Pain is subjective and is defined as an unpleasant sensory or emotional experience associated with actual or potential tissue damage.1 Animals greatly vary in their behavioral responses to pain. Immediate responses include vocalization and attempts to escape, whereas longer term responses also include loss of appetite or lack of grooming.1 Most of these responses are very difficult to assess in ectothermic vertebrates, which often remain motionless and have a behavioral repertoire that is considerably more difficult to interpret than that of mammals. In mammals, pain also elicits consistent physiologic responses, including increased heart rate and blood pressure,2 altered breathing patterns,2 and increased plasma concentrations of epinephrine3 and cortisol.3,4 Administration of analgesics consistently reduces these behavioral and physiologic responses in mammals.2,5 It follows that such physiologic responses would provide a useful means of assessing the sensation of pain in amphibians and reptiles.
Pain and pain management in nonmammalian vertebrates remain poorly understood. Results of several studies indicate that reptiles,6–8 fish,9 and birds10 react to painful stimuli and that analgesia relieves pain, yet the basic physiologic response to painful stimuli has not been described to our knowledge. In a survey,11 98.4% of 367 members of the Association of Reptile and Amphibian Veterinarians stated that they believe reptiles can feel pain. Nonetheless, only 39% of those veterinarians reported using analgesics during surgical procedures in reptiles. According to results of the same survey, butorphanol (a mixed opioid receptor agonistantagonist) and NSAIDs are the most commonly used analgesics for reptiles,11 even though their effects on reptiles are largely unknown.
The aim of the study reported here was to characterize the physiologic responses of ball pythons (Python regius) following a minor surgical procedure, to assess whether physiologic variables can be used as a measurable indicator of pain, and to assess whether preoperative administration of butorphanol and the NSAID meloxicam affects postoperative physiologic responses.
Materials and Methods
Animals—Fifteen ball pythons of undetermined sex, with a mean ± SEM body weight of 0.5 ± 0.06 kg (1.1 ± 0.14 lb), were obtained from commercial suppliers and housed in temperature-controlled (30°C [86°F]) 25 × 30 × 20-cm terrariums within the animal care facility at Aarhus University. Snakes were fed mice once or twice a week and had free access to clean water. Food was withheld 5 days before experiments. Experimental protocols were approved by the Danish Experimental Animal Inspectorate.
Analgesia and anesthesia—Snakes were randomly allocated to 3 groups of 5 snakes each. A control group received an IM injection of 1 mL of isotonic saline (0.9% NaCl) solution, a second group was treated with meloxicam (0.3 mg/kg [0.14 mg/lb], IM), and the third group received butorphanol (5 mg/kg [2.3 mg/lb], IM) 3 hours before surgery. The IM injections were given in the right side of the dorsal portion of the tail. Snakes were maintained in a climatic chamber at 30°C for 24 hours prior to surgery and kept there for 72.5 hours after the procedure.
For induction of anesthesia, snakes were placed in an induction chamber containing 5% isoflurane for 20 minutes until tactile reflexes decreased so the glottis could be intubated with soft rubber tubing for artificial ventilation.a The isoflurane concentration was then maintained at 4% in room airb until snakes lost the righting reflex and local rippling of the ventral scales when snakes were lightly touched along the midline was no longer evident. The isoflurane concentration was subsequently reduced to 2% during the remainder of the procedure.12 Snakes were positioned on a heating mat to maintain body temperature at 28° to 30°C (82.4° to 86°F) during surgery.
Surgical procedure—The painful stimulus chosen was surgical cannulation of the vertebral artery, which is a procedure commonly used in physiologic research to obtain blood samples from undisturbed snakes during experiments. Briefly, a 4-cm longitudinal incision was made cranial to the heart on the left side of the ventral scales. The vertebral artery was located and cannulated with a polyethylene catheterc containing heparinized saline solution (50 U/mL). Following occlusive fixation in the artery, the catheter was tunneled into and directed out of the skin on the dorsum of the snake, and the incision was closed with interrupted everting sutures. Finally, the catheter was secured via 2 sutures on the dorsum of the snake. The surgical procedure lasted approximately 30 minutes, after which administration of isoflurane was discontinued. Artificial ventilation was continued until the snakes resumed spontaneous ventilation. Snakes were maintained on the heat mat for 1 hour and then transferred to the climatic chamber. Measurements of physiologic variables were obtained during the subsequent 72.5 hours.
Hematologic analysis—Blood samples (0.2 to 0.3 mL each) were collected from catheters at 0.5, 1.0, 1.5, 2.0, 2.5, 4.5, 6.5, 20.5, 24.5, 48.5, and 72.5 hours after surgery concluded. Subsequently, plasma was separated and stored immediately at −80°C (112°F) for later analysis. The remaining RBCs from each blood sample were diluted in saline solution and injected back into the snakes through the catheter. At 1.5 and 72.5 hours, an additional blood sample (0.3 mL) was collected for measurements of arterial pH, PaCO2, PaO2, and Hct. Blood pH was measured at 30°C by means of a capillary pH electrode connected to a pH meter.d Values of PaO2 were measured at 30°C by use of an electrode.e Plasma concentrations of CO2 were measured as described elsewhere.13 Plasma bicarbonate concentrations were calculated as CtCO2 – (PCO2 × αCO2), where CtCO2 is the total plasma concentration of CO2 and αCO2 is the plasma solubility coefficient for CO2, which has a value of 0.0366 mmol/L.14 The Henderson-Hasselbalch equation was used to calculate PaCO2 as follows: PaCO2 = CtCO2/(αCO2[1 + 10(pH – pK′)]), where pK′ represents the value determined for Python molurus (pK′ = −0.0763 × pH + 6.7283).15 Hematocrit was determined in duplicate as the fractional red cell volume after centrifugationf at 20,000 × g for 3 minutes.
Determination of plasma concentrations of catecholamines and cortisol—Plasma concentrations of epinephrine and norepinephrine were determined via high-pressure liquid chromatography. Plasma cortisol concentration was determined by use of a time-resolved fluoroimmunoassay,g an electronic counter,h and commercial softwarei in accordance with a protocol provided by the manufacturer of the immunoassay kit. All plasma samples were measured for each hormone in duplicate.
Determination of heart rate and blood pressure—After each blood sample was collected, the catheter was connected to a pressure transducerj for determination of blood pressure and heart rate, allowing a 30-minute interval before each measurement was obtained, except at 0.5 hours and 1.5 hours. The signal was amplified by means of a preamplifier that was built in-house, and data were collected via a computerized acquisition system.k The transducer was calibrated daily against a static water column.
Behavioral observations—Snakes were observed when undisturbed and when handled for catheter manipulations. Specifically, snakes were monitored for degree of activity and their tendency to protect their wound, compared with their typical behavior (curled up under their drinking tray).
Statistical analysis—In the control group, plasma catecholamine and cortisol concentrations, blood pressure, heart rate, and blood gas values from 0.5 hours through 48.5 hours after surgery were compared with values obtained at 72.5 hours by means of 1-way repeated-measures ANOVA. Values at 72.5 hours were selected to represent baseline values because it was presumed that the variables measured would have returned to regular values by this time. For Hct, the value at 1.5 hours was used as a baseline measurement.
Values for variables in the 3 treatment groups at all time points were compared by means of 2-way repeated-measures ANOVA. A value of P ≤ 0.05 was used to indicate a significant difference. Summary data are presented as mean ± SEM.
Results
Hematologic analysis—In general, values of arterial blood gases were not affected by surgical cannulation of the vertebral artery, regardless of whether snakes were treated beforehand with saline solution, butorphanol, or meloxicam (Table 1). There was, however, a significant reduction in Hct during the experiment. Values for 2 snakes at 72.5 hours are missing because of technical difficulties.
Mean ± SEM hematologic values in ball pythons that received a preoperative dose of saline (0.9% NaCl) solution (1 mL, IM; control snakes), meloxicam (0.3 mg/kg [0.14 mg/lb], IM), or butorphanol (5 mg/kg [2.3 mg/lb], IM) before surgical cannulation of the vertebral artery, as measured 1.5 and 72.5 hours after surgery.
Variable | Saline solution | Meloxicam | Butorphanol | |||
---|---|---|---|---|---|---|
1.5 h (5) | 72.5 h (4) | 1.5 h (5) | 72.5 h (4) | 1.5 h (5) | 72.5 h (5) | |
pH | 7.57 ± 0.03 | 7.67 ± 0.04 | 7 61 ± 00 | 7 68 ± 0 03 | 7 69 ± 0 05 | 7 65 ± 0 03 |
Bicarbonate (mmol/L) | 15.8 ± 0.7 | 15.9 ± 0.4 | 13.0 ± 0.8 | 16.2 ± 1.6* | 16.7 ± 1.0 | 19.2 ± 2.2 |
Paco2(mm Hg) | 16.0 ± 2.4 | 13.0 ± 1.5 | 12.4 ± 1.3 | 13.1 ± 1.9 | 13.0 ± 0.9 | 16.4 ± 1.3 |
Pao2(mm Hg) | 60.3 ± 10.0 | 68.9 ± 11.7 | 45.9 ± 10.1† | 44.7 ± 7.4 | 32.1 ± 9.8 | 69.6 ± 12.0 |
Hct(%) | 20 ± 0.8 | 16 ± 1.9* | 20 ± 0.8 | 15 ± 1.2* | 19 ± 0.9 | 14 ± 1.4* |
Values in parentheses indicate number of snakes evaluated.
Value is significantly (P ≤ 0.05) different between time points.
Value for control snakes is significantly (P≤0.05) different from that of treated snakes at the given time point.
Plasma concentrations of catecholamines and cortisol—At 72.5 hours after surgical cannulation of the vertebral artery was performed, mean ± SEM plasma concentration of epinephrine in control snakes was 1.9 ± 0.6 nM. Plasma epinephrine concentrations generally did not change during the 72.5-hour postoperative period, regardless of the preoperative treatment (Figure 1). Plasma norepinephrine concentrations remained low (< 0.1 ± 0 nM) during the entire experiment in all 3 groups (data not shown). Plasma cortisol concentrations did not change significantly during the postoperative period in any of the groups, although concentrations in control snakes rose slightly from 69 ± 21 ng/mL to 107 ± 48 ng/mL at 1.5 hours after surgery.
Blood pressure and heart rate—Blood pressure remained remarkably stable during the postoperative period, and there were no differences between effects of the 3 experimental treatments. Mean blood pressure at 72.5 hours (baseline value) was 7.14 ± 0.3 kPa, and this value was not significantly different from values at 0.5 through 48.5 hours (Figure 2). Mean heart rate, which initially significantly increased, decreased significantly during the first hour of the postoperative period in the control snakes. Similar but insignificant changes in mean heart rate were evident in snakes treated with butorphanol or meloxicam. There were no significant differences in heart rate among the 3 groups. Baseline heart rate at 72.5 hours was 35 ± 3 beats/min.
Behavioral observations—In general, the behavior of the snakes was not measurably different before and after surgery. Snakes were observed curled up under their water tray and stayed there during sample collection. However, when the incision was touched, snakes protected the site by changing position or moving away. No behavioral differences among groups were detected.
Discussion
In the study reported here, the effects of surgery on postoperative physiologic variables in reptiles were investigated. The results indicated that, following a transient increase, heart rate, blood pressure, and concentrations of stress hormones returned to stable baseline values within 24.5 hours after surgical cannulation of the vertebral artery in ball pythons. Notably, we found that preoperative administration of 2 different analgesics, meloxicam and butorphanol, did not have an effect on these physiologic variables. In all groups, heart rates were increased for the first hour postoperatively but decreased to baseline values 2.5 hours after surgery. Plasma epinephrine concentrations increased slightly but insignificantly at 2.5 hours, as did plasma cortisol concentrations by 1.5 hours, which slowly returned to initial values within the next 20.5 hours. Thus, cardiovascular changes did not correspond to changes in plasma concentrations of epinephrine or cortisol.
Snakes were affected by anesthesia during the first hours after surgery and were not considered fully recovered from anesthesia until 2.5 hours after surgery. The transient but insignificant increase in plasma epinephrine concentration that coincided with recovery of snakes from anesthesia may have reflected loss of the analgesic effect of isoflurane. The increase in plasma catecholamine concentrations, however, was modest in comparison with the 3- to 5-fold increase that was detected after handling of another species of snake, the black racer (Coluber constrictor).16 In anesthetized pythons, injections of 3 nmol of bradykinin/kg (1.4 nmol of bradykinin/lb) caused a 10-fold rise in plasma concentrations of norepinephrine but did not affect plasma concentrations of epinephrine,17 which suggests that these hormones were released from sympathetic neurons rather than adrenal glands. In our study, the increased plasma epinephrine concentrations at 2.5 hours (with unchanged plasma norepinephrine concentrations) indicated that epinephrine was being released from the adrenal medulla. Nonetheless, it remains unclear whether the low concentrations of epinephrine measured at 0.5 hours in our study were attributable to the anesthetic or whether concentrations of catecholamines simply do not increase in response to surgery in ball pythons.
The increases in heart rate and blood pressure during the first hour occurred despite low concentrations of circulating epinephrine, and these increases were likely to have resulted from increased sympathetic tone of the heart. Isoflurane usually depresses heart rate and blood pressure in reptiles18,19 and would therefore have caused opposite effects. In mammals, blood pressure and heart rate typically increase during the postoperative period,2,20 and these responses are often interpreted as indicators of pain and used to determine the need for analgesia.2 Nevertheless, it has been proposed that the heart rate in mammals rarely increases as a result of pain when other obvious signs of distress are lacking.21 Because the snakes in our study were still recovering from anesthesia during the first hours after surgery, it was not possible to reconcile the tachycardia with behavioral observations, and it remains unknown therefore whether increased heart rate can be used as an indicator of pain in reptiles.
In general, the surgery had little effect on blood gas values. Pain and shock often induce hyperventilation, which would have resulted in decreased PaCO2 and caused PaO2 and pH to increase. The stable values for blood gas measurements indicated that the snakes were ventilated adequately during surgery and that their ventilation was not affected after surgery. The significant reduction in Hct as the experiment progressed was evident in all snakes regardless of whether preoperative analgesics were administered and was likely caused by frequent blood sampling and intraoperative blood loss.
Postoperative stress has received little attention in reptiles, and information about concentrations of stress hormones during the postoperative period is not available. The low plasma epinephrine concentrations that we detected in snakes during anesthesia and immediately after surgery differed from the situation in mammals, in which plasma catecholamine concentrations increase markedly within the first minutes, followed by gradual decrease toward baseline concentrations several hours after a painful procedure.22,23 In contrast, the increase in plasma cortisol concentration was detected early (1.5 hours) after surgery in the snakes in our study. Plasma cortisol concentrations in mammals also increase 1 hour after surgery, and that increase is used as an indicator of pain in mammals.3,4 Plasma cortisol concentration in ball pythons has not been reported, but plasma concentration of corticosterone, the predominant reptilian glucocorticoid, reportedly increases from 40 to 60 ng/mL during container restraint in ball pythons.24 The increase in plasma concentrations of glucocorticoids, corticosterone, and cortisol when snakes are exposed to stressful and painful procedures suggests that plasma concentrations of these hormones could be used in assessment of pain in ball pythons.
We were unable to detect discernable effects of pre-operative administration of meloxicam or butorphanol on physiologic variables in snakes after surgery. In reptiles, the absorption time and half-life of both drugs are unknown. However, because of the slower metabolic rate of reptiles versus mammals, both drugs are likely to be absorbed and metabolized slower than they are in mammals. This would imply that both drugs were in effect at the time of surgery.
Researchers in other studies8,19,l also failed to detect an analgesic effect of butorphanol in reptiles, whereas the analgesic potential of meloxicam, to our knowledge, has not been investigated. In mammals, preoperative treatment with butorphanol results in a lowering of the plasma cortisol concentration (except at 1 hour after surgery), compared with the value obtained before surgery (baseline). On the other hand, treatment with meloxicam before surgery results in an increase in plasma cortisol concentration during the first 3 hours after surgery, and afterward, the concentration decreases to lower than baseline values.25
The results of our study indicated that surgery in ball pythons is associated with a transient increase in blood pressure, heart rate, and plasma cortisol concentration. Clearly, more research on pain and pain management in reptiles is needed.
HI 665 ventilator (tidal volume of 50 mL/kg [22.7 mL/lb] at 10 breaths/min), Harvard Apparatus, Holliston, Mass.
Fluotec, Cyprane Ltd, Keighley, England.
PE50, SIMS Portex Ltd, Hythe, England.
PH radiometer (PHM73) connected to Blood Micro System (BMS2), Radiometer, Copenhagen, Denmark.
Oxygen radiometer connected to Blood Micro System (BMS2), Radiometer, Copenhagen, Denmark.
Haemofuge, Heraeus Sepatech GmbH, Osterode/Harz, Germany.
Delfia Cortisol 1244-060, PerkinElmer Life and Analytical Sciences, Turku, Finland.
Victor3 1420 multilabel counter, PerkinElmer Life and Analytical Sciences, Hvidovre, Denmark.
Workout, version 1.0, PerkinElmer Life and Analytical Sciences, Hvidovre, Denmark.
Model Px600, Baxter-Edward, Irvine, Calif.
AcqKnowledge MP 100, BioPac Systems, Santa Barbara, Calif.
Fleming JG, Robertson AS. Use of thermal threshold test response to evaluate the antinociceptive effects of butorphanol in juvenile green iguanas (Iguana iguana) (abstr), in Proceedings. Annu Conf Am Assoc Zoo Vet 2006;279.
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