Abstract / Summary
Abstract Secondary injury following ischemic traumatic brain injury (TBI) is heavily driven by neurovascular dysfunction involving endothelial damage, astrocyte reactivity, inflammation, and hypoxic stress, among other contributing pathophysiological processes. Here, we establish a mechanistically informative in vitro ischemic TBI model that isolates endothelial-astrocyte crosstalk using human brain microvascular endothelial cells (HBMECs) and astrocytes. Using this platform, we evaluated melatonin and oxygen nanobubbles (ONBs) as a combinatorial therapeutic strategy. Fabricated ONBs exhibited a mean hydrodynamic diameter of 78.35 ± 0.42 nm, a negative surface charge of −37.86 ± 1.32 mV, and sustained oxygen retention (56.38 ± 0.86 mg/L initially). Mechanical needle-scratch injury combined with hypoxia induced primary endothelial injury characterized by reduced viability and increased expression of inflammatory and hypoxia-associated markers, quantified by quantitative real-time PCR (qRT-PCR). Injured HBMECs subsequently triggered astrocyte reactivity marked by elevated GFAP (1.93-fold; p < 0.0001), NLRP3 (3.21-fold; p < 0.0001), NF-κβ signaling (1.62-fold; p < 0.0001), and HIF-1α (2.28-fold; p < 0.0001), while reactive astrocytes propagated a secondary wave of endothelial degeneration, establishing a bidirectional injury loop. Melatonin alone only partially mitigated injury-associated signaling, whereas the melatonin-ONBs combination restored astrocytic and endothelial gene expression to near-baseline levels. These findings highlight a multimodal therapeutic approach that effectively suppresses inflammatory, apoptotic, and hypoxic signaling in ischemic TBI.