Electrochemical monitoring of early astrocytic responses underlying the synergistic effect of extracellular matrix softening and hypoxia

Traumatic brain injury (TBI) disrupts central nervous system homeostasis, leading to extracellular matrix (ECM) softening and localized hypoxia and thus contributing to astrocytic activation and sustained neuroinflammation. But the interplay between ECM softening and hypoxia in regulating astrocytic activation and response remains elusive. To understand this, we developed an in vitro model incorporating tunable-stiffness hydrogels and a precise oxygen-control system to simulate the mechanical and hypoxic microenvironment of TBI. We characterized the activation-related proteins and cytokine production of astrocytes under the in vitro model, and found that soft ECM and hypoxia independently promote astrocytic activation and synergistically activate astrocytes via HIF-1α/YAP-NF-κB signaling, resulting in astrocytic redox imbalance and neuroinflammation. We further used scanning electrochemical microscopy (SECM) to track the dynamic changes in glutathione (GSH) efflux and membrane integrity in live astrocytes in situ under pathophysiological conditions of ECM softening and hypoxia. The SECM results show that the combined ECM softening and hypoxia progressively impair cellular membrane integrity and promote GSH efflux of astrocytes, corresponding to the early changes in astrocytic function and indicative of an early activation-primed state to exacerbate secondary injury of astrocytes. Last, we found that the lovastatin (a neuroprotective agent) treatment can effectively attenuate astrocytic membrane impairment and decrease GSH efflux, proving the potential of lovastatin to mitigate inflammation and preserve neuroregulatory function. Our work observes the in situ and early state changes of astrocytes under a combined mechanical-hypoxic microenvironment for the first time. The findings offer mechanistic insights into TBI pathogenesis and highlight promising strategies for early therapeutic intervention.