However, the MWM data indicate that Enzalutamide CYP17 inhibitor donepezil does not make animals in the isoflurane+donepezil and donepezil groups more clever than the control mice. The Nilotinib mechanism responsible for these effects is unknown. Kakinuma found that ChAT levels in the ventricular myocardium increased after donepezil treatment, which was accompanied by an increase in ChAT promoter activity. More studies should be performed to determine the detailed mechanism for these effects. Although Zivin found that AChE mRNA in the brain increased significantly after 28 days of donepezil treatment, we did not find any changes in AChE protein levels among the four groups. Alpha7-nAChRs are one of the major functional nAChR subtypes in the brain, and these receptors play an important role in learning and memory. Although Takadatakatori showed the upregulation of a7-nAChRs in primary culture rat cortical neurons after chronic donepezil treatment, we did not observe any significant changes in a7-nAChRs levels between groups. With the two-electrode voltage-clamp technique, Jackson demonstrated that isoflurane and halothane inhibited acetylcholine-evoked currents of a7-nicotinic acetylcholine receptors in Xenopus oocytes in a reversible and concentration-dependent manner. Studies are needed to examine a7-nAChRs after donepezil and volatile anesthetic exposure in vivo. Six-hour isoflurane exposure in 30% oxygen resulted in stable blood pressure and heart rate, normal oxygenation, adequate PCO2 and moderate acidosis. Similarly, Szczesny demonstrated that 0.8�C1.3% isoflurane exposure for 6.5 hours resulted in a stable mean blood pressure and heart rate in mice. With an oximeter probe, Ewald found that oxygen saturation remained at,97% at anesthesia levels of 0.9%�C1.25% isoflurane. Because it is quite difficult to acquire a sufficient volume of arterial blood from mice without anesthesia for analysis, there is currently no record of normal blood gas values for mice. For this reason, many studies do not assess blood gases. Therefore, we do not 
know how to define and evaluate the effects of acidosis in mice. However, in the present study, no animals died after six hours of isoflurane exposure, which indicated that even if some minimum physiological changes occurred, they were of little clinical relevance. While this study has provided some interesting data, it also has limitations. To observe the long-term effects of isoflurane exposure, we only performed the behavioral and biochemical tests two weeks after isoflurane exposure, as did other studies, thus we do not know the acute effects of isoflurane exposure in aged mice. Saab found that in adult mice exposed to 1.3% isoflurane for 1 h, contextual fear memory persisted for 24 hours after isoflurane exposure. Our previous study showed that repeated isoflurane exposure improved spatial memory. Additional studies are needed to demonstrate the acute effects of isoflurane on the aged mice. In the present study, we only performed our behavioral and biochemical tests after donepezil pretreatment and isoflurane exposure; therefore, more studies should be performed to assess the causal relationship between the behavior and alterations in ChAT levels. The present study used aged mice, but caution should be paid to transferring the preventative effects of donepezil to other subjects. Although we demonstrated that donepezil prevented the isoflurane-mediated decrease in ChAT levels, more studies should be performed before donepezil can be clinically used to treat POCD. In conclusion, isoflurane exposure for six hours impaired the spatial memory of aged mice.