A novel method for inducing focal ischemia in vitro

Abstract

Current in vitro models of stroke involve applying oxygen-glucose deprived (OGD) media over an entire brain slice or plate of cultured neurons. Thus, these models fail to mimic the focal nature of stroke as observed clinically and with in vivo rodent models of stroke. Our aim was to develop a novel in vitro brain slice model of stroke that would mimic focal ischemia and thus allow for the investigation of events occurring in the penumbra. This was accomplished by focally applying OGD medium to a small portion of a brain slice while bathing the remainder of the slice with normal oxygenated media. This technique produced a focal infarct on the brain slice that increased as a function of time. Electrophysiological recordings made within the flow of the OGD solution ("core") revealed that neurons rapidly depolarized (anoxic depolarization; AD) in a manner similar to that observed in other stroke models. Edaravone, a known neuroprotectant, significantly delayed this onset of AD. Electrophysiological recordings made outside the flow of the OGD solution ("penumbra") revealed that neurons within this region progressively depolarized throughout the 75 min of OGD application. Edaravone attenuated this depolarization and doubled neuronal survival. Finally, synaptic transmission in the penumbra was abolished within 50 min of focal OGD application. These results suggest that this in vitro model mimics events that occur during focal ischemia in vivo and can be used to determine the efficacy of therapeutics that target neuronal survival in the core and/or penumbra.

Ischemic stroke: experimental models and reality

“…On the one hand, lack of perfused vessels in brain slices clearly represents an artificial situation that on the other hand allows separating the ischemic effects on neuronal tissue from those due to actions on the cerebrovascular system [28]. Since all in vitro systems primarily mimic the situation of global cerebral ischemia, there are attempts to more closely model the situation of ischemic stroke, i.e., focal cerebral ischemia [101]. To this end, OGD medium is only focally applied to the brain slice while the rest is kept in normal oxygenated media.…”

Modeling Ischemic Stroke In Vitro: Status Quo and Future Perspectives

“…Richard et al ., have built upon this model to provide focal OGD achieved by perfusing OGD aCSF over a brain slice in a focal stream, while the remainder of the slice is perfused with normal aCFS. Using this model in rodent brain slices, Richard et al ., have demonstrated rapid neuronal depolarization in the anoxic core, followed by progressive depolarization in the “penumbra” region 18 .…”

Therapeutic Use of Creatine in Brain or Heart Ischemia: Available Data and Future Perspectives

“…The central part of this area (“core”) is severely ischemic. Here, anoxic depolarization (AD) occurs soon after ischemia and neurons die rapidly by necrosis . By contrast, in the more peripheral region (“penumbra”) of the ischemic area, neurons are exposed to a lesser degree of ischemia and die mostly by apoptosis, free radicals injury, recurrent periinfarct depolarizations, and excitotoxicity .…”

“…Here, anoxic depolarization (AD) occurs soon after ischemia and neurons die rapidly by necrosis. [2][3][4][5] By contrast, in the more peripheral region (''penumbra'') of the ischemic area, neurons are exposed to a lesser degree of ischemia and die mostly by apoptosis, 6 free radicals injury, 7 recurrent periinfarct depolarizations, 8 and excitotoxicity. 9 In turn, these various mechanisms of neuronal damage worsen energy failure by damaging, among others, the mitochondria and further decreasing the production of Adenosine Triphosphate (ATP).…”