Inhibition of Firefly Luciferase by General Anesthetics: Effect on In Vitro and In Vivo Bioluminescence Imaging

Abstract

Bioluminescence imaging is routinely performed in anesthetized mice. Often isoflurane anesthesia is used because of its ease of use and fast induction/recovery. However, general anesthetics have been described as important inhibitors of the luciferase enzyme reaction.AimTo investigate frequently used mouse anesthetics for their direct effect on the luciferase reaction, both in vitro and in vivo.Materials and Methodsisoflurane, sevoflurane, desflurane, ketamine, xylazine, medetomidine, pentobarbital and avertin were tested in vitro on luciferase-expressing intact cells, and for non-volatile anesthetics on intact cells and cell lysates. In vivo, isoflurane was compared to unanesthetized animals and different anesthetics. Differences in maximal photon emission and time-to-peak photon emission were analyzed.ResultsAll volatile anesthetics showed a clear inhibitory effect on the luciferase activity of 50% at physiological concentrations. Avertin had a stronger inhibitory effect of 80%. For ketamine and xylazine, increased photon emission was observed in intact cells, but this was not present in cell lysate assays, and was most likely due to cell toxicity and increased cell membrane permeability. In vivo, the highest signal intensities were measured in unanesthetized mice and pentobarbital anesthetized mice, followed by avertin. Isoflurane and ketamine/medetomidine anesthetized mice showed the lowest photon emission (40% of unanesthetized), with significantly longer time-to-peak than unanesthetized, pentobarbital or avertin-anesthetized mice. We conclude that, although strong inhibitory effects of anesthetics are present in vitro, their effect on in vivo BLI quantification is mainly due to their hemodynamic effects on mice and only to a lesser extent due to the direct inhibitory effect.

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“…The depth and location of the tumor as well as the thickness or color of the animal’s skin can alter the bioluminescent signal (Baba et al, 2007), as well as the choice of anesthesia used (Keyaerts et al, 2012). The route of administration and the timing of imaging after D-luciferin injections can also impact the signal.…”

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“…Working stock solutions can be stored for a limited time at -80°C in light proof container but substrate should be stored as a powder long term. Substrates are administered at a weight-dependent dose [53]. The increase in demand for BLI has increased the number of suppliers of substrate, which has reduced the initial prohibitive high cost.…”

“…However, dosage should be optimized by individual investigators and varies between animal models. Notably anesthetics can have different effects on the blood system, which can alter the BLI results [53]. Anesthetics can also potentially have an effect on the long-term health of the animal.…”

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“…Pentobarbital was found to have a less inhibitory effect. The possible reasons for this inhibitory effect might be attributable to the hemodynamic effects of anesthetics [135] and the binding of anesthetics to the domain that regulates the opening and closing of the enzymatic pocket, and to the inhibition of the binding of D-luciferin to luciferase [136]. Concentrations of serum proteins may also have negative effects on BLI signal intensity.…”