3D cellular-based stroke model to investigate the protective mechanism of hypoxic postconditioning

Abstract

Stroke is the leading cause of disability and the second leading cause of mortality worldwide. However, due to the brain's intrinsic complexity and its poor accessibility, no effective treatments have been established to block secondary reperfusion injury and restore the brain functions after cerebral ischemia, necessitating the development of novel therapeutic strategies. For that, it is crucial to construct an appropriate experimental model that accurately recapitulates the pathophysiological events in cerebral ischemia while facilitating intuitive and rapid assessments. 3D spheroids composed of brain cells have emerged as a promising model to mimic the normal and pathological brain. It provides greater experimental simplicity than animal models and represents more densely packed neurons and close cell-cell interactions amongst various constituent cells of the 3D brain than 2D models. Nevertheless, a spheroidal brain-disease model of cerebral ischemia has not been reported to date. In this study, we aimed to investigate an ultra-low attachment (ULA) surface-mediated formation of 3D cortical spheroids using primary rat cortical cells to recapitulate the cerebral ischemic responses in stroke by oxygen-glucose deprivation-reoxygenation (OGD-R) treatment. The primary principle of existing cerebral ischemia therapies is the reperfusion of the ischemic brain tissue that remains salvageable. Accordingly, there is a lack of clinically approved therapies to block secondary reperfusion injury in cerebral ischemia. Hypoxic postconditioning (HPC) has been reported to protect the brain against secondary reperfusion injury and enhance long-term functional recovery under various protocols. However, the complete mechanisms underlying brain protection by HPC are far from clear. Notably, the role of astrocytes and microglia in mediating the neuroprotective effect of HPC remains elusive. In this study, we aim to investigate the optimal protocols for HPC in the established 3D cortical stroke model using cortical spheroids and ultimately unravel the effect of HPC on the activation status of astrocytes and microglia and their potential crosstalk.