The Role Of Oxidative Stress-mediated Loss Of Phenotype Of Parvalbumin Interneurons In Repeated Neonatal Ketamine Exposures-induced Long-term Cognitive Impairments And The Underlying Mechanism

Objective:With the development of surgical techniques,more and more babies,infants,and toddlers are subjected to surgery with general anesthesia at home and abroad annually.Ketamine,a noncompetitive N-methyl-D-aspartate(NMDA)receptor ion channel blocker,is a commonly used anesthetic for pediatric patients.Extensive bodies of preclinical studies have suggested that repeated neonatal ketamine exposures induce cognitive impairments such as learning and memory dysfunction.However,the precise mechanisms underlying this neurotoxic effect of repeated neonatal ketamine exposures remain unclear.Loss of phenotype of parvalbumin(PV)interneurons has been implicated in impaired behavior and cognition associated with a number of major neuropsychiatric disorders,including Alzheimer’s disease(AD),schizophrenia,etc.Although the factors triggering dysfunction of PV interneurons are diverse,oxidative stress is regarded as one of the contributing factors because of the high metabolic requirements of these interneurons.Considering that ketamine treatment can induce a persistent increase of superoxide in brain due to activation of nicotinamide adenine dinucleotide phosphate(NADPH)oxidase in vitro,we raised a hypothesis that repeated neonatal ketamine exposures might mediate loss of phenotype of PV interneurons and consequently contribute to long-term cognitive impairments.The present study aimed to investigate the role of the alteration of PV expression in the pathogenesis of repeated neonatal ketamine exposures,and to study whether NADPH oxidase is involved in this process.Methods:Male Sprague Dawley(SD)rats at postnatal day 6(P6)rats were randomly assigned to the following four groups:control + vehicle group;control + Apo group;ketamine + vehicle group;and ketamine + Apo group.Animals in the ketamine groups received ketamine(75 mg/kg)intraperitoneally daily for 3 consecutive days from P6 to P8.For the interventional study,pups were treated with a NADPH-oxidase 2(NOX2)inhibitor,apocynin(Apo).Learning and memory abilities of rats(n = 48)were tested by the open field,fear conditioning,and Morris water maze on P40,P42-44,and P50-56,respectively.For histological and biochemical assays,a separate cohort of rats(n = 140)was killed on P8 or P60 and the brain tissues were harvested.The determination of the contents of hippocampal and prefrontal cortex glutamic acid decarboxylase 67(GAD67),PV,NOX2/gp91phox,and Nox4 were detected by Western Blot(WB).The measurement of NOX2/gp91phox mRNA level was conducted by quantitative real-time polymerase chain reaction(q-PCR).The fluorescence intensities of 8-hydroxyl-2’-deoxyguanosine(8-OH-dG),PV and NOX2/gp91phox in hippocampus and prefrontal cortex were detected by immunofluorescence.Electron transport chain(ETC)enzymatic activities and adenosine triphosphate(ATP)level tests were performed.Results:In fear conditioning test,the freezing time to context was decreased after repeated ketamine exposures in the Ket + vehicle group compared with the control +vehicle group(P<0.05),whereas the freezing time was increased in the Ket + Apo group compared with the Ket + vehicle group(P<0.05).In the MWM training test,a longer escape latency was observed after repeated ketamine exposures from days 3 to 5 in the Ket + vehicle group than in the control+ vehicle group(P<0.05),whereas the escape latency was shorter from days 3 to 5(P<0.05)in the Ket +Apo group than in the Ket + vehicle group.In the MWM probe trial,the target quadrant time in the Ket + vehicle group was shorter than the control + vehicle group(P<0.05).In the Western Blot,the contents of hippocampal and prefrontal cortex NOX2/gp91phox were increased,and the contents of hippocampal and prefrontal cortex PV and GAD67 were decreased in the Ket + vehicle group compared with the control + vehicle group(P<0.05)at P8 as well as P60;whereas the contents of hippocampal and prefrontal cortex PV and GAD67 were increased in the Ket + Apo group compared with the Ket+ vehicle group(P<0.05).In the q-PCR,the mRNA of hippocampal and prefrontal cortex NOX2/gp91phox were increased in the Ket + vehicle group compared with the control + vehicle group(P<0.05)at P8,whereas the contents of hippocampal and prefrontal cortex NOX2/gp91phox were increased in the Ket + Apo group compared with the Ket + vehicle group(P<0.05).In the immunofluorescence,an increase of 8-OH-dG staining in the prefrontal cortex and hippocampus was observed in the Ket+ vehicle group compared with the control + vehicle group(P<0.05),whereas administration of Apo attenuated DNA oxidation in neonatal rats at P8(P<0.05).In addition,double-immunofluorescence staining was performed for the PV and gp91phox/NOX2.Most PV immunoreactive interneurons were stained for gp91phox/NOX2 in the ketamine-exposed rats.However,no difference was detected in the open field test,the percent of the freezing time to tone in the fear conditioning test,and the contents of hippocampal and prefrontal cortex NOX4,enzymatic activities of complexes Ⅰ and Ⅲ,and ATP levels among the four groups(P>0.05).Conclusion:In the present study,we showed that repeated ketamine exposures resulted in an increase in NOX2 and subsequent enhanced oxidative stress,which in turn caused loss of phenotype of PV interneurons in the hippocampus and prefrontal cortex and ultimately led to the neurobehavioral impairments later in life.