Apelin-36 is protective against N-methyl-D-aspartic-acid-induced retinal ganglion cell death in the mice

Retinal ganglion cell death in glaucoma is caused at least in part by a large Ca²⁺ influx through N-methyl-D-aspartic acid (NMDA) receptors. Apelin is a peptide originally found in the tissue extracts of bovine stomach. Recent studies have been shown that apelin protects against the ischemic-reperfused injury in the brain. We examined whether apelin had protective effects on the NMDA-induced retinal ganglion cell (RGC) death using B6.Cg-TgN(Thy1-CFP)23Jrs/J transgenic mice, which express the enhanced cyan fluorescent protein in RGCs in the retina, in vivo. The mice were anesthetized by ketamine and xylazine, and NMDA (40 nmol/eye) was intravitreally injected. We evaluated the effects of apelin-13, [Glp¹]-apelin-13, a potent agonist of apelin receptor, and apelin-36 on the NMDA-induced retinal ganglion cell death. NMDA-induced retinal ganglion cell loss was clearly seen 7 days after NMDA injection. Intravitreal apelin-36 (0.33 nmol/eye), but not apelin-13 (1 nmol/eye) nor [Glp¹]-apelin-13 (1 nmol/eye), simultaneously injected with NMDA significantly reduced the cell loss. The protective effect of apelin-36 was not reduced by ML221 (0.1 nmol/eye; 5-[(4-Nitrobenzoyl)oxy]-2-[(2-pyrimidinylthio)methyl]-4H-pyran-4-one), an apelin receptor antagonist, GF109203X (0.03 nmol/eye), a protein kinase C inhibitor, U0126 (0.2 nmol/eye), a MAPK/ERK kinase inhibitor, LY294002 (0.1 nmol/eye), a phosphoinositide 3-kinase inhibitor, Akti 1/2 (0.05 nmol/eye), an Akt inhibitor, or 4,5,6,7-tetrabromobenzotriazole (0.2 nmol/eye), a casein kinase-2 inhibitor. In addition, human apelin-36 did not affect the kainic-acid (20 nmol/eye)-induced ganglion cell death. The present study suggests that apelin-36 protects against the NMDA-induced ganglion cell death independently of the activation of apelin receptor in the murine retina in vivo.