Towards Identifying a Molecular Activator of Spreading Depolarization Generated by the Ischemic Brain

Spreading depolarizations (SDs) are waves of mass depolarization that propagate through gray matter following Na+/K+-ATPase (NKA) failure because of stroke, traumatic brain injury or sudden cardiac arrest. SDs expand the initial site of neuronal injury and worsen clinical outcomes. The molecular events underlying SD initiation and propagation are not well understood. In this rodent study, we hypothesized that gray matter stressed by oxygen/glucose deprivation (OGD) releases a compound(s) that promotes SD, which we term a spreading depolarization activator (SDa). We used rat brain slices incubated in artificial cerebrospinal fluid (aCSF) and subjected to OGD to release a putative SDa. The aCSF was collected either prior to ("Pre-SD aCSF") or 10 min after initiation of OGD conditions ("Post-SDOGD aCSF"). These solutions were then separately superfused over a healthy, naive (non-stressed) brain slice. Post-SDOGD aCSF (with re-normalized O2 and glucose) evoked SD in 82.35% of the naive brain slices (n = 17) whereas Pre-SD aCSF evoked no SD in 10 naive slices. Then to investigate the NKA as a potential target of the SDa, we used a hemolysis assay, comparing the effects of Pre- or Post-SDOGD aCSF on red blood cell (RBC) lysis and compared it to the known hemolytic effect of the NKA-specific inhibitor, palytoxin. Post-SDOGD aCSF evoked neither swelling nor lysis of RBCs on its own. However, when a sub-threshold concentration (0.01-0.02 nM) of the specific NKA inhibitor palytoxin (PLTX) was added, a striking "priming" effect was observed, whereby Post-SDOGD aCSF evoked a highly significant increase in both RBC swelling and then hemolysis, compared to Pre-SD aCSF. High pressure liquid chromatography (HPLC) experiments show a several-fold increase in released molecules post-SD vs pre-SD. Overall, this study provides support for SDa release capable of inducing SD-associated swelling in brain slices and, when combined with a trace amount of PLTX, swelling/hemolysis of RBCs caused by NKA inhibition. A greater understanding of the molecular events underlying SD should identify novel targets to reduce recurrent SD-evoked neuronal injury under ischemic conditions.