We used tadpoles to address the question of whether isoflurane could produce tolerance in a vertebrate. We chose to use tadpoles because week-long exposures to high concentrations of anesthetic are possible in this animal, they have many of the same neurotransmitters (e.g., γ-aminobutyric acid type A, glycine, glutamate, acetylcholine) and anatomic substrates (forebrain, hindbrain, spinal cord) as more complex vertebrates, and most importantly tadpoles have been used as a model organism to study mechanisms of anesthetic action (17
The isoflurane concentrations we used were equivalent to 0.4 to 1 MAC in mice or rats. Keeping sufficient numbers of rats or mice healthy while exposed continuously to these concentrations of isoflurane for 7 days would have posed significant husbandry (and ethical) issues. For example, the animals would need to be fed and hydrated while anesthetized, and body temperature and airway patency would have to be monitored and maintained around the clock. These issues were not present in developing tadpoles. Although the isoflurane concentration we used reduced spontaneous movement in tadpoles, in their first week of development tadpoles derive nutrition from their yolk sac and have none of the aforementioned care requirements.
Our tadpoles were raised in an aqueous environment at room temperature (averaging 22°C in our laboratory). Because the potency of volatile anesthetics delivered in the gas phase increases as temperature decreases (21
), the gas phase concentrations we report place a lower limit on the MAC fraction of isoflurane we applied. However, even without a correction for temperature, ours are the highest concentrations that have been used in any study of anesthetic tolerance in vertebrates. At the highest exposure, tadpoles were exposed to approximately three times their anesthetic EC50
Although we found statistically significant effects of prolonged treatment with isoflurane on the concentration of isoflurane required to prevent movement in Xenopus laevis tadpoles, this effect was small and varied from at most a 7.4% increase in EC50 (with exposure to 0.98% isoflurane for one week) to a 16.2% decrease in EC50 (with exposure to 1.52% isoflurane). This decrease is in the opposite direction expected for tolerance. Possibly, the presence of isoflurane throughout development led to compensation for the presence of isoflurane. Because the observed effects are small and there was no consistent direction to the change in EC50 over a range of isoflurane concentrations, these effects are probably of little biological significance. Given the small and inconsistent effects we observed at one week, it is doubtful that shorter exposures, which might correspond to the duration of surgical procedures would produce measurable effects. We conclude that tolerance does not develop in tadpoles with exposure to isoflurane. Our results are in accord with other published studies that fail to show tolerance to halogenated clinical anesthetics at lower concentrations or with shorter exposures.
At the two higher concentrations of isoflurane, some animals died in the course of the study. Might the loss of these animals have biased our results, by selecting for animals with isoflurane EC50s that promoted survival? Although we cannot exclude this, we think selection, if present, would have biased the data in favor of producing tolerance. We reasoned that animals that were particularly sensitive to isoflurane, with lower EC50s, should have succumbed to exposure to isoflurane, rather than ones that were more resistant to isoflurane and had higher EC50s. This would have enriched the remaining population in animals with higher EC50s. That there was no increase in EC50 at the highest concentration, and only small increases at lower concentrations, in the face of possible selection, strengthens our conclusion that tolerance did not develop.
We did not design our study to evaluate possible toxic effects of isoflurane during development; however, developing Xenopus laevis
tadpoles are an established model for these effects. The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) model, and modifications thereof (22
), use procedures similar to ours. The FETAX test concludes on day 4, at which time the number of abnormalities and deaths are totaled and compared between treatment groups. We determined the cumulative number of abnormalities and deaths in each group by day 4 and asked whether there were differences between groups in this measure. Although at the two higher concentrations, some morphological abnormalities were present while none were present at the two lower concentrations, and there was a greater frequency of deaths in the control (no isoflurane) group, the total number of deaths and abnormalities combined did not differ between any of the isoflurane exposure groups and the control group over this span of time (P > 0.05). However, by day 6 and day 7/8, there were significantly more abnormalities and deaths in the two highest isoflurane exposure groups compared to control, but not in the lowest isoflurane exposure group. While this might suggest a toxic or teratogenic effect to high or prolonged exposures to isoflurane, the clinical relevance of this finding is questionable since surgical procedures during preganancy do not expose developing human fetuses to isoflurane over such a large fraction of their development. The finding of toxicity from isoflurane in developing Xenopus laevis
tadpoles would be of concern if there were a short but crucial time when exposure to isoflurane is deleterious in humans, but this is a situation for which there is currently no evidence.
A variety of microorganisms respond to long term exposure to anesthetics by changing the composition of their membranes (23
). This is presumably part of a general response to changing environmental conditions, which allows the organism to compensate for perturbations in membrane properties produced by chemicals in the environment. This response might be considered to produce “tolerance” in these organisms. We did not investigate membrane compositional changes in tadpoles reasoning that because vertebrates regulate their internal milieu, there would be no selection to maintain such a mechanism. The lack of tolerance of Xenopus laevis
tadpoles to isoflurane in this study suggests either that we were correct in thinking that this mechanism does not occur in Xenopus laevis
, or that the mechanism occurs but has little functional effect.
In summary, we provide the first description of week-long exposures of vertebrates to surgical anesthetic concentrations of isoflurane, and the first report of such exposures in developing vertebrates. We conclude that tolerance to isoflurane does not occur in developing Xenopus laevis tadpoles. Taken together with studies in other organisms, the development of tolerance to ethanol but not isoflurane indicates that molecular mechanisms shared with isoflurane probably cannot account for the development of tolerance to ethanol.