ABSTRACT
Objective To investigate the effects of sevoflurane ( Sevo ) on dendritic development and the expression of collapsin response mediator proteins ( CRMP ) in the hippocampus of developing rats. Methods Twenty-four neonatal Sprague Dawley (SD) rats at postnatal day 7 (P7) were randomly divided into control group or sevoflurane group ( 12 rat pups for each group) .Rats in the control group were exposed to air for 4 h,whereas rats in the sevoflurane group were exposed to 2.8%sevoflurane for 4 h.The hippocam-pus of some rats were collected,and the expressions of CRMP1,CRMP2 and CRMP4 proteins and phospho-rylation of CRMP2 protein at Ser522,Thr514 and Thr555 were detected by Western blot 6h after exposure ( n=6) .The rest rats were housed till P30,the expression of CRMP1,CRMP2 and CRMP4 proteins in the hip-pocampus were detected by Western blot ( n=6) and the morphology changes of dendrites in the dentate gy-rus ( DG) of hippocampal neurons were detected by Golgi-Cox Staining ( n=6) .Results The expression of CRMP1,CRMP2 and CRMP4 proteins of rats at P7 in the sevoflurane group was decreased by 35.0%( P=0.004) ,27.5%( P=0.015) and 12.0%( P=0.003) ,respectively,and the phosphorylation of CRMP2 pro-tein at Ser522 and Thr514 in the sevoflurane group were increased by 68.3%( P<0.01) ,74.5%( P<0.01) , respectively,6 h after exposure compared with control rats.However,the phosphorylation of CRMP2 protein at Thr555 was not significantly changed after sevoflurane exposure.At P30,both total dendrite length ( P=0.001) and the dendrites length at level 2 and 3 ( P=0.033, P<0.01,respectively) were shorter and the dendritic branching at 120,140 and 160 μm rings in Sholl analysis were less ( P=0.009, P=0.028, P=0.048,respectively) for rats in the sevoflurane group,compared with control rats.There were no significant changes at the expressions of CRMP1,CRMP2 and CRMP4 proteins.Conclusion Sevoflurane inhibits the development of dendrites in the hippocampal DG area of developing rats,which may be related to inhibition of CRMP1,CRMP2 and CRMP4 proteins expression and hyperphosphorylation of CRMP2 Ser522 and Thr514.
ABSTRACT
Aim To investigate the effect of isoflurane on the phosphorylation of p38 mitogen-activated protein kinase (MAPK)in the hippocampus of neonatal rats, and the effect of p38 MAPK pathway on isoflurane-in-duced neuronal apoptosis.Methods Forty-eight neo-natal rats on postnatal day 7 were assigned randomly into four groups:DMSO group (group Air +DMSO), p38 MAPK inhibitor SB203580 group (group Air +SB20 ),isoflurane +DMSO group (group Iso +DM-SO),and isoflurane +SB203580 group (group Iso +SB20 ).Rats were exposed to air or isoflurane (volume fraction of 0.01 1 )for 4h.The p38 inhibitor SB203580 (20 nmol)or DMSO (volume fraction of 0.1 )5μl was intraventricularly administered 30 min before the expo-sure.The brains of some rats in each group were per-fused and embedded by paraffin 6h after the exposure. Neuronal apoptosis in the hippocampal CA1 area was detected by terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL)(n =6). The hippocampal tissues of the other rats in each group were dissected 6h after the exposure,and the protein expressions of phospho-p38 (p-p38 ),p38,cleaved caspase-3,phospho-NF-κB (p-NF-κB ),Bcl-2 and Bax were detected by Westem blot (n =6).Results The number of TUNEL positive cells in the hippocam-pal CA1 region in group Iso +DMSO increased by 4.8 fold compared with that in group Air +DMSO (P <0.01 ),while the number of TUNEL positive cells in group Iso +SB20 decreased by 3 /5 compared with that in group Iso +DMSO (P <0.01 ).The protein expres-sion of cleaved caspase-3 in group Iso +DMSO signifi-cantly increasd (P =0.003)compared to that in group Air +DMSO,which was significantly decreasd in group Iso +SB20 (P =0.007 ).In addition,isoflurane also increased the protein expression of p-p38,p-NF-κB and Bax,decreased the level of Bcl-2,and reduced the ratio of Bcl-2 /Bax compared with control animals (P <0.01 ,P =0.004,P <0.01 ,P <0.01 ,P <0.01 ,respectively).Howerver,SB203580 partly at-tenuated the isoflurane-induced protein change above. Conclusion Isoflurane induces neuroapoptosis in neo-natal rat hippocampus by the activation of p38 MAPK pathway.
ABSTRACT
Objective To evaluate the effects of sevoflurane on proteome in the cortices of neonatal rats.Methods Thirty neonatal rats at postnatal day 7 (6 rats each litter,5 litters in total) were randomly assigned into 2 groups (n =15 each):control group (C group) and sevoflurane group (S group).The rats were exposed to air and 1.8 ％ sevoflurane for 4 h in C and S groups,respectively.One rat from each litter was chosen in each group at the end of anesthesia and the puncture needle was inserted into the left ventricle via the chest wall.Arterial blood samples were then collected for blood gas analysis and for determination of blood glucose.One rat from each litter was sacrificed in each group at 3 and 72 h after the end of anesthesia,and their cortices were then dissected.Two-dimensional differential in-gel electrophoresis (2-D DIGE) was used to identify patterns of protein expression in cortices cross-labeled with different CyDyes.The differentially expressed proteins were analyzed by using matrixassisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).Results Acid-base imbalance,anoxia or lycopenia were not found at 3 h after the end of anesthesia in both groups.The analysis showed there were 6 differentially expressed proteins at 3 h after the end of anesthesia in S group compared with C group.Among the 6 proteins,the expression of 4 proteins (class 2 c beta-tubulin,neuron-specific class Ⅲ beta-tubulin,CRMP-1 and CRMP-4) which belonged to cytoskeleton/neuronal growth proteins was down-regulated,the expression of 1 protein (ATP synthase beta subunit) which belonged to hydrolyses and transferases was down-regulated,and the expression of 1 protein (guanine nucleotide binding protein beta1) which belonged to signal transduction proteins was up-regulated (P ＜ 0.05).No significant changes in protein expression were identified at 72 h after 1.8％ sevoflurane anesthesia (P ＞ 0.05).Conclusion 1.8％ sevoflurane-induced 4 h anesthesia can induce short-time changes in the expression of proteins which are related to neuronal migration,differentiation,energy metabolism and signal transduction in cortices of neonatal rats,which may contribute to its neurodegenerative effects in brains of rats during the development period.
ABSTRACT
Objective To investigate the effects of the c-Jun N-terminal kinase (JNK)pathway on isoflurane induced neuronal apoptosis and the proteins expression of phospho-JNK,Bcl-2 and Bax in the hippocampi of neonatal rats.Methods Forty-eight neonatal rats at postnatal day 7 (P7) were randomly assigned into 4 groups:DMSO control group (group D),SP600125 control group (group SP30),isoflurane + DMSO group (group Iso +D),isoflurane + SP600125 group (group Iso + SP30).Rats were exposed to air (control group) or 1.1％ isoflurane (isoflurane group) for 4 h.The JNK inhibitor SP600125 at 30 μg or 12％ DMSO 5 μl was intraventricularly administered 20 min before the exposure.The brains of some rats in each group were perfused and embedded by paraffin 6 h after the exposure.Neuronal apoptosis in the hippocampi CA1 area was detected by TUNEL (n =6).The fresh hippocampi of other rats in each group were dissected 6 h after the exposure and the proteins expression of phospho-JNK,Bcl-2 and Bax were detected by Western blot (n =6).One way ANOVA were used for data analysis among groups.Results The number of TUNEL positive cells in the hippocampal CA1 regions in group Iso +D (135.72 ±21.26 per mm2) increased by 5 folds compared with group D (24.07 ± 1.35 per mm2) (P＜0.01) ;while the number of apoptotic cells in group Iso + SP30 (42.49 ± 5.56 per mm2) decreased by 84％ (P ＜ 0.05)compared with group Iso + D.The expression of phospho-JNK p46 kd in group Iso + D increased by 44.1％ (P ＜0.01),while both phospho-JNK at p46kd and at p54kd in group Iso + SP30 decreased significantly (P＜0.05,P ＜0.01) compared with group Iso + D.The protein expression of Bax increased 1.5 folds (P＜0.05) and Bcl-2 decreased by 42.2％ (P＜0.05) in group Iso + D compared to group D;while SP600125 significantly decreased expression of Bax (P ＜0.05) and increased expression of Bcl-2 (P＜0.01).Conclusion JNK activation contributes to isoflurane-induced neuroapoptosis in the developing brain.Maintaining Bcl-2 expression and inhibiting Bax expression may be involved in the neuroprotective effects of SP600125.