Volatile anesthetics and surgical procedures are known to produce acute and subacute alterations in the brain and peripheral circulation that can impair neurological function. The long-term neurological consequences of combined isoflurane (Iso) anesthesia and surgery, and the mechanisms that sustain postoperative neurological dysfunction, remain incompletely characterized. This study examined chronic behavioral, transcriptomic, and circulating extracellular vesicle (EV) changes after Iso with laparotomy, testing the hypothesis that persistent EV-mediated inflammatory signaling contributes to sex-dependent long-term brain dysfunction.
Young adult male (M) and female (F) C57BL/6 mice underwent either 4 hours of 2% isoflurane exposure combined with laparotomy (Iso/OP) or a sham procedure. Animals were allowed to recover, and outcomes were assessed 12 weeks after the intervention. Behavioral assays evaluated olfactory and cognitive function and included odor memory (OM), buried food, Y‑maze, and active avoidance tests. Olfactory bulbs (OB) and hippocampi (HI) were harvested for bulk RNA sequencing to profile transcriptomic changes. Plasma EVs were isolated and characterized by NanoFCM for particle number and size distribution, and EV protein cargo was profiled using Olink proteomics. To test EV bioactivity, isolated EVs were injected into the hippocampus of naive male mice, and hippocampal cytokine/chemokine responses were measured 24 hours later.
At 12 weeks post-intervention, both male and female Iso/OP mice exhibited olfactory impairments compared with sham-treated controls, but deficits were larger in females. Female Iso/OP mice showed reduced odor recognition in the OM test and longer latencies to locate buried food. In hippocampal-dependent spatial working memory assessed in the Y‑maze, female Iso/OP mice demonstrated greater deficits characterized by more arm returns and fewer alternations relative to Sham/F mice, whereas Iso/OP/M mice performed similarly to male controls. In associative learning measured by the active avoidance test, female but not male Iso/OP mice showed impaired performance, with fewer avoided trials and more escape responses. Thus, chronic behavioral abnormalities after Iso/OP were sex-dependent and particularly pronounced in females across olfactory and hippocampal-dependent tasks.
RNAseq profiling of OB and HI tissue revealed sex-divergent transcriptomic changes after Iso/OP. Altered pathways included synaptic signaling, neurodevelopmental processes, extracellular matrix components, stress-response pathways, and chemotaxis-related genes. The findings indicate region-specific and sex-specific molecular remodeling that accompanied the observed long-term behavioral deficits. The source reports these pathway-level alterations but does not list specific differentially expressed genes or quantitative fold-change values in the summary.
Plasma EV analysis showed that Iso/OP reduced total EV particle numbers in both sexes and altered EV size distributions. The change was notable in the 40–100 nm EV fraction, which showed a prominent reduction after Iso/OP. Olink proteomics of purified EVs identified distinct profiles according to sex and experimental condition; several EV-associated proteins displayed opposing expression patterns between males and females following Iso/OP. The proteomic results indicate sex- and condition-specific shifts in EV cargo that could influence peripheral-to-central signaling.
To assess whether circulating EVs from Iso/OP animals carry biologically active inflammatory signals, the investigators injected Iso/OP-derived EVs into the hippocampi of naive male recipient mice. Twenty-four hours after injection, hippocampal cytokine and chemokine remodeling was observed and depended on the donor sex of the EVs, confirming that EVs from Iso/OP animals can induce region-specific inflammatory responses in recipient brain tissue.
Four-hour exposure to 2% isoflurane combined with laparotomy produced chronic, sex-dependent neurological deficits in young adult mice that persisted at least 12 weeks. Behavioral impairments included olfactory dysfunction in both sexes and greater hippocampal-dependent cognitive deficits in females. These long-term phenotypes were accompanied by sex-divergent transcriptomic remodeling in olfactory bulb and hippocampus and by persistent alterations in circulating extracellular vesicles—both in particle abundance and cargo composition. Functional testing demonstrated that Iso/OP-derived EVs can elicit donor sex-dependent inflammatory signaling in recipient hippocampus, supporting a model in which altered circulating EVs contribute to sustained neuroinflammation and long-term brain dysfunction after anesthesia and surgery.
The results emphasize the need to consider sex as a biological variable in studies of postoperative neurological outcomes and to investigate circulating EVs as mediators or biomarkers of chronic postoperative brain changes.
The study was supported by the National Institute on Aging (R01AG077541, RF1AG093965) and the National Institute of Neurological Disorders and Stroke (R01NS145443). The authors declared no competing interests. The source provides pathway- and assay-level summaries but does not report full gene lists, individual protein identities or quantitative effect sizes in the summary; those details would require reference to the full preprint or supplementary material.