Stroke is a devastating neurological disorder and a leading cause of death and long-term disability. Despite many decades of research, there are still very few therapeutic options for patients suffering from stroke or its consequences. This is partially due to the limitations of current research models, including traditional in vitro models which lack the three-dimensional (3D) architecture and cellular make-up of the in vivo brain. 3D spheroids derived from primary postnatal rat cortex provide an in vivo-relevant model containing a similar cellular composition to the native cortex and a cell-synthesized extracellular matrix. These spheroids are cost-effective, highly reproducible, and can be produced in a high-throughput manner, making this model an ideal candidate for screening potential therapeutics. To study the cellular and molecular mechanisms of stroke in this model, spheroids were deprived of glucose, oxygen, or both oxygen and glucose for 24 hours. Both oxygen and oxygen-glucose deprived spheroids demonstrated many of the hallmarks of stroke, including a decrease in metabolism, an increase in neural dysfunction, and an increase in reactive astrocytes. Pretreatment of spheroids with the antioxidant agent N-acetylcysteine (NAC) mitigated the decrease in ATP seen after 24 hours of oxygen-glucose deprivation. Together, these results show the utility of our 3D cortical spheroid model for studying ischemic injury and its potential for screening stroke therapeutics.

Purpose Ischemic brain injury occurs when there is reduced or complete disruption of blood flow to a brain region, such as in stroke or severe traumatic brain injury. Even short interruptions can lead to devastating effects including excitotoxicity and widespread cell death. Despite many decades of research, there are still very few therapeutic options for patients suffering from brain ischemia. Methods We developed an in vitro brain ischemia model using our previously established 3D spheroids derived from primary postnatal rat cortex. These spheroids provide an in vivo-relevant model containing a similar cellular composition to the native cortex and a cell-synthesized extracellular matrix. This model is cost-effective, highly reproducible, and can be produced in a high-throughput manner, making it an ideal candidate for screening potential therapeutics. To study the cellular and molecular mechanisms of stroke in this model, spheroids were deprived of glucose, oxygen, or both oxygen and glucose for 24 h. Results Both oxygen and oxygen-glucose deprived spheroids demonstrated many of the hallmarks of ischemic brain injury, including a decrease in metabolism, an increase in neural dysfunction, breakdown in the neurovascular unit, and an increase in reactive astrocytes. Pretreatment of spheroids with the antioxidant agent N-acetylcysteine (NAC) mitigated the decrease in ATP after oxygen-glucose deprivation, was partially neuroprotective, and enhanced the expression of laminin. Conclusion This 3D cortical spheroid model provides a platform for studying ischemic injury and has the potential for screening therapeutics. Keywords 3D in vitro models • Brain ischemia • Screening • Central nervous system