| Part Ⅰ Effects of single or multiple exposures of propofol on long-term learning and memory in neonatal ratsObjective: To explore effects of single or multiple exposures of propofol on long-term learning and memory in neonatal rats.Methods: P7(postnatal day 7)Sprague-Dawley male rat pups weighing 13~18g were randomly divided into 4 groups: Control,pups underwent the same environmental conditions as other groups did,but did not experience any procedure;Vehicle,pups received equal numbers and volumes of intraperitoneal injections of 0.1%DMSO;P(single),pups received two intraperitoneal injections of 5m L/kg 0.1%DMSO at P7 and P8,followed by50mg/kg propofol at P9;P(multiple),pups received three intraperitoneal injections of 50mg/kg propofol from P7 to P9.Before full recovery,some pups were used for transcardial arterial blood gas analysis.The others were used forMorris Water Maze test from P36 to P41 to evaluate long-term spatial learning and memory function.Results: During anesthesia,there were no significant differences in blood gas analysis among the groups(P>0.05).Compared with the control group,the swimming speeds,escape latencies and platform crossing times in P(single)group were not significant different;multiple propofol exposures increased the escape latencies and decreased the platform crossing times(P<0.05);there were no significant differences in escape latencies or platform crossing times between vehicle group and control group(P>0.05).Conclusion: Single exposure of propofol in neonatal rats did not affect the long-tem learning or memory,while multiple exposures of propofol impaired the long-term learning and memory.Part Ⅱ The mechanism of long-term learning and memory impairment after multiple but not single exposure of propofol in neonatal ratsObjective: To explore the underlying mechanism of long-term learning and memory impairment after multiple but not single exposure of propofol in neonatal rats.Methods: P7 Sprague-Dawley male rat pups were randomly divided into 4 groups: Control,pups underwent the same environmental conditions as other groups did,but did not experience any procedure;Vehicle,pups received equal numbers and volumes of intraperitoneal injections of 0.1%DMSO;P(single),pups received two intraperitoneal injections of 5m L/kg 0.1%DMSO at P7 and P8,followed by 50mg/kg propofol at P9;P(multiple),pups received three intraperitoneal injections of 50mg/kg propofol from P7 to P9.The rats were sacrificed at P9(5h after recovery),P14,P21 or P35,their left brains were used for activated caspase-3 immunohistochemistry and Nissl staining to observe the neuronal apoptosis and neuronal density respectively,and their right brains were used for synaptophysin immunofluorescence to observe the synaptic density in the hippocampal CA1 region and Pr L region.Results:In P(single)group,the activated caspase-3 positive neurons in the hippocampal CA1 region and Pr L region were significantly increased(P<0.05),and the neuronal density was decreased(P<0.05)at P9,which were no differences at P14,P21 or P35 compared with the control,the synaptophysinpositive puncta were not different from the control at P9,P14,P21 or P35;in P(multiple)group,the activated caspase-3 positive neurons in the hippocampal CA1 region and Pr L region were significantly increased(P<0.05),the neuronal density and synaptophysin positive puncta were decreased at P9,P14,P21 or P35(P<0.05).There were no significant differences in all the data between vehicle group and control group(P>0.05).Conclusion:Single exposure of propofol in neonatal rats only induced transient neuronal apoptosis and neuronal loss in the hippocampus and prefrontal cortex;while multiple exposures of propofol could induce persistent neuronal apoptosis,neuronal loss and synaptic deficits,which may contribute to long-term learning and memory impairment.Part Ⅲ The molecular mechanism of TNF-α mediating multiple exposures of propofol-induced neurotoxicity in the developing brainObjective: To explore the molecular mechanism of TNF-α mediating multiple exposures of propofol-induced neurotoxicity in the developing brain.Methods: P7 Sprague-Dawley male rat pups were randomly divided into 3 groups: Control,pups underwent the same environmental conditions as propofol group did,but did not experience any procedure;Vehicle,pups received equal numbers and volumes of intraperitoneal injections of0.1%DMSO;Propofol,pups received three intraperitoneal injections of 50mg/kg propofol from P7 to P9,rat pups were sacrificed on P7,P8,P9(6h after anesthesia),P10 and P11,the cerebral spinal fluid in the lateral cerebral ventricle was collected for ELISA to detect the s TNF-α level,the hippocampus and prefrontal cortex were dissociated for Western blot analysis to detect the s TNF-α level.In another experiment,P7 rat pups were randomly divided into 4groups: Control;Sham,pups received an equal volume of artificial cerebral spinal fluid intracerebroventricularly on P7,and they underwent the same environmental conditions without propofol exposure;Propofol;ETN,ETN(5μg/2μL)was intracerebroventriculary 30 min before propofol exposure on P7,which was followed by the other two intraperitoneal injections of propofol on P8 and P9.Some rats were sacrificed at P9(5h after recovery)or P35,their left brains were used for activated caspase-3 immunohistochemistry and Nisslstaining to observe the neuronal apoptosis and neuronal density,and their right brains were used for synaptophysin immunofluorescence to observe the synaptic density in the hippocampal CA1 region and Pr L region.The others were used for Morris Water Maze test from P36 to P41 to evaluate long-term spatial learning and memory function.In the third experiment,P7 rat pups were randomly divided into 3 groups: Control;Vehicle and Propofol,rat pups were sacrificed on P9(6h after anesthesia)and their brains were used for double immunofluoroscence to explore the cellular source of propofol-increased TNF-α.Results: The TNF-α levels of cerebral spinal fluid,hippocampus and prefrontal cortex in propofol group were significant increased at P7,P8,P9 and P10(P<0.05),and it returned to the control level on P11(P>0.05);in propofol group,the activated caspase-3 positive neurons in the hippocampal CA1 region and Pr L region were significantly increased(P<0.05),the neuronal density and synaptophysin positive puncta were decreased at P9 or P35(P<0.05),the escape latencies were significant increased(P<0.05)and the platform crossing times were significant decreased(P<0.05);after ETN administration,there were no changes of activated caspase-3 positive neurons,neuronal density,synaptophysin positive puncta,escape latencies or platform crossing times after propofol anesthesia compared with control(P>0.05);there were no differences in all the data between the sham group and the control group(P>0.05).Afterpropofol anesthesia,TNF-α was expressed in microglia either in the hippocampus or PFC,but it was only expressed in neurons in the PFC.Conclusion: Propofol increased the release of TNF-α in the developing brain,and TNF-α may mediate multiple exposure of propofol-induced developmental neurotoxicity.Microglia are the main source of TNF-α after propofol anaesthesia,while TNF-α synthesis in neurons is brain-region selective. |
PDF Full Text Request