Investigating the Initiation of Spreading Depolarizations

Abstract

Ischemic stroke is one of the most prevalent, devastating and therapeutically challenging medical conditions. Following brain ischemia, failure of the Na+/K+ ATPase is linked with a massive cationic influx through an unidentified channel that drives spreading depolarization (SD). With focal stroke, SD can devastate neurons in the ischemic core as well as recur and expand the affected area for hours and even days after the initial incident. Despite the critical role of SD in ischemic brain pathophysiology, the link between Na+/K+ ATPase failure and the opening of the channel that drives SD onset is unclear. This study proposes that an SD activator (SDa) is released by metabolically stressed gray matter and drives SD genesis. We hypothesize that an unidentified SDa can be identified, is likely not glutamate and may originate from microglia. The role of glutamate in inducing SD was evaluated to determine if glutamate could be the SDa. Oxygen glucose deprivation (OGD)-induced SD in live brain slices was shown to persist treatment with glutamate receptor antagonists kynurenate and MK-801, although a delay in SD onset was observed. Glutamate application to live brain slices also showed that it did not initiate SD, but in some slices, glutamate promoted a slower localized signal spread surrounding the weight securing the slice. Microglia were also investigated as a source of the SDa; however, blockade of the channel KCa3.1 with TRAM-34, as well as blockade of M1 microglial activation with minocycline did not significantly affect SD initiation. Finally, high pressure liquid chromatography (HPLC) was utilized to characterize superfusate from brain slices that had undergone SD, while matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) was used to analyze SD-related compounds in fresh frozen brain slices. Size exclusion HPLC identified a ~2 kDa peak that was associated with SD and may contain an SDa. MALDI-IMS analysis of brain slices exposed to OGD revealed three compounds (m/z 593, m/z 610 and m/z 620) that are associated with SD genesis and propagation. Further characterization may yield a novel therapeutic target to mitigate neural damage during acute brain ischemia.