Recently, the Canadian Council on Animal Care published Guidelines on the Care and Use of Fish in Research, Teaching and Testing, in which it is stated, “Little is known about the effect of analgesic drugs on fishes.”1 Thus, we initiated a research program with the goal of providing data regarding the effects and efficacy of potential analgesics in fish. Initially, a comprehensive investigation2 of the pharmacokinetics of morphine in fish was performed. That study revealed that significant interspecific variations exist in the pharmacokinetics of morphine among fish and that, compared with findings in mammals, the disposition of morphine in fish is slower by approximately 1 order of magnitude.
To our knowledge, there are only 3 credible published reports3-5 regarding the efficacy of morphine as an analgesic agent in fish, and there appear to be no reports of studies in which the effects of morphine on the cardiovascular system of ectotherms were examined. Data on the cardiovascular effects of analgesics in fish are needed for several reasons. Fish cardiorespiratory physiology is an extremely active area of research,6-9 and the requirement for analgesic use in these types of studies could have important implications. The cardiovascular and respiratory systems of fish are sensitive to numerous abiotic and biotic factors7 (including anesthetic agents10), and alterations in cardiovascular function are likely to influence several other physiologic systems of interest to researchers. Finally, morphine affects the cardiovascular system of animals differently. For example, morphine administration depresses cardiac output in rabbits and dogs11,12 but causes an increase in cardiac output and tachycardia in horses.13 Thus, it is impossible to predict how morphine administration would affect cardiorespiratory function in fishes.
The purpose of the study reported here was to assess the cardiorespiratory effects of morphine and evaluate whether morphine blocks cardiac responses to a noxious stimulus in winter flounder. Experiments were performed to investigate the short-term effects of morphine administered IP and IV on cardiac output and heart rate of winter flounder, and on the basis of those findings, an additional experiment was conducted to investigate the long-term effects of morphine administration on cardiac function and respiratory rate. To assess the potential analgesic efficacy of morphine, responses to a noxious stimulus in fish that were or were not treated with the drug were evaluated. Winter flounder were used in our experiments because numerous aspects of the cardiovascular physiology of this species are currently being studied14 and because the ventral aorta is easily exposed, making placement of Doppler flow probe cuffs for the measurement of cardiac output uncomplicated.
Material and Methods
Approval was obtained from the Animal Care Committees at the University of Guelph and at Memorial University of Newfoundland, and all fish were treated according to the Canadian Council on Animal Care guidelines. Flounder (Pseudopleuronectes americanus) (mean weight, 599 ± 20 g) were caught by divers with hand nets in Conception Bay, Newfoundland, Canada, at a depth of 4 to 6 m. The fish were initially transported to the Ocean Sciences Centre at the Memorial University of Newfoundland and held for 3 weeks in a tank (2 × 2 × 0.5 m deep) that was supplied with sea water (8°C; pH, 8.0 to 8.1) at 10 L/min. Thereafter, they were transferred to a similar-sized tank in the center's Aquaculture Research and Development Facility, and the temperature was raised to 10°C over a period of 2 weeks. The flounder were fed diced herring (1% body weight/feeding) twice a week; however, food was withheld for at least 12 hours prior to surgery.
Forty-two flounder were anesthetized individually by use of 0.2 g of tricaine methanesulfonate/L of watera and weighed after removing as much superficial water as possible. Thereafter, they were transferred to a surgical table where their gills were irrigated with chilled (approx 4°C) and oxygenated sea water containing 0.1 g of tricaine methanesulfonate/L.
All fish were subsequently fitted with a cuff-type Doppler flow probeb around the ventral aorta, and the Doppler flow probe leads were sutured to the dorsal surface of each fish at 2 sites. In addition, depending on the experiment, flounder were fitted with 1 or more cannulae. For examination of the short-term effects of morphine on cardiac performance, most fish were fitted with an IP polyethylene cannulac (PE 50; volume capacity, 0.2 mL), although 2 fish were fitted with a cannula in the postcardinal vein2 instead to examine whether the route of administration influenced the results. To evaluate the long-term effects of morphine on cardiorespiratory function, flounder were fitted with an IP cannula for drug injection and a respiratory cannulac (PE 60; volume capacity, 0.2 mL) was placed inside the buccal cavity15; the pressure signal from the latter was used to measure respiratory frequency. Finally, for testing the efficacy of morphine as an analgesic agent, fish were fitted with an IP cannula for drug injection and an SC cheek polyethylene cannulac (PE 20; volume capacity, 0.07 mL) to deliver the noxious (acetic acid) or control (saline [0.9% NaCl] solution) stimulus without otherwise disturbing the fish. We chose acetic acid as the noxious stimulus because it is used in standard pain tests in mammals16 and acetic acid stimulates nociceptors in the facial region of fish.4
The surgical procedures were completed within 20 minutes. Flounder were then placed into an experimental chamber (a darkened plastic container [50 × 30 × 25 cm]) receiving sea water (10°C) at 5 L/min, and recovery from anesthesia was facilitated by directly flushing the gills with fresh sea water for approximately 1 minute. The fish were subsequently allowed at least 12 hours to recover prior to any experiments.
Three experiments were performed. In the first experiment, fish were allocated to 1 of 3 treatment groups: 1 group received saline solution but did not receive the noxious stimulus (no analgesic–control stimulus group [n = 10 fish]), 1 group received saline solution and the noxious stimulus (no analgesic–noxious stimulus group [9]), and the third group received morphine and the noxious stimulus (analgesic–noxious stimulus group [10]). Cardiac output was recorded prior to, and for the first 50 minutes after, administration of the IP injection (saline or morphine), and these data were used to investigate the effects of morphine on cardiovascular function. After 50 minutes, the fish received a cheek injection of either noxious or innocuous stimulus, and cardiac output was recorded for the following 60 minutes; these data were used to investigate the analgesic properties of morphine.
In the second experiment, 2 fish were injected IV with morphine and cardiac output was recorded for 6 hours; the recording time in this experiment was longer because heart rate and cardiac output were still high at 50 minutes after injection in fish in the first IP experiment. Finally, in the third experiment, cardiac output, heart rate, and respiratory frequency were recorded continuously for the first 2 hours and for 45 minutes at 24, 48, and 72 hours after flounder were injected IP (8 fish received morphine, and 3 fish received saline solution). The third experiment investigated the effects of morphine on respiratory rate as well as on cardiovascular variables for a longer period of time because heart rate and cardiac output were still high 6 hours after the IV injection in the second experiment. Data for the first 100 minutes after the morphine injection IP from this third experiment were used as findings from an analgesic–control stimulus group to explain the gradual increase in cardiac output detected in the analgesic–noxious stimulus group from the first experiment.
On the morning of all 3 experiments, syringes containing morphine sulfated (pH, 3.22) or pH-adjusted saline solution (pH, 3.22) were prepared and connected to the IP or IV cannula prior to recording. In the first experiment, syringes containing saline solution or 5% acetic acide were also prepared and connected to the appropriate cheek cannula. The Doppler flow probe leads and the respiratory cannula were then connected to the flow meter and pressure transducer, respectively, prior to the recording.
After recording resting values for 30 minutes, a dose of 40 mg of morphine sulfate/kg (1.56 ± 0.06 mL, IP), 17 mg of morphine sulfate/kg (0.5 mL, IV), or saline solution (1.63 ± 0.07 mL, IP, and 0.5 mL, IV) was injected slowly (over a period of 30 to 40 seconds) through the IP or IV cannula. Fish that were to receive the noxious stimulus were injected with 200 μL of 5% acetic acid in saline solution 50 minutes after the IP injection of saline solution or morphine. Fish that were not to receive the noxious stimulus were injected with saline solution (200 μL; an innocuous control stimulus) 50 minutes after the initial injection of saline solution or morphine. At the end of all experiments, the fish were euthanized by use of an overdose of tricaine methanesulfonate (0.3 g/L), the flow probe was removed, and the correct positioning of all cannulae was confirmed via dissection. Tissue samples were collected after the first experiment in the area where the cheek cannula had been inserted. These tissue samples were fixed in formalin and subsequently examined for histopathologic changes related to the injection of the noxious stimulus.
Data and statistical analyses—To measure cardiac output and heart rate, the Doppler flow probe leads were connected to a directional pulsed Doppler flow meterf that was interfaced with a data acquisition systemg and a computer software program.h The buccal cannula was connected to a pressure transducer that was interfaced with the data acquisition system to measure respiratory rate. The pressure transducer was calibrated daily with a column of water, with zero pressure set to the surface of the water in the plastic container.
In all experiments, cardiac output data were normalized to the mean of values recorded at 20, 15, 10, and 5 minutes prior to injection (immediately prior to injection was designated as time 0) because Doppler flow signals only provide a relative measure of cardiac output. Heart rate and respiratory frequency at all measurement intervals were calculated by measuring the time required for 20 peaks to appear in the flow and pressure traces, respectively.
For data comparisons, 2-way ANOVAsi were performed (ie, for the first 50 minutes of the first experiment and for the second and third experiments). When significant main effects were identified, Tukey post hoc tests were used to compare variables for the morphine and saline solution treatment groups at each time point and Dunnett post hoc tests were used to compare within treatment values with preinjection (time 0) values. A value of P < 0.05 was considered significant.
Results
Treatment of fish with saline solution did not result in any significant changes in cardiac function in the first experiment or cardiorespiratory function in the third experiment (Figures 1 and 2). Furthermore, there was no apparent effect of the route of morphine administration (IP or IV) on changes in cardiac function between the first and second experiment.
Changes in heart rate (A and B) and cardiac output (C and D) in winter flounder injected IP with morphine (black circles;n=10;AandC) or pH-adjusted saline (0.9% NaCl) solution (white circles; 19; A and C) in the first of 3 experiments or injected IV with morphine in a second experiment (n = 2; B and D); recordings were made for 50 and 400 minutes after injection, respectively. The vertical lines through 0 on the x-axis indicate the time of injection. Fish in panels A and C received 40 mg of morphine/kg, IP, or 1.63 mL of saline solution, IP. Fish in panels B and D received 17 mg of morphine/kg, IV. Data are presented as mean value ± SE. aSignificant (ANOVA withTukey's post hoc tests; P < 0.05) difference between treatments at this time point. bValue in morphine-treated fish at this time point was significantly (ANOVA with Dunnett post hoc tests; P < 0.05) different from baseline (time 0) value.
Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.592
Changes in heart rate (A and B) and cardiac output (C and D) in winter flounder injected IP with morphine (black circles;n=10;AandC) or pH-adjusted saline (0.9% NaCl) solution (white circles; 19; A and C) in the first of 3 experiments or injected IV with morphine in a second experiment (n = 2; B and D); recordings were made for 50 and 400 minutes after injection, respectively. The vertical lines through 0 on the x-axis indicate the time of injection. Fish in panels A and C received 40 mg of morphine/kg, IP, or 1.63 mL of saline solution, IP. Fish in panels B and D received 17 mg of morphine/kg, IV. Data are presented as mean value ± SE. aSignificant (ANOVA withTukey's post hoc tests; P < 0.05) difference between treatments at this time point. bValue in morphine-treated fish at this time point was significantly (ANOVA with Dunnett post hoc tests; P < 0.05) different from baseline (time 0) value.
Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.592
Changes in heart rate (A and B) and cardiac output (C and D) in winter flounder injected IP with morphine (black circles;n=10;AandC) or pH-adjusted saline (0.9% NaCl) solution (white circles; 19; A and C) in the first of 3 experiments or injected IV with morphine in a second experiment (n = 2; B and D); recordings were made for 50 and 400 minutes after injection, respectively. The vertical lines through 0 on the x-axis indicate the time of injection. Fish in panels A and C received 40 mg of morphine/kg, IP, or 1.63 mL of saline solution, IP. Fish in panels B and D received 17 mg of morphine/kg, IV. Data are presented as mean value ± SE. aSignificant (ANOVA withTukey's post hoc tests; P < 0.05) difference between treatments at this time point. bValue in morphine-treated fish at this time point was significantly (ANOVA with Dunnett post hoc tests; P < 0.05) different from baseline (time 0) value.
Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.592
Changes in respiratory frequency (Rf; A and B), heart rate (C and D), and cardiac output (E and F) in winter flounder injected IP with morphine (black circles;n=8) or pH-adjusted saline solution (white circles; 3) in the third of 3 experiments; after injection, variables were recorded for 120 minutes (A, C, E) and after 1, 2, and 3 days (B, D, and F). Injections were administered at 0 minutes. Fish received either 40 mg of morphine/kg, IP, or 1.63 mL of saline solution, IP. Data are presented as mean value ± SE. See Figure 1 for key.
Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.592
Changes in respiratory frequency (Rf; A and B), heart rate (C and D), and cardiac output (E and F) in winter flounder injected IP with morphine (black circles;n=8) or pH-adjusted saline solution (white circles; 3) in the third of 3 experiments; after injection, variables were recorded for 120 minutes (A, C, E) and after 1, 2, and 3 days (B, D, and F). Injections were administered at 0 minutes. Fish received either 40 mg of morphine/kg, IP, or 1.63 mL of saline solution, IP. Data are presented as mean value ± SE. See Figure 1 for key.
Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.592
Changes in respiratory frequency (Rf; A and B), heart rate (C and D), and cardiac output (E and F) in winter flounder injected IP with morphine (black circles;n=8) or pH-adjusted saline solution (white circles; 3) in the third of 3 experiments; after injection, variables were recorded for 120 minutes (A, C, E) and after 1, 2, and 3 days (B, D, and F). Injections were administered at 0 minutes. Fish received either 40 mg of morphine/kg, IP, or 1.63 mL of saline solution, IP. Data are presented as mean value ± SE. See Figure 1 for key.
Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.592
Morphine injection in the 3 experiments significantly affected all variables measured (heart rate, cardiac output, and respiratory frequency); however, the pattern of response of each variable was quite different (Figures 1 and 2). Immediately after injection of morphine, there was a large (approx 20 breaths/min) transient increase in respiratory rate in the third experiment, compared with the saline-solution–injected group, but this value returned to baseline value within 5 minutes. Compared with the saline-solution–injected group, injection of morphine also resulted in an immediate but transient drop in heart rate (bradycardia) that was concomitant with an approximately 40% decrease in cardiac output in all 3 experiments; however, the influence of morphine was clearly different for these 2 variables after this time point. For heart rate, the bradycardia was followed by an immediate increase in heart rate (by as much as 15 beats/min greater than baseline values) that was maintained for approximately 30 minutes. Thereafter, heart rate decreased by 5 beats/min during the following 2 to 3 hours and remained at this level for 48 hours after injection. In contrast, cardiac output did not return to baseline value until approximately 50 minutes after the initial bradycardia, and this variable continued to increase until it peaked (approx 20% greater than baseline value) at approximately 100 minutes after injection and was maintained at this level for the duration of measurement (72 hours).
The acetic acid injection in the first experiment was clearly a noxious stimulus because it denatured the muscle of the cheek (area of damage, approx 1 cm in diameter; Figure 3) and resulted in a significant increase in cardiac output (approx 10%) for an interval of approximately 5 minutes (Figure 4). Prior IP administration of morphine (40 mg/kg) in the first experiment completely abolished the cardiac output response to the noxious stimulus. Furthermore, the gradual increase in cardiac output that followed the initial bradycardia in the analgesic–noxious stimulus group was not caused by the acetic acid injection, but was a result of morphine administration alone. In contrast to cardiac output, heart rate was unaffected by the noxious stimulus and remained constant during the experimental period in all 3 treatments (data not shown).
Representative photomicrographs of sections of cheek tissue at the site of SC injection of 200 μL of saline solution (control stimulus; A) or 200 μL of 5% acetic acid in saline solution (noxious stimulus; B) in winter flounder from the first experiment. Notice that the muscle after administration of the control stimulus is apparently normal, whereas the muscle after administration of the noxious stimulus is denatured. H&E stain; bar = 1 mm.
Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.592
Representative photomicrographs of sections of cheek tissue at the site of SC injection of 200 μL of saline solution (control stimulus; A) or 200 μL of 5% acetic acid in saline solution (noxious stimulus; B) in winter flounder from the first experiment. Notice that the muscle after administration of the control stimulus is apparently normal, whereas the muscle after administration of the noxious stimulus is denatured. H&E stain; bar = 1 mm.
Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.592
Representative photomicrographs of sections of cheek tissue at the site of SC injection of 200 μL of saline solution (control stimulus; A) or 200 μL of 5% acetic acid in saline solution (noxious stimulus; B) in winter flounder from the first experiment. Notice that the muscle after administration of the control stimulus is apparently normal, whereas the muscle after administration of the noxious stimulus is denatured. H&E stain; bar = 1 mm.
Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.592
Cardiac output in winter flounder that received an IP injection of pH-adjusted saline solution (1.63 ± 0.07 mL) or morphine (40 mg/kg) followed by an SC injection in a cheek with either 200 μL of 5% acetic acid in saline solution (noxious stimulus) or saline solution (control stimulus) or no stimulus. Data (mean values ± SE) are from the no analgesic–control stimulus group (A; n = 10), the no analgesic–noxious stimulus group (B; 9), and the analgesic–noxious stimulus group (C; 10) in the first experiment, and fish that received morphine treatment with no stimulus in the third experiment (D; 8).The arrows at time 0 minutes indicate IP injection, and those at time 50 minutes indicate cheek injection administrations. *Value after cheek injection was significantly (P < 0.05) increased, compared with values from time 0 to time 50 minutes. See Figure 1 for key. Sal = Saline solution. CS = Control stimulus. NS = Noxious stimulus. M = Morphine.
Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.592
Cardiac output in winter flounder that received an IP injection of pH-adjusted saline solution (1.63 ± 0.07 mL) or morphine (40 mg/kg) followed by an SC injection in a cheek with either 200 μL of 5% acetic acid in saline solution (noxious stimulus) or saline solution (control stimulus) or no stimulus. Data (mean values ± SE) are from the no analgesic–control stimulus group (A; n = 10), the no analgesic–noxious stimulus group (B; 9), and the analgesic–noxious stimulus group (C; 10) in the first experiment, and fish that received morphine treatment with no stimulus in the third experiment (D; 8).The arrows at time 0 minutes indicate IP injection, and those at time 50 minutes indicate cheek injection administrations. *Value after cheek injection was significantly (P < 0.05) increased, compared with values from time 0 to time 50 minutes. See Figure 1 for key. Sal = Saline solution. CS = Control stimulus. NS = Noxious stimulus. M = Morphine.
Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.592
Cardiac output in winter flounder that received an IP injection of pH-adjusted saline solution (1.63 ± 0.07 mL) or morphine (40 mg/kg) followed by an SC injection in a cheek with either 200 μL of 5% acetic acid in saline solution (noxious stimulus) or saline solution (control stimulus) or no stimulus. Data (mean values ± SE) are from the no analgesic–control stimulus group (A; n = 10), the no analgesic–noxious stimulus group (B; 9), and the analgesic–noxious stimulus group (C; 10) in the first experiment, and fish that received morphine treatment with no stimulus in the third experiment (D; 8).The arrows at time 0 minutes indicate IP injection, and those at time 50 minutes indicate cheek injection administrations. *Value after cheek injection was significantly (P < 0.05) increased, compared with values from time 0 to time 50 minutes. See Figure 1 for key. Sal = Saline solution. CS = Control stimulus. NS = Noxious stimulus. M = Morphine.
Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.592
Discussion
In the present study, the IV and IP administration of morphine in fish resulted almost immediately in the development of bradycardia of approximately 5 minutes' duration. The immediacy of the response to IV injection is easy to explain. For example, in a study17 of the effects of morphine on cardiomyocytes of neonatal rats, the drug caused a negative inotropic effect. Thus, there are opioid receptors sensitive to morphine in mammalian hearts. If receptors similar to those in mammalian hearts (ie, opioid receptors) exist in the fish myocardium, injection of morphine into the postcardinal vein would be expected to have an effect on the heart within seconds. As for the fish treated with morphine IP, no clear explanation can be given at this point for the significant transient decrease in cardiac output and heart rate that occurred so soon after injection of morphine. This response was not attributable to the pH of the morphine solution (pH, 3.22) because the saline solution used for injections in other fish was adjusted to a pH of 3.22. However, it is possible that morphine circulated through the liver and to the heart within seconds after the drug was injected and directly affected cardiac function within that short time frame. On the basis of findings of our previous study,2 it is known that morphine absorption into the blood is extremely rapid because the drug is detectable in the postcardinal vein quickly. Furthermore, in our previous pharmacokinetic study2 and in the present study, the IP cannulae were positioned adjacent to the liver.
Although IP injection of morphine in fish induced tachypnea immediately, respiratory rate quickly returned to baseline level and did not change significantly during the next 72 hours. The lack of a long-term effect of morphine on the respiratory rate of flounder is in contrast to the respiratory depression detected in some mammals (eg, dogs18) following morphine administration.
Injection of morphine directly into a flounder's blood stream or IP also resulted, after the initial bradycardia, in a slow and steady increase in cardiac output. However after 100 minutes, no further changes in cardiac output were detected; values of cardiac output were approximately 20% greater than baseline and remained constant for the duration of the experiment (72 hours). Similarly, heart rate initially increased 30% to 45%, compared with the value before injection, and then oscillated near 20% greater than the preinjection value during the next 48 hours. Such increases in cardiac output and heart rate following morphine administration are not detected in humans or dogs. In humans with uncomplicated acute myocardial infarction, administration of morphine does not alter cardiac output.19 In infants receiving mechanical ventilation, bolus injections of morphine did not affect cardiac output and did not have any cardiovascular effects.20 Following injection of 1 to 3 mg of morphine/kg in conscious dogs, an initial increase (20%) in cardiac output was detected, but cardiac output returned to values that were not significantly different from control values after 10 minutes.21 In contrast, injection of morphine into horses13 resulted in an initial increase in heart rate and cardiac output 5 minutes after morphine administration, which was followed by a gradual decrease in these variables, such that values returned to baseline after 15 minutes and were significantly lower than baseline values by 60 minutes after injection.
From the aforementioned studies, it is difficult to discern whether the inconsistency in the cardiovascular response to morphine among species is attributable to interspecific differences in the effect of morphine on heart function or to the different doses and routes of administration used. In humans, the clinical target plasma morphine concentration for treatment of pain is 0.06 mg/L.22 In contrast, in our previous experiment,2 the estimated plasma morphine concentration in winter flounder 72 hours after receiving an IP injection with morphine was 1.22 mg/L. In the present study, we chose to evaluate a dose of 40 mg of morphine/kg because the experiments were run in conjunction with the pharmacokinetic study,2 and a high morphine dose was required to ensure that morphine was detectable in plasma from the flounder for 4 days. Clearly, future investigations should examine whether the cardiovascular response of the winter flounder to morphine is dose dependent and whether the response is consistent among fish. No recommendations regarding the use of morphine as an analgesic in fish can be given until its effects on the cardiovascular system are known.
Nociceptor response to application of acid and injection of acetic acid has long been used as a standard noxious stimulus in mammals and amphibians. For example, acetic acid injected into the paw of rodents elicits a repeatable and easily measured behavioral response.16 Rats that received an injection of 50 μL of 0.6% acetic acid immediately flinched and displayed licking behaviors of approximately 20 to 40 minutes' duration and a diminished response for 180 minutes. Finally, the analgesic potencies of 11 opioids in Northern grass frogs (Rana pipiens) were assessed by use of the acetic acid test,23 and results indicated that the application of acetic acid to the thigh region of conscious frogs caused vigorous wiping motions along the treated limb. Moreover, the wiping response was successfully blocked by analgesics such as morphine but was restored by the administration of naloxone, an opioid antagonist. A similar test has been used previously in fish4,5; 100 μL of 0.1% acetic acid was injected into the lip of rainbow trout and resulted in an increase in respiratory rate. In the experiments of the present study, SC injection of 5% acetic acid into the cheek of flounder elicited a rapid but short-lived (approx 5 minutes) increase in cardiac output. The brevity of the response to the acetic acid stimulus is interesting and somewhat puzzling because the acetic acid injection resulted in complete denaturation of the muscle of the cheek at the injection site. Thus, we expected a more long-term response because of the extensive tissue damage. However, more importantly, administration of morphine 50 minutes prior to the administration of acetic acid completely blocked the cardiovascular response to the noxious stimulus. This result is consistent with findings of other studies,4,5 which indicate that morphine acted as an analgesic when administered before acetic acid injection into the lip of rainbow trout. This provides further evidence that morphine, or similar drugs, could be used in fish experiments to reduce the effects of noxious stimuli or the discomfort induced by surgery or other procedures.
In the present study, morphine administration blocked the cardiac response to a noxious stimulus (acetic acid injection) in winter flounder but also caused notable long-term increases in cardiac output and heart rate. These data provide strong indirect evidence that fish hearts have opioid receptors and suggest that morphine may not be a suitable analgesic for use in fish because of its adverse effects. However, a definitive conclusion as to the applicability of morphine as an analgesic in fish awaits further investigation. First, we used relatively high doses of morphine in the present study (17 and 40 mg/kg), and it is possible that the changes in cardiac function following injection could be avoided by use of lower morphine doses that are still effective at blocking the responses to noxious stimuli in fish. Second, there is considerable interspecific variability in the mammalian cardiovascular response to morphine administration, and thus, the suitability of this drug as an analgesic may be species dependent.
ABBREVIATIONS
MS-222, Syndel Laboratories, Vancouver, BC, Canada.
Doppler flow probe (lumen, 1.6 mm; 20-MHz crystal), Iowa Doppler Products, Iowa City, Iowa.
Polyethylene tubing, Clay Adams, Division of Becton, Dickinson & Co, Parsippany, NJ.
Morphine sulfate, Sabex, Boucherville, QC, Canada.
Glacial acetic acid (laboratory grade), Fisher Scientific Co, Fair Lawn, NJ.
Doppler flow meter, model 545C-4, Bioengineering, University of Iowa, Iowa City, Iowa.
Data acquisition system, MP100, BIOPAC Systems Inc, Goleta, Calif.
AcqKnowledge software, version 3.7.2, BIOPAC Systems Inc, Goleta, Calif.
MINITAB Release 12.1, Minitab Inc, State College, Pa.
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