Intramuscular infiltration of a local anesthetic, lidocaine, does not result in adverse behavioural side effects in rainbow trout.

Fish are a useful animal model for research, but our improvement in some aspects of their welfare has not kept pace with their increased popularity for this use. For example, researchers rarely use analgesics. We evaluated the side effects of lidocaine, a local anesthetic widely used in human and veterinary medicine. We infiltrated lidocaine on each side of the dorsal fin (total 20 mg/kg) of young rainbow trout (15 fish per group) compared with infiltration with an equal volume of saline. We monitored behaviour of individual trout during the 4-hour trial. Food was presented 5 times during the trial (30 min, 1 h, 2 h, 3 h, 4 h after infiltration) and we analyzed behaviour for 1 minute before and after food presentation. Behaviour of Saline-Infiltrated trout compared with trout that received no infiltration showed that infiltration in and of itself had no statistically significant effects on trout behaviour. However, there were many statistically significant effects of Lidocaine-Infiltrated trout compared with Saline-Infiltrated trout; none of the side-effects were adverse.

Evaluation of tissue changes following intramuscular infiltration of lidocaine in rainbow trout Oncorhynchus mykiss.

Rainbow trout Oncorhynchus mykiss were infiltrated with either saline or lidocaine adjacent to the dorsal fin to assess histopathological changes. Infiltration was done as if it were being used as a local anaesthetic. Tissue lesions and associated tissue healing were examined over a period of 30 days. Most changes occurred at the cranial site of where the solution was first infiltrated. The infiltration of a dose of 10 mg kg-1 of lidocaine appears to have damaged the skeletal muscle and connective tissues more than a similar volume of saline, especially during the first 15 days. The primary changes included haemorrhage, inflammation and muscle degeneration and necrosis. By day 30 post-infiltration inflammatory lesions were either nearly or completely absent, signs of myofibre regeneration were noted in only one fish. This experiment shows local anaesthetics and saline can produce localized tissue damage, especially during the first 2 weeks post infiltration. Care should be taken to allow the fish to heal for at least 30 days and probably more, no matter the solution administered, especially if giving repeated injections or infiltrations at the same site.

The dose-response relation for the antinociceptive effect of morphine in a fish, rainbow trout.

There have been suggestions that analgesics be used by fish researchers. But in the absence of dose-response data for morphine, this suggestion seems imprudent. The purpose of the present study was to develop a dose-response relationship in fish using six doses of morphine. The response (movement of the fins or tail) to a noxious stimulus (electrical shock to the face region) was monitored before and after a dose of morphine intraperitoneally (i.p.). The i.p. dose of morphine ED(50) in rainbow trout was 6.7 ± 0.8 mg/kg (n = 12 at each dose). The plasma morphine concentration EC(50) was 4.1 ± 1.5 mg/L. In a second experiment, rainbow trout tested with equal amounts of morphine and naloxone (30 mg/kg) showed that the antinociceptive effect of morphine was blocked by naloxone. It has been suggested that stress-induced analgesia has been a confounding factor in some fish studies. However, plasma cortisol levels in our study indicated that stress was not a confounding factor in the present experiments. The ED(50) for morphine in fish was higher than that reported for humans or other mammals. Our observation showing a dose-response relation for morphine using a noxious stimulus supports arguments for its effectiveness as an antinociceptive drug in fish.

Pharmacokinetics of morphine and its metabolites in freshwater rainbow trout (Oncorhynchus mykiss).

In this study, we injected morphine sulfate IP into rainbow trout and measured the concentration of morphine and all potential metabolites in plasma using LC-MS/MS at a series of times after the injection. The pharmacokinetics of morphine were similar to those previously reported for seawater-acclimated rainbow trout, i.e. they were about one order of magnitude slower than in similarly sized mammals. The only metabolite of morphine present in the plasma was morphine-3-beta-D-glucuronide (M3G); morphine-6-beta-D-glucuronide (M6G) was not detected. M3G gradually increased after the morphine injection, peaked about 2 days later, then gradually decreased. In mammals, M3G plasma levels exceed morphine levels extremely rapidly, i.e. in less than an hour, regardless of dose, route of administration, or species. In trout, it took 2 days for M3G levels to exceed morphine levels. This is the first study of the metabolites of morphine in any ectotherm. We conclude that trout can metabolize morphine, but at a rate much slower than in mammals.

Gill morphology of the mangrove killifish (Kryptolebias marmoratus) is plastic and changes in response to terrestrial air exposure.

Amphibious mangrove killifish, Kryptolebias marmoratus (formerly Rivulus marmoratus), are frequently exposed to aerial conditions in their natural environment. We tested the hypothesis that gill structure is plastic and that metabolic rate is maintained in response to air exposure. During air exposure, when gills are no longer functional, we predicted that gill surface area would decrease. In the first experiment, K. marmoratus were exposed to either water (control) or air for 1 h, 1 day, 1 week, or 1 week followed by a return to water for 1 week (recovery). Scanning electron micrographs (SEM) and light micrographs of gill sections were taken, and morphometric analyses of lamellar width, lamellar length and interlamellar cell mass (ILCM) height were performed. Following 1 week of air exposure, SEM indicated that there was a decrease in lamellar surface area. Morphometric analysis of light micrographs revealed that there were significant changes in the height of the ILCM, but there were no significant differences in lamellae width and length between any of the treatments. Following 1 week of recovery in water, the ILCM regressed and gill lamellae were similar to control fish, indicating that the morphological changes were reversible. In the second experiment, V(CO(2)) was measured in fish continuously over a 5-day period in air and compared with previous measurements of oxygen uptake (V(O(2))) in water. V(CO(2)) varied between 6 and 10 micromol g(-1) h(-1) and was significantly higher on days 3, 4 and 5 relative to days 1 and 2. In contrast to V(O(2)) in water, V(CO(2)) in air showed no diurnal rhythm over a 24 h period. These findings indicate that K. marmoratus remodel their gill structures in response to air exposure and that these changes are completely reversible. Furthermore, over a similar time frame, changes in V(CO(2)) indicate that metabolic rate is maintained at a rate comparable to that of fish in water, underlying the remarkable ability of K. marmoratus to thrive in both aquatic and terrestrial habitats.

A test of biochemical symmorphosis in a heterothermic tissue: bluefin tuna white muscle.

To test predictions of biochemical symmorphosis, we measured the activity of seven consecutive glycolytic enzymes at three positions along the heterothermic white muscle of the bluefin tuna. Biochemical symmorphosis predicts that adjustments in sequential enzyme concentrations along a thermal gradient should occur as a function of the thermal sensitivity of the enzymes to ensure that no one enzyme in the pathway is in excess at any point along the gradient. We found no evidence for adjustments in enzyme quantity or quality along the thermal gradient, as well as no evidence for the prediction that the more temperature-sensitive enzymes would exhibit more dramatic compensation. Conservation of glycolytic flux in the cold exterior and warm interior muscle may be achieved by the near insensitivity of glyceraldehyde-3-phosphate dehydrogenase to temperature in this tissue. This may have the added benefit of moderating flux during seasonal or transient changes in the thermal gradient. According to the strictest application of biochemical symmorphosis, such a mechanism represents adequate, yet suboptimal design.

Passive stretching does not protect against acute contraction-induced injury in mouse EDL muscle.

A popular part of many athletes pre-game regime is to stretch. We examined whether a pre-injury stretching protocol could prevent acute contraction-induced injury. The in situ extensor digitorum longus (EDL) muscle of an anesthetized mouse (80 mg/kg intra-peritoneal) was used. Damage to the muscle from eccentric contraction-induced injury was quantified by the deficit in tetanic force production, and was not confounded by metabolic fatigue. The force deficits resulting from eccentric contractions alone (E) were compared with the force deficits resulting from a protocol that consisted of a stretch before the eccentric contractions (S + E). The pre-injury stretch was performed to 5% L0 strain, at a velocity of 0.5 mm/s. The muscle was held in the stretch position for 1 min, then slowly released. Eccentric contraction protocols (excursion > or = 24% L0) resulted in pronounced force deficits that increased with the excursion amplitude of the eccentric contraction. The eccentric contractions also resulted in an average right shift of 2 +/- 0.53% in the length-force relationship (t-test, P = 0.0001). The regression lines for the E (eccentric contraction only) and S + E (stretch and eccentric contractions) treatments did not differ from one another for either force deficit (ANCOVA, P = 0.82) or work deficit (ANCOVA, P = 0.12). Therefore, the pre-injury stretch protocol did not reduce the force deficit or the work deficit resulting from contraction-induced injury.

The capacity of mdx mouse diaphragm muscle to do oscillatory work.

1. Mdx mice were used as a model for Duchenne muscular dystrophy; both lack dystrophin. It was hypothesized that the mdx condition would have a marked effect on the ability of diaphragm muscle from mdx mice to do active net work and generate power. This hypothesis was tested using the work-loop technique. 2. Specific twitch force, specific tetanic force and maximum power were all significantly less in diaphragm strips from mdx mice than those from control mice. 3. In all preparations muscle length at which maximum power was achieved (Lw) was about 8% less than that at which maximum tetanic force was achieved (L0), both in mdx and control muscle. 4. The isometric force-length curve for mdx muscle was steeper on both sides of the plateau. Similarly, the curve relating net work per cycle to muscle length was steeper for mdx muscle on both sides of the plateau. 5. Maximum power of mdx muscle was achieved at a lower strain than for control muscle; maximum power occurred at a strain of 10.2% for mdx and 14.7% for control. Further increases in strain caused a marked decrease of power production in mdx muscle, whereas they caused a smaller decrease in control muscle. 6. In summary, at muscle lengths longer than Lw and at high strains, performance of mdx muscle was compromised relative to that of control muscle. Work and power were compromised more than isometric force.

No evidence for homeoviscous adaptation in a heterothermic tissue: tuna heat exchangers.

Many poikilotherms are known to adjust the membrane composition of their cells in response to a temperature change so that membrane fluidity, and therefore function, is conserved. Such compensatory changes in membrane composition are considered "homeoviscous adaptations." In this study, we examined a heterothermic tissue, the visceral rete mirabile of the bluefin tuna, for evidence of homeoviscous adaptation. We measured the proportions of phospholipid fatty acids and phospholipid head groups as a function of position along the rete thermal gradient, which has been estimated to be approximately 10 degrees C. We found no effect of position along the rete on the composition of either phospholipid fatty acids or head groups. Our results were unexpected in light of our previous demonstration of compensation of metabolic enzyme activity in the same tissue. The lack of evidence for a homeoviscous response may be due to the fluctuating nature of the thermal gradient along the visceral retia; i.e., membranes may be adapted to a eurythermal existence rather than being fine-tuned to a particular temperature.

Crystal growth and molecular modeling studies of inhibition of struvite by phosphocitrate.

The inhibition by phosphocitrate of struvite crystal formation and growth has been examined in the present study. Crystal growth in a gel matrix was controlled by phosphocitrate in a dose-dependent manner. The effects of inhibition were followed using scanning electron microscopy, optical microscopy, and single crystal X-ray analysis. The presence of phosphocitrate induced very strong, crystal face specific inhibition of struvite, leading to total cessation of crystal growth when sufficient concentration of the inhibitor was made available. Crystal growth studies and results from molecular modeling indicated strong affinity of phosphocitrate to (101) faces of struvite. This in turn led to an alteration in the expression of these faces and the development of a characteristic arrowhead struvite morphology. Similar changes were not observed in the presence of identical concentrations of citrate, acetohydroxamic acid, and N-sulfo-2 amino tricarballylate (an analog of phosphocitrate), emphasizing the unique interaction of phosphocitrate with the struvite crystal lattice.

Enzyme adaptation along a heterothermic tissue: the visceral retia mirabilia of the bluefin tuna.

We measured enzyme activities along a heterothermic tissue, the visceral retia mirabilia of the bluefin tuna, to test current theories of enzyme temperature adaptation. The heterothermic tissue model is ideal for the study of fundamental temperature adaptation because it eliminates confounding effects of whole animal acclimation. Enzymes were measured at six positions along the rete at four temperatures (15, 20, 25, and 30 degrees C). Five enzymes (aspartate aminotransferase, citrate synthase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, and pyruvate kinase) exhibited a significant positive compensatory effect, with activity at the cold end of the rete 1.2-3.1 times higher than at the warm end. Two enzymes (alanine aminotransferase and lactate dehydrogenase) exhibited no significant compensation. On the basis of activation energies of enzymes along the rete, differences in activity were due to differences in enzyme concentration and not isozymes or enzyme modification. Analysis of the compensatory responses of the enzymes in light of their thermal sensitivities leads us to conclude that the pentose phosphate shunt is especially enhanced at the cold end of the rete.

The pattern of stimulation influences the amount of oscillatory work done by frog muscle.

1. A doublet is the interjection of an additional action potential at the beginning of a regular motoneuron discharge pattern. The present experiments were designed to estimate the magnitude of the effect of doublets on the capacity of isolated frog sartorius muscle to do work during oscillatory length changes. 2. For the work loop method, the muscle was subjected to sinusoidal length changes at 4 Hz. Work was calculated from the loop formed when force was plotted against length. Work done was positive when the muscle was shortening and was negative when the muscle was lengthening; net work was the difference. 3. Adding pulses at the start of the stimulus pulse train increased isometric force and markedly increased net work per cycle. Adding one pulse increased the net work by about 52%, but increased isometric force by only 24%. 4. Eighty per cent of the maximum net work was achieved by adding only three pulses at the start of a low-frequency pulse train, i.e. 80% of the work achieved with seventeen pulses could be achieved with only six pulses. 5. The maximum net work per stimulus pulse was achieved with a stimulus train that consisted of five pulses with two being additional initial pulses at 5 ms interpulse intervals, i.e. a triplet. 6. The results of the present study imply that doublet stimulation patterns may be important to reduce the fatigue that occurs during artificial neuromuscular stimulation of skeletal muscle.

Effect of phase of stimulation on acute damage caused by eccentric contractions in mouse soleus muscle.

Eccentric contractions (activation during muscle lengthening) can cause muscle damage. The effect of phase of stimulation on the extent of muscle damage was studied by using the work-loop method. For the work-loop method, the muscle was subjected to sinusoidal length changes at 2 Hz. The muscle was activated at different times during the imposed length-change cycle; this time is called the phase of stimulation. Work was calculated from the loop formed when force was plotted against length. Work done was positive when the muscle was shortening and was negative when the muscle was lengthening; net work was the difference. One complete length-change cycle was 100 (i.e., given as a percentage of the cycle); shortening occurred from 25 to 75. The muscle did the most net work when stimulated at phase 20, that is, when activation started just before shortening. Damage was defined as a decrease in work. Significant damage occurred after a single trial of three consecutive eccentric contractions; the muscle did less positive and less net work because of the damage. Maximal damage occurred at phases 90 and 0, the center of the lengthening part of the length-change cycle (work decreased 10%). Negligible damage occurred at phases 20-40. Negative work (work required to lengthen the muscle) also decreased because of the damage. Eccentric contractions caused much more damage than concentric contractions during oscillatory work.

Comparative contractile dynamics of calling and locomotor muscles in three hylid frogs.

Isometric twitch and tetanus parameters, force-velocity curves, maximum shortening velocity (Vmax) and percentage relaxation between stimuli (%R) across a range of stimulus frequencies were determined for a muscle used during call production (the tensor chordarum) and a locomotor muscle (the sartorius) for three species of hylid frogs, Hyla chrysoscelis, H. versicolor and H. cinerea. The call of H. chrysoscelis has a note repetition rate (NRR) approximately twice as fast as the call of H. versicolor (28.3, 42.5 and 56.8 notes s-1 for H. chrysoscelis and 14.8, 21.1 and 27.4 notes s-1 for H. versicolor at 15, 20 and 25 degrees C, respectively). Hyla cinerea calls at a very slow NRR (Approximately 3 notes s-1 at 25 degrees C). Hyla versicolor evolved from H. chrysoscelis via autopolyploidy, so the mating call of H. chrysoscelis is presumably the ancestral mating call of H. versicolor. For the tensor chordarum of H. chrysoscelis, H. versicolor and H. cinerea at 25 degrees C, mean twitch duration (19.2, 30.0 and 52.9 ms, respectively), maximum isometric tension (P0; 55.0, 94.4 and 180.5 kN m-2, respectively), tetanic half-relaxation time (17.2, 28.7 and 60.6 ms, respectively) and Vmax (4.7, 5.2 and 2.1 lengths s-1, respectively) differed significantly (P < 0.05) among all three species. The average time of tetanic contraction to half-P0 did not differ significantly between H. chrysoscelis (14.5 ms) and H. versicolor (15.8 ms) but was significantly longer for H. cinerea (52.6 ms). At 25 degrees C, Vmax differed significantly among the sartorius muscles of H. chrysoscelis, H. versicolor and H. cinerea (5.2, 7.0 and 9.8 lengths s-1, respectively) but mean twitch duration (29.5, 32.2 and 38.7 ms, respectively), P0 (252.2, 240.7 and 285.1 kN m-2, respectively) and tetanic half-relaxation time (56.3, 59.5 and 60.7 ms, respectively) did not differ significantly. The average time of contraction to half-P0 did not differ significantly between H. chrysoscelis (23.7 ms) and H. versicolor (22.9 ms) but was significantly shorter for H. cinerea (15.6 ms). The only consistent contractile differences found in this study between the calling muscle and locomotor muscle of H. chrysoscelis, H. versicolor and H. cinerea were that the calling muscles generated less tension and their force-velocity relationship was much more linear. These differences may be attributable to ultrastructural differences between calling and locomotor muscles.(ABSTRACT TRUNCATED AT 400 WORDS)

Scanning electron microscopy and molecular modeling of inhibition of calcium oxalate monohydrate crystal growth by citrate and phosphocitrate.

Binding of citrate and phosphocitrate to calcium oxalate monohydrate crystals has been studied using scanning electron microscopy (SEM) and molecular modeling. Phosphocitrate structure has been resolved using low temperature X-ray analysis and ab initio computational methods. The (-1 0 1) crystal surface of calcium oxalate monohydrate is involved in binding of citrate and phosphocitrate, as shown by SEM and molecular modeling. Citrate and phosphocitrate conformations and binding energies to (-1 0 1) faces have been obtained and compared to binding to another set of calcium-rich planes (0 1 0). Difference in inhibitory properties of these compounds has been attributed to better coordination of functional groups of phosphocitrate with calcium ions in (-1 0 1). Relevance of this study to design of new calcium oxalate monohydrate inhibitors is discussed.