Presentation description

Acute ischemic stroke (AIS) increases plasma ceramides, which correlate with the prevalence and severity of neurobehavioral and motor deficits in patients. Here, we sought to determine cause and effect. To model AIS, we challenge C57BI6 mice with 60-min transient middle cerebral artery occlusion + 23 hr reperfusion (tMCAO + R). Compared to sham-operated mice, we found AIS increases ceramides in the plasma and cerebral tissue. Furthermore, among the myriad cell types contained in cerebral homogenates, endothelial cells (ECs) make the greatest contribution to AIS -induced ceramide generation. We hypothesized that AIS -induced cerebral EC ceramide generation is necessary and sufficient to worsen AIS outcomes. Dihydroceramide desaturase 1 (DES1) is the enzyme that catalyzes inert dihydroceramides to produce toxic ceramides. The gene that encodes DES1 is Degs1. In a "loss of EC ceramide" model, male Degs1fl/fl mice were given (i) AAV-BR1-Cre intravenously to selectively deplete Degs1 in cerebral ECs (n=3) or (ii) AAV-BR1-Null virus (n=3). Fourteen days later, cerebral tissue was collected, and the EC and non-EC -enriched fractions were isolated. As predicted, Degs1 mRNA expression was lower in the EC-enriched fraction obtained from Degs1fl/fl mice that received AAV-BR1-Cre vs. AAV-BR1-Null virus. In a different cohort, AIS -induced infarct volume and neurobehavioral and motor deficits were mitigated in Degs1fl/fl mice 14 days after receiving AAV-BR1-Cre (n=2) vs. AAV-BR1-Null virus mice (n=2). In a "gain of cerebral EC ceramide" model, delivering AAV-BR1-Cre to mice harboring a fusion construct comprising the 3 SPT subunits (SPTLC1, SPTLC2, SPTSSA, aka fSPT mice) elevated SPTLC1 and SPTLC2 mRNA expression in cerebral ECs, whereas deploying AAV-BR1-Null was without effect. In a different cohort, AIS outcomes were more severe when EC ceramide generation was amplified. Together, these preliminary findings suggest that AIS -induced EC ceramide generation is necessary and sufficient to worsen AIS outcomes.