Ketamine Potentiates Hippocampal Neurodegeneration And Persistent Learning And Memory Impairment Through The PKCγ-ERK Signaling Pathway In The Developing Brain

Objective: Ketamine, an N-methyl-D-aspartate (NMDA) receptorantagonist, is widely used as a general pediatric anesthetic. Recent studiessuggest that ketamine may enhance neuronal apoptosis in developing rodentsand nonhuman primates. The main goals of this study are to determinewhether ketamine causes hippocampal neurodegeneration in the postnatal day7rats and investigate the effects of ketamine on protein kinase Cγ(PKCγ),extracellular signalregulated kinase (ERK)1/2and Bcl-2expression inhippocampi and behavioral deficits in adulthood.Methods: Postnatal day7Sprague–Dawley rats were randomly dividedinto four groups (one control and three treatment groups) and the dosagelevels were defined as0,25,50and75mg/kg body weight. ketamine wasgiven intraperitoneal, once a day and for3consecutive days.After treatment with ketamine, the rats were decapitated and thehippocampi were isolated for detection of neuronal apoptosis by TUNEL．Thenumber of TUNEL-stained hippocampal neurons at regions CA1, CA2, CA3and gyrus dentatusIn order to determine whether ketamine leads to hippocampalneurodegeneration by the PKCγ-ERK signaling, we tested the effect ofketamine on some proteins includings p-PKCγ,t-PKCγ, p-ERK1/2, t-ERK1/2and Bcl-2in different groups. The expression levels of PKCγ,ERK1/2andBcl-2in hippocampal neural cell were measured by Western-blot.At2months of age, learning and memory were tested by the MorrisWater Maze.Results:3.1The number of TUNEL-stained hippocampal neurons at regions CA1, CA2, CA3and gyrus dentatusThere were a number of apoptosis positive cells in control group, group k1，k2and k3at regions CA1, CA2, CA3and gyrus dentatus, while the number ofapoptotic cell per mm~2in group k3at regions CA1and gyrusdentatus(47.10±9.31and39.40±9.82) was more than those in control group(25.70±5.75and27.30±5.37)(P <0.01and P <0.01). Data were shown asTable1and Fig.1.3.2Effect of the different doses of ketamine on the PKCγ-ERK signalingby western blottingIn order to determine whether ketamine leads to hippocampalneurodegeneration by the PKCγ-ERK signaling, we tested the effect ofketamine on some proteins includings p-PKCγ,t-PKCγ, p-ERK1/2, t-ERK1/2and Bcl-2in different groups. Compared with the control group, ketamineinduced a mild depression in PKCγ and ERK1/2phosphorylation andexpression of Bcl-2with the increasing dose, whereas, it did not significantlyaffect t-PKCγ and t-ERK expression levels. Data were shown as Table2andFig.2.3.3Effects of ketamine on learning and memory ability by Morris WaterMaze testAt age of60days, the control group and the rats that underwent thedifferent doses of ketamine were trained in the Morris water maze. Thelatency and distance for rats to find the platform in either Trial1or Trial2were shown in Fig.3. As indicated in Trial1of Fig.3, compared with thecontrol group, group k3took longer to find the hidden platform on the fourthand fifth day training days. One way ANOVA showed that there was asignificant difference （P <0.01and P <0.05） between the fourth and fifthtraining days. Dunnett’s post hoc test showed that there are differencesbetween group k3and control group on day4(P <0.01) and5(P <0.05).Furthermore, compared with the control group, group k3took longer swimdistance to find the hidden platform on the fourth and fifth training days. Oneway ANOVA showed that there was a significant difference（P<0.01and P < 0.01）in the fourth and fifth day training days. Dunnett’s post hoc test showedthat there are differences between group k3and control group on day4(P<0.01) and5(P <0.01). These data revealed that75mg/kg ketamine had causedsignificant learning ability impairment, and this impairment started fromtraining day4.In the probe trial, the platform was removed and the animal was placedinto quadrant2, which is opposite to the target quadrant (quadrant4). Datawere recorded including the time spent in crossing the platform, the time thatan animal spent on the target quadrant and the number of times that the sameanimal crossed the former platform area. In the120s of the probe trial, groupk2and k3spent longer time to cross the platform than the control group. Oneway ANOVA showed that there was a significant difference (P <0.01) in theprobe trial. Dunnett’s post hoc test showed that there were significantdifferences in group k2, k3vs. control group (P <0.05and P <0.01,respectively, Table5and Fig.4A).During the120s of the probe trial, thecrossing times of group k3was significantly less than animals in control group(ANOVA P<0.05; Dunnett’s post hoc test: P<0.05; Table5and Fig.4B).Furthermore, animals in control group spend significantly more time in thetarget quadrant than rats in group k3(ANOVA: P <0.01; Dunnett’s post hoctest: P <0.01; Table5and Fig.4C)Conclusion:In addition, The results showed that high doses of ketamine suppressedp-PKCγ, p-ERK1/2and Bcl-2expression. In contrast, ketamine did notsignificantly affect t-PKCγ and t-ERK expression levels. The high dose ofketamine administrated growing spurt in the brain may cause the learning andmemory impairments in adults. Therefore, these data demonstrate that the highdose of ketamine administration in the developing brain may result inhippocampal neurodegeneration and persistent learning and memoryimpairment, which is closely associated with the PKCγ-ERK signalingpathway.