Abstract 16749: ET B Receptor Agonist, IRL-1620, Decreased Cerebral Ischemic Damage by Decreasing Fission and Increasing Fusion of Mitochondria in Rat

Abstract

Introduction: We have shown that endothelin B (ET B ) receptor agonist, IRL-1620, provides neuroprotection by inhibiting apoptosis in rats. Mitochondrial dysfunction has been implicated in the cell death following ischemia. Hypothesis: IRL-1620 prevents neuronal death by altering mitochondrial activity. Methods: We determined the effect of IRL-1620 on mitochondrial dysfunction following cerebral ischemia. Male Sprague-Dawley rats underwent permanent middle cerebral artery occlusion (MCAO). Following surgery, rats received intravenous injections of vehicle or IRL-1620 (5 μg/kg, i.v.) at 4, 6, and 8 hr post-occlusion on day 0; similar treatment of vehicle or IRL-1620 was repeated on day 3 and 6 post-MCAO. Animals were sacrificed at 24 hr and day 7 following occlusion and brains processed to evaluate expression of mitochondrial dysfunction markers (DRP-1, MFN-2 and ATP5A) using immunofluorescence and western blot. Results: Animals treated with IRL-1620 showed significant improvement in all neurological and motor function tests compared with vehicle-treated group. At 24 h and day 7 following MCAO, a significant increase in DRP-1 (a key mediator of mitochondrial fission) and decrease in MFN-2 (key mediator of mitochondrial fusion) was observed in the vehicle-treated group compared to sham group. IRL-1620 treatment significantly [F(5,18)=10.5, p<0.001] decreased (-53%) expression of DRP-1 and significantly [F(5,18)=25.78, p<0.0001] increased (165%) expression of MFN-2 in MCAO rats compared to vehicle treated rats. There were no changes in ATP5A expression levels in the brains of sham, vehicle, and IRL-1620 treated rats 24 h post occlusion. A significant [F(5,18)=11.38, p=0.011] increase (70%) in ATP5A in the vehicle group was observed on day 7 compared to sham. However, IRL-1620 significantly [F(5,18)=11.38, p=0.0009] decreased (-53%) the expression of ATP5A in MCAO rats compared to vehicle. Immunofluorescence studies corroborated Western blot results showing an increase of DRP1 and a decrease of MFN-2 positive cells in the vehicle group after 24 hr and day 7 compared to sham group. These changes were reversed in IRL-1620 treated rats. Conclusions: Stimulation of ET B receptors with IRL-1620 is neuroprotective in a rat model of cerebral ischemia by decreasing (DRP-1) fission and increasing (MFN-2) fusion of mitochondria.