Prolonged Zero-Nicotine E-Cigarette Exposure Disrupts Blood–Brain Barrier Integrity and Promotes Neuroinflammation: Evidence from In-Vitro and In-Vivo Models

Electronic cigarettes (e-cigarettes), particularly nicotine-free (0%) formulations, are often perceived as safer alternatives to nicotine-containing products. However, the progressive and mechanistic basis of blood-brain barrier (BBB) injury following acute and sub-chronic 0% exposure remains poorly researched. In this study, we investigated whether short-term and prolonged 0% e-cigarette exposure alters BBB integrity using complementary in-vitro and in-vivo models. Using a well-established astrocyte-bEnd.3 co-culture BBB model, 24-hour exposure significantly reduced transendothelial electrical resistance (TEER) without increasing sodium fluorescein (NaF) permeability. Following prolonged 5-day exposure, TEER remained significantly reduced and was accompanied by increased NaF permeability. In contrast, permeability to 4 kDa and 70 kDa FITC-dextran tracers remained unchanged. Immunocytochemistry (ICC) demonstrated reduced claudin-5 mean fluorescence intensity (MFI) after 24 hours, along with reduced ZO-1 and claudin-5 MFI and disrupted junctional morphology following 5-day exposure. Western blot (WB) analysis additionally demonstrated a significant reduction in claudin-5 expression after 5 days. To assess translational relevance, male and female CD-1 mice were exposed to 0% aerosols for 7 or 14 days, with 7-day exposures producing no significant changes in BBB tight junction (TJ) proteins, inflammatory cytokines, or plasma cardiac troponin-l levels. 14-day exposures reduced ZO-1 expression in both sexes, occludin expression in males, and elevated pro-inflammatory cytokines IL-17A, GM-CSF, MCP-1, TNF-α, and IL-1α without affecting plasma troponin-1. Collectively, these findings demonstrate that repeated 0% e-cigarette exposure promotes progressive BBB dysfunction and a neuroinflammatory environment, highlighting the need to consider BBB vulnerability as a critical endpoint in 0% e-cigarette exposure toxicology.