Evaluation of effects of CYP450 derived eicosanoids in cerebral ischemia

Stroke and cardiac arrest (CA) both are characterized by severe fall in the cerebral blood flow (CBF) locally or globally at the onset of the event with subsequent prolonged reductions observed after initiation of reperfusion. These CBF derangements may lead to secondary neuronal damage and worse functional outcomes. Currently there is limited understanding of the causative factors of CBF dysregulation after cerebral ischemic injury. Among the many known vasoactive substances, CYP450 derived metabolites of arachidonic acid (AA), called CYP eicosanoids were shown to act as potent regulators of microvascular tone. The main objective of this research was to investigate the role of CYP eicosanoids in the cerebral ischemic injury using in vitro and in vivo models of ischemia. In order to characterize the regional and temporal distribution of CYP eicosanoids in the brain after ischemic injury, sophisticated lipidomics based analytical methods were developed and validated. Studies in in vitro neuronal cultures indicated that 11,12-EET (vasodilatory) exhibited concentration-dependent neuroprotective effects whereas 20-HETE (vasoconstrictive) exhibited concentration-dependent toxic effects in a hypoxia-reoxygenation injury model.We have used two pharmacological inhibitors of CYP eicosanoid pathway for in vivo studies namely t-AUCB, an inhibitor of soluble epoxide hydrolase (sEH) that converts EETs to inactive DHETs (dihydroxyeicosatrienoic acid), and HET0016 that inhibits 20-HETE synthesis. The in vivo studies iv in animal model of focal ischemia with t-AUCB showed the following key findings: i) significant reduction in the infarct size by specifically increasing the ratio of EET/DHET in the cortex; ii) significant improvement in the short-term functional outcome after acute administration; iii) marginal improvement in CBF around the infarcted area during the post-ischemic reperfusion, and iv) dose-dependent significant increase in the neuronal survival in a hypoxia-reoxygenation injury model of primary rat cortical neuronal cultures. Our studies in animal model of global ischemia showed a specific pattern of CBF dysregulation during the reperfusion phase that is dependent on the duration of the initial injury itself. Our lipidomics analysis of regional and temporal distribution of AA metabolites in the rat brain revealed a significant imbalance in the tissue levels of these metabolites, and their association with the pathological changes in CBF in the cortex post resuscitation from different durations of CA. The studies with acute administration of HET0016 showed improvement in the cortical CBF during the early reperfusion phase after 12 min CA by selectively inhibiting 20-HETE levels in the cortex. Further, we showed significant reduction in neuronal death, and improved short-term behavioral performance after a single low dose administration of HET0016. In conclusion, these results showed the evidence that CYP eicosanoids are associated with CBF dysregulation after ischemic injury in both focal and global ischemia, and that alteration of CYP eicosanoids levels in brain tissue with pharmacological inhibitors improves short-term functional outcome through vascular and non-vascular mechanisms. Further research is warranted in order to design an effective therapeutic strategy for administering pharmacological inhibitors of CYP eicosanoid pathway to rectify the CBF dysregulation and improve the long-term functional outcomes after stroke injury.