Intranasal rosmarinic acid reduces cognitive and hippocampal damage from repeated neonatal isoflurane exposure by regulating apoptotic/ oxidative/inflammatory responses, and heat-shock and 14-3-3 proteins

Abstract

Repeated or prolonged exposure to general anesthetics like isoflurane (ISO) during neurodevelopment can lead to long-term neurocognitive and behavioral deficits, particularly because pediatric brains lack adequate antioxidant defenses, and no preventive therapies currently exist. Rosmarinic acid (RA), a polyphenolic compound with antioxidant and neuroprotective properties, has not yet been evaluated for mitigating ISO-induced toxicity. In this study, Wistar albino rat pups were exposed to ISO (1.5% in 30% oxygen/air, 3-h) on postnatal days (P)7 +P9 + P11, and the protective effects of intranasal RA (25 mg/kg) pretreatment (1-h before anesthesia) were investigated for the first time. Control groups received either oxygen alone or RA before oxygen exposure. On P12, hippocampal tissue was examined for detecting acute neuronal apoptosis, oxidative stress, inflammation, and stress-related proteins using histopathology and immunoblotting. Cognitive performance was assessed using Morris Water Maze tests that evaluated spatial learning (P28–P32) and both short- and long-term memory (P33, P60, P90). Repeated ISO exposure impaired learning and memory, increased anxiety-like behaviors, and caused hippocampal damage, along with elevated pro-apoptotic markers (Bax/Bcl-2 ratio, cleaved caspase-3, PARP1 fragments), redox imbalance and inflammation (reduced SOD1; increased GPX1, 4HNE, NF-κB-p65, TNF-α). ISO also disrupted stress signaling by reducing p-HSF1, Hsp90, and Hsp60 levels, while raising Hsp70 and decreasing 14–3–3 isoforms. RA pretreatment countered these effects by restoring antioxidant and stress-response proteins, reducing inflammation and apoptosis, and maintaining neuronal integrity and cognitive function. No harmful effects were observed in the RA-only group. These findings suggest that intranasal RA pretreatment may be a preventive strategy against anesthesia-related neurotoxicity in pediatric patients.