Phosphatidylcholine (PC) specific-phospholipase C (PC-PLC) hydrolyzes PC to generate phosphocholine and diacylglycerol. Activation of PC-PLC may affect the neural cell cycle after stroke. Post-mitotic neurons are able to enter into the cell cycle after stroke, but die instead of proliferating. Our data show up-regulation of cyclin-dependent kinase after oxygen-glucose deprivation (OGD)/reoxygenation in primary cortical neuronal cultures - evidence of cell cycle entry. Activated microglia/macrophages produce significant amounts of reactive oxygen species (ROS) able to oxidize unsaturated fatty acids of phospholipids, resulting in oxidized PC (OxPC). OxPC is a component of oxidized LDL (OxLDL), one of the ligands of scavenging receptor CD36. CD36 is elevated in cerebral ischemia and mediates tissue damage. OxLDL induces PC-PLC expression in other disease states, while the PC-PLC inhibitor D609 reduces expression. Hypothesis: PC-PLC stimulation through OxPC leads to neural cell proliferation and may differentially affect neuronal and non-neuronal cells after stroke. D609 may block the cell cycle, attenuating neuronal death and glia proliferation after stroke. Supporting our hypothesis, D609 reduced (a) cerebral infarction, (b) OxPC-protein adduct, and (c) PC-PLC activity after 1 hr transient middle cerebral artery occlusion (tMCAO) in spontaneously hypertensive rat (SHR). Aim 1: Investigate the cell-specific role of PC-PLC in neuronal death after OGD in primary neuronal cultures and during proliferation of primary microglia. Primary neuronal cultures will be subjected to 2 hr OGD/22 hr reoxygenation; the effect of D609 and/or PC-PLC neutralizing antibodies on OxPC-protein adduct and PC-PLC expression will be determined. The role of OxPC in induction of PC-PLC expression and cell cycle proteins will be assessed in normal primary cultures of neurons and microglia using POVPC, a synthetic OxPC. Since D609 reduces the expression of other scavenging receptors, the effect of D609 on CD36 expression will be investigated in microglia cultures. Aim 2. Investigate the role of PC-PLC after tMCAO in SHR. Our data shows induction of PC-PLC in the IC after tMCAO. This aim investigates the role OxPC, PC-PLC and CD36 and how D609 modulates these responses in stroke. We will use immunohistochemistry to determine cell-specific expression of PC-PLC and OxPC after stroke. PC-PLC neutralizing antibodies will be administered icv to SHR to assess the role of PC-PLC in stroke.
|Effective start/end date||07/01/2011 → 09/01/2011|