Long-Term Effects of Isoflurane Anesthesia and Surgery: Sex-Associated Neurological Dysfunction and Alterations in Circulating Extracellular Vesicles

Purpose: It is well established that volatile anesthetics and surgery induce acute and subacute changes in the cellular and molecular landscape of the brain and peripheral circulation and can impair neurological function. However, the chronic neurological sequelae of isoflurane (Iso) anesthesia combined with surgical operation (OP), as well as the underlying mechanisms of postoperative neurological dysfunction, remain poorly understood. Methods: Young adult male (M) and female (F) C57BL/6 mice underwent 4 h of 2% Iso plus laparotomy or sham treatment. At 12 weeks, olfactory and cognitive function were assessed by odor memory, buried food, Y-maze, and active avoidance tests. Olfactory bulbs (OB) and hippocampi (HI) were collected for RNAseq, while plasma extracellular vesicles (EVs) were isolated, characterized by NanoFCM, and profiled by Olink proteomics. Lastly, EVs were injected into the HI of naïve male mice, and cytokine/chemokine responses were measured 24 h later. Results: Both sexes showed olfactory impairment after chronic Iso/OP, with greater deficits in females. Iso/OP mice, especially females, exhibited impaired odor recognition in the OM test and longer latencies to locate buried food. Female mice also showed greater hippocampal-dependent spatial working memory deficits in the Y-maze, with more arm returns and fewer alternations than Sham/F mice, whereas Iso/OP/M mice performed similarly to controls. Likewise, female, but not male, Iso/OP mice displayed impaired associative learning in the active avoidance test, evidenced by fewer avoided trials and more escape responses. These long-term behavioral abnormalities were accompanied by sex-divergent transcriptomic remodeling in the OB and HI, including altered synaptic, neurodevelopmental, extracellular matrix, stress-response, and chemotaxis-related pathways. Iso/OP reduced plasma EV particle numbers in both sexes and shifted EV size distributions, with prominent reductions in the 40-100 nm EV fraction. Proteomics revealed distinct sex-and condition-specific EV profiles, with several EV-associated proteins showing opposing sex-dependent expression patterns. Hippocampal injection of Iso/OP-derived EVs induced donor sex-dependent cytokine remodeling, confirming inflammatory bioactivity. Conclusions: Four-hour isoflurane (Iso) exposure combined with laparotomy in young adult mice induces chronic, sex-dependent neurological deficits with distinct transcriptomic remodeling across brain subregions. Persistent alterations in circulating EV abundance and inflammatory cargo may drive chronic neuroinflammation and long-term brain dysfunction.