Special Challenges in PET Imaging of Ectothermic Vertebrates.

The bulk of biomedical positron emission tomography (PET)-scanning experiments are performed on mammals (ie, rodents, pigs, and dogs), and the technique is only infrequently applied to answer research questions in ectothermic vertebrates such as fish, amphibians, and reptiles. Nevertheless, many unique and interesting physiological characteristics in these ectothermic vertebrates could be addressed in detail through PET. The low metabolic rate of ectothermic animals, however, may compromise the validity of physiological and biochemical parameters derived from the images created by PET and other scanning modalities. Here, we review some of the considerations that should be taken into account when PET scanning fish, amphibians, and reptiles. We present specific results from our own experiments, many of which remain previously unpublished, and we draw on examples from the literature. We conclude that knowledge on the natural history and physiology of the species studied and an understanding of the limitations of the PET scanning techniques are necessary to avoid the design of faulty experiments and erroneous conclusions.

Acidosis counteracts the negative inotropic effect of K+ on ventricular muscle strips from the toad Bufo marinus.

Strenuous activity is associated with acidosis, increased extracellular potassium concentration ([K+]o), and elevated levels of circulating catecholamines. Acidosis and elevated [K+]o are normally considered harmful to cardiac function, and a high sympathetic tone on the heart may lead to arrhythmia. During activity, however, the heart must be able to increase rate and strength of contraction. While the individual effects of [K+]o, acidosis, and adrenaline on contractile properties of cardiac muscle have been characterized for some ectothermic species, less information is available on their interactions. Here we examine the isolated and combined effects of [K+]o, acidosis, and adrenaline on ventricular muscle strips from the toad Bufo marinus. This study showed that increased [K+]o significantly reduced twitch force, while lactic acid significantly increased twitch force and more than counteracted the negative inotropic effects of elevated [K+]o. There was no inotropic effect of Na-lactate (neutralized lactic acid), which suggests that lactic acid stimulated twitch force through reduced pH and not by serving as a substrate. Adrenaline had a positive effect on twitch force in all preparations. Irrespective of treatment, twitch force decreased as stimulation rate increased. During high [K+]o, there was a severe reduction in maximal frequency of toad ventricular strips that was not alleviated by lactic acidosis and/or adrenaline, which suggests that high [K+]o influences twitch force and maximal rate by different mechanisms. In vivo levels of lactic acid, [K+]o, adrenaline, and heart rate previously observed during forced activity in Bufo did not significantly affect the contractile properties of heart muscle strips in vitro. Thus, although [K+]o significantly decreased twitch force, this detrimental effect was more than counteracted by the positive inotropic effect of lactic acid and adrenaline.

Cardiorespiratory effects of forced activity and digestion in toads.

Digestion and physical activity are associated with large and sometimes opposite changes in several physiological parameters. Gastric acid secretion during digestion causes increased levels of plasma bicarbonate ([HCO-3](pl)), whereas activity leads to a metabolic acidosis with increased lactate and decrease in plasma bicarbonate. Here we describe the combined effects of feeding and activity in the toad Bufo marinus to investigate whether the increased bicarbonate buffering capacity during digestion (the so-called alkaline tide) protects the acid-base disturbance during activity and enhances the subsequent recovery. In addition, we describe the changes in arterial oxygen levels and plasma ion composition, as well as rates of gas exchange, heart rates, and blood pressures. Toads were equipped with catheters in the femoral artery and divided into four experimental regimes: control, digestion, forced activity, and forced activity during the postprandial period (N=6 in each). Digestion induced a significant metabolic alkalosis with increased [HCO-3](pl) that was completely balanced by a respiratory acidosis; that is, increased arterial Pco(2) (P(a)co(2)), so that arterial pH (pH(a)) did not change. Forced activity led to a substantial reduction in pH(a) by 0.43 units, an increase in plasma lactate concentration by 12.5 mmol L(-1), and a reduction in [HCO-3](pl) of similar magnitude. While digesting animals had higher P(a)co(2) and [HCO-3](pl) at rest, the magnitude and duration of the changes in arterial acid-base parameters were similar to those of fasting animals, although the reduction in pH(a) was somewhat lower (0.32 units). In conclusion, while recovery from the acidosis following exercise did not seem to be affected by digestion, the alkaline tide did slightly dampen the reduction in pH(a) during activity.

Effects of anaesthesia on blood gases, acid-base status and ions in the toad Bufo marinus.

It is common practice to chronically implant catheters for subsequent blood sampling from conscious and undisturbed animals. This method reduces stress associated with blood sampling, but anaesthesia per se can also be a source of stress in animals. Therefore, it is imperative to evaluate the time required for physiological parameters (e.g. blood gases, acid-base status, plasma ions, heart rate and blood pressure) to stabilise following surgery. Here, we report physiological parameters during and after anaesthesia in the toad Bufo marinus. For anaesthesia, toads were immersed in benzocaine (1 g l(-1)) for 15 min or until the corneal reflex disappeared, and the femoral artery was cannulated. A 1-ml blood sample was taken immediately after surgery and subsequently after 2, 5, 24 and 48 h. Breathing ceased during anaesthesia, which resulted in arterial Po(2) values below 30 mmHg, and respiratory acidosis developed, with arterial Pco(2) levels reaching 19.5+/-2 mmHg and pH 7.64+/-0.04. The animals resumed pulmonary ventilation shortly after the operation, and oxygen levels increased to a constant level within 2 h. Acid--base status, however, did not stabilise until 24 h after anaesthesia. Haematocrit doubled immediately after cannulation (26+/-1%), but reached a constant level of 13% within 24 h. Blood pressure and heart rate were elevated for the first 5 h, but decreased after 24 h to a constant level of approximately 30 cm H2O and 35 beats min(-1), respectively. There were no changes following anaesthesia in mean cellular haemoglobin concentration, [K+], [Cl-], [Na+], [lactate] or osmolarity. Toads fully recovered from anaesthesia after 24 h.