P–JNK expression of the rat retina in NMDA–induced neurotoxicity.

Abstract

Abstract: : Purpose:To investigate the biochemical mechanism by which neuronal death in the rat retina after NMDA activation, we examined the localization of phosphorylated–c–Jun N–terminal kinase (p–JNK) expression, which is indicated to participate in apoptotic pathway, in the rat retina after intravitreal injection of NMDA. Methods:8–week–old male Wistar rats were used as subjects. A single 5–µl injection of 40 mM NMDA was administered intravitreally into one eye, and PBS was used as a control. The rats were fixed by perfusion with 4% paraformaldehyde/0.1 M PB, and eyes were enucleated at 1, 3, 6, 12, and 24 hours after injection. TUNEL method was performed to examine localization of the apoptotic cells. P–JNK localization was examined by fluorescent double labeling with various antibodies as well as TUNEL method. Changes in p–JNK expression were also examined by Western blot analysis. Results:TUNEL positive cells were localized in nerve fiber layer (NFL) at 3 hours, and they were observed in NFL, retinal ganglion cell layer (RGCL) and inner nuclear layer (INL) at 6,12,18,and 24 hours. Western blot analysis showed that expression of p–JNK started to increase at 1 hour after NMDA injection, and kept high from 3 to 6 hours in the retina. Immunohistochemistry showed that NMDA induced dramatical increase in the number of p–JNK positive cells. Double labeling showed the colocalization of p–JNK with TUNEL positive cells in NFL, RGCL and INL. Furthermore, p–JNK positive cells were identified at least as ganglion cells in RGCL , astrocytes in NFL and Müller cells in INL. Conclusions:NMDA–treatment induced expression of p–JNK in ganglion cells in RGCL, astrocytes in NFL, and Müller cells in INL, mostly in coincidence with apoptotic cells. These data suggest the involvement of p–JNK in NMDA–induced neurotoxicity on ganglion cells by a direct pathway and/or indirect one through glial cells.

Keywords: apoptosis/cell death • retina • ganglion cells