In the present study, we demonstrated that sevoflurane affects the A1 receptor at the neuromuscular junction and delays sugammadex-induced recovery from rocuronium-induced neuromuscular blockade. Adenosine acts as an A1 agonist at a low concentration (300 nM), and as an A2A agonist at high concentration (> 1 µM) . Furthermore, it has been demonstrated that enflurane and sevoflurane have the ability to activate adenosine A1 receptors in an in-vitro culture of rat hippocampus . Aminophylline is a nonselective antagonist of the adenosine receptor , and it can decrease the sedation effects of sevoflurane [5, 18], but not the anaesthesia induced by desflurane . Sevoflurane is one of the most potent volatile anaesthetics that potentiates the effect of neuromuscular blocking agents . The present study results are in consistent with these findings. Sugammadex has no effect on the neuromuscular junction and ACh release. Sugammadex-induced recovery from rocuronium-induced neuromuscular blockade is dependent on the relative concentration of rocuronium and ACh at the neuromuscular junction. Thus, the T1 twitch response to the indirect nerve stimulation reappears in the presence of a large amount of ACh molecules at the neuromuscular junction, because the ACh molecules have a greater chance to attach to AChR than rocuronium. When the action of the receptor or channel related to the release of ACh was modulated and the release of ACh in the neuromuscular presynaptic membrane was reduced, there was a delay in recovery from neuromuscular blockade . In the present study, the modulation of adenosine receptor with CADO and sevoflurane, delayed T1 recovery and resulted in a low recovery index compared with those of the control (no receptor modulation). We speculated that the modulation of the A1 receptor at the presynaptic membrane of the neuromuscular junction by sevoflurane could partially induce delayed recovery from neuromuscular blockade. We did not change the current for phrenic nerve stimulation and thus, the magnitude of indirect stimulation to the phrenic nerve might not have changed. However, in this study, the amount of ACh released per indirect stimulation might have reduced during sugammadex-induced recovery from neuromuscular blockade because of the activation of the A1 receptor in the neuromuscular junctions by sevoflurane. Furthermore, sugammadex binds to rocuronium only outside the neuromuscular junction, and there is a concentration difference in rocuronium between the neuromuscular junction and organ bath; consequently, rocuronium is removed from the neuromuscular junction. In the present study, the decrease in rocuronium concentration at the neuromuscular junction was thought to be similar among all groups because we used equimolar dose of sugammadex and rocuronium in the organ bath. Rocuronium molecules were able to bind to the AChRs at the postsynaptic membrane because the amount of ACh released was less at the same stimuli that caused presynaptic A1 receptor modulation by sevoflurane. This might have resulted in the delayed recovery from rocuronium-induced neuromuscular blockade in the present study.
The present study had some limitations. First, this was an ex-vivo study. We extracted phrenic nerve-hemidiaphragm tissue specimens after sacrificing SD rats. During this phase, although we handled the specimen in the Krebs buffer solution with 95% O2/CO2 gas bubbling, transient hypoxemia and tissue damage were inevitable. Furthermore, pharmacokinetic component was abolished during all phases of the experiment. To compensate for these limitations, we cautiously performed the following steps. (1) tissue specimens were extracted and immersed in a Petri dish and organ bath containing oxygenated Krebs buffer solutions throughout the experiment to minimize tissue hypoxia. (2) Maintaining the concentration of sevoflurane in the organ bath was another challenge. In the clinical setting, sevoflurane is supplied using an exclusive vaporizer and a closed circuit system, and its concentration is expressed as vol%. However, in the present study, it was difficult to develop a closed circuit system because frames, electrods, and strings connected to the force transducer were out from the orifice of organ bath. Furthermore, scavenging system of our laboratory was not suitable for use with volatile anesthetics. Instead, to minimize air pollution in the laboratory, we had to find alternative method simulating a closed circuit sustem to apply sevoflurane. We sealed the organ bath with a flexible film and added sevoflurane intermittently to achieve the desired concentration in the Krebs buffer solution. In a clinical experiment, it took about 40 min for sevoflurane to equilibrate between blood and muscle component and make effect on muscle [20, 21]. However, in some studies, sevoflurane was only needed 10 min for sevoflurane to make effect . In our experiment, we shortened this reaction time to 10 min by applying sevoflurane directly to the environment of tissue specimen. This was quite different approach comparing with the clinical setting. Moreover, as sevoflurane is volatile, it is very difficult to maintain the designated concentration of sevoflurane in the organ bath, and fluctuation in its concentration was inevitable. The dose of sevoflurane was selected as 400–500 µM at the point of initiation of recovery. This was determined before the main experiment by performing a pilot study and by referring to a previous study . Second, this was a functional study, not an immunochemistry study. We performed this experiment by measuring the tension generated by diaphragm contraction. That is, we deduced the results in an indirect manner, as the tension is thought to be driven by the ACh concentration differences at the neuromuscular junction. For accurate data, timely measurement of the amount of ACh released during serial indirect stimulation is required. However, we could not find ideal method for this. The ACh concentrations should be measured repeatedly at 20 s interval. We performed a conventional functional study that is commonly used in neuromuscular studies. To obtain more convincing results, a more suitable immunohistochemical study should be conducted in the future.