Maternal E-Cigarette Exposure Exacerbates Neonatal Ischemic Brain Injury Through the miR-30c-5p-Neurod1 Axis

Background: Neonatal ischemic stroke is an important cause of mortality and long-term neurological morbidity in infants. Maternal smoking and e-cigarette exposure are recognized risk factors for adverse neonatal outcomes, but the mechanisms underlying increased susceptibility to ischemic brain injury remain unclear. This study investigated whether dysregulation of the miR-30c-5p-Neurod1 axis contributes to this susceptibility. Methods: Pregnant rats were exposed to e-cigarette aerosols from embryonic day 4 to 20. Studies were conducted in male offspring on postnatal day 10. Baseline molecular changes were assessed using microRNA sequencing, quantitative reverse-transcription polymerase chain reaction, Western blotting, RNA-induced silencing complex (RISC) and luciferase reporter assays. Ischemic injury was evaluated following transient middle cerebral artery occlusion (tMCAO). Flow cytometry and molecular analyses were performed to characterize associated cellular changes. The effects of in vivo miR-30c-5p mimic administration, Neurod1 knockdown, and miR-30c-5p inhibition were also investigated. Results: Maternal e-cigarette exposure increased tMCAO-induced infarct size and was associated with increased apoptosis and senescence-associated changes, together with reduced expression of markers of neurogenesis and synaptic development. Under baseline conditions, microRNA profiling and molecular analyses revealed increased miR-30c-5p expression and decreased Neurod1 mRNA and protein levels in the neonatal brain. RISC and luciferase reporter assays supported an interaction between miR-30c-5p and Neurod1 mRNA. In vivo administration of a miR-30c-5p mimic reduced Neurod1 expression and increased infarct size, whereas Neurod1 knockdown independently exacerbated ischemic injury and associated cellular changes. Conversely, inhibition of miR-30c-5p restored Neurod1 expression, reduced infarct size, and ameliorated associated pathological changes. Conclusions: Maternal e-cigarette exposure is associated with baseline dysregulation of the miR-30c-5p-Neurod1 axis and increased susceptibility to neonatal ischemic brain injury in male rats. These findings suggest that this regulatory axis may contribute to the effects of prenatal e-cigarette exposure on the developing brain and may increase susceptibility to neonatal ischemic stroke.