Effect Of GM1on PI3K/AKT Signaling Pathway In The Hippocampal Neurone Of Rat With Mild Hypothermic Cardiopulmonary Bypass

Cardiopulmonary bypass (CPB) has been used to assist cardiac surgeryand developed in clinical application fastly. Despite technologicalimprovements with CPB, brain injury after CPB still is one of clinicallycommon and serious complications. The so-called brain injury from CPB isknown as postoperative central nervous system (CNS) dysfunction. Aspectrum of postoperative CNS dysfunction both acute and persistent occursin a proportion of patients after cardiac surgical procedures, including braindeath, stroke, subtle neurologic signs, and neuropsychologic impairment.Postoperative neurocognitive decline (POCD) is the most frequently reportedform of brain injury in the cardiac surgery setting. Studies indicate earlyPOCD rates from50%to70%within the first postoperative week. Therefore,the understanding of the pathophysiologic mechanisms of these disorders,protective strategies and diagnostic techniques are highly concerned.Monosialoganglioside1(GM1) is a glycosphingolipid present in most cellmembranes, displays antioxidant, neuroprotective properties, and participates in multiple physiological function. Previous studies suggested that GM1preconditioning played brain protection through increasing synaptictransmission and plasticity in the hippocampus, increasing superoxide dismutaseactivities, reducing intracellular calcium and malondialdehyde levels. Butanimal studies suggested that neuronal apoptosis occured in hippocampus at1hand6h after moderate hypothermic CPB. Evidence shows that GM1couldactive PI3K/Akt signaling pathway so as to prevent cell apoptosis. Therefore,the purpose of our research is to investigate the mechanism and effect of GM1on the hippocampal neurone of rat with mild hypothermic CPB.Experiment1: To establish the rat model of mild hypothermic CPB.Objective: To explore the feasibility of establishing a rat model of mildhypothermic CPB.Methods: SPF-class adult male SD rats（n=20）were cannulated forcardiopulmonary bypass. Blood was drained from the fight atrium via jugularvein catheter into the CPB circuit and returned to the right carotid artery. Theminiature CPB was primed with6%hetastarch without blood. CPB wasweaned after60min. During the whole process, the rectal temperature,electrocardiograph, hemodynamics and blood-gas analysis were observed.Results: Success rate of model was85percent. The vital signs ofexperimental rats were stable during CPB, biochemical index were normalduring CPB.100%experimental rats could survive to3h after CPB.Conclusion: The rat model of mild hypothermic CPB is easy to beperformed. This model is suitable for studying clinically relevant problemsconcerning CPB.Experiment2: To study the effects of GM1on PI3K/Akt signalingpathway in the hippocampal neurone of rat with mild hypothermic CPB. Objective: To study the protective effects and underlying mechanisms ofGM1on the hippocampal neurone of rat under mild hypothermic CPB.Methods: Healthy adult male SD rats (n=18) were randomly assigned toblank control group (C group), monosialoganglioside1therapy group (Ggroup) and cardiopulmonary bypass group (CPB group). All operations of Cgroup were the same with the other2groups, no CPB. G group was subjectedto CPB with GM120mg/kg added to the priming solution. CPB group wassubjected to CPB with the same volume of saline with G group added to thepriming solution. At3h after CPB, all rats were euthanized to collect the lefthippocampal tissues for detection. Changes in hippocampal neuronal ultrastructure were determined with electron microscopy, neuronal apoptosis wasdetected by TUNEL, Caspase-3positive cells of hippocampus weredetermined by immunohistochemistry and hippocampus expression of the Akt,Caspase-3, phospho-AktSer473and phospho-GSK3βSer9were assessed byWestern blot.Results1. Changes in hippocampal neuronal ultra structure were determined withelectron microscopy.C group: The cell nucleus was round, nucleolus and nuclear membranewere clear. The euchromatin in nucleus was well distributed. Cytoplasm wasrich in the mitochondria, ribosomes, rough endoplasmic reticulum, lysosomes,etc.G group: Nuclear membrane was not clear in some regions. There weremore abnormal euchromatin in nucleus. There were more mitochondria,ribosomes, rough endoplasmic reticulum in the cytoplasm, but mitochondrialcristae reduced.CPB group: The cell nucleus was suborbicular with no nucleolus in it.Nuclear membrane was not clear in some regions. The heterochromatin condensed and marginated in nucleus. Most of the cytoplasm dissolved. Thenuclear chromatin, a few abnormal side focused. The left mitochondrialcristae reduced or vacuolarly degenerated.2. Hippocampal neuronal apoptosisCompared with C group, the neuronal apoptosis of G and CPB groupincreased (P<0.05). The neuronal apoptosis of CPB group (IA,13.95±1.20)was significantly higher in the neuronal apoptosis of G group (IA,8.08±0.80)(P<0.05).3. Caspase-3positive cellsCompared with C group, Caspase-3positive cells of G and CPB groupincreased (P<0.05). Compared with CPB group, the IA of Caspase-3positivecells of G group decreased by34.71%(P<0.05).4. Akt, pAktSer473, pGSK3βSer9and Caspase-3protein level.There was no significant difference in Akt (P>0.05). The pAktSer473andpGSK3βSer9in the C group were significantly higher than in CPB group andlower than in G group (P<0.05). The Caspase-3protein level of G group wassignificantly higher than in C group and lower than in CPB group (P<0.05).Conclusions1.Ultra-structural changes consistent with apoptosis occurred inhippocampal neurone of rat with mild hypothermic cardiopulmonary bypass.2.GM1attenuates hippocampal neuronal apoptosis by mechanismsinvolving PI3K/Akt signaling pathway, upregulation of the pAktSer473,inhibited GSK-3β activities and decreased expression of the Caspase-3protein.