Lack of postexposure analgesic efficacy of low concentrations of eugenol in zebrafish.

OBJECTIVE: To test the postexposure analgesic efficacy of low doses of eugenol in zebrafish. STUDY DESIGN: Prospective experimental study. ANIMALS: A total of 76 large adult zebrafish (Danio rerio). METHODS: Fish swimming behavior (median velocity, freeze time, high-speed swimming and distance moved in the vertical direction) was recorded in a 1.6 L video arena before and after exposure to eugenol (0, 1, 2, 5, 10 and 20 mg L-1). In a second experiment, fish were anesthetized with 2-phenoxy-ethanol and treated with an injection of 5% acetic acid (noxious stimulus), and then exposed to 0, 1, 2 and 5 mg L-1 eugenol. The fish swimming behavior was also recorded. RESULTS: The higher doses (10 and 20 mg L-1) reduced the median velocity, high-speed swimming and distance moved in the vertical direction, and increased the freeze time. Zebrafish behavior was not altered by eugenol (1, 2 and 5 mg L-1) after noxious stimulation. CONCLUSIONS AND CLINICAL RELEVANCE: The change in the behavior of zebrafish associated with a noxious stimulus can be monitored and is a good model for studying analgesia in fish. Eugenol (10 and 20 mg L-1) induced zebrafish sedation. The response after a noxious stimulus was not affected by postexposure to lower doses, and thus we cannot recommend its use as an analgesic.

Updated Review of Fish Analgesia.

Analgesics are an integral part of routine pain management in mammals, yet their use in fish is still limited. Some recommendations on the use of analgesics in fish are currently in the literature; however, information on the properties of analgesic drugs in most fish species is still scarce and sometimes misleading. The present review of information on the use of analgesics in fish was thus compiled to help clinicians make an informed decision as to which drug and dose to use. The main agents that have been investigated are opioids, NSAID, and local anesthetics, primarily in rainbow trout and zebrafish. There is presently no overwhelming evidence of efficacy for most analgesics in fish, although beneficial effects on behavior and physiologic parameters have been reported in many instances, especially associated with morphine administration. Furthermore, most analgesics did not result in significant adverse side effects. Thus, analgesics could be administered whenever it is considered that an animal might experience pain, given that the drugs appear not to cause harm and may be beneficial. However, caution must be advised because 1) important interspecies variation has been reported and 2) unforeseen effects could affect experimental results. Further research is needed to investigate analgesic use in fish. This should be accompanied by research aimed at improving our knowledge of the various species of fish. The current lack of a validated approach to assessing pain in fish limits our ability to evaluate the efficacy of analgesics in fish.

Aspects of morphine chemistry important to persons working with cold-blooded animals, especially fish.

The relative amounts of the different forms of morphine, and many other pharmacologic agents, depend on temperature and pH. Some forms are more efficacious because they are uncharged and can penetrate lipid membranes more easily than the charged forms. Persons who administer pharmacologic agents to ectotherms (that is, cold-blooded animals) should consider the effect of temperature on the relative amounts of the different forms of drugs. For example, the fraction of morphine present in the uncharged form is twice as high in a fish or frog at 5 degrees C as in a mammal at 37 degrees C. Moreover, because the pH of blood, plasma, and tissues of ectotherms is higher when they are held at lower temperatures, the combined effect of temperature and pH on the speciation of pharmacologic agents also should be considered. In addition, the total solubility of morphine and other pharmacologic agents depends on temperature and pH. The purpose of this overview is to describe how temperature and pH influence the solubility and speciation of morphine.

Cardiorespiratory effects and efficacy of morphine sulfate in winter flounder (Pseudopleuronectes americanus).

OBJECTIVE: To assess the cardiorespiratory effects of morphine sulfate and evaluate whether morphine blocks cardiac responses to a noxious stimulus in winter flounder. ANIMALS: 42 winter flounder (Pseudopleuronectes americanus) that were acclimated at 10 degrees C. PROCEDURES: Each fish was fitted with a Doppler flow probe around the ventral aorta; cannulae were placed for injection of drug or saline (0.9% NaCl) solution and assessments of respiration. Selected cardiorespiratory variables were measured in morphine-injected (40 mg/kg, IP [n = 18] or 17 mg/kg, IV [2]) or saline solution-injected (1.6 mL [22]) fish at various intervals. Heart rate and cardiac output (CO) were also measured in flounder that were injected with saline solution (n = 19) or morphine (10) and received a noxious or innocuous stimulus (injection of 5% acetic acid or saline solution SC into a cheek) 50 minutes later. RESULTS: Morphine administration promptly induced marked bradycardia (and a concomitant reduction in CO), followed by prolonged (> 48 hours) increases in CO and heart rate. Morphine injection only transiently affected respiratory rate. Application of a noxious stimulus to control flounder resulted in a significant (10%) but transient (< 5 minutes' duration) increase in CO, which was completely blocked by prior administration of morphine. CONCLUSIONS AND CLINICAL RELEVANCE: Although morphine blocked the response to a noxious stimulus in fish, its cardiovascular effects might preclude its use in many research situations. Investigation of the dose dependency of these cardiovascular effects and their interspecific variation is required to determine the applicability of morphine for use in fish.

Uses and Doses of Local Anesthetics in Fish, Amphibians, and Reptiles.

Local anesthetics are an integral part of routine pain management in mammals, yet their use is relatively limited in fish, amphibians and reptiles. These animals frequently undergo potentially painful surgical procedures and therefore could possibly benefit from those drugs. Some recommendations are currently available in the literature concerning analgesic use in these animals. However the pharmacological properties, safety and often efficacy of local anesthetic drugs have not been investigated yet in fish, amphibians, or reptiles. This review compiled current information concerning the use of those agents in fish, reptiles and amphibians to help clinicians make an informed decision as to which dose and drug to use. The resulting literature search showed that the literature concerning use of local analgesics in fish and amphibians is very limited while the literature for reptiles is more extensive. We found few experimental studies evaluating the efficacy of local anesthetics. Further studies would provide additional information for developing guidelines to improve the welfare of fish, amphibians and reptiles.

Effect of TRIS and bicarbonate as buffers on anesthetic efficacy of tricaine methane sulfonate in Zebrafish (Danio rerio).

Tricaine methane sulfonate (TMS), often called MS-222, is the most common anesthetic used with fishes. Because it is very acidic (pKa about 3) it must be neutralized especially when used in soft fresh water. Much of the literature on fish anesthetics recommends neutralizing with bicarbonate. However, much of the zebrafish literature uses the protocol in "The Zebrafish Book" that recommends neutralizing with TRIS. Three considerations when comparing these buffers are: first, TRIS has the advantage that the pH tends to remain constant, whereas the pH of solutions containing bicarbonate tends to increase as CO2 diffuses from the water to air; second, the CO2 produced by bicarbonate may have some sedative effects in and of itself; and third, there is some evidence that the efficacy of TMS changes with pH. In the present study, we compared the efficacy of TMS using these two buffers and show that there is no substantial difference in anesthetic properties in zebrafish.

Parameters influencing the dissolved oxygen in the boundary layer of rainbow trout (Oncorhynchus mykiss) embryos and larvae.

We investigated the influence of oxygen demand (developmental stage) and supply (hypoxia, water flow rate, the chorion and body movements) on the oxygen concentration within the boundary layer next to the chorion of embryos or skin of larvae of rainbow trout (Oncorhynchus mykiss). Oxygen microelectrodes were used to measure dissolved oxygen (DO) within the boundary layer of trout embryos and larvae. As the embryos and larvae developed, the DO gradient and the thickness of the boundary layer increased. The DO concentration within the boundary layer next to the chorion or skin surface decreased as the DO concentration in the free-stream water decreased. A decrease in water flow rate increased the magnitude of the gradient and thickness of the boundary layer. In normoxia, the DO in the perivitelline fluid inside the chorion was 16+/-3.0% saturation at 31 days post fertilization, indicating that the chorion was a significant barrier to oxygen diffusion. The number of body movements did not change when embryos were exposed to hypoxia before hatching, but after hatching, hypoxia resulted in a decrease in body movements of the larvae. Taken together, our data indicate that the oxygen boundary layer around trout embryos and larvae depends on both the oxygen demand and supply. The factors that significantly impacted boundary layer oxygen were developmental stage, free-stream oxygen levels, water flow rate, and the presence of the chorion.

Removal of the chorion before hatching results in increased movement and accelerated growth in rainbow trout (Oncorhynchus mykiss) embryos.

We investigated the effects of the chorion on movement and growth in rainbow trout (Oncorhynchus mykiss) embryos. To test if the chorion restricts movement and growth before hatching, we manually removed the chorion 3-6 days before the natural time of hatching (dechorionated) and compared movement, growth and oxygen consumption in dechorionated embryos and in embryos whose chorions remained intact until the time of hatching (chorionated). Dechorionated embryos exhibited 36 times more movement before hatching compared with intact embryos. By 10 h post-hatch there was no difference in the number of movements between the two groups. At the time of hatching [30 days post-fertilization (d.p.f.)], dechorionated embryos had a significantly greater embryonic body dry mass compared with chorionated embryos, which persisted up to 45 d.p.f. At first feeding (50 d.p.f.) there was no significant difference in embryonic body dry mass between the two groups. Dechorionated embryos had a significantly greater embryonic body protein content after hatching (32, 33 d.p.f.) compared with chorionated embryos. Despite the differences in movement and growth, there were no significant differences in oxygen consumption between chorionated and dechorionated embryos. Furthermore, there was no correlation between the number of movements and oxygen consumption in rainbow trout embryos (chorionated, dechorionated, and hatched). Taken together, the data indicate that rainbow trout embryos have the capacity to be relatively active before hatching, but that the chorion restricts or inhibits movement. Moreover, precocious activity in pre-hatch embryos is correlated with accelerated growth and higher protein content, suggesting that the exercise training effect observed in adult salmonids is also present in early developmental stages.

Pharmacokinetics of morphine in fish: winter flounder (Pseudopleuronectes americanus) and seawater-acclimated rainbow trout (Oncorhynchus mykiss).

We made a single intraperitoneal (IP) injection of morphine sulfate (40 mg/kg) into winter flounder and seawater acclimated rainbow trout at 10 degrees C and then followed its disposition by measuring the change in plasma morphine concentration for 100 h using a morphine specific ELISA. Disposition also was followed for 6h after a single IV injection of 7.5mg morphine sulfate in winter flounder. Plasma morphine reached a maximum within an hour post-injection IP and then decreased in a bi-exponential fashion with a rapid distribution phase followed by a slower elimination phase. The disposition was slower in flounder than in trout even though the fish were held at the same temperature. For example, plasma clearance was 76 mL h(-)(1) kg(-)(1) in the flounder but was almost twice as much in the trout (153 mL h(-)(1) kg(-)(1)) and mean residence time was 27.9h in the flounder but was 7.0 h in the trout. The present study is the first comprehensive pharmacokinetic analysis for any analgesic in an ectotherm, and our results show that: 1) significant intra-specific variation exists between fishes: and 2) the disposition of morphine in fish is approximately one order of magnitude slower than it is in mammals. These differences may be due in part to mass specific differences in cardiac output.

Expression of four glutamine synthetase genes in the early stages of development of rainbow trout (Oncorhynchus mykiss) in relationship to nitrogen excretion.

The incorporation of ammonia into glutamine, catalyzed by glutamine synthetase, is thought to be important in the detoxification of ammonia in animals. During early fish development, ammonia is continuously formed as yolk proteins and amino acids are catabolized. We followed the changes in ammonia and urea-nitrogen content, ammonia and urea-nitrogen excretion, glutamine synthetase activity, and mRNA expression of four genes coding for glutamine synthetase (Onmy-GS01-GS04) over 3-80 days post fertilization and in adult liver and skeletal muscle of the rainbow trout (Oncorhynchus mykiss). Both ammonia and urea-nitrogen accumulate before hatching, although the rate of ammonia excretion is considerably higher relative to urea-nitrogen excretion. All four genes were expressed during early development, but only Onmy-GS01 and -GS02 were expressed at appreciable levels in adult liver, and expression was very low in muscle tissue. The high level of expression of Onmy-GS01 and -GS03 prior to hatching corresponded to a linear increase in glutamine synthetase activity. We propose that the induction of glutamine synthetase genes early in development and the subsequent formation of the active protein are preparatory for the increased capacity of the embryo to convert the toxic nitrogen end product, ammonia, into glutamine, which may then be utilized in the ornithine-urea cycle or other pathways.

A 2 week routine stretching programme did not prevent contraction-induced injury in mouse muscle.

Most athletes stretch as part of their training regimen and it is commonly believed that this practice prevents muscle injury. We tested this belief using an animal model, in situ mouse extensor digitorum longus (EDL) muscle. One lower hindlimb was slowly stretched for 1 min on alternate days for 12 days; the other leg served as a control. The mouse was lightly anaesthetized during the stretching protocol (isofluorane). Both legs were tested in situ by measuring maximum isometric force and maximum work before and after an eccentric contraction that was designed to cause a contraction-induced injury. The difference between a contraction before and after (i.e. the deficit) was used as a measure of damage caused by the eccentric contraction. There was a threshold for force deficit at a peak to peak eccentric excursion amplitude of 19.5 % (i.e. L(o) +/- 9.75 %, where L(o) is muscle length at peak isometric force). There was a significant increase in force deficit, work deficit, and curve shift with an increase in eccentric excursion amplitude above the threshold. There was no statistical difference in the force deficit, work deficit, or curve shift between the stretched leg and the control leg (P > 0.05). A routine stretching programme, at least at the intensities employed in this experiment, did not prevent contraction-induced injury in the in situ mouse EDL muscle.