A Study Of Mechanisms Underlying Isoflurane Induced Neurotoxicity

Backgroud Isoflurane is a commonly used anaesthetic agent in adults as well as infant patients to maintain general anesthesia in various types of surgery.There is convincing evidence showing that isoflurane exposure may be linked with various forms of neuronal disorder.In early postnatal developing rodents model,it has been demonstrated that isoflurane exposure cause widespread neuronal apoptosis and subsequent behavioral impairment.These observations have raised increasing concern regarding the side effects of anesthesia in infants and children.Isoflurane is thought to perform its function through multiple mechanisms,including agonist action on GABAA receptor and antagonist action on the NMDA receptor.However,the underlying mechanism of isoflurane induced neurotoxicity is not quite understood.In the recent study,it has been showed that neuron cells exposured to isoflurane result in increased neuronal apoptosis through the mitochondria dependent apoptosis pathway.This study was designed to determine whether mitochondrial adenosine triphosphate-sensitive potassium channel(mitoKATP),which is directly activated by isoflurane,and signal transducer and activator of transcription(STAT3)are involved in the neurotoxicity induced by isoflurane.Methods U251 human neuroglioma cells and seven-day-old mice were treated with isoflurane for 6 h.JC-1 was used to determine the changes in mitochondrial transmembrane potential(△Ψm),and intracellular ROS was measured using carboxy-H2DCFDA.The levels of ATP and NAD+/NADH were repeated measured during isoflurane treatment.Apoptosis was evaluated by flow cytometric analysis of Annexin V/PI,immunohistochemical staining for activated caspase-3,and western blot analysis for apoptosis related proteins such as activated caspase-3,cytosolic cytochrome C,Bcl-2,BAX and survivin.The intellicage tests were performed to evaluate the memory of mice four weeks after isoflurane exposure.The protective effect of 5-HD,a mitoKATP channel blocker,was evaluated in vitro and in vivo.Western blot,RT-PCR and histopathologic analysis were performed to determine the influrance of isoflurane on STAT3 expression.Plasmid containing human wild-type STAT3,siRNA and specific inhibitor of STAT3 were used to investigate potential functions of STAT3 in isoflurane induced oxidative damage and apotosis.MG-132,a proteasome inhibitor and FK506,a calcineurin inhibitor,were used to examine the mechanism underlying isoflurane-induced degradation of STAT3 and apoptosis.Western blot analysis were processed for the assessment of ubiquitined total protein,STAT3 and apoptosis related proteins levels.Results Isoflurane exposure led to depolarization of △Ψm,ROS formation and apoptosis in U251 cells.And these effects were blocked by 5-HD,a specific mitoKATP channel antagonist.Moreover,the dissipation of △Ψm was also abrogated by N-acetylcysteine(NAC),a free radical scavenger.The levels of ATP and NAD+/NADH increased at first and decreased subsequently during the treatment of isoflurane for 6h.Pretreatment with 5-HD maintained the levels of ATP and NAD+/NADH radio during the isoflurane exposure as the levels of naive cells or mice.While the levels of ATP and NAD+/NADH radio increased at first and but not decreased then if pretreatment with NAC.In animal studies,mice exposed to isoflurane had widespread neuroapoptosis and impaired cognitive function in the intellicage test.During the place learning phase the mice exposured to isoflurane did not show a significant difference until the fourth day.Nevertheless,when it turns to the reversal learning phase,isoflurane-treated mice showed an obvious learning disability to recognize their new assigned correct corner.5-HD pretreatment could protect against isoflurane-induced apoptosis and cognitive impairment.Compared to the control group,isoflurane exposure significantly decreased the protein levels of STAT3,pY705-STAT3 and its downstream protein both in the U251 cells and in the neonatal mice.The histologic evaluation indicated that application of isoflurane induced a significant downregulation of STAT3 and pY705-STAT3 levels in the developing brain,especially in the cortex and CA1 region of hippocampus.Immunofluorescence evaluation showed that there was a reduction of STAT3 in the U251 cells after isoflurane exposure,especially in the nucleus.Western blot analysis showed that total STAT3 protein levels in the U251 cells transfected with STAT3 plasmid significantly increased compared to the negative control vector transfected cells and untransfected cells.RT-PCR demonstrated that STAT3 and its target genes such as cyclin D1,Mcl-1,survivin,and Bcl-xl genes were up-regulated.STAT3,pY705-STAT3 and the accumulation of STAT3 in mitochondria were uninfluenced after isoflurane exposure in the cells transfected with STAT3 plasmid.Overexpression of STAT3 prevented the release of cytochrome c into the cytoplasm and resulted in less apoptosis after isoflurane exposure.Whereas cells pretreatment with STAT3 siRNA or STA21,which inhibits STAT3 dimerization and DNA binding activity,were more vulnerable to isoflurane induced oxidative damage and apoptosis.Application of isoflurane caused in an upregulation of STAT3 transcription.Western blot analysis showed that isoflurane induced degradation of total ubiquitined protein.MG 132 prevented degradation of total ubiquitined protein but aggravated the apoptosis level.FK506 could protected cells against apoptosis and STAT3 degradation induced by isoflurane without influence to other proteins.ConclusionsIsoflurane induces △Ψm disruption and intracellular ROS accumulation via opening of mitoKATP channel.MitoKATP channel opening lead to a decrease in △Ψm,which subsequently result in the decreased driving force of H+ influx to produce ATP at site V,therefore,the compensatory mitochondrial respiration could be activated.This leads to increased production of ATP、NAD+/NADH and ROS.ROS may further stimulate opening of the mitoKATp channel and form a positive feedback.While once the accumulation of ROS exceed the tolerance of cell,it may lead to oxidative injury,followed by the reduction of ATP and NAD+/NADH levels and ultimately cause neurotoxicity.5-HD exerts a protective effect against the apoptogenic action of isoflurane.Intellicage is a effective method to detect the mice with impaired cognitive function after exposed to isoflurane.Long-term neurocognitive function was indeed impaired in mice after subjected to isoflurane.The deficit in a less demanding task is rather subtle,or just appears as learning slowly,but can be more pronounced if increase the task difficulty.Isoflurane exposure induces impairment of STAT3 signaling both in vitro and in vivo.STAT3 is an important target underlying isoflurane-induced cytotoxicity.Neuroprotective mechanism mediated by STAT3 involves its canonical activity as a nuclear transcription factor and its direct action in the mitochondria,such as inhibiting oxidative stress and preventing the opening of mPTP.Post-translational regulation participate in the mechanism for isoflurane-induced STAT3 downregulation.Isoflurane accelerate the degradation of ubiquitined protein.FK506 specifically prevented STAT3 degradation and may be a potential agent for prevention and treatment of cognitive dysfunction induced by isoflurane or other neurodegenerative diseases.