Factors affecting theprotective effect of morphine preconditioning on murine hippocampal neurons againstanoxia-reoxygenation injury

：Objective To investigatethe factors affecting the protective effects of morphine preconditioning on murinehippocampal neurons against anoxia/reoxygenation (A/R) injury and the underlyingmechanisms.Methods Hippocampal slices (400 μm thick) were prepared usinghippocampi isolated from decapitated mice. A/R injury was simulated in vitro usingartificial cerebral spinal fluid (ACSF) deprived of O_2 and glucose for 20 min followed byreoxygenation and glucose supply for 2 h. The experiment was performed in 4 parts: I .Theslices were incubated with 5 different concentrations of morphine (0.1, 0.3, 0.5, 1.0,3.0, 10.0 /μmol/L) for 30 min at 30 min before A/R; Ⅱ.The slices were incubated with morphine 3.0 /μmol/L for 5 different periodsof time (5, 15, 30, 45, 60 min) at 30 min before A/R; Ⅲ. The slices were incubatedwith morphine 3.0 μmol/L for 30 min followed by A/R at 6 differentintervals (0, 5, 15,30,60, 120 min); Ⅳ. The slices were incubatedwith (a) chelerythrine (a non-selective PKC antagonist) 10 /μmol/L or (b) eVl-2(a selective nPKCe isoform antagonist) 2 μmol/L or (c) AIP 2 μmol/L(a selective CaMK Ⅱ antagonist) or (d) MK-801 10μmol/L (a non-competitive NMDA receptor blocker) for 30 min and then for another30 min together with morphine 3.0 μmol/L before A/R at 30 mininterval. The survival rates of the hippocampal neurons were assessed by TTC staining.Results Neuronal survival rates were significantly higher in morphine preconditioninggroups which preconditioned with morphine (0.5-10.0 μmol/L) for 15-60 min at aninterval of 0-60 min before A/R than in A/R group. Increase in neuronal survival rateinduced by morphine preconditioning was partially blocked by chelerythrine or eV1-2or AIP or MK-801. Conclusion Preconditioning with appropriate concentrations of morphine(0.5-10.0 μmol/L) for appropriate period of time (15-60 min) at appropriateinterval (within 60 min) before A/R can protect hippocampal neurons against A/R injurythrough activation of nPKCe, NMDA receptor and CaMKⅡ. Key words: Morphine; Ischemicpreconditioning; Hippocampus; Neurons; Reperfusion injury