Brief, repeated isoflurane anaesthesia elicits sex-dependent behavioural, cerebrovascular, and microglial alterations in mice

Abstract General anaesthetics are indispensable tools in biomedical research, yet growing evidence shows that their effects on brain function extend beyond the transient loss of consciousness for which they are used. Although numerous preclinical studies have examined the impact of a single anaesthetic exposure, far less is known about the cumulative consequences of repeated administration protocols, despite their widespread use in longitudinal imaging, behavioural testing, and disease model studies. Here, we show that brief, repeated isoflurane exposures induce alterations in behaviour, cerebrovascular patterns, and microglial phenotype in adult CD1 mice, with several responses exhibiting pronounced sex specificity. Repeated anaesthesia suppressed locomotor activity selectively in females and impaired ethologically important maintenance behaviours, including nesting and burrowing, in both sexes. These behavioural effects were accompanied by sex-dependent changes in cerebral blood flow dynamics, with females displaying elevated cerebrovascular flow, yet males showing higher reactivity to isoflurane exposure. At the molecular level, repeated isoflurane exposure altered endothelial marker expression without modifying capillary density and shifted microglia toward a hyporeactive state. Together, these data reveal that even short, routine anaesthetic events produce biologically meaningful and sex-dependent effects on neural, vascular, and immune systems. These results indicate that the anaesthetic regimen is an often overlooked but influential factor that deserves careful consideration in pre-clinical neuroscience. Competing Interest Statement The authors have declared no competing interest.