Effect Of Ketamine On Brain Cortical Apoptosis Of Neonatal Rats, Investigation Of Its Mechanism, And Prevention Of Shenfu Infusion

PART ONEEffect of Ketamine on Brain Apoptosis of Cortical and Hippocampal Areas of Neonatal RatsOBJECTIVEThis stydy was conducted to investigate the effects of ketamine on neuronal apoptosis of the cortical and hippocampal areas in the neonatal rat brain.METHODSFifteen 7-day-old Sprague-Dawley rats were randomly divided into 3 groups(n=5 each), and received intraperitoneal(i.p) injection with saline(control), keamine 20 mg/kg and ketamine 80 mg/kg.10 min after injection,0.5ml blood were drawn from left cardiac ventricle by 1ml skin test needle and sent for performing blood gas analyse.24h after injection, body weight inceasing of three groups were compared. Apoptotic neurons were observed by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and the Caspase-3 expression was detected by immunohistochemical method.RESULSThere were no difference on blood gas results and body weight increasing between three groups.Compared with the control group, the TUNEL positive cells were increased but no obvious difference(P>0.05), the Karyopyknosis neuron number and the Caspase-3 positive cells were significantly increased in 20mg/kg ketamine group(P<0.05), the TUNEL positive cells, the Karyopyknosis neuron number and the Caspase-3 positive cells were significantly increased in 80mg/kg ketamine group(P<0.05).The apoptotic cells, the Karyopyknosis neuron number and the Caspase positive cells in ketamine group were mostly seen in cortical area, not in hippocampal area.CONCLUSIONKetamine can induce neuronal apoptosis in cortical area of neonatal rat in vivo. Ketamine induce apoptosis of the cortical neurons in neonatal rat brain in vivo in a dose-related mannar. Apoptosis induced by low doses of ketamine are minor, whereas high doses of ketamine are more serious. Apoptosis induced by Ketamine have no relation with the nutritional intake and hypoxia of the neonatal rats. Apoptosis of the hippocampus induced by Ketamine were not so significant in vivo.PART TWOInvestigation of Mechanism of Ketamine Induced Apoptosis of Primary Cultured Cortical Neurons of New Born Rats in vitro OBJECTIVEThis stydy was conducted to investigate the mechanism of apoptosis induced by ketamine of primary cultured cortical neurons of new born rats in vitro.METHODSCortical neurons was isolated from new-born SD rats and cultured for 6 days, Cerebral cortex of 24h-newborn SD rat is isolated and cortical neurons is primary cultured in vitro, cortical neurons were identified by tetanus toxin labeled with fluorescent dye which is specifically binded to neurons. Inverted phase contrast microscope and fluorescent staining Hochest33258 were used to observe morphological changes of apoptosis; lactate dehydrogenase (lactate dehydrohenase, LDH) release rate were used to indicate cell death rate; apoptosis rate were detected by counting neurons staining with Hoches33258, Akt and GSK-3βphosphorylation level were detected and semiquantitative analysed with the Western Blotting.Primary cultured cortical neurons were treatment with 100μM ketamine for 24 hours.Morphological changes of neurons and apoptotic rates at different time points were compared with the control group. GSK-3βand Akt phosphorylation were detected 0,0.5,1,6,12,18 hours after adding 100μM ketamine and compared with the control group. Cortical neurons were treated with 1,10,100μM ketamine respectively for 0.5 hour, phosphorylation of Akt were compared in these different concentrations of ketamine. Effects of three concentrations (5μM,25μM and 50μM) of GSK-3βspecific small molecule inhibitor SB-415286 on ketamine-induced cortical neurons apoptosis and release of LDH relative were compared. Effects of two concentrations (5ng/ml and 50ng/ml) of IGF-1 on ketamine-induced cortical neuronal apoptosis rates and GSK-3βand Akt phosphorylation levels were compared.RESULS Cortical neurons of neonatal rat were cultured for 6 days in vitro and were identified by immunofluorescence staining. Neuronal purity was more than 90%.12 hours after 100μM ketamine treatments, cortical neurons began to emerge morphological changing, cell bodies became round and neuronal processes broken. Accompanied with treatment time increasing, neurons body became shrinked and rounded, the structure became unclear, boundaries became vague, neuronal processes became uncontinuous; 24 hours after treatment neuronal bodies became even smaller, vacuolization, processes and network structure disappeared. After treatment with 100μM ketamine, cortical neuronal apoptosis increased in a time-dependent manner. 24 hours after treatment, neuronal apoptosis rate were up to near 41.8%, There is statistically significant differences when compared with the control group. Neruonal LDH release increasing appeard 18 hours after 100μM ketamine treatment,24 hours after treatment, neuronal relative release of LDH was about 2 times of the control group.0.5,1,3,6 and 12 hours after treatment of 100μM ketamine, GSK-3βphosphorylation level on Ser9 sites of cortical neurons were 81.6±3.36%,57.0±2.24%,53.2±1.92%,51.6±2.30% and 52.6±3.04% of the control group. There was statistical difference when compared with 0 hr control group, whereas 18 hours after treatment GSK-3βphosphorylation level gone up to 89.2±2.59, reflecting that 1 to 18 hours after ketamine adding GSK-3βwas continued to be activated. After three concentrations (5μM,25μM and 50μM) of SB-415286 pre-incubated for 2 hours, neuronal apoptosis rates were reduced to 51.2±2.8%,40.1±3.5% and 34.9±3.3%, compared with the ketamine group the difference was statistically significance (P <0.05), suggesting the activity of GSK-3P inhibition antagonized ketamine-induced neuronal apoptosis.0.5h after 100μM ketamine treatment, Akt Ser473 phosphorylation level of cortical neurons significantly decreased to 67.2±2.42% of the control group, and remained to be low at 1,6,12 hours (49.6±3.01%,30.8±1.94% and 35.3±2.87% respectively), then recovered to 60.4±2.33% at 18h, there was statistically significant differences when compared with the control group (P<0.05), reflecting Akt activity was continued to be suppressed 1 to 18 hours after adding of ketamine.After 5ng/ml and 50ng/ml of IGF-1 pre-incubated for 2 hours, cortical neurons were treated with 100μM ketamine for 24 hours. Compared with the ketamine group, neuronal apoptosis rate were lower(27.8±0.99% and 14.7±1.37% respectively), suggesting that activation of Akt can antagonize ketamine-induced apoptosis in cortical neurons; On the same condition, GSK-3 (3 Ser9 phosphorylation were 199±9.2% and 212±11.5% of the control group, Akt Ser473 phosphorylation levels were 229±11.2%, and 232±17.1% of the control group. Consistency of changing of GSK-3βSer9 phosphorylation with Akt Ser473 phosphorylation, suggests that GSK-3βand Akt are involved in the ketamine-induced apoptosis of cortical neurons. Ketamine may inhibit Akt, thereby activating GSK-3(3, eventually inducing apoptosis in cortical neurons.CONCLUSIONKetamine induced the new-born SD rats' cerebral cortical neurons apoptosis through inhibiting Akt, then activating GSK-3β.PART THREEEffects of Shenfu Injection on Neuron Apoptosis Caused by Ketamine in Cortical area of Neonatal Rat's BrainOBJECTIVE This stydy was conducted to investigate the effects of Shenfu injection on neuron apoptosis caused by ketamine in cortical area of neonatal rat's brain.METHODS15 seven-day old Sprague Dawley rats were randomly divided into three groups(n=5 each):Control group(Group C) receiving intraperitoneal (i. p) injection of saline, ketamie group(Group K) receiving 80mg/kg, and Shenfu injection pretreatment plus ketamine group(Group SK). Apoptotic neurons were observed by the terminal deoxynucleotidyl transferase·mediated dUTP nick-end labeling(TUNEL)and the Caspase-3 expression was detected by immunohistochemical method.RESULSCompared with C group, there are more pyknosis neurons in K group and SK group, but there is no significant difference (p>0.05) between three gruops. Compared with Group C, there is significant increase in apoptosis rate of cerebral cortical neurons in Group K and Group SK (p<0.05); compared with Group K, apoptosis rate of cerebral cortical neurons decrease significantly in Group SK (p<0.05). Compared with Group C, there are significant more Caspase-3 positive neurons in Group K and Group SK (p<0.05); compared with Group K, Caspase-3 positive neurons decrease significantly in Group SK (p<0.05)CONCLUSIONOur results suggest Shenfu injection may decrease the incidence of ketamine induced apoptosis of cerebral cortical neurons of seven-day old rats, and inhibiting caspase-3 activation may be one of its mechanisms. TO SUMMARIZEThe first:Ketamine can induce neuronal apoptosis in cortical area of neonatal rat in vivo. Ketamine induce apoptosis of the cortical neurons in neonatal rat brain in vivo in a dose-related mannar. Apoptosis induced by low doses of ketamine are minor, whereas high doses of ketamine are more serious. Apoptosis induced by Ketamine have no relation with the nutritional intake and hypoxia of the neonatal rats. Apoptosis of the hippocampus induced by Ketamine were not so significant in vivo.The second:Ketamine can induce apoptosis of cultured cortical neurons of neonatal rat in vitro. Ketamine increased apoptosis of neonatal cortical neurons, accompanied by morphological changes characterized by cell shrinkage, nuclear condensation or fragment formation. Insulin-like growth factor-1 completely abolished the ketamine-induced apoptosis in neonatal rat cortical neurons. Ketamine decreased Akt phosphorylation levels. Ketamine induced apoptosis by activating GSK-3βin neonatal rat cortical neuronsThe Third:Shenfu injection may decrease the incidence of ketamine induced apoptosis of cerebral cortical neurons of seven-day old rats, and inhibiting caspase-3 activation may be one of its mechanisms.