Understanding of pain in reptiles has expanded greatly within the past 10 years, but some weaknesses remain, particularly with respect to species differences in response to analgesics, identification of optimal analgesics, and determination of the most appropriate analgesic dosages. This is primarily due to the wide diversity of anatomic, physiologic, and metabolic characteristics in reptile species. Whereas a few studies1–3 have revealed specific neuroanatomic structures associated with pain in reptiles, more recent studies4–9 have focused on the behavioral responses to various noxious stimuli across several reptile species.
Studies of nociception and antinociception have been performed in a variety of reptile species, with responses measured to thermal, chemical, or mechanical noxious stimuli. For example, behavioral responses to chemical noxious stimuli (ie, formalin or capsaicin) have been evaluated in Speke hinge-back tortoises (Kinixys spekii),6,7 ball pythons (Python regius),8 and crocodiles,9 before and after analgesics were administered. Although evidence from some of these studies suggests that μ-opioid receptor agonists attenuate the nociceptive response, these noxious chemicals can cause severe inflammation and tissue damage, hindering accurate measurement of a nociceptive response.
Studies4,5,10,11 in which responses were measured after application of a noxious thermal stimulus (hind limb withdrawal apparatus) have yielded consistent results for various reptile species, with responses similar to those in mammals.10 This type of test has been successfully used for red-eared sliders,4,11 bearded dragons,5 corn snakes,5 and green iguanas.12 When the hind limb withdrawal latency test is used, responses can be measured before and after administration of saline (0.9% NaCl) solution or analgesics.13,14
Opioids are commonly used for pain management in veterinary medicine. Morphine, a pure μ-opioid receptor agonist from this class, is a commonly used analgesic; however, it can cause several adverse effects, such as respiratory depression. Butorphanol, a drug commonly recommended for reptile analgesia,15,16 is a κ-opioid receptor agonist and μ-opioid receptor antagonist with no evidence of the analgesic efficacy and respiratory depression observed with morphine.4 In reptiles, the effects of morphine administration differ by species. For example, snakes appear to be much more refractory to μ-opioid analgesics than lizards and chelonians.4,5
Tegus (Salvator merianae) are omnivorous lizards that inhabit a wide variety of ecosystems throughout South America. Tegus are common components of the pet trade, zoological institutions, and anatomy and physiology studies.17–20 However, the authors are unaware of any reported studies in which tegus were used to evaluate the antinociceptive efficacy of analgesics. Therefore, the objective of the study reported here was to evaluate the antinociceptive efficacy of single doses of morphine sulfate and butorphanol tartrate after IM administration in tegus.
Materials and Methods
Animals
Six juvenile (12- to 24-month-old) tegus of unknown sex with a mean ± SD body weight of 1,484 ± 473 g (range, 505 to 2,125 g) were used in the study. Tegus had been provided by the Biosciences Institute of São Paulo State University. Health status was verified through physical examinations and routine laboratory tests (CBC and plasma biochemical analysis) in accordance with established standards.21 Individual identification and body temperature measurements were determined by microchipsa implanted SC between the scapulae.
Before the study began, all tegus were allowed to acclimate to their housing in a room with a controlled temperature (25° to 30°C), humidity (range, 50% to 60%), and photoperiod (12 hours of light and 12 hours of darkness) for approximately 3 months. They were maintained in round fiberglass tanks (1.35 m in diameter and 0.73 m in height) with wood shavings as a substrate; plastic containers within the tanks provided sheltered hiding places. Heat and light were provided by 250-W incandescent lamps, which provided higher temperatures (40° to 45°C) at a basking site. Tegus were fed a diet of ground beef heart supplemented with calcium and vitamin D3b 3 times/wk; fresh water was provided ad libitum. The study protocol was approved by the Committee of Ethics in Animal Experimentation of the University of São Paulo (protocol No. 14.1.112.74.2) and the Environment Ministry (SISBIO No. 42845).
Treatments
In a crossover study design, tegus were randomly assigned to treatment order by use of a random number generator,c with a minimum washout period of 15 days between treatments. Each of 5 treatments was administered IM in a forelimb: saline solution (0.5 mL; control treatment), morphine sulfated (5 or 10 mg/kg), and butorphanol tartratee (5 or 10 mg/kg).
Antinociception testing
The antinociceptive efficacy of morphine and butorphanol was evaluated by hind limb withdrawal latency testing, which involved application of a thermal noxious stimulus to the plantar surface of the hind limbs. Observers were unaware of treatment received. The noxious thermal stimulusf was provided by exposing the hind limb to a radiant heat source set at an intensity of 70 (range, 238 to 252 mW/cm2). This heat source provided an exposure temperature range of 45° to 47°C for a maximum of 30 seconds, which prevented tissue burns. For this testing, each tegu was restrained in an acrylic box (60 × 13 × 14 cm) with opaque barriers that prevented tegus from seeing each other. The tegus were positioned to stand on the surface of the plantar test apparatus.
Following a 5-minute acclimation period, the heat source was aimed at the plantar surface of 1 hind limb and the noxious thermal stimulus applied. Limb withdrawal latency (interval to limb withdrawal from the thermal stimulus) was automatically measured by the device at baseline (0 hours) and 0.5, 1, 2, 3, 4, 6, 12, and 24 hours after treatment administration. At each assessment point, 2 limb withdrawal measurements were made and recorded, and if the values differed from each other by > 2 seconds, a third measurement was performed. From these measurements, a mean withdrawal time was determined for each assessment point. To prevent tissue damage, the hind limb exposed to the noxious thermal stimulus was alternated between measurements, with 2 minutes separating exposures.
Statistical analysis
Statistical analysis was performed with the aid of statistical software.g Normality of data distribution was evaluated with the Shapiro-Wilk test, revealing a normal distribution. Body temperatures and mean hind limb withdrawal latencies were compared by means of 2-way repeated-measures ANOVA, and the Bonferroni post hoc test was used for within-group comparisons with baseline values. For between-group comparisons at each assessment point, 2-way ANOVA was used, followed by application of the Tukey post hoc test. Values are reported as mean ± SD. Differences were considered significant at values of P < 0.05.
Results
At baseline (0 hours), tegus had similar mean hind limb withdrawal latencies (overall mean ± SD, 8.8 ± 2.9 seconds) for IM administration of saline solution, morphine at 5 and 10 mg/kg, and butorphanol at 5 mg/kg. However, values for butorphanol at 10 mg/kg (mean ± SD, 18 ± 10 seconds) were much higher (P < 0.01) than the values for the other treatments at that assessment point (Figure 1). With saline solution, no significant difference from baseline was identified in mean hind limb withdrawal latencies measured after treatment administration, except at the 12-hour assessment point (mean ± SD, 16.29 ± 3.36 seconds). With morphine at 5 mg/kg, mean hind limb withdrawal latencies were greater than at baseline between 0.5 and 1 hour and at 12 hours after administration, and with morphine at 10 mg/kg, values were greater than at baseline between 1 and 12 hours after administration. With butorphanol at 5 and 10 mg/kg, mean hind limb withdrawal latencies did not change significantly from baseline after administration.
Mean ± SEM hind limb withdrawal latencies (interval to limb withdrawal from a thermal noxious stimulus applied to the plantar surface of the hind limb) of 6 tegus (Salvator merianae) before (0 hours; baseline) and 0.5, 1, 2, 3, 4, 6, 12, and 24 hours after IM administration of each of 5 treatments in random order in a crossover study design. Treatments included saline (0.9% NaCl) solution (0.05 mL; CON), morphine at 5 mg/kg (MOR5) or 10 mg/kg (MOR10; panel A), and butorphanol tartrate at 5 mg/kg (BUT5) or 10 mg/kg (BUT10; panel B). A minimum 15-day washout period was provided between treatments. Observers were unaware of treatment received. Notice that the x-axis is not continuous. *Value differs significantly (P < 0.05) from baseline.
Citation: American Journal of Veterinary Research 78, 9; 10.2460/ajvr.78.9.1019
Because of some variability in mean hind limb withdrawal latencies at baseline, postadministration values were also normalized to baseline values and plotted (Figure 2). With morphine at 5 mg/kg, mean hind limb withdrawal latencies were significantly greater than control (saline solution) values by 0.5 hours and remained high for at least 12 hours after administration. With morphine at 10 mg/kg, mean hind limb latencies were significantly greater than control values at 1 hour and remained that way for at least 12 hours after administration. No significant differences from control values were detected at any point after administration for butorphanol at 5 or 10 mg/kg.
Mean ± SEM change from baseline in hind limb withdrawal latencies at various points for the tegus in Figure 1. Dotted horizontal line denotes no change from baseline. *Value differs significantly (P < 0.05) from the respective control value. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 78, 9; 10.2460/ajvr.78.9.1019
Although attempts were made to keep the ambient temperature fairly constant throughout the experiment, the mean body temperatures of the tegus varied. Mean body temperatures for all treatments combined were significantly different from baseline at the 3-, 4-, and 12-hour assessment points (Figure 3).
Mean ± SEM body temperature (as measured via a microchipa implanted SC between the scapulae) for all treatments at various points for the tegus in Figure 1. Dotted horizontal lines represent the optimal temperature range for the species (32° to 38°C). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 78, 9; 10.2460/ajvr.78.9.1019
Discussion
Findings of the present study suggested that antinociception in tegus is better achieved with a μ-opioid receptor agonist (ie, morphine sulfate). Butorphanol tartrate at the doses evaluated provided no antinociception in tegus. This is consistent with results of other studies indicating that μ-opioid receptor agonists (eg, morphine and hydromorphone) are most effective at minimizing nociceptive responses and pain in lizard and chelonian species.4–6,10 The choice to administer morphine and butorphanol IM at 5 and 10 mg/kg was made on the basis of the doses used in a study5 involving bearded dragons.
Appropriate treatment of pain can minimize the physiologic changes initiated by noxious stimuli, thereby typically leading to lower morbidity and mortality rates associated with trauma and surgery.14 Although the efficacy of morphine and butorphanol for antinociception has been evaluated in a few reptile species,4,5 there are clear species differences with respect to effective drugs and dosages, which are largely attributable to species diversity within the class Reptilia and vastly different physiologic adaptations.
Evaluation of pain in reptiles for experimental purposes is a complex process, particularly with respect to the type of noxious stimulus used. The validity of application of a thermal noxious stimulus has been questioned, given that reptiles are ectothermic and can develop skin burns from heat sources in terrariums. Furthermore, a few desert reptile species have a high tolerance to heat.22 However, many reptile species have experimentally received this type of stimulus, and all had avoidance responses similar to mammals, regardless of species.23 The plantar hind limb withdrawal test used in the present study was the same as that used in other studies with red-eared sliders,4,11 bearded dragons, and corn snakes,5 in which these reptiles had consistent withdrawal responses to the thermal noxious stimulus, with and without opioid administration.
Comparison between findings of the present and previous studies reveals variability in nociceptive responses, depending on the species examined. Reptiles that have adapted to extreme climates, such as bearded dragons that inhabit desert areas, generally have longer baseline withdrawal latencies5 and, therefore, smaller variations in latency durations. On the other hand, reptiles from aquatic environments with little fluctuation in temperature, such as red-eared sliders, have shorter withdrawal latencies and therefore larger variations in latency durations.4
In preparation for the present study, and to standardize a thermal stimulus for tegus, we performed a preliminary evaluation of the plantar test by use of an infrared intensity of 80 (range, 260 to 280 mW/cm2), with a cutoff period of 35 seconds. Twenty-four hours after the stimulus, a dermal lesion was identified on the plantar surface of a tegu's limb. The intensity and duration of the thermal stimulus were thus reduced to 70 and 30 seconds, respectively, and no more lesions were observed in any tegu.
Although withdrawal responses to the noxious thermal stimulus were measureable and rapid during baseline data collection for all treatments in the present study, variability existed in the overall mean limb withdrawal latency value between treatments at 0 hours. With saline solution (9.4 ± 2.0 seconds), morphine at 5 mg/kg (6.5 ± 1.5 seconds), morphine at 10 mg/kg (8.1 ± 3.3 seconds), and butorphanol at 5 mg/kg (11.0 ± 2.9 seconds), mean ± SD values were statistically similar, but that of butorphanol at 10 mg/kg (17.5 ± 9.9 seconds) was 86%, 166%, 116%, and 59% longer, respectively. Baseline variability in results achieved with the plantar hind limb withdrawal apparatus has been observed in other reptile species, and this may be a function of species or individual differences or circadian rhythms altering responses at particular times of the day.5
Other than at 12 hours after administration, IM administration of saline solution and butorphanol at 5 and 10 mg/kg in the tegus of the present study failed to yield any significant differences from baseline or even between treatments (Figure 2). These results corroborate the findings of other studies involving red-eared sliders4 and bearded dragons.5 In both studies, the use of high butorphanol doses (28 and 20 mg/kg) led to no increase in hind limb withdrawal latencies, compared with baseline values or results achieved with saline solution. Findings of the present study supported the hypothesis that κ-opioid receptor agonists such as butorphanol are not effective antinociceptive drugs or analgesics in reptiles. Although we recognize that the antinociceptive testing method used involved only a thermal noxious stimulus, which may limit generalizability of the data, our findings suggested that butorphanol at the doses evaluated fails to increase limb withdrawal latencies in healthy tegus.
Morphine administration at 5 mg/kg led to 203% and 193% increases from baseline in hind limb withdrawal latencies at 0.5 and 1 hours, respectively, and to 204% at 12 hours. An increase in limb withdrawal latencies at 12 hours after morphine administration might have been associated with the decrease in body temperature observed in this test condition and was also observed with the control treatment. The increase in the limb withdrawal latencies between 0.5 and 1 hour after morphine administration corroborated reported findings for bearded dragons.5 In bearded dragons, no increase from baseline in hind limb withdrawal latencies was observed at 2, 4, and 8 hours after low-dose morphine administration (1 to 5 mg/kg, IM), but increases were observed at 12 and 24 hours.5 In red-eared sliders, the antinociceptive effect of morphine (1.5 and 6.5 mg/kg, IM) endured for 24 hours after administration.4 The prolonged effect of morphine in this chelonian species may be associated with differences in opioid receptor distribution or may be due to species differences in response to the thermal noxious stimulus, as previously discussed.
With morphine administration at 10 mg/kg in the present study, an increase from baseline in hind limb withdrawal latencies was observed in tegus from 1 to 12 hours after administration, with increases ranging over time from 112% to 225%. The increase in values observed at 12 hours may have also been attributable to decreases in body temperature, which were documented. Similar results have been reported for red-eared sliders and bearded dragons,4,5 and respiratory depression was associated with the use of morphine in those species. We made no attempt to measure respiratory depression in tegus; however, we presume that respiratory depression following morphine administration is a valid concern in all reptile species. Findings of the present study lent support to the hypothesis that μ-opioid receptor agonists such as morphine are the most effective antinociceptive or analgesic opioids for use in reptiles, but respiratory depression should be considered a potential adverse effect of these drugs.
To avoid the influence of body temperature on drug metabolism and antinociceptive responses to the thermal stimulus, tegus were kept in a controlled environment. However, the presence of basking (40° to 45°C) sites and free access to regions with higher temperatures contributed to body temperature variability during the study. It should be noted that although body temperatures differed among treatments, they were all maintained within the optimal range for the species (32° to 38°C).24
In a previous study,24 body temperature of lizards varied throughout the day in a parabolic manner. Early in the day, at approximately 6:00 am, body temperatures ranged from 26° to 27°C and peaked at approximately 4:00 pm (33° to 34°C), with a decrease to 26° to 27°C by approximately 12:00 am owing to the lizards’ burrowing behavior. This same circadian rhythm in body temperature was identical to the variation observed in the present study. Body temperature was measured at 12:00 pm in the present study, and maximum and minimum body temperatures were achieved at 4 hours (mean ± SD, 36.2 ± 0.5°C) and 12 hours (29.1 ± 0.9°C) after treatment administration, respectively. However, we recognize that body temperature variability may have influenced opioid metabolism in the tegus, even though the body temperature range was within the optimum range for this species.
The results of the study reported here indicated that morphine, but not butorphanol, provided antinociception, as measured via use of a thermal noxious stimulus, when administered IM at 5 or 10 mg/kg in tegus. These data supported the hypothesis that μ-opioid receptor agonists, but not κ-opioid receptor agonists, provide antinociception in reptiles, regardless of species.
Acknowledgments
This manuscript represents a portion of a thesis submitted by Dr. Leal to the Animal Biosciences Graduate Program, School of Animal Science and Food Engineering, University of São Paulo, as partial fulfillment of the requirements for a Master of Science degree.
Dr. Carregaro's research was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (grant Nos. 2014/10452-9 and 2014/03247) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (grant No. 304566/2015-2).
Footnotes
Lifechips 134kw Biotermico Allflex, Joinville, Santa Catarina, Brazil.
Repti calcium, Zoo Med Laboratories Inc, San Luis Obispo, Calif.
RANDOM.ORG, Randomness and Integrity Services Ltd, Dublin, Ireland. Available at: www.random.org/. Accessed Jan 3, 2015.
Dimorf, Cristália Produtos Químicos Farmacěuticos Ltd, São Paulo, Brazil.
Torbugesic, Fort Dodge Animal Health, Fort Dodge, Iowa.
Ugo Basile plantar analgesia instrument (Hargreaves apparatus), Ugo Basile Co, Comerio, Varese, Italy.
Graphpad Prism, version 7, Graphpad Software Inc, San Diego, Calif.
References
1. Liang YF, Terashima S, Zhu AQ. Distinct morphological characteristics of touch, temperature, and mechanical nociceptive neurons in the crotaline trigeminal ganglia. J Comp Neurol 1995;360:621–633.
2. ten Donkelaar HJ, de Boer-van Huizen R. A possible pain control system in a non-mammalian vertebrate (a lizard, Gekko gecko). Neurosci Lett 1987;83:65–70.
3. Xia Y, Haddad GG. Major difference in the expression of delta and mu-opioid receptors between turtle and rat brain. J Comp Neurol 2001;436:202–210.
4. Sladky KK, Miletic V, Paul-Murphy J, et al. Analgesic efficacy and respiratory effects of butorphanol and morphine in turtles. J Am Vet Med Assoc 2007;230:1356–1362.
5. Sladky KK, Kinney ME, Johnson SM. Analgesic efficacy of butorphanol and morphine in bearded dragons and corn snakes. J Am Vet Med Assoc 2008;233:267–273.
6. Wambugu SN, Towett PK, Kiama SG, et al. Effects of opioids in the formalin test in the Speke's hinged tortoise (Kinixys spekii). J Vet Pharmacol Ther 2010;33:347–351.
7. Dahlin J, Kanui TI, Wambugu SN, et al. The suspended formalin test: a method designed for studying formalin-induced behaviour in the Speke's hingeback tortoise (Kinixys spekii). Scand J Lab Anim Sci 2012;39:11–15.
8. Williams CJA, James LE, Bertelsen MF, et al. Tachycardia in response to remote capsaicin injection as a model for nociception in the ball python (Python regius). Vet Anaesth Analg 2016; 43;429–434.
9. Kanui TI, Hole K, Miaron JO. Nociception in crocodiles—capsaicin instillation, formalin and hot plate test. Zoolog Sci 1990;7:537–540.
10. Mans C, Lahner LL, Baker BB, et al. Antinociceptive efficacy of buprenorphine and hydromorphone in red-eared slider turtles (Trachemys scripta elegans). J Zoo Wildl Med 2012;43:662–665.
11. Sladky KK, Kinney ME, Johnson SM. Effects of opioid receptor activation on thermal antinociception in red-eared slider turtles (Trachemys scripta). Am J Vet Res 2009;70:1072–1078.
12. Fleming GJ, Robertson SA. Assessments of thermal antinociceptive effects of butorphanol and human observer effect on quantitative evaluation of analgesia in green iguanas (Iguana iguana). Am J Vet Res 2012;73:1507–1511.
13. Hargreaves K, Dubner R, Brown F, et al. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988;32:77–88.
14. Sladky KK, Mans C. Clinical analgesia in reptiles. J Exot Pet Med 2012;21:158–167.
15. Read MR. Evaluation of the use of anesthesia and analgesia in reptiles. J Am Vet Med Assoc 2004;224:547–552.
16. Schnellbacher R. Butorphanol J Exot Pet Med 2010;19:192–195.
17. Klein W, Abe AS, Andrade DV, et al. Structure of the posthepatic septum and its influence on visceral topology in the tegu lizard Tupinambis merianae (Teiidae: Reptilia). J Morphol 2003;258:151–157.
18. Andrade DV, Brito SP, Toledo LF, et al. Seasonal changes in blood oxygen transport and acid-base status in the tegu lizard, Tupinambis merianae. Respir Physiol Neurobiol 2004;140:197–208.
19. Skovgaard N, Galli G, Abe A, et al. The role of nitric oxide in regulation of the cardiovascular system in reptiles. Comp Biochem Physiol A Mol Integr Physiol 2005;142:205–214.
20. Tattersall GJ, Leite CAC, Sanders CE, et al. Seasonal reproductive endothermy in tegu lizards. Sci Adv 2016;2:e1500951.
21. Diethelm G, Stein G. Hematologic and blood chemistry values in reptiles. In: Mader DR, ed. Reptile medicine and surgery. 2nd ed. Philadelphia: Saunders Elsevier, 2005; 1103–1118.
22. Mosley C. Pain and nociception in reptiles. Vet Clin North Am Exot Anim Pract 2011;14:45–60.
23. Sladky KK, Mans C. Clinical anesthesia in reptiles. J Exot Pet Med 2012;21:17–31.
24. Andrade DV, Sanders C, Milsom WK, et al. Overwintering in tegu lizards. In: Barnes BM, Carey HV, eds. Life in the cold: evolution, mechanisms, adaptation, and application. Fairbanks, Alaska: Institute of Arctic Biology, University of Alaska, 2004; 339–348.
