Effect Of CREB Signaling Pathway On Neurotoxicity To Hippocampus Of Developing Rat Caused By Ketamine

BackgroundKetamine, a noncompetitive N-methyl-D-aspartate (NMDA) receptor ion channelblocker, has been used as a general pediatric anesthetic for surgical procedures ininfants and toddlers.Ketamine may cause widespread and dose-dependent apoptoticneurodegeneration during development. After been exposed to the NMDA receptorantagonists, the animals are found to exhibit deficits in hippocampal synaptic functionand persistent memory and learning impairments at a later age.It is recognized that synaptic NMDA receptor-induced membrane depolarizationplays a vital role in providing and sustaining neurotrophic support by activation ofpro-survival protein kinase signaling pathways. In particular, there is compelling invitro evidence for the trophic role of synaptic NMDA receptors via coupling toextracellular signal-regulated protein kinase (ERK1/2), calcium/calmodulin-dependentkinaseâ…£(CaMKâ…£) and cAMP-responsive element binding protein (CREB). CREB isa central transcription factor that mediates cyclic AMP (cAMP) and calcium-dependentgene expression and Ser-133 phosphorylation is considered to be a critical event thatmediates the initiation of transcription, since mutation of Ser-133 to alanine abolishes transcription.The NMDA receptor (NMDAR) is an ionotropic glutamate receptor that isspecifically activated by the selective agonist, NMDA (N-methyl d-aspartate). NMDAreceptor activation leads the opening of an ion channel which allows the flow of Na⁺and K⁺ and Ca²⁺. Calcium influx through NMDARs drives the activation of manydownstream signaling molecules, which underlie many physiological processes such assynpatic plasticity, and learning and memory.In vitro the trophic role of synapticNMDA receptors couples with the cAMP-responsive element binding protein(CREB) .The purposes of this study are to determine whether treatment of developinghippocampus neurons cultures with ketamine results in increase of neurotoxicity andthe contribution of CREB signaling pathway. CREB regulates transcription of manypro-survival genes, including BDNF and Bcl-2. The ability of calcium influx to inducetranscription of brain-derived neurotrophic factor (BDNF) is regulated by the route ofcalcium entry into the cell, by the pattern of phosphorylation induced on thetranscription factor cAMP-response element (CRE) binding protein (CREB).Stimulations activate CREB mainly through increased intracellular cAMP and Ca²⁺(Figure 1). Ca²⁺/CREB/CBP-dependent gene regulation might be a shared mechanismcriticalin both long-term synaptic plasticity and neuronal survival.ThecAMP-responsive element-binding protein (CREB) pathway has been involved in 2major cascades of gene expression regulating neuronal function. The first one presentsCREB as a critical component of the molecular switch that controls long-lasting formsof neuronal plasticity and learning. The second one relates CREB to neuronal survivaland protection.In this paper we studied neurotoxicity to developing hippocampal neuronscaused by ketamine in vivo and in vitro from the effects on neuronal survival, synapseformation, synaptic plasticity and long-term learning and memory.We tested hypothesises that (1) ketamine induces the the apoptosis of developing hippocampusneurons in vitro and in vivo (2) ketamine spoiled synaptogenesis in vitro and in vivo(3) ketamine disrupted memory and learning at a later age. (4) ketamine decreased thecalcium influx of developing neurons by antagonized NMDA receptor calcium channel(5) ketamine down regulated the phosphorylation of CREB on Ser-133 and (6)ketamine reduced expressions of downstream genes of CREB ( BDNF ,Bcl-2 andsynaptophysinI). Methods and Results1. Effect of intravenous general anesthetics on intracellular Ca²⁺ of ratdeveloping hippocampal neurons in vitroMethods: On the fifth day of cultured ,hippocampal neurons were co-incubated with10μmol/L Fluo-4 AM for 30 min at 37℃. Excess dye was washed out with threerinses of DMEM. Fluorescence imaging of intracellular Ca²⁺ was performed to anumber of neurons selected when the hippocampal neurons were exposed to 150μmol /L ketamine, 3μmol / L midazolam or 10μmol / L propofol respectively by confocallaser microscope.Results: After exposed to 150μmol/L ketamine, the intracellular calcium intensity ofthe neurons decreased from (987±307) to (766±226)(P＜0.05). While exposed to3μmol/ L midazolam or 10μmol/ L propofol, the cytosolic Ca²⁺ of the neuronsincreased significantly ,fluorescence intensity of the neurons exposed to midazolamincreased from (1707±514)to (2663±572) (P＜0.05), fluorescence intensity of theneurons exposed to propofol increased from (1057±353) to (1749±708) (P＜0.05).2. Effects of ketamine on neuronal survival of rat developinghippocampal neurons in vitroMethods After cultured primary hippocampal neurons for 5 days, the neurons weredevided into two groups, each group had six well of culture plates: group C (controlgroup) and the group K (co-culture the neurons with the neuronbasel mediumincluding 1 000μmol/L ketamine for 3 h). Detected the apoptosis of the neurons byAnnexin V-PI Apoptosis Kit on flow cytometryResults Compared to group C,the apoptosis of the neurons from group K increased significantly.3. Effects of ketamine on synaptogenesis of rat developing hippocampalneurons in vitroMethods After been cultured for 5 days , primary hippocampal neurons weredevided into group C and the group K.After been intervented, the neurons werecultured to the 14th day continuously then expressions of SynaptophysinI of neuronsin both groups were detected by double-immunofluorescence methods. Greenfluorescence the expression of Synaptophysin I and blue fluorescence representedDAPI.Results Compared with the group C, density and synaptic connections of neurons ingroup K declined significantly and expression of synapsin I reduced statistically.4. Effects of ketamine on CREB signaling pathway of rat developinghippocampal neurons in vitroMethods After been cultured for 5 days, primary hippocampal neurons were devidedinto group C and the group K .After been intervented, expression of p-CREB in bothgroups were measured by immunocytochemistry ,then expressions of the BDNF andBcl-2 in both groups were detected by RT-PCR.Results Compared with the group C,expressions of p-CREB, Bcl-2 and BDNF of theneurons from K proup all decreased statistically (P＜0.05) .5. Preconditioning with NMDA antagonised the effects of ketamine onphosphorylation of CREB on Set-133 of rat developing hippocampalneurons. Methods DIV5 hippocampal neurons were divided into six groups: group C(Control group), group N₁ ( the neurons were co-culture with the neuronbaselmedium including 2μmol/L NMDA for 4 h ), group N₂ ( the neurons were co-culturedwith the neuronbasel medium including 10μmol/L NMDA for 4 h ), group N₁ +K ( theneurons were co-culture with the neuronbasel medium including 2μmol/L NMDA for1 h then the neurons were co-culture with the neuronbasel medium including 1000μmol/L ketamin for 3 h), group N₂ +K (the neurons were co-cultured with theneuronbasel medium including 10μmol/L NMDA for 1 h then the neurons wereco-cultured with the neuronbasel medium including 1000μmol/L ketamin for 3 h) andgroup K ( the neurons were co-cultured with the neuronbasel medium including 1 000μmol/L ketamine for 3 h) and expressions of CREB and p-CREB in each groupwere detected by Western-blot.Results Compared to group C, there were no statistically difference in expression ofCREB among six groups,expression of p-CREB in group N₂ increased significantly,expressions of p-CREB in group N₁+K, group N₂+K and group K all decreasedstatistically. Compared to group K, expressions of p-CREB in group N₂+K increasedstatistically.6. Effects of ketamine on apoptosis rate in hippocampus of developingratMethods Fifteen 7-day-old rats were randomly devided into three groups, group C(control group), group K₁, clinically relevant concentrations of ketamine (10mg/kgsubcutaneous injection) and group K₂ , superclinical concentration of ketamine(20mg/kg subcutaneous injection) . Repeat Subcutaneous administrations for 7 times,each time interval 90min. After administration for 1d, rats were decapitated and frozensections of brain were made.TUNEL Assay Kits was used to detecte the apoptosis of the neurons.Results Compared to group C, apoptosis of neurons in both groups increasedstatistically, from (0.82±1.66) %to (1.55±2.25) % (P＜0.05) and (2.77±2.42)% (P＜0.05) respectively.7.Effects of ketamine on synaptogenesis of rat hippoeampus during developingMethods Thirty 7-day-old rats were randomly derided into three groups, group C,K₁ andK₂, rats were administrated as former. Skin color of rats and whether had performance ofhypoxia were observed carefully after treatment of neonatal rats with ketamine. Duringanesthesia low concentration of oxygen (oxygen flow: 2L/min) were given. Afterrecovery from anesthesia, the rats were returned to mother rats for continue feeding.Afterbeen administrated for 1 week,immuno -histochemistry was used to measur theexpression of Synaptophysin I (the marker of Presynaptic membrane) and Western-blotwas used to detecte the expression of PSD95 (marker of postsynaptic membrane ). IODand relative gray value represented the expressions of synapsin I and PSD95 respectively.Results Between group C and K₁ the expressions of Synaptophysin I and PSD95 had nostatistically difference. Compared to group C ,the expressions of Synaptophysin I andPSD95 in group K₂ both decreased significantly. (P＜0.05) .8. Effects of ketamine on CREB signaling pathway of rat hippocampus duringdevelopingMethods Thirty 7-day-old rats were randomly devided into three groups, group C,K₁and K₂, rats were administrated as former. Double-immunofluorescence methods wereused to measure the expression of p-CREB, then RT-PCR was used to detecte theexpressions of the BDNF and Bcl-2 on the second day after intervention.Results Compared to group C, expressions of the p-CREB,Bcl-2 and BDNF ofneurons in group K1 and K2 both descended statistically (P＜0.05), IOD of CREB in group Kland K2 decreased from (2627.86±345.29) to (1607.98±97.63) and(967.58±61.24 ) ( P＜0.05 ),respectively. Relative gray value of BDNF decreased from(1.355±0.147) to (1.241±0.004) (P＜0.05) and ( 0.863±0.009 ) (P＜0.05) respectively.Relative gray value of Bcl-2 decreased from ( 0.790±0.008 ) to ( 0.740±0.034 ) (P＜0.05)and (0.560±0.014) (P＜0.05) respectively.9. Effects of ketamine on learning and memory of developing rat after maturityMethods Thirty 7-day-old rats were randomly devided into three groups, group C,K₁ andK₂, and rats were administrated as former. After recovery from anesthesia, the rats werewere returned to mother rats for continue feeding .After been administrated for 6 weeks,Morris watermaze was used to test learning and memory function of rats in each group.After been trained for 7d continuously, escape latency, times of creossed the formerplatform and the ratio of time and distance in target quadrant of rats were tested by placenavigation test and spatial probe test.Results Compared with group control,latency to find the platform in group K₂increased from ( 5.12±3.24 ) to ( 10.83±6.48 ) (P＜0.05 ) ,whereas between group C andgroup K₁, latency to find the platform had no difference statistically. There are nostatistically difference in times of crossed the former platform and the ratio of time anddistance in target quadrant among three groups.10. Statistical analysisData are presented as mean±standard deviation (SD), Calcium fluorescence intensityusing paired t test , apoptosis rate ,the expressions of p-CREB,BDNF and Bcl-2 in vitrousing t tests,and the other results using ANOVA by SPSS 13.0. A value of P＜0.05 wasconsidered as statistical significance. CONCLUSIONS and SUMMARY1. Developmental neurotoxicity in hippocampal neurons induced by ketamine wereconfirmed by researches in vitro (cultured primary neurons)and in vivo (animalexperiments) through two aspects of neurotoxicity, survival and neurogenesis ofneurons.2. Effects of ketamine on NMDA receptor and intracellular Ca²⁺ upstream of CREBwere confirmed by researches on cultured primary hippocampal neurons in vitro:ketamine antagonisted NMDA receptor ion channel then reduced calcium influx.Effects of ketamine on expressions of p-CREB,BDNF and Bcl-2 were confirmed bothin vitro and in vivo.3. Researches on long-term learning and memory function of clinically relevantconcentrations of ketamine and superclinical concentration of ketamine conformedthat only superclinical concentration of ketamine can lead to learning and memoryfunction impairment partially.