| BackgroundIt was estimated that6million children (including1.5million infants) undergo surgery and anesthesia each year in the USA alone. Although there was no relevant data in China, the number of Chinese children received general anesthesia and surgery was much larger than that in USA on the fact of large number of Chinese population. The development of human brain begins in the early pregnant stage and ends several years after birth. It is generally accepted that brain growth spurt in humans was between the last month of gestation and first6months after birth. Therefore it is a hot issue whether general anesthesia could exert adverse effect on children’s intelligence and long-lasting deficit in learning and memory.In clinical trials, children who received general anesthesia in infant period represented cognitive impairment in adulthood. This indicated that general anesthetics could damage central nervous system in infants’period. In rodents, exposure to general anesthetics such as isoflurane, sevoflurane, devoflurane, ketamine and propofol in the period of peak brain growth induced damage of synaptic plasticity, neuroapoptosis and other neurodegenerative changes, finally impaired learning and memory in adulthood.Inhalational anesthetics are extensively used in clinical anesthesia. In recent reports, isoflurane induced imbalance of cytoplasmic Ca2+and subsequently lead to neuroapoptosis in the mitochondrial pathway. However, the mechanism involved in the effect of isoflurane mitochondrial structure and function is unclear. Supposed that infants exposed to general anesthetics was indispensable, it is a challenge to elucidate the exact mechanism involved in isoflurane-induced abnormal structure and function of mitochondria and investigate the effective interventions for the prophylaxis and treatment of this disorder.Mitochondria undergo dynamical fusion and fission events which maintain steady mitochondrial morphology. This process is also called mitochondrial dynamics. The rates of fusion and fission are regulated by several large GTPase molecules (OPA1, mfn1, mfn2, drp-1, mff and FIS1). Drp-1binds to the mitochondrial fission factor (mff) located on the outer membrane of mitochondrial and then promotes mitochondrial division, while OPA1, mfnl and mfn2potentiate mitochondrial fusion. Under physiological condition, the events of fusion and fission not only determine the shape and size of mitochondria but also regulate mitochondrial distribution and function. It was reported that dysregulation of fusion and fission of mitochondria in neurons affected the neurite growth, synaptic plasticity, long-term potentiation and even induced neuroapoptosis.The molecules regulated mitochondrial dynamics were associated with neuroapoptosis and synaptic mis-interconnection in neurodegenerative diseases such as Alzheimer’s disease and Huntington’s disease. In the early stage of apoptosis, the activity of drp-1was upregulated and promoted mitochondrial fragmentation in mammalian cells. These changes lead to enlargement of mitochondrial cristae and increase of mitochondrial membrane permeability which facilitated the release of cytochrome C from mitochondrial cristae to the cytoplasm and initiated the mitochondrial apoptotic pathway. Amyloid-beta protein and huntingtin induced upregulation of drp-1and downregulation of mfiil and mfn2and promoted excessive mitochondrialfission and fragmentation which contributed to neuroapoptosis, damage of synaptic connections and long-lasting cognitive disorder. In summary, abnormal mitochondrial fission is an early demonstration of apoptosis; imbalance of mitochondrial fusion and fission underlie the pathophysiological mechanism of neurodegenerative diseases.Ca2+in neuronal cytoplasm regulated the mitochondrial dynamics in several signaling pathway by the way that influence the expression and activity of the regulating proteins.With the nocuous stimuli, the level of cytoplasmic Ca2+was elevated, and then calcineurin was activated which dephosphorylated the637serine of drp-1. This change enhanced the activity of drp-1and promoted the fragmentation of mitochondrial and ultimately disturbed the interconnection of synapse and formation of neural circuit. Our previous results demonstrated that clinically relevant concentration of isoflurane elevated the level of cytoplasmic Ca2+and triggered mitochondrial apoptotic pathway in developmental neurons. However, it remains to be elucidated that isoflurane induced dysregulation of mitochondria dynamics via calcineurin signaling pathway.In this study, the dynamic changes of mitochondrial morphology the regulating proteins were detected in neurons and rats. Also, pathways of cell signaling transduction relevant to the imbalance of mitochondrial dynamics induced by isoflurane were investigated. All the designed experiments are conducted to elucidate the role of mitochondrial dynamics disruption in the developmental neurotoxicity triggered by isoflurane and provide a new prospective for prophylaxis and medication of the neurotoxitiy. Methods and Results1. Effects of isoflurane on the mitochondrial morphological changes and mitochondrial membrane potential of rat developmental hippocampal neurons in vitroMethods Neurons cultured5day in vitro(DIV) were randomly assigned to control group (group C) and isoflurane treated group (group I). Group I was exposed to1.5%isofluane carried by5%CO2-8%O2-87%N2for6hours, while group C was exposed to carrier air for6h. After isoflurane exposure, neurons were fixed by2.5%glutaraldehyde and then treated by a series of special steps. And then eletron microscope was used to detect mitochondrial morphology. DIV3neurons were transfected with CellLight(?) Mitochondria-RFP BacMan2.0and fixed with4%paraform and detected mitochondrial changes with confocal laser scanning microscope (CLSM) immediately the anesthesia ended.Results Compared with group C, the length of mitochondria in group I was significantly shorter (P<0.05).2. Effects of isoflurane on mitochondrial membrane potential ((?)m) of rat developmental hippocampal neurons in vitroMethods Neurons DIV5were randomly assigned to control group (group C) and isoflurane treated group (group I). The treatment of neurons was the same as aforementioned. Neurons was harvested and inbubated with JC-1for30min at37℃and subject to detection with flow cytometer and confocal laser scanning microscope (CLSM).Results Compared with group C,(?)m in group I was significantly depolarized with detection of CLSM and flow cytometer(p<0.05). 3. Effects of isoflurane on proteins that regualted mitochondrial dynamics of rat developmental hippocampal neurons in vitroMethods Neurons DIV5were randomly assigned to control group (group C) and isoflurane treated group (group I). The treatment of neurons was the same as aforementioned. Immediately at the end of isofluane exposure, total RNA and protein was extracted respectively for Western Blot (WB) and real-time PCR detection.Results Compared with group C, the expression of drp-1, mff, FIS1, OPA1, mfn1and mfn2was no staticstical difference in group I at the level mRNA or protein (P>0.05).4. Effects of isoflurane on the expression of phospholyrated drp-1(ser637) of rat developmental hippocampal neurons in vitroMethods Neurons DIV5were randomly assigned to control group (group C) and isoflurane treated group (group I). The treatment of neurons was the same as aforementioned. At the end of isoflurane anesthesia, total protein was extracted by RIPA for detection p-drp-1(ser637). And also the expression of p-drp-1(ser637) was assayed by double-immunofluorescence methods. Red fluorescence represented p-drp-1(ser637).Results Compared with the group C, the expression of p-drp-1(ser637) significantly decreased and the positive stained neurons and red fluorescence drammatically declined (P<0.05).5. Effects of inhibition of drp-1on isoflurane-induced(?)m depolarization of rat developmental hippocampal neurons in vitroMethods Neurons DIV5were randomly divied into four groups:Control group (group C),isoflurane treated group (group I), mdivi-1pretreated and isoflurane expousre group (group M+I) and only mdivi-1treated group (group M). Group C was exposed to carrier air for6hours;group I was exposed to1.5%isofluane carried by5%CO2-8%O2-87%N2for6h; group M+I treated with5μM mdivi-1for2h and then exposed to1.5%isoflurane; group M was treated by5uM mdivi-1and exposure to carrier air for6hours. Neurons was harvested and inbubated with JC-1for30min at37℃and subject to detection with flow cytometer and confocal laser scanning microscope (CLSM).Results Compared with the group C, the (?)mingroup M and group M+I did not declined significantly(P>0.05); while the (?)m in group Idecreased significantly (P<0.05). Compared with groupI, the (?)ningroup M+I depolarized significantly (P<0.05).6. Effects of mdivi-1on isoflurane-induced mitochondrial morphological changes of rat developmental hippocampal neuronsin vitroMethods Neurons DIV5were randomly divied into group C,group I, group M+I and group M. The detailed treatment processure was refered to setion Methods in Part5. DIV3neurons were transfected with CellLight(?) Mitochondria-RFP BacMan2.0and fixed by4%paraform and detected mitochondrial changes with confocal laser scanning microscope (CLSM) immediately the anesthesia ended. After isoflurane exposure, neurons were fixed by2.5%glutaraldehyde and sent for detection mitochondrial morphology with eletron microscope.Results Compared with the group C, the diameter of mitochondria was not significantly different in group M and group M+I (P>0.05), while it was much smaller in group I (P<0.05). Compared with groupI, the diameter of mitochondria in group M+I was significantly larger(P<0.05).7. Effects of mdivi-1on neuronal viability, theexpression of cyto-C and AC3of rat developmental hippocampal neuronsin vitroMethods Neurons DIV5were randomly divided into group C,group I, group M+I and group M. The detailed treatment processure was refered to setion Methods in Part5. The expression of cyto-C was detected at the levels of protein after isoflurane treatment. The expression of AC3was detected in a double-stained immunocytochemistric way. Results Compared with the group C, all the detected parameters were not significantly different in group M and group M+I (P>0.05). Compared with the group I, the expression of cyto-C was downregulated significantly in group M+I (P<0.05).The number of AC3-positive neurons was reduced in group M+I (P<0.05).8. Effects of isoflurane on activity and expression of calcineurin(CaN) of rat developmental hippocampal neuronsin vitroMethods Neurons DIV5were randomly assigned to control group (group C) and isoflurane treated group (group I). The treatment of neurons was the same as aforementioned in Part.1. Immediately at the end of isofluane exposure, the total protein was extracted for WB and the activity of CaN was assayed by the commecially kit following the manufacture’s instructions.ResultsAfter1.5%isoflurane treatment, the activity and expression of CaN increased significantlly in neurons(P<0.05).9. Effects of FK506(inhibitor of CaN) on the isoflurane-induced downregualtion of p-drp-1(ser637), abnomal mitochondrial morphology and depolarized (?)mof rat developmental hippocampal neuronsin vitroMethods Neurons DIV5were randomly divied into four groups:Control group (group C),isoflurane treated group (group I), FK506pretreated and isoflurane expousre group (group F+I) and only FK506treated group (group F). Group C was exposed to carrier air for6hours;group I was exposed to1.5%isofluane carried by5%CO2-8%O2-87%N2for6hours; group F+I treated with5nM FK506for2hours and then exposed to1.5%isoflurane; group F was treated by5nMFK506and exposure to carrier air for6hours. Neurons was harvested and inbubated with JC-1for30min at37℃and subject to detection with flow cytometer and CLSM.Results Compared with the group C, diameter was not significantly different in group F and group F+I (P>0.05), while it significantly changed in group I (P<0.05). Compared with groupI, the diameter of mitochondria in group F+I was significantly larger (P<0.05); the (?)mingroup F+I increased significantly (P<0.05); the downregulation of p-drp-1(ser637) was significantly upregulated (P<.05).10. Effects of FK506on the expression of cyto-C and AC3of rat developmental hippocampal neuronsin vitroMethods Neurons DIV5were randomly divided into group C,group I, group M+I and group M. The detailed treatment processure was refered to setion Methods in Part9. The expression of cyto-C was detected at the protein level after isoflurane treatment.The expression of AC3was detected in a double-stained immunocytochemistric way.Results Compared with the group C, all the detected parameters were not significantly different in group F and group F+I (P>0.05). Compared with the group I, the expression of cyto-C was downregulated significantly in group F+I (P<0.05); The number of AC3-positive neurons was reduced in group M+I (P<0.05).11. Effects of isoflurane on the mitochondrial morphological changes in CA1of PND5ratsMethods PND5rats were randomly assigned to group C and group I. Group I was exposure to1.5%isoflurane for6h carried by30%O2-70%N2,while group C exposed to carrier air. At the end of anesthesia, CA1in hippocampus was gently dissected and transferred to2.5%glutaraldehyde for2h fixation and send for eletron microscopy imaging and analysis.Results Compared with the group C, the diameter of mitochondria was significantly reduced in group I and mitochondrial in group I was significantly condensed(P<0.05).12. Effects of isoflurane on proteins that regualted mitochondrial dynamics and p-drp-1in hippocampi of PND5rats MethodsPND5rats were randomly assigned to group C and group I. The detailed treatment information was refered to Part.11. At the end of anesthesia, hippocampi were dissected for total protein and RNA extraction.ResultsCompared with group C, the expressions of drp-1, mff, FIS1, OPA1, mfn1and mfn2were no staticstical difference in group I at the level mRNA (P>0.05);but the expression of p-drp-1(ser637) significantly decreased(P<0.05).13. Effects of mdivi-1on isoflurane-induced neuronal apoptosis and the expression of cyto-Cin hippocampi of PND5ratsMethods PND5rats were randomly divided into group C,group I, group M+I and group M. Group C and group I were treated as delineted Part.11. Group M+I and group Mreceived a dose of5μg/kg mdivi-1by intraperitoneal injection2h before isoflurane treatment. After anesthesia, hippocampi was dissociated for detection ofcyto-C. And ratswere decapitated and frozen sections of brainwere made to stain the positive active caspse3neurons.Results Compared to group C, there were no statistical difference in expression of cyto-C and the amount of active caspase3-positive neurons in group M+I and group M(P>0.05). Compared with group I, the expression cyto-C was downregulated significantly in group M+I (P<0.05) and the number of active caspase3-positive neurons was smaller(P<0.05).14. Effects of mdivi-1on isoflurane-induced long-term mitochondrial changes and deficit in learning and memoryMethods PND5rats were randomly divided into group C,group I, group M+I and group M.The detailed treatment processure was refered to setion Methods in Part13. After isofluane exposure, rats were sent to cages until they were fully awake and raised to60days after their birth when Morris water maze was used to assess spatial learning and memory.Results Compared with group C, the latency was significantly longer and the probe time wasshorter in group I from the third training dayto the lasttraining day (P<0.05). However, these changes were reversed in groupM+I (P<0.05).15. Statistical analysesAll datawerepresentedandgraphedas the mean±EM. The Statistical Package for the Social Sciences20software was used for the statistical analyses. Data acquired fromthe detection of protein and mRNA was analyzed with an analysis of variance (ANOVA), followed by a least square difference (LSD) multiple comparison test. Data collected from the spatial acquisition trials were analyzed using a repeated measures ANOVA (the different treatments were the between groups factors and time was the repeated measures factor), followed by a post-hoc test to compare four groups. Differences were deemed statistically significant if P<0.05.Summaries1. It was verified that clinical relevant concentration isoflurane induced imbalance of mitochondrial dynamics in in vitro and in vivo.2. It was confirmed that isoflurane activated calcineurin which dephosphorylated drp-1at serine-637. The dephosphorylated drp-1translocated to mitochondria our membrane and promoted excessive mitochondrial fission even fragmentation.3. Pretreatment with mdivi-1, an inhibitor of drp-1, mitigated isoflurane-induced neuroapoptosis and long-lasting cognitive impairment in in vitroandin vivo. ConclusionsElevated cytoplasmic Ca2+induced by Isoflurane via activating GABAA or facilitating IP3and RyR receptors increased the activity of calcineurin which dephosphorylated drp-1at serine-637.The dephosphorylated drp-1translocated to mitochondria our membrane and promoted excessive mitochondrial fission even fragmentation, which increased the permeability of mitochondrial transition pore. Thus cyto-C tethered in mitochondrial cristae released and initiated the mitochondrial apoptototic pathway of developmental neurons. However, mdivi-1, an inhibitor of drp-1, alleviated isoflurane-induced neuroapoptosis in vitro and in vivo and improved long-lasting cognitive function. |
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