Abstract WP299: PGE2 EP1 Receptor Blockade Prevents Hemin-Induced Neurotoxicity In An In Vitro Model of Intracerebral Hemorrhage
Background: Intracerebral hemorrhagic (ICH) stroke is defined by the rupture of intracranial artery that leads to the formation of an hematoma. Subsequently, neuronal damage is associated with the lysis of red blood cells which release hemoglobin. Hemoglobin breakdown product is the prooxidant toxic hemin. We and others have documented the various toxic or protective actions of the prostaglandin receptors in ischemic stroke. In this study, we aim to investigate the PGE2 EP1 G-protein coupled receptor’s contribution to hemin-induced neurotoxicity as an in vitro ICH model.
Methods: Postnatal primary neuronal cultures were used to investigate hemin-induced neurotoxicity and the PGE2 EP1 receptor role. WT and EP1-/- neurons were treated with vehicle and hemin (12.5-100uM) for 18h and cell viability measured using LDH and calceinAM/EthD-1 assays.
Results: Hemin increased neuronal cytotoxicity when treated with 50 (25.0±2.2), 75 (34.7±4.5) and 100uM (50.1±4.2) compared to vehicle (16.5±1.5) measured using LDH assay. Hemin at 25 (75.1±9.4), 50 (61.5±2.8), 75 (52.0±4.7) and 100uM (38.0±8.9) decreased calceinAM positive neurons compared to vehicle (96.8±4.8). Hemin also increased EthD-1 positive neurons compared to vehicle. Compared to WT neurons, hemin exposure at only 75 (28.3±5.1) and 100uM (44.3±0.8) showed significant cytotoxicity in EP1-/- neurons and had no significant difference in the amount of calceinAM positive neurons compared to vehicle. To further address the mechanism responsible for EP1 receptor mediated hemin-neurotoxicity, calcium-imaging experiments were conducted. Preliminary data using Fluo-8AM show significantly decreased average fluorescence intensity in EP1-/- neurons (3,248±435) compared to WT neurons (6,735±1,234) when treated with 1uM PGE2.
Conclusions: Together, the results suggest that the EP1 receptor, likely through its regulation of calcium signaling, appears to aggravate the hemin-induced neurotoxicity. Ongoing experiments include detailed investigation of the regulation and the source of calcium in neurons. We also aim to determine if the EP1 receptor or the other PGE2 receptors would be unique therapeutic target(s) against hemolysis induced by various ICH preclinical models.
- © 2012 by American Heart Association, Inc.