Effect And Mechanism Of Sevoflurane Exposure Mediated Autophagy In The Fetal Rat Neurotoxicity During The Midtrimester

Introduction: With the continuous improvement of surgical and diagnostic techniques,more and more fetal interventions have been performed,and the midtrimester has been regarded as a safe period for pregnancy surgery.The continuous increase of the number of patients also put forward higher requirements for pregnancy anesthesia.In order to meet the need of open fetal surgery,the use of inhaled anesthetics has become the preferred method of intrauterine surgery.A large number of animal studies have shown that commonly used general anesthesia drugs can cause cognitive impairment in immature brains.Although sedation or general anesthesia processes are generally considered safe,but people come to realize that,the relative safe operation period is not necessarily safe for fetal nervous system development,because the midtrimester is a critical period of the nervous system development.There is a wide range of neurogenesis in the brain during this period.During this period,the fetal brain is susceptible to interference from outside environmental factors,and subtle changes can lead to abnormalities in the forward nervous system.The previous work of our research group: gestation day 14(G14)rats exposed to the most commonly used inhalation anesthetic,sevoflurane,in 2% and 3.5% two different concentration for 2 hours,and the development of the nervous system and the ability of learning and memory in offspring were observed.The results showed that 3.5% sevoflurane had the effect of inducing learning and memory impairment in the offspring rats,while the offspring in 2% sevoflurane group did not show impaired learning and memory function.The dysfunction of learning and memory was caused by the increased apoptosis and the inhibited proliferation of neural stem cells(NSCs)by 3.5% sevoflurane.With the deepening understanding of general anesthetic neurotoxicity,people found the mechanisms of anesthetic induced neurotoxicity,including mitochondrial damage,intracellular Ca2+ balance disorders,etc.,and these factors can lead to the activation of autophagy in cells.Autophagy is a cellular process,that is a kind of highly conservative self-degradation,can participate in a variety of physiological or pathological processes in the cells,such as nutrition deprivation,stress,aging or infection,although autophagy can remove harmful damaged proteins and organelles,but excessive autophagy could lead to type ? programmed cell death(PCD).Interfering with the PCD process can also affect the normal development of the nervous system.This study explored the effects of sevoflurane anesthesia with different concentration on autophagy levels in fetal brains.In addition to maintaining cell homeostasis,autophagy is involved in midtrimester neural development,and it is necessary for normal brain development,Autophagy is involved in the development of the nervous system,including cell proliferation and differentiation.Interfering with autophagy would affect the process of neurogenesis,leading to abnormal neural development.As the mechanisms of PCD,autophagy and apoptosis have complex interactions.In certain cases,autophagy promotes cell survival and disturbs apoptosis.In other cases,autophagy could synergize with apoptosis and accelerate cell death.When autophagy is overactive,cell damage,mitochondrial dysfunction and protein degradation were caused,leading to inability to maintain the basic function of the cell.There is rare research on relationship between autophagy and apoptosis in the anesthetic neurotoxicuty at present,so this study discussed the role of autophagy in midtrimester sevoflurane exposure induced NSC apoptosis and proliferation.Akt/m TOR as a negative autophagy regulation pathway has been widely recognized.As a tumor suppressor gene,PTEN is closely related to neural development,and is ubiquitous in human and mouse developing brain,and is an important intrinsic molecule of neurogenesis and regeneration.PTEN inhibits PI3 K phosphorylation,and thus blocks Akt and its downstream kinase activity.PTEN can inhibit Akt/m TOR pathway and increase autophagy.Ethanol regulated the activity of autophagy through the m TOR pathway in newborn rats.Therefore,this experiment investigated whether sevoflurane regulated autophagy levels through PTEN/Akt/m TOR signaling pathway.Materials and Methods: 1.Sprague Dawley rats were selected as experimental animals,and G14 rats were treated with anesthesia drugs,inhaling 2% or 3.5% sevoflurane.The expressions of LC3B?,Beclin-1 and p62 were detected 2 hours,12 hours,24 hours and 48 hours after anesthesia,transmission electron microscopy(TEM)was used to observe autophagy structures in the NSCs,and LC3 B and the NSC marker Nestin were stained with immunofluorescence.2.The autophagy inhibitor,3-MA was intraperitoneally in to G14 rats 1 hour before gas exposure.The levels of autophagy markers and the anti-apoptotic proteins,Bcl-2,were measured.The number of TUNEL and Nestin positive cells was counted.3.1 hour before gas exposure,3-MA was intraperitoneally in to G14 rats.Nestin level was measured with Western blot and immunohistochemistry.Ki67 immunofluorescence was performed to detected cell proliferation.4.The expression of PTEN/Akt/m TOR pathway proteins in each group was detected with Western blot.The expression of autophagy related markers was detected with Western blot and immunofluorescence double staining,TUNEL and Nestin double staining was used to detect apoptosis,and Nestin immunohistochemistry and Ki67 immunofluorescence were used to detect cell proliferation.5.After application of 3-MA or bp V,the spatial learning and memory function of the offspring was tested by Morris water maze test.The relative quantity of hippocampus CA1 area neurons was measured with Neu N immunohistochemistry.Results: 1.The autophagy in the fetal rats was activated by 3.5% sevoflurane anesthesia.The expression of LC3B? and Beclin-1 was increased,and p62 was decreased.The LC3 B and Nestin positive cells showed punctate pattern in the brain sections from the 3.5% sevoflurane anesthesia group,whereas the others showed diffused green fluorescence.2.Administration of the autophagy inhibitor 3-MA reduced 3.5% sevoflurane induced autophagy and the apoptosis of NSCs: compared with the 3.5% sevoflurane anesthesia group,the Bcl-2 expression was increased,and the number of TUNEL and Nestin positive cells was decreased.3.After the application of the autophagy inhibitor 3-MA,the proliferation inhibition of NSCs was alleviated: the reduction of Nestin expression and the decrease of Ki67 positive cell rate were alleviated after using 3-MA.4.The mechanism of autophagy activation was the increased PTEN expression,and the decreased p-Akt/Akt and m TOR expression caused by 3.5% sevoflurane exposure.After application of the PTEN inhibitor,bp V,the expressions of p-Akt/Akt and m TOR were increased,3.5% sevoflurane induced autophagy and NSC apoptosis were reduced,and decreases of Nestin expression and the Ki67 positive cell rate were alleviated.5.After the 3-MA and bp V were applied,the offspring rats showed improved learning and memory function and neural development compared with the offspring of 3.5% sevoflurane anesthesia group: the escape latency was shortened,platform crossing times were increased,and the neuron density of hippocampus CA1 area was increased.Conclusions: Our results demonstrate that 2 hours 3.5% sevoflurane exposure at G14 induced excessive autophagy in the fetal brain via the PTEN/Akt/m TOR pathway,2% sevoflurane did not show the effect;Autophagy or PTEN inhibition reversed 3.5% sevoflurane anesthesia-induced NSC apoptosis,proliferation decline,memory deficits and neuron loss.