Neurosretoid Protects Against Ketamine-induced Neuroapoptosis Of The Developing Cortical Neurons In Rats

Globally, there were about1.5million infants and young childrenundergoing general anesthesia because of various reasons such as surgery orinterventional therapy each year. For a long time, whether or not generalanesthesia would affect the development of infant’s brain has been the focus ofattention of family members and anesthesiologists. The conventional belief isthat, as long as there was no cerebral hypoxia or other relevant consequencesduring the anesthesia, it would not affect the brain development of infants andyoung children. However, neuronal cell death after general anesthesia hasrecently been demonstrated in neonatal animal models. The possibility ofanesthesia-induced neurotoxicity in human neonates or infants has led toserious questions about the safety of pediatric anesthesia.Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, iswidely used in surgical anesthesia of infants and young children now. TheNMDA receptor is a kind of central nervous system excitatory amino acidionotropic receptors, with many different allosteric regulatory sites and Ca2+high degree of permeability. In the central nervous system, NMDA receptorsare mainly distributed in the cerebral cortex and hippocampus. Studies havefound that the NMDA receptor is closely related to the development of centralnervous system, controlling neuronal differentiation, migration and survival,and playing an important role in the formation and maintenance of learningand memory. Recent studies have found that developing neurons can beinduced to activate pathways resulting in cell death at the peak of the centralnervous system growth and development, when their synaptic development,dendritic branching, and remodeling activities are interfered with. Manystudies have confirmed that it is of great significance for the NMDA receptorto maintain moderate excitement for the survival of neurons in the critical period of brain development. If the activity of NMDA receptors was changed,it would affect the development and function of the central nervous system.An increasing number of animal studies have suggested that ketaminecauses neurodegeneration during early development. Studies in vitro have alsoshown that ketamine administration induces apoptosis in cultured neurons.The exact mechanism triggering ketamine-induced apoptosis has not yet beenfully elucidated by now. Previous studies have shown that injection ofketamine could cause up-regulation of the NMDA receptor in the rat brainduring its developmental stages, resulting in a wide range of dose-dependentapoptosis. In addition, as a noncompetitive antagonist of the NMDA receptor,ketamine can act on PCP binding sites of the NMDA receptor, blocking theNMDA receptor coupled calcium channels, reducing calcium influx, andreducing the concentration of intracellular calcium. This can antagonize theregulation of nervous system development by glutamate, aspartate and otherexcitatory amino acids. Recent studies showed that ketamine inducedneuroapoptosis by inhibiting Ca2+oscillations in neurons.With respect to anesthesia-induced brain injury in neonates, the NIH, theFDA, and the International Anesthesiology Research Society (IARS) haveurged researchers not only to define the scope of the problem but also todevelop therapeutic strategies that target prevention. Although many strategies,including the use of activity-dependent neuroprotective protein peptidefragment NAPVSIPQ (NAP), vitamin D, lithium, erythropoietin, L-carnitine,nicotinamide, and Clonidine, are known to have neuroprotective effectsagainst ketamine-induced apoptosis both in vivo and in vitro, their efficacyand safety need to be further verified for uses targeting the developing brain.Steroids hormones and their metabolites within the central nervous system(CNS) are commonly defined as neuroactive steroids or neurosteroids. Theycan be synthesized de novo in the CNS by glial cells and neurons. Neuroactivesteroids mainly include pregnenolone (PREG), dehydroepiandrosterone(DHEA), their sulfate derivatives pregnenolone sulfate (PREGS) anddehydroepiandrosterone sulfate (DHEAS), and progesterone (PROG), 5α-dihydroprogesterone (5α-DHP),3α,5α-tetrahydroprogesterone(allopregnanolone/AP), deoxycorticosterone (DOC),tetrahydrodeoxycorticosterone (THDOC), estradiol (E2), etc. The studyshowed that neurosteroids played an important role as endogenous modulatorsin the development of CNS and exerted neuroprotection.Estradiol is a kind of neurosteroids, which has the potential for promotingsurvival and function of neurons. Recently, Trickler et al reported thatketamine attenuated17β-estradiol level and increased testosterone level andketamine had been shown to be neurotoxic in early life stages of zebrafish.PI3K-Akt signaling is very important for promoting neuronal survival.Ketamine induces apoptosis by blocking NMDA receptor activity anddecreasing levels of p-AKT. Previous studies reported that estradiol treatmentof neonatal rats inhibited neuroapoptosis induced by the NMDAR antagonistMK-801and anesthetic cocktails including midazolam, isoflurane, and nitrousoxide. More recently, Ramezani and colleagues suggested that17β-estradiolcould protect the developing rat cerebellum from ethanol-inducedneurotoxicity and improve behavioral deficits. The results from our laboratoryhave shown that Aβ25-35induces apoptosis in primary cultured cortical neuronsaccompanying the change of the methabolism of neurosteroids. However,whether ketamine induces apoptosis in primary cultured cortical neuronsaccompanied by the change of the methabolism of neurosteroids and whether17β-estradiol can protect rat cortical neurons from ketamine-induced apoptosisremain relatively elusive. In our study, an in vitro ketamine-induced cellapoptosis model was established based the primary cultured rat corticalneurons. Based on this model, the HPLC-MS/MS assay was applied toinvestigate the changes of chief neurosteroids, PREG, PROG and17β-estradiol levels in cultured neurons induced by ketamine cytotoxicity.Afterwards,17β-estradiol, whose level found obviously decreased byketamine treatment, was chosen as intervening substances to investigateitsprotective effects against ketamine-induced neuroapoptosis. Then whetherPI3K-Akt signaling takes part in neuroptotection of17β-estradiol against ketamine-induced neuroapoptosis was studied. The study is to add valuableefforts for the protective effects of17β-estradiol against ketamine-inducedneuroapoptosis.The following methods were applied in this thesis:1Cortical neurons of newborn SD rat were cultured.Cortical neurons were grown for7days before experimentation. Invertedmicroscope was used to observe morphological changes of cultured neurons.2Grouping and drug treatments①Ketamine induces injuries in primary cultured cortical neurons:different concentrations of ketamine treatment for24h100μM ketamine treatment for different time②Effect of ketamine on neurosteroidogenesis in primary rat corticalneurons:control, ketamine, cholesterol, cholesterol+ketamine③Effects of17β-estradiol treatment on neuroapoptosis induced byketamine exposure:control, ketamine,17β-estradiol+ketamine④17β-estradiol exerts neuroprotective effects against ketamine-inducedneuroapoptosis and long-term behaviour deficts in developing brain:control, veichle-control,17β-estradiol, ketamine,17β-estradiol+ketamine⑤Study on the mechanism of17β-estradiol against ketamine-inducedneuroapoptosis of the developing cortical neurons:control, ketamine,17β-estradiol+ketamine,17β-estradiol+ketamine+LY2940023Sampling and quantitationTreated neurons were collected, after cell lysis, supernatant proteinconcentrations were quantitatived by multifunctional enzyme markinstrument.4Neuron viability assay5Apopyosis of neurons indentified by Hoechst233258dying Apoptosis were identified by nuclear morphometry under fluorescencemicroscope. The apoptosis was calculated by5randomly selected eye fields.6Apoptotic neurons were detected using the TUNEL assayApoptotic neurons were identified by TUNEL assay under invertedicroscopy. The apoptosis was calculated by5randomly selected eye fields.7Immunohistochemical Staining for cleaved caspase-3Apoptotic cells were identified by immunohistochemical Staining forcleaved caspase-3under inverted icroscopy. The apoptotic cells (per0.01mm2)in the prefrontal cortex were counted8Western Blot Analysis for p-Akt, Akt, cleaved-caspase-3, Bcl-2expression9Determination of the concentrations of neurosteroids in culture mediausing HPLC-MS/MS assay10Morris water maze test11Statistical AnalysisThe data were expressed as mean±standard deviation (SD). The meanescape latency of the Morris water maze behavioral tests collected during thetraining days were analyzed by repeated-measure analysis of variance(ANOVA). Other data were statistically analyzed by one-way ANOVA,followed by between-group comparisons using LSD post hoc test. Statisticalsignificance was concluded with a value of P<0.05for all analyses. Dataanalyses were performed with the SPSS software version13.0Results:1Ketamine induces injuries in primary cultured cortical neurons1.1Ketamine decreased neuron viability dose and time dependentlyThe MTT assay was used to evaluate the viability of neurons. Ketaminedecreased cell viability dose and time dependently. Compared withvehicle-control group, the neuron viability of ketamine treated group (24h)significantly (P<0.01for ketamine100μM and1000μM) decreased.Compared with control group, the neuron viability of100μM ketaminesignificantly decreased (P<0.05for24h, P<0.01for48h).1.2Morphoolgical alteration of neurons The newly inoculated cortical neurons were round, small, translucent,and in a single suspension uniform distribution. After8h, the cells started toadhere to the culture plates, and most of neurons had adhered in24h. Theirmorphologies were fusiform, triangular, with different length of slenderprotrusions. On the3rd day, the cells were well-rounded and full, and theprominences were elongated and enlarged more significantly and connected tothe network, sparse connections had come into being. Cortical neuronscultured for7days, were generally bipolar or multipolar cells under invertedmicroscopy, most of them with triangular soma,and usually aggregated withtheir branching dendrites interconnected as network. Compared with controlgroup,24h after100μM ketamine treatment, numbers of neurons decreasedobviously, with reduced refraction, axons break, and granuliform pieces.1.3Ketamine influences on neuroapoptosis identified by TUNEL assay andHoechst33258staining.Compared with control group, ketamine treatment increased neuronsapoptosis signficantly (P<0.01).2Effect of ketamine on neurosteroidogenesis in primary rat cortical neurons2.1Influence on neurons viabilityCompared with control group, neuron viability decreased significantly inketamine-treated group (P<0.01), while cholesterol treatment made nodifference with controls (P>0.05). But, in ketamine+cholesterol group, neuronviability was obviously higher than ketamine treatment alone (P<0.01)2.2Influence on neuron apoptosisWe use TUNEL assay and Hoechst33258staining to identified neuronapoptosis in different treatment group. Compared with control group,apoptosis rate increased significantly in ketamine-treated group (P<0.01),while cholesterol treatment made no difference with controls (P>0.05). But, inketamine+cholesterol group, apoptosis rate was obviously lower thanketamine treatment alone (P<0.01).2.3Influence on neurosteroidgenesisAbsence of the necessary substance, cholesterol, no neurosteroids found in control and ketamine alone groups. Compared with cholesterol treatmentalone, no changes in PREG level were observed after treatment with ketamine,while PROG increased significantly (P<0.05) and17β-estradiol decreasedgreatly (P<0.01).3Effects of17β-estradiol treatment on neuroapoptosis induced by ketamineexposure3.1Protective effects of17β-estradiol against ketamine-induced cell death incultured cortical neurons.We used the MTT assay to assess the changes in neuron viability.Compared with the control group, neurons exposed to100μM ketamine for24h exhibited a significant decrease in neuron viability (P<0.01). Toinvestigate the possible neuroprotective effects of17β-estradiol againstketamine-induced cell death, we used an MTT assay to study the changes inviability after the neurons were co-incubated with ketamine and differentconcentrations (0.001,0.01,0.1and1μM) of17β-estradiol for24h.Co-incubation with17β-estradiol resulted in a significant andconcentration-dependent enhancement of survival. The maximal rescueoccurred at a17β-estradiol concentration of0.1μM.3.217β-estradiol treatment reduces neuroapoptosis induced by exposure toketamine.To determine the amount of apoptosis that occurs after differenttreatments, we carried out TUNEL assays, in the control group, only a fewapoptotic cells were seen, but the number of apoptotic cells increased afterexposure to100μM ketamine for24h (P<0.01). This increase was preventedby the addition of17β-estradiol (P<0.01). To further investigate theantiapoptotic effect of17β-estradiol, we used Hoechst33258staining todetermine the amount of neuron apoptosis, and similar results were obtained.3.317β-estradiol exerts neuroprotective effects against ketamine-inducedneuroapoptosis in brain PFC.We use immunohistochemical staining for cleaved caspase-3toinvestigate the neuroprotective effects of17β-estradiol against ketamine-induced neuroapoptosis in PFC. Compared to control group, arobust degenerative reaction was detected in PFC of ketamine-treatedgroup(P<0.01). Administration of17β-estradiol only had no influence on theamount of ongoing physiological apoptosis. However, when17β-estradiol wascoadministered with ketamine, it significantly amelioratedneuroapoptosis-induced by ketamine exposure (P<0.01).3.417β-estradiol improved long-term behavioral deficits in Morris water mazetestAt60day of age, all rats were trained in the Morris water maze to testlearning and memory abilities. Briefly, rats received four training trials dailyfor five consecutive days. All animals showed a progressive decline in theescape latency and path length with training. The escape latency and pathlength of ketamine treatment group were more than the vehicle controls on thethird, fourth and fifth training days (P<0.01), while those of the17β-estradiolin combination with ketamine treatment group were significantly lower thanketamine treatment group (P<0.01). On test day6, rats were subjected to aprobe trial, ratio of time spent in the target quadrant and the number ofcrossings over previous platform locations in ketamine group were fewer thanveichle-control group (P<0.01), while those of17β-estradiol in combinationwith ketamine treatment group were more than ketamine treatment group(P<0.01).4Study on the mechanism of17β-estradiol against ketamine-inducedneuroapoptosis of the developing cortical neurons4.1Effects of different treatments on pAkt expressionWe measured the changes in pAkt in neurons exposed to17β-estradiolalone, pAkt level remained elevated for at least4h when only17β-estradiolwas added to the culture.When17β-estradiol and ketamine were added tocultures simultaneously, p-Akt level remained elevated for at least4h.Wesuspect that17β-estradiol can attenuate the ketamine-induced down-regulationof pAkt by activating the PI3K pathway. To ascertain this, we treated cellswith10μM LY294002, a PI3K inhibitor. Compared with control group, pAkt level decreased when neurons were exposed to ketamine alone for2h(P<0.01). Compared with ketamine-treated group, pAkt increasedsignificantly in cells treated with both ketamine and17β-estradiol for2h(P<0.01), while a1-hr pre-treatment with LY294002abolished the17β-estradiolinduced up-regulation of p-Akt (P<0.01).4.217β-estradiol enhances Bcl-2expression through the PI3K/Akt pathwayWestern blot analysis detected decreased Bcl-2expression in neurons ofthe ketamine group compared with those in the control group (P<0.01).However, Bcl-2expression increased in cortical neurons treated with17β-estradiol after24hr of ketamine exposure (P<0.01). The effect of17β-estradiol treatment was inhibited by LY294002pre-treatment (P<0.01).4.317β-estradiol prevents apoptosis in ketamine-treated neurons by activatingthe PI3K pathway and blocking caspase-3activityCompared with control group, the expression of cleaveded caspase-3incells exposed to ketamine for24h increased significantly (P<0.01).Co-incubation of cells with both17β-estradiol and ketamine significantlydecreased the up-regulation of cleaved caspase-3induced by exposure toketamine alone (P<0.01). Consistent with the critical role of PI3K inmediating17β-estradiol induced neuroprotection, the addition of LY294002tothe medium reversed the effect of17β-estradiol on cleaved caspase-3expression in neurons (P<0.01).4.4Effect of LY294002on neuroprotection of17β-estradiolCompared with control group, neuron viability decreased significantly inketamine-treated group (P<0.01), while only LY294002treatment made nodifference with controls (P＞0.05). But, in ketamine+17β-estradiol group,neuron viability was obviously higher than ketamine treatment alone (P<0.01),the addition of LY294002to the medium reversed the effect of17β-estradiolon neuron viability (P<0.01).We used TUNEL assay and Hoechst33258staining to identified neuron apoptosis in different treatment groups.Compared with control group, apoptosis rate increased significantly inketamine-treated group (P<0.01), while ketamine+17β-estradiol treatment group, apoptosis rate was obviously lower than ketamine treatmentalone(P<0.01). The addition of LY294002to the medium reversed the effectof17β-estradiol on neuron apoptosis (P<0.01).Conclusions1The inhibitions of ketamine to the immature cortical neurons areconcentration-and time-dependent.2Ketamine-induced neuron apoptosis is accompanied by increasedlevel of PROG and decreased level of17β-estradiol.317β-estradiol protects primary cultured rat cortical neurons fromketamine-induced apoptosis.417β-estradiol attenuates ketamine-induced neuroapoptosis and long-term behavioral deficits in developing rats.517β-estradiol protects primary cultured rat cortical neurons fromketamine-induced apoptosis, in part through the PI3K/Akt/signalling pathway,with the increase of Bcl-2expression and the decrease of cleaved-caspase-3expression.