Effects Of Limb Remote Ischemic Preconditioning On Neuronal Apoptosis And Learning Memory Induced By Ketamine In Developing Brain Of Rats

Objective To study the effects of limb remote limb ischemic preconditioning(RIPC)on the apoptosis of neurons in the cerebral cortex and hippocampal CA1 region and learning memory impairment of the neonatal rats induced by ketamine anesthesia.Methods a total of 96 Sprague-Dawley rats on postnatal day 5,Weighing 8-12 g,were randomly divided into four groups: control group(C group),limb remote ischemic preconditioning group(RIPC group),ketamine group(KTM group),limb remote ischemic preconditioning + ketamine group(RIPC+KTM group).In group C,normal saline(8 ml/kg)was intraperitoneally injected on postnatal day 7,six times at an interval of 2 hours.RIPC group were subjected to limb ischemia for 5 minutes and reperfusion for 5 minutes for 4 cycles on postnatal day 5,and then injected with the same volume of saline after 48 hours.KTM group was intraperitoneally injected with ketamine 20mg/kg(2.5 mg/ml diluted with physiological saline,injection volume 8 ml/kg),once every 2 hours after administration for 6 times;Rats in RIPC+KTM group were subjected to remote ischemic preconditioning on postnatal day 5 and underwent the same treatment with the ketamine group after 48 hours.The parts of Rats(18 rats in each group)in the four groups were sacrificed to cardiac perfusion on postnatal day 8.The morphological and quantitative changes of pyramidal neurons in cerebral cortex and hippocampal CA1 region of rats were observed by HE staining.In the meantime,transmission electron microscopy was used for observing neurons ultrastructure.Immunohistochemistry and western blot analysis of cleaved-caspase-3?Bax and Bcl-2 expression and Tunel staining in cortex and hippocampal CA1 region were used to assess neural cell apoptosis.Learning and memory abilities were tested at the age of 60 days by Morris water maze test.Results Compared with group C,The number of nerve cells in the KTM group and RIPC+KTM group decreased,the apoptosis rate increased,and the expression of cleaved caspase-3? Bax protein increased,while the expression of Bcl-2 decreased(P <0.05).There was no statistically significant difference between the C group and RIPC group(P >0.05);Compared with the RIPC group,the number of nerve cells in the KTM group and RIPC+KTM group also decreased,the apoptosis rate increased,and the expression of cleaved caspase-3? Bax protein increased,while the expression of Bcl-2 decreased(P <0.05);Compared with group KTM,the number of neurons increased,the apoptosis rate and the expression of cleaved caspase-3? Bax protein decreased,while the expression of Bcl-2 increased in the RIPC+KTM group(P <0.05).Transmission electron microscope indicated that the endoplasmic reticulum of RIPC group was slightly swollen,and there was no significant change in the rest of the structure compared with group C,and damage of cerebral cortex and hippocampal CA1 neuronal subcellular organelle in KTM group and RIPC+KTM group were severer compared with group C;Compared with the RIPC group,the results of the KTM group and the RIPC+KTM group were as same as comparing with C group;RIPC+KTM group caused less damaged than that in KTM group.The results of Morris water maze test showed that escape latency and path length in KTM and RIPC+KTM treated rats were elevated compared with C group and RIPC group(P <0.05);But in the RIPC+KTM group,they were lower than KTM group(P >0.05).The C and RIPC groups showed similar values(P >0.05).The results of probe trial experiment showed that times to reach the platform were increased and the numbers of crossings over previous platform locations were reduced for the KTM group in comparison with C and RIPC values(P <0.05),whereas co-administration of RIPC with ketamine significantly enhanced these values in comparison with those of the KTM group,There was no statistically significant difference between the C group and RIPC group(P >0.05).Conclusion Repeated exposure to ketamine anaesthesia induced abnormalities in the morphology of neurons and neurons loss as well as neuroapoptosis and impairs spatial learning and memory.In addition,RIPC could protect neurons from ketamine-associated deleterious effects in the cerebral cortex and the hippocampal CA1 region of the developing rat,reducing long-term behavioral deficits as the animal matures.