Effect of ketamine on glutamate induced apoptosis in cultured rat cerebellar granule neurons / 中华麻醉学杂志

ABSTRACT

Objective To investigate if ketamine protects cultured rat cerebellar granule neurons (CGNs) from apoptosis induced by glutamate and its possible mechanism of signal transduction. Methods Primarily cultured CGNs prepared by enzymatical digestion of cerebellum isolated bom 7-8 day old SD rats were randomly divided into 8 groups (1) control group (C); (2) glutamate group (G) in which CGNs were incubated with 300 ?mol?L-1 glutamate; (3) (4) (5) ketamine groups (K1 , K2, K3) in which CGNs were incubated with ketamine 10 (K1 ) 100 (K2) or 1 000 (K3) ?mol ? L-1 for 1h before glutamate was added; (6) Ly 294002 - K3 group (LK3) in which CGNs were incubated with 20 ?mol?L-1 Ly 294002 (a specific phosphatidyl-inositol 3-kinase inhibitor) for 30 min before ketamine 1 000 ? mol ? L-1 and glutamate were added as in group K3; (7) Ly 294002 group (L) in which CGNs were incubated with Ly 294002 20?mol ? L-1 and (8) Ly 294002-glutamate group (LG) in which CGNs were incubated with Ly 294002 20 ?mol ? L-1 for 30 min before glutamate was added. After 20 h incubation the apoptosis in CGNs was detected by Hoechst 33258 nucleus staining, phase-contrast microscopy and DNA fragmentation agarose gel electrophoresis. The neuronal survival was determined by fluorescein diacetate (FDA) staining.Results Glutamate 300 ?mol ? L-1 induced apoptosis in CGNs as characterized by cytoplasmic blebbing, heterochromatic clumping, condensation of nuclear chromatin and a typical apoptotic DNA ladder revealed by agarose gel electrophoresis. Ketamine improved the survival of CGNs incubated with glutamate (300 ?mol ? L-1) and blocked the glutamate-induced apoptosis in a concentration-dependent manner. Ly 294002 (20 ?mol ? L-1) antagonized the anti-apoptotic effects of ketamine. Conclusion Ketamine can protect CGNs from apoptosis induced by glutamate. Phosphatidyl-inositol 3-kinase/Akt pathway may be involved in the antiapoptotic action of ketamine.