Abstract
Traumatic brain injury (TBI) frequently leads to chronic neurovascular dysfunction, yet mechanistic insights into human-specific responses have been limited by the absence of long-term, multicellular in vitro models. Here, we report a five-cell-type human neurovascular culture system, comprising endothelial cells, astrocytes, pericytes, microglia, and neurons, engineered within a 3D scaffold to study injury-induced remodeling over multiple weeks. This PENTA-culture platform recapitulates hallmark features of the neurovascular unit and enables dissection of cell-specific contributions to vascular repair and degeneration. Upon mechanical trauma, cultures exhibit a biphasic response marked by acute endothelial disintegration, mitochondrial stress, and glial activation, followed by a delayed and incomplete repair. Confocal and proteomic analyses reveal persistent disruptions in tight junction organization, elevated TDP-43 and APP expression, and altered angiogenic and immunomodulatory signaling involving Tie2 and JAK/STAT pathways. Compared to simpler culture systems, the inclusion of microglia and neurons enhances post-injury cytokine resolution and junctional recovery, underscoring the importance of neuroimmune crosstalk. This system offers a mechanistically rich, human-relevant model for studying chronic neurovascular dysfunction and therapeutic revascularization.