C) (p = 0.001). Temporal changes for ST

163

were not significant in Period 1 (p = 0.08) and 2 (p = 0.57) (Fig. 3).

RI PT

159

Repeatability of TT was recorded over Period 1 (p = 0.83) and 2 (p = 0.07) (Fig. 3).

165

Reproducibility of TT between Period 1 (57.4 ± 3.8°C) and 2 (57.3 ± 4.3°C) was

166

remarkable (p = 0.86). There were two and zero false-positive responses during sham

167

testing in Period 1 and 2, respectively (10% in total). In one of them, the observer

168

recognized immediately that the remote control was activated accidentally and the observed

169

behavior was not a typical TT response.

M AN U

SC

164

170 Phase II

172

Skin temperature was significantly increased at 2, 4 and 8 hours after the administration of

173

morphine when compared with baseline value and at 4 and 8 hours when compared with

174

saline 0.9% (Fig. 4). It was observed that the bearded dragons consistently adopted a

175

resting posture with the head flat on the surface beneath them (floor or wood branch) at 4-

176

12 hours after morphine administration. The skin of some of these individuals was slightly

177

darker than usual.

EP

AC C

178

TE D

171

Thermal thresholds were significantly increased at 2, 4 and 8 hours after morphine

179

administration when compared with baseline values (Bonferroni correction p < 0.05).

180

Thermal thresholds were significantly increased at 2 and 4 hours in the morphine treatment

181

when compared with saline 0.9% treatment. Mean maximum TT after the administration of

ACCEPTED MANUSCRIPT

morphine was 67.7 ± 0.7°C at 4 hours (Fig. 5). Mean maximum TT after saline

183

administration was 60.4 ± 3.1°C at 8 hours. Significant differences in TT when compared

184

with baseline assessments were not observed after saline 0.9% administration. Tissue

185

damage was not observed in any bearded dragon during the entire study.

RI PT

182

186 Discussion

188

Thermal nociceptive threshold testing was repeatable, reproducible and well tolerated

189

without causing tissue damage in unrestrained bearded dragons. The antinociceptive effects

190

of morphine were observed. Research using the thermal threshold device may be useful for

191

the evaluation of different opioid regimens (doses, routes of administration, drugs) and to

192

provide evidence-based recommendations for the treatment of clinical pain in bearded

193

dragons. The antinociceptive effects of a drug in the laboratory setting are commonly

194

extrapolated to the clinical setting where analgesic protocols become species-specific.

195

Nevertheless, it may be argued that clinical pain is much more complex and laboratorial

196

methods should involve a comprehensive evaluation of nociception using more than one

197

type of stimulus (Steagall et al. 2007), which could be a limitation of this study.

198

Furthermore, the relationship between antinociception and clinical analgesia is unknown in

199

reptiles and clinical studies are required to validate if our results can be extrapolated to

200

bearded dragons with naturally-occurring painful conditions.

M AN U

TE D

EP

AC C

201

SC

187

Nociceptive models involve the assessment and quantification of sensory

202

sensitivity for the study of pain in a controlled manner. Thermal, mechanical or electrical

203

nociceptive stimuli are applied until a behavioral response is observed (i.e. threshold) (Le

204

Bars et al. 2001). The use of thermal nociception withdrawal latency (TWL) was originally

ACCEPTED MANUSCRIPT

reported in crocodiles (Kanui et al. 1990; Kanui & Hole 1992) and more recently in

206

bearded dragons (Pogona vitticeps), corn snakes (Pantherophis guttulatus) (Sladky et al.

207

2008) and yellow bellied and red-eared slider turtles (Trachemys scripta scripta and

208

Trachemys scripta elegans) (Sladky et al. 2007, 2009; Baker et al. 2011; Giorgi et al. 2014,

209

2015a, b). The use of TT nociception has been reported in green iguanas (Fleming &

210

Robertson 2012), and validated for use in cats, horses, chickens and rabbits (Dixon et al.

211

2002; Hothersall et al. 2011; Love et al. 2011; Barter & Kwiatkowski 2013). Animals are

212

unrestrained in their normal habitat and can display normal behavior; an acclimation period

213

is required as it was performed in the present study (Dixon et al. 2002). However, the use of

214

a TT probe implies a tactile stimulus compared with the selective stimulus provided by

215

radiant heat in TWL (Le Bars 2001). No publication investigating the repeatability nor the

216

reproducibility of TWL or TT in reptiles were found. Phase I of this study aimed to address

217

this issue. Repeatability relates to the degree of agreement between findings in successive

218

evaluations. Reproducibility relates to the degree of agreement between findings in a

219

different period of time or by different evaluators. Reproducibility between different

220

observers was not assessed; however mean TT values were compared between periods 1

221

and 2 as previously reported in cats (Dixon et al. 2002). The small size of bearded dragons

222

prevented the application of an inflatable cuff that maintains a consistent contact between

223

probe and skin (Dixon et al. 2002) and may have reduced repeatability and reproducibility

224

in Phase I.

AC C

EP

TE D

M AN U

SC

RI PT

205

225

Sham testing was used to assess specificity of the behavioural response to thermal

226

stimulation and similarly to what has been reported in rabbits and cats while studying if the

227

model is subject of observer bias (Dixon et al. 2002; Barter & Kwiatkowski 2013). Sham

ACCEPTED MANUSCRIPT

testing revealed some false-positive reactions during testing. Kicking or other escape

229

behavior was not recorded during the preliminary study or when the TT probe was

230

attached, but not activated, to the thigh of the animal using bandage material. Therefore,

231

false-positive responses were unlikely to be associated with poor conditioning or simply

232

bandage application. In the authors’ experience, false-positive responses to nociceptive

233

stimulation can occur with spontaneous movement in cats, dogs and horses. Acclimation,

234

appropriate experimental conditions, adequate observer experience/training and larger

235

sample sizes may reduce individual variability to thermal stimulation and false positive

236

responses using this model in bearded dragons.

M AN U

SC

RI PT

228

Thermal threshold and cut-off temperatures were higher in bearded dragons when

238

compared with other species. Mean TT was 57.4°C, 40.0°C and 43.3°C in bearded dragons,

239

cats and broiler chickens, respectively (Dixon et al. 2002; Hothersall et al. 2011). In our

240

study, a cut-off temperature of 55°C such as used in green iguanas (Fleming & Robertson

241

2012) did not evoke a consistent response to thermal stimulation. A final cut-off of 68°C

242

did not cause tissue damage. The onset, magnitude and duration of action of morphine on

243

thermal antinociception could be recorded. Differences in reptile skin structure and function

244

may be habitat-related and could explain variations in cut-off temperatures and

245

susceptibility to skin damage using thermal nociceptive devices (Klein & Gorb 2012). For

246

example, species that are found in tropical arid and semi-arid environments including

247

bearded dragons might have habitat-related skin adaptations that could result in high TT

248

(Sladky et al. 2008). In addition, following the completion of the study, skin biopsies of one

249

bearded dragon that did not respond to thermal stimulation with a cut-off of 62°C revealed

250

a superficial fungal infection. It has been hypothesized that shedding may influence TT

AC C

EP

TE D

237

ACCEPTED MANUSCRIPT

responses in bearded dragons (Sladky et al. 2008). Therefore, species-specific cut-off

252

temperatures and experimental devices should be determined and any underlying skin

253

condition should be investigated prior to the start of a study.

RI PT

251

The dose of morphine (10 mg kg-1) was chosen based on a previous study using the

255

TWL that resulted in thermal antinociception (Sladky et al. 2008). Sedation was not

256

evaluated in the present study and it is not clear how it would have affected nociceptive

257

testing and morphine-induced antinociception. However, previous studies showed that

258

sedation scores did not influence TT after the administration of dexmedetomidine in cats

259

(Slingsby & Taylor 2008). Further studies are warranted to address the effects of sedation

260

on nociceptive testing in bearded dragons.

M AN U

SC

254

Differences in ST were observed in both phases of the study, however ST

262

differences did not seem to alter TT. The ectothermic nature of bearded dragons may

263

explain these differences since animals were unrestrained which allowed behavioral

264

thermoregulation during testing. In Phase II, significant increases in ST were recorded as

265

well as color changes after the administration of morphine. Morphine-induced dose-

266

dependent hyperthermia has been reported in lizards (Kavaliers et al. 1984) but the

267

mechanism by which hyperthermia is induced is not clear. Indeed, changes in skin color are

268

considered to be means of thermoregulation in many reptiles and darker coloration is

269

associated with heat gain and increases in body temperature (de Velasco & Tattersall 2008).

270

The small number of animals is a limitation of this study, especially considering the

AC C

EP

TE D

261

271

large individual variability in TT. This number was originally based on similar TT studies

272

in cats and green iguanas (Fleming & Robertson 2012; Steagall et al. 2015). It was deemed

273

to be acceptable since significant differences in Phase II and remarkable repeatability and

ACCEPTED MANUSCRIPT

reproducibility of Phase I were detected. Finally, although experimental nociceptive testing

275

provides an objective assessment of drug effects, more than one type of stimulus should be

276

used in an attempt to address the complexity of clinical pain (Steagall et al. 2007).

277

Furthermore, the relationship between antinociception and clinical analgesia is unknown in

278

reptiles. Clinical studies in bearded dragons with naturally occurring painful conditions are

279

required to support our results.

280

In conclusion, TT testing was repeatable, reproducible, and well tolerated in unrestrained

281

bearded dragons. Sham testing resulted in rare false-positive responses. Thermal

282

antinociception was detected following morphine administration using the TT device.

283

Study of opioid regimens using TT may be useful for providing evidence-based

284

recommendations for the treatment of clinical pain in bearded dragons. However, the

285

clinical relevance in this species requires further investigation.

286

TE D

M AN U

SC

RI PT

274

Acknowledgements

288

Funding was provided by the Bourse de Recherche du Zoo de Granby en Santé de la Faune

289

and by the Fonds de Recherche Zoetis of the Université de Montréal. Dr Cedric B Larouche

290

and Dr Graham Zoller for technical assistance. Topcat Metrology for technical advice. Dr.

291

Beatriz Monteiro is a recipient of the Vanier Canada graduate scholarship.

AC C

292

EP

287

293

Authors’ contributions

294

Generation of hypothesis and study design, funding and grant writing: ÉLC, ÉT, PVS.

295

Experimental study: ÉLC, BPM, JA, PVS. Statistical analysis: ÉLC, PVS. Manuscript

296

writing: all authors.

ACCEPTED MANUSCRIPT

297

References

298

Baker BB, Sladky KK, Johnson SM (2011) Evaluation of the analgesic effects of oral and subcutaneous tramadol administration in red-eared slider turtles. J Am Vet Med Assoc

300

238, 220–227.

303 304 305

New Zealand white rabbits. J Am Assoc Lab Anim Sci 52, 44–47.

SC

302

Barter LS, Kwiatkowski A (2013) Thermal threshold testing for evaluation of analgesics in

Dixon MJ, Robertson SA, Taylor PM (2002) A thermal threshold testing device for evaluation of analgesics in cats. Res Vet Sci 72, 205–210.

M AN U

301

RI PT

299

Fleming GJ, Robertson SA (2012) Assessments of thermal antinociceptive effects of

306

butorphanol and human observer effect on quantitative evaluation of analgesia in green

307

iguanas (Iguana iguana). Am J Vet Res 73, 1507–1511.

Gibbons PM, Klaphake E, Carpenter JW (2012) Reptiles. In: Exotic Animal Formulary (4th

309

edn). Carpenter JW, Marion CJ (eds). Elsevier Health Sciences, USA. pp. 83–182.

310

Giorgi M, de Vito V, Owen H et al. (2014) PK/PD evaluations of the novel atypical opioid

313 314 315 316 317

EP

312

tapentadol in red-eared slider turtles. Med Weter 70, 530–535. Giorgi M, Lee H-K, Rota S et al. (2015a) Pharmacokinetic and pharmacodynamic assessments of tapentadol in yellow-bellied slider turtles (Trachemys scripta scripta)

AC C

311

TE D

308

after a single intramuscular injection. J Exotic Pet Med 24, 317–325.

Giorgi M, Salvadori M, De Vito V et al. (2015b) Pharmacokinetic/pharmacodynamic assessments of 10 mg/kg tramadol intramuscular injection in yellow-bellied slider

turtles (Trachemys scripta scripta). J Vet Pharmacol Ther 38, 488–496.

318

Hothersall B, Caplen G, Nicol CJ et al. (2011) Development of mechanical and thermal

319

nociceptive threshold testing devices in unrestrained birds (broiler chickens). J

ACCEPTED MANUSCRIPT

323 324 325 326 327 328 329

Pharmacol Ther 15, 101–103.

RI PT

322

Kanui TI, Hole K (1992) Morphine and pethidine antinociception in the crocodile. J Vet

Kanui TI, Hole K, Miaron JO (1990) Nociception in crocodiles: capsaicin instillation, formalin and hot plate tests. Zoolog Sci 7, 537–540.

Kavaliers M, Courtenay S, Hirst M (1984) Opiates influence behavioral thermoregulation

SC

321

Neurosci Methods 201, 220–227.

in the curly-tailed lizard, Leiocephalus carinatus. Physiol Behav 32, 221–224. Klein M-CG, Gorb SN (2012) Epidermis architecture and material properties of the skin of

M AN U

320

four snake species. J R Soc Interface 9, 3140–3155.

Kummrow MS, Tseng F, Hesse L, Court M (2008) Pharmacokinetics of buprenorphine

330

after single-dose subcutaneous administration in red-eared sliders (Trachemys scripta

331

elegans). J Zoo Wildl Med 39, 590–595.

335 336 337 338

TE D

334

53, 597–652.

Love EJ, Murrell J, Whay HR (2011) Thermal and mechanical nociceptive threshold testing

EP

333

Le Bars D, Gozariu M, Cadden SW (2001) Animal models of nociception. Pharmacol Rev

in horses: a review. Vet Anaesth Analg 38, 3–14. Norton TM, Cox S, Nelson SE Jr et al. (2015) Pharmacokinetics of tramadol and O-

AC C

332

desmethyltramadol in loggerhead sea turtles (Caretta caretta). J Zoo Wildl Med 46,

262–265.

339

Sladky KK, Mans C (2012) Clinical analgesia in reptiles. J Exotic Pet Med 21, 158–167.

340

Sladky KK, Miletic V, Paul-Murphy J et al. (2007) Analgesic efficacy and respiratory

341 342

effects of butorphanol and morphine in turtles. J Am Vet Med Assoc 230, 1356–1362. Sladky KK, Kinney ME, Johnson SM (2008) Analgesic efficacy of butorphanol and

ACCEPTED MANUSCRIPT

343 344

morphine in bearded dragons and corn snakes. J Am Vet Med Assoc 233, 267–273. Sladky KK, Kinney ME, Johnson SM (2009). Effects of opioid receptor activation on thermal antinociception in red-eared slider turtles (Trachemys scripta).

346

Am J Vet Res 70, 1072–1078.

347

RI PT

345

Slingsby LS, Taylor PM (2008) Thermal antinociception after dexmedetomidine

administration in cats: a dose-finding study. J Vet Pharmacol Ther 31, 135–142.

349

Steagall PV, Monteiro BP, Lavoie AM, Troncy E (2015) Preliminary investigation of the

350

thermal antinociceptive effects of codeine in cats. J Feline Med Surg 17, 1061-1064.

M AN U

351

SC

348

Steagall PV, Taylor PM, Brondani JT et al. (2007) Effects of buprenorphine, carprofen and

352

saline on thermal and mechanical nociceptive thresholds in cats. Vet Anaesth Analg 34,

353

344–350.

354

de Velasco JB, Tattersall GJ (2008) The influence of hypoxia on the thermal sensitivity of skin colouration in the bearded dragon, Pogona vitticeps. J Comp Physiol B 178, 867–

356

875.

EP

358

AC C

357

TE D

355

ACCEPTED MANUSCRIPT

Figure legends

360

Figure 1. Thermal threshold (TT) device with a digital display (A) connected by a cable to

361

the probe (B). Arrow indicates the heater element.

362

RI PT

359

Figure 2. Probe (arrow) of the thermal threshold device placed on the medial surface of the

364

thigh of a bearded dragon and secured using bandage material.

SC

363

365

Figure 3. Skin temperature (ST) and thermal threshold (TT) (means ± standard deviations)

367

in five bearded dragons of four thermal nociceptive stimulations (performed twice, 2 weeks

368

apart; Phase I, Periods 1 and 2).]

369

M AN U

366

Figure 4. Skin temperature (ST) (mean ± standard deviation) in five bearded dragons

371

before (baseline) and after intramuscular administration of morphine (10 mg kg-1) or an

372

equivalent volume of saline. *Significantly different from baseline (p < 0.05).

373

†Significantly different from saline at the same time point (p < 0.05).

EP

374

TE D

370

Figure 5 Thermal threshold (TT) (mean ± standard deviation) in five bearded dragons

376

before (baseline) and after intramuscular administration of morphine (10 mg kg-1) or an

377

equivalent volume of saline. *Significantly different from baseline (p < 0.05).

378

†Significantly different from saline at the same time point (p < 0.05).

379

AC C

375

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT