Neonatal Sevoflurane Research Articles

Rosiglitazone Promotes Oligodendrocyte Development and Myelin Formation of Repeated Neonatal Sevoflurane Exposure via PPARγ Signaling.

Sevoflurane is one of the most commonly used general anesthetics for children and infants. Recent research indicates that repeated exposure to sevoflurane in neonates induces cognitive and fine motor deficits. Peroxisome proliferator-activated receptor-γ (PPARγ) agonists have garnered significant attention as potential therapies for a variety of neurological conditions. In this research, we evaluated whether pretreatment with rosiglitazone in neonatal mice could address myelination defects, cognitive impairment, and fine motor dysfunction via PPARγ. The mice were exposed to 3% sevoflurane for 2h on postnatal days 6-8 (P6-P8). Behavioral tests were conducted from P29 to P34. Additionally, we evaluated morphological and functional changes related to myelin. Our results showed that rosiglitazone pretreatment significantly ameliorated the cognitive and fine motor impairments of repeated neonatal sevoflurane exposure. In addition, rosiglitazone pretreatment promoted oligodendrocyte precursor cells (OPCs) differentiation and myelination. This suggests that rosiglitazone may be used in clinical settings to enhance the security of neonatal sevoflurane exposure. Furthermore, PPARγ and fatty acid synthase (FASN) may be mediators for rosiglitazone, which alleviates myelination defects, cognitive impairment, and fine motor dysfunction.

Complement C1q-mediated microglial synaptic elimination by enhancing desialylation underlies sevoflurane-induced developmental neurotoxicity

BackgroundRepeated neonatal sevoflurane exposures led to neurocognitive disorders in young mice. We aimed to assess the role of microglia and complement C1q in sevoflurane-induced neurotoxicity and explore the underlying mechanisms.MethodsNeonatal mice were treated with sevoflurane on postnatal days 6, 8, and 10, and the Morris water maze was performed to assess cognitive functions. For mechanistic explorations, mice were treated with minocycline, C1q-antibody ANX005, and sialidase-inhibitor N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (NADNA) before sevoflurane exposures. Western blotting, RT-qPCR, Golgi staining, 3D reconstruction and engulfment analysis, immunofluorescence, and microglial morphology analysis were performed. In vitro experiments were conducted in microglial cell line BV2 cells.ResultsRepeated neonatal sevoflurane exposures resulted in deficiencies in learning and cognition of young mice, accompanied by microglial activation and synapse loss. Sevoflurane enhanced microglia-mediated synapse elimination through C1q binding to synapses. Inhibition of microglial activation and phagocytosis with minocycline significantly reduced the loss of synapses. We further revealed the involvement of neuronal sialic acids in this process. The enhanced activity of sialidase by sevoflurane led to the loss of sialic acids, which facilitated C1q binding to synapses. Inhibition of C1q with ANX005 or inhibition of sialidase with NADNA significantly rescued microglia-mediated synapse loss and improved neurocognitive function. Sevoflurane enhanced the engulfment of BV2 cells, which was reversed by ANX005.ConclusionsOur findings demonstrated that C1q-mediated microglial synaptic elimination by enhancing desialylation contributed to sevoflurane-induced developmental neurotoxicity. Inhibition of C1q or sialidase may be a potential therapeutic strategy for this neurotoxicity.

Neuroligin1 in excitatory synapses contributes to long-term cognitive impairments after repeated neonatal sevoflurane exposures

BackgroundRepeated sevoflurane exposures in neonatal rats may lead to neuronal apoptosis affecting long-term cognitive function, the mechanism is unknown. Neuroligin1 (NL1) is essential for normal excitatory transmission and long-term synaptic plasticity in the hippocampus of intact animals. Herein, we explore the role of NL1 in hippocampal excitatory synapses on long-term cognitive impairments induced by repeated sevoflurane exposures in neonatal rats. MethodsFrom postnatal day six (P6) to P8, neonatal rats were exposed to 30% oxygen or 3% sevoflurane +30% oxygen for 2 h daily. Rats from each litter were randomly assigned to five groups: control group (Con), native control adeno-associated virus (NC-AAV) group (Con + NC-AAV), sevoflurane group (Sev), sevoflurane + recombinant RNAi adeno-associated virus targeting NL1 downregulation (NL1−-AAV) group (Sev + NL1−-AAV) and control + recombinant RNAi adeno-associated virus targeting NL1 upregulation (NL1+-AAV) group (Con + NL1+-AAV). Animals were injected with NC-AAV or NL1-AAV into the bilateral hippocampal CA1 area and caged on P21. From P35 to P40, behavioral tests including open field (OF), novel object recognition (NOR), and fear conditioning (FC) tests were performed to assess cognitive function in adolescent rats. In another experiment, rat brains were harvested for immunofluorescence staining, western blotting, co-immunoprecipitation, and real-time polymerase chain reaction (PCR). ResultsWe found that the mRNA and protein levels of NL1 were substantially higher in the Sev group than in the Con group. Immunofluorescence showed that NL1 and PSD95 were highly colocalized in hippocampal CA1 area and vesicular GABA transporter (vGAT) around neurons decreased after repeated sevoflurane exposures. Co-immunoprecipitation showed that the amount of PSD95 with NL1 antibody was significantly increased in the Sev group compared to the Con group. These rats had a poorer performance in the NOR and FC tests than control rats when they were adolescents. These results were reversed by NL1−-AAV injection into the CA1 area. NL1+-AAV group was similar to the Sev group. ConclusionWe have demonstrated that repeated neonatal sevoflurane exposures decreased inhibitory synaptic inputs (labelled by vGAT) around neurons, which may influence the upregulation of NL1 in hippocampal excitatory synapses and enhanced NL1/PSD95 interaction, ultimately leading to long-term cognitive impairments in adolescent rats. Injecting NL1−-AAV reversed this damage. These results suggested that NL1 in excitatory synapses contributes to long-term cognitive impairments after repeated neonatal sevoflurane exposures.

The effect of sevoflurane exposure on cell-type-specific changes in the prefrontal cortex in young mice.

Sevoflurane, the predominant pediatric anesthetic, has been linked to neurotoxicity in young mice, although the underlying mechanisms remain unclear. This study focuses on investigating the impact of neonatal sevoflurane exposure on cell-type-specific alterations in the prefrontal cortex (PFC) of young mice. Neonatal mice were subjected to either control treatment (60% oxygen balanced with nitrogen) or sevoflurane anesthesia (3% sevoflurane in 60% oxygen balanced with nitrogen) for 2 hours on postnatal days (PNDs) 6, 8, and 10. Behavioral tests and single-nucleus RNA sequencing (snRNA-seq) of the PFC were conducted from PNDs 31 to 37. Mechanistic exploration included clustering analysis, identification of differentially expressed genes (DEGs), enrichment analyses, single-cell trajectory analysis, and genome-wide association studies (GWAS). Sevoflurane anesthesia resulted in sociability and cognition impairments in mice. Novel specific marker genes identified 8 distinct cell types in the PFC. Most DEGs between the control and sevoflurane groups were unique to specific cell types. Re-defining 15 glutamatergic neuron subclusters based on layer identity revealed their altered expression profiles. Notably, sevoflurane disrupted the trajectory from oligodendrocyte precursor cells (OPCs) to oligodendrocytes (OLs). Validation of disease-relevant candidate genes across the main cell types demonstrated their association with social dysfunction and working memory impairment. Behavioral results and snRNA-seq collectively elucidated the cellular atlas in the PFC of young male mice, providing a foundation for further mechanistic studies on developmental neurotoxicity induced by anesthesia.

SynCAM1 deficiency in the hippocampal parvalbumin interneurons contributes to sevoflurane-induced cognitive impairment in neonatal rats.

Sevoflurane is widely used for general anesthesia in children. Previous studies reported that multiple neonatal exposures to sevoflurane can induce long-term cognitive impairment in adolescent rats, but the underlying mechanisms were not defined. Postnatal day 6 (P6) to P8 rat pups were exposed to 30% oxygen with or without 3% sevoflurane balanced with air. The Y maze test (YMT) and Morris water maze (MWM) tests were performed in some cohorts from age P35 to assess cognitive functions, and their brain samples were harvested at age P14, 21, 28, 35, and 42 for measurements of various molecular entities and invivo electrophysiology experiments at age P35. Sevoflurane exposure resulted in cognitive impairment that was associated with decreased synCAM1 expression in parvalbumin (PV) interneurons, a reduction of PV phenotype, disturbed gamma oscillations, and dendritic spine loss in the hippocampal CA3 region. Enriched environment (EE) increased synCAM1 expression in the PV interneurons and attenuated sevoflurane-induced cognitive impairment. The synCAM1 overexpression by the adeno-associated virus vector in the hippocampal CA3 region restored sevoflurane-induced cognitive impairment, PV phenotype loss, gamma oscillations decrease, and dendritic spine loss. Our data suggested that neonatal sevoflurane exposure results in cognitive impairment through decreased synCAM1 expression in PV interneurons in the hippocampus.

7,8-Dihydroxyflavone ameliorates cognitive impairment induced by repeated neonatal sevoflurane exposures in mice through increasing tau O-GlcNAcylation

BackgroundSevoflurane, one of the most commonly used general anesthetics for pediatric anesthesia, has recently gained significant attention in both preclinical and clinical settings due to its potential neurotoxicity in the developing brain. Tau phosphorylation, induced by sevoflurane, is recognized as one of the major causes of neurotoxicity. 7,8-dihydroxyflavone (DHF), a TrkB receptor agonist, has been reported to exhibit potential neuroprotective effects against tauopathies. In this study, our objective was to investigate whether DHF could provide neuroprotective effects against sevoflurane-induced neurotoxicity and explore the underlying molecular mechanisms. MethodsSix-day-old mice were subjected to 2 h of anesthesia with 3 % sevoflurane, with or without pretreatment of DHF (5 mg/kg/day, i.p.) for 3 consecutive days. Autonomic motor ability was assessed by open-field test, while learning and memory abilities were evaluated by the fear conditioning test. Western blotting was conducted to measure the levels of t-TrkB, p-TrkB, tau, and phosphorylated tau. Additionally, a co-immunoprecipitation assay was performed to investigate the interaction between O-GlcNAcylation and tau. ResultsRepeated neonatal sevoflurane exposures resulted in reduced freezing time during the context and cued fear conditioning tests in adulthood. However, pretreatment with DHF restored the freezing time to the level of the control group, indicating that DHF effectively alleviated cognitive impairments induced by neonatal sevoflurane exposure. We also observed that repeated neonatal sevoflurane exposures increased tau phosphorylation while decreasing tau O-GlcNAcylation. However, DHF pretreatment rebalanced the tau O-GlcNAcylation/phosphorylation ratio by enhancing the interaction between tau and O-GlcNAcylation. ConclusionOur findings demonstrate that DHF effectively ameliorates sevoflurane-induced cognitive impairment in developing mice by restoring the balance between tau O-GlcNAcylation and phosphorylation. Therefore, this study suggests that DHF has the potential to be a therapeutic agent for treating cognitive impairment associated with anesthetics, such as sevoflurane.

Melatonin-mediated mitophagy protects against long-term impairments after repeated neonatal sevoflurane exposures

BackgroundMelatonin is known to have protective effects in aging, neurodegenerative disorders and mitochondria-related diseases, while there is a poor understanding of the effects of melatonin treatment on mitophagy in neonatal cognitive dysfunction after repeated sevoflurane exposures. This study explores the protective effects of melatonin on mitophagy and cognition in developing rats exposed to sevoflurane. MethodsPostnatal day six (P6) neonatal rats were exposed to 3 % sevoflurane for 2 h daily from P6 to P8. In the intervention groups, rats received 3-Methyladenine (3-MA) intracerebroventricularly from P6 to P8 and melatonin intraperitoneally from P6 to P8 following water drinking once daily from P21 to P41, respectively. Behavioral tests, including open field (OF), novel object recognition (NOR), and fear conditioning (FC) tests, were performed to assess cognitive function during young adulthood. In another experiment, rat brains were harvested for biochemical, histopathological, and electron microscopy studies. ResultsRats exposed to sevoflurane showed disordered mitophagy and mitochondrial dysfunction as revealed by increased mitophagy marker proteins (microtubule-associated protein 1 light chain 3 (LC3) II/I, and parkin), decreased autophagy marker protein (sequestosome 1 (P62/SQSTM1)), electron transport chain (ETC) proteins and ATP levels. Immunofluorescent staining of LC3 was co-localized mostly with a neuronal marker and microglial marker but was not co-localized with a marker for astrocytes in rats exposed to sevoflurane. These rats had poorer performance in the NOR and FC tests than control rats during young adulthood. Melatonin treatment reversed the abnormal expression of mitophagy proteins, mitochondrial energy metabolism, the activity of microglia, and impaired cognition. These ameliorations were blocked by an autophagy inhibitor, 3-MA, except for the activation of microglia. ConclusionWe have demonstrated that melatonin inhibits microglial activation by enhancing mitophagy and finally significantly reduces sevoflurane-induced deficits in cognition in neonatal rats. These results suggest that melatonin might be beneficial if considered when the anesthesia must be administered at a very young age.

Apamin, an SK2 Inhibitor, Attenuated Neonatal Sevoflurane Exposures Caused Cognitive Deficits in Mice through the Regulation of Hippocampal Neuroinflammation.

Cognitive dysfunction induced by anesthesia in the infant is a crucial clinical issue that is still being debated and the focus of concern for the parents. However, the mechanism of cognitive decline caused by anesthesia and the corresponding treatment methods remain unclear. Postnatal day 7 (PND7) C57BL/6 mice included in the study were randomly divided into a control group (Control), a group with repeated exposure to sevoflurane (Sevo), and an Apamin intervention group (Sevo + Apamin). Apamin (0.5 μL at the concentration of 100 nmol/L) was injected into the bilateral hippocampus of mice. qRT-PCR, enzyme-linked immunosorbent assay (ELISA), and western blotting assay were used to evaluate the protein levels in the hippocampus. Object location memory (OLM) and novel object recognition (NOR) tasks, as well as elevated plus maze and contextual and cued fear conditioning tasks were used to evaluate the cognitive function of mice. Apamin mitigated sevoflurane-induced cognitive impairment of mice, sevoflurane-induced neuronal injury, and sevoflurane-induced activation of microglial in the hippocampus of the mice. Apamin inhibited M1-type polarization but promoted M2-type polarization of microglia after neonatal sevoflurane exposures in the hippocampus. In conclusion, Apamin attenuates neonatal sevoflurane exposures that cause cognitive deficits in mice through regulating hippocampal neuroinflammation.

Lactate Improves Long-term Cognitive Impairment Induced By Repeated Neonatal Sevoflurane Exposures Through SIRT1-mediated Regulation of Adult Hippocampal Neurogenesis and Synaptic Plasticity in Male Mice.

Repeated neonatal exposures to sevoflurane induce long-term cognitive impairment that has been reported to have sex-dependent differences. Exercise promotes learning and memory by releasing lactate from the muscle. The study tested the hypothesis that lactate may improve long-term cognitive impairment induced by repeated neonatal exposures to sevoflurane through SIRT1-mediated regulation of adult hippocampal neurogenesis and synaptic plasticity. C57BL/6 mice of both genders were exposed to 3% sevoflurane for 2h daily from postnatal day 6 (P6) to P8. In the intervention experiments, mice received lactate at 1g/kg intraperitoneally once daily from P21 to P41. Behavioral tests including open field (OF), object location (OL), novel object recognition (NOR), and fear conditioning (FC) tests were performed to assess cognitive function. The number of 5-Bromo-2'- deoxyuridine positive (BrdU+) cells and BrdU+/DCX+ (doublecortin) co-labeled cells, expressions of brain-derived neurotrophic factor (BDNF), activity-regulated cytoskeletal-associated protein (Arc), early growth response 1 (Egr-1), SIRT1, PGC-1α and FNDC5, and long-term potentiation (LTP) were evaluated in the hippocampus. Repeated exposures to sevoflurane induced deficits in OL, NOR and contextual FC tests in male but not female mice. Similarly, adult hippocampal neurogenesis, synaptic plasticity-related proteins and hippocampal LTP were impaired after repeated exposures to sevoflurane in male but not female mice, which could rescue by lactate treatment. Our study suggests that repeated neonatal exposures to sevoflurane inhibit adult hippocampal neurogenesis and induce defects of synaptic plasticity in male but not female mice, which may contribute to long-term cognitive impairment. Lactate treatment rescues these abnormalities through activation of SIRT1.

Neonatal sevoflurane exposure induces long-term changes in dendritic morphology in juvenile rats and mice.

General anesthetics are potent neurotoxins when given during early development, causing apoptotic deletion of substantial number of neurons and persistent neurocognitive and behavioral deficits in animals and humans. The period of intense synaptogenesis coincides with the peak of susceptibility to deleterious effects of anesthetics, a phenomenon particularly pronounced in vulnerable brain regions such as subiculum. With steadily accumulating evidence confirming that clinical doses and durations of anesthetics may permanently alter the physiological trajectory of brain development, we set out to investigate the long-term consequences on dendritic morphology of subicular pyramidal neurons and expression on genes regulating the complex neural processes such as neuronal connectivity, learning, and memory. Using a well-established model of anesthetic neurotoxicity in rats and mice neonatally exposed to sevoflurane, a volatile general anesthetic commonly used in pediatric anesthesia, we report that a single 6 h of continuous anesthesia administered at postnatal day (PND) 7 resulted in lasting dysregulation in subicular mRNA levels of cAMP responsive element modulator (Crem), cAMP responsive element-binding protein 1 (Creb1), and Protein phosphatase 3 catalytic subunit alpha, a subunit of calcineurin (Ppp3ca) (calcineurin) when examined during juvenile period at PND28. Given the critical role of these genes in synaptic development and neuronal plasticity, we deployed a set of histological measurements to investigate the implications of anesthesia-induced dysregulation of gene expression on morphology and complexity of surviving subicular pyramidal neurons. Our results indicate that neonatal exposure to sevoflurane induced lasting rearrangement of subicular dendrites, resulting in higher orders of complexity and increased branching with no significant effects on the soma of pyramidal neurons. Correspondingly, changes in dendritic complexity were paralleled by the increased spine density on apical dendrites, further highlighting the scope of anesthesia-induced dysregulation of synaptic development. We conclude that neonatal sevoflurane induced persistent genetic and morphological dysregulation in juvenile rodents, which could indicate heightened susceptibility toward cognitive and behavioral disorders we are beginning to recognize as sequelae of early-in-life anesthesia.

Prolonged exposure of neonatal mice to sevoflurane leads to hyper-ramification in microglia, reduced contacts between microglia and synapses, and defects in adult behavior.

Prolonged exposure to general anesthetics during development is known to cause neurobehavioral abnormalities, but the cellular and molecular mechanisms involved are unclear. Microglia are the resident immune cells in the central nervous system and play essential roles in normal brain development. In the study, postnatal day 7 (P7) C57BL/6 mice were randomly assigned to two groups. In the sevoflurane (SEVO), mice were exposed to 2.5% sevoflurane for 4 h. In the control group, mice were exposed to carrier gas (30% O2/70% N2) for 4 h. Fixed brain slices from P14 to P21 mice were immunolabeled for ionized calcium-binding adapter molecule 1 (IBA-1) to visualize microglia. The morphological analysis of microglia in the somatosensory cortex was performed using ImageJ and Imaris software. Serial block face scanning electron microscopy (SBF-SEM) was performed to assess the ultrastructure of the microglia and the contacts between microglia and synapse in P14 and P21 mice. The confocal imaging of brain slices was performed to assess microglia surveillance in resting and activated states in P14 and P21 mice. Behavioral tests were used to assess the effect of microglia depletion and repopulation on neurobehavioral abnormalities caused by sevoflurane exposure. The prolonged exposure of neonatal mice to sevoflurane induced microglia hyper-ramification with an increase in total branch length, arborization area, and branch complexity 14 days after exposure. Prolonged neonatal sevoflurane exposure reduced contacts between microglia and synapses, without affecting the surveillance of microglia in the resting state or responding to laser-induced focal brain injury. These neonatal changes in microglia were associated with anxiety-like behaviors in adult mice. Furthermore, microglial depletion before sevoflurane exposure and subsequent repopulation in the neonatal brain mitigated anxiety-like behaviors caused by sevoflurane exposure. Our experiments indicate that general anesthetics may harm the developing brain, and microglia may be an essential target of general anesthetic-related developmental neurotoxicity.

Role of posttranslational modifications in memory and cognitive impairments caused by neonatal sevoflurane exposure.

With the advancement of technology, increasingly many newborns are receiving general anesthesia at a young age for surgery, other interventions, or clinical assessment. Anesthetics cause neurotoxicity and apoptosis of nerve cells, leading to memory and cognitive impairments. The most frequently used anesthetic in infants is sevoflurane; however, it has the potential to be neurotoxic. A single, short bout of sevoflurane exposure has little impact on cognitive function, but prolonged or recurrent exposure to general anesthetics can impair memory and cognitive function. However, the mechanisms underlying this association remain unknown. Posttranslational modifications (PTMs), which can be described roughly as the regulation of gene expression, protein activity, and protein function, have sparked enormous interest in neuroscience. Posttranslational modifications are a critical mechanism mediating anesthesia-induced long-term modifications in gene transcription and protein functional deficits in memory and cognition in children, according to a growing body of studies in recent years. Based on these recent findings, our paper reviews the effects of sevoflurane on memory loss and cognitive impairment, discusses how posttranslational modifications mechanisms can contribute to sevoflurane-induced neurotoxicity, and provides new insights into the prevention of sevoflurane-induced memory and cognitive impairments.

Long Term Neurodevelopmental Outcomes after Sevoflurane Neonatal Exposure of Extremely Preterm Children: A Cross-Sectional Observationnal Study.

Sevoflurane, a volatile anesthetic, is used when extremely preterm neonates (EPT) undergo painful procedures. Currently, no existing studies analyze sevoflurane’s long-term effects during the EPT’s immediate neonatal period. Our primary objective was to compare the EPT’s neurocognitive development regardless of any sevoflurane exposure prior to 45 weeks corrected gestational age (GA). We analyzed those live discharges, less than 28 weeks GA, who were either exposed, unexposed, and/or multiply exposed to sevoflurane before 45 weeks GA. All data were obtained from a cross-sectional multicenter study (GPQoL study, NCT01675726). Children, both exposed and non-exposed to sevoflurane, were sampled using a propensity-guided approach. Neurological examinations (Touwen), cognitive and executive functions (WISC IV, NEPSY, Rey figure), and assessments when the children were between 7 and 10 years old, were correlated to their neonatal sevoflurane exposure. There were 139 children in the study. The mean gestational age was 26.2 weeks (±0.8) GA and the mean birth weight was 898 g (±173). The mean age of their evaluation was 8.47 years old (±0.70). Exposure to sevoflurane to the mean corrected age 27.10 (3.37) weeks GA had a significant correlation with cerebral palsy (adjusted odds ratio (aOR): 6.70 (CI 95%: 1.84–32.11)) and other major disorders (cerebral palsy and/or severe cognitive retardation) (aOR: 2.81 [95% CI: 1.13–7.35]). Our results demonstrate the possibility of long-term effects on EPT infants who had a sevoflurane exposure before 45 weeks corrected GA. However, these results will require further confirmation by randomized controlled trials.

Melatonin pretreatment alleviates the long-term synaptic toxicity and dysmyelination induced by neonatal Sevoflurane exposure via MT1 receptor-mediated Wnt signaling modulation.

Sevoflurane (Sev) is one of the most widely used pediatric anesthetics. The major concern of neonatal repeated application of Sev is its potential long‐term impairment of cognition and learning/memory, for which there still lacks effective treatment. At the cellular level, Sev exerts toxic effects in multiple aspects, making it difficult for effective interference. Melatonin is a pineal hormone regulated by and feedbacks to biological rhythm at physiological condition. Recent studies have revealed significant neuroprotective effects of exogenous melatonin or its agonists under various pathological conditions. Whether melatonin could prevent the long‐term toxicity of Sev remains elusive. Here, we report that neonatal repeated Sev exposure up‐regulated MT1 receptor in hippocampal neurons and oligodendrocytes. Pretreatment with melatonin significantly alleviated Sev‐induced synaptic deficiency, dysmyelination, and long‐term learning impairment. Both MT1‐shRNA and MT1 knockout effectively blocked the protective effects of melatonin on synaptic development, myelination, and behavior performance. Interestingly, long‐lasting suppression of Wnt signaling, instead of cAMP/PKA signaling, was observed in hippocampal neurons and oligodendrocytes after neonatal Sev exposure. Pharmacologically activating Wnt signaling rescued both the long‐term synaptic deficits and dysmyelination induced by Sev. Further analysis showed that MT1 receptor co‐expressed well with β‐catenin and Axin2 and bound to β‐catenin by its C‐terminal. Melatonin pretreatment effectively rescued Sev‐induced Wnt suppression. Wnt signaling inhibitor XAV939 significantly compromised the protective effects of melatonin. Taken together, our data demonstrated a beneficial effect of melatonin pretreatment on the long‐term synaptic impairment and dysmyelination induced by neonatal Sev exposure, and a novel MT1 receptor‐mediated interaction between melatonin and canonical Wnt signaling, indicating that melatonin may be clinically applied for improving the safety of pediatric Sev anesthesia.

Repeated neonatal sevoflurane induced neurocognitive impairment through NF-\u03baB-mediated pyroptosis

BackgroundExposure to general anesthesia (GA) during the postnatal period is associated with neuroinflammation and long-term neurocognitive impairment in preclinical and clinical settings. Pyroptosis is a novel type of programmed cell death that, along with inflammation, has been found to play an important role in the mechanism of diverse neurological diseases. However, its roles in GA-induced neuroinflammation and neurocognitive impairment in the developing brain have not been investigated.MethodsRats at postnatal day 6 or primary hippocampal neurons at 9 days in vitro received 3% sevoflurane for 2 h daily for three consecutive days. A pharmacological inhibitor of nuclear factor (NF)-κB (BAY 11-7082) was administered to suppress NF-κB activation. Histological and biochemical analyses were performed to assess the pyroptosis as well as neuronal and synaptic damage both in vivo and in vitro. In addition, behavioral tests were performed to evaluate neurocognitive ability in rats.ResultsRepeated sevoflurane exposure activated NF-κB-mediated pyroptosis and neuroinflammation in the hippocampus in developing rats, damaged the neuronal morphology and synaptic integrity, and induced neurocognitive impairment in rats. BAY 11-7082 treatment suppressed the activation of pyroptosis, attenuated the neuronal and synaptic damage, and ameliorated the neurocognitive impairment induced by repeated sevoflurane administration to developing rats.ConclusionsRepeated sevoflurane GA may induce neuroinflammation and neurocognitive impairment in developing rats via the activation of NF-κB-mediated pyroptosis. Our findings characterize a novel role of pyroptosis as a potential therapeutic target in neuroinflammation after repeated neonatal GA.

Gut microbiota mediates cognitive impairment in young mice after multiple neonatal exposures to sevoflurane.

Multiple exposures to anesthesia may increase the risk of cognitive impairment in young children. However, the mechanisms underlying this neurodevelopmental disorder remain elusive. In this study, we investigated alteration of the gut microbiota after multiple neonatal exposures to the anesthetic sevoflurane and the potential role of microbiota alteration on cognitive impairment using a young mice model. Multiple neonatal sevoflurane exposures resulted in obvious cognitive impairment symptoms and altered gut microbiota composition. Fecal transplantation experiments confirmed that alteration of the microbiota was responsible for the cognitive disorders in young mice. Microbiota profiling analysis identified microbial taxa that showed consistent differential abundance before and after fecal microbiota transplantation. Several of the differentially abundant taxa are associated with memory and/or health of the host, such as species of Streptococcus, Lachnospiraceae, and Pseudoflavonifractor. The results reveal that abnormal composition of the gut microbiota is a risk factor for cognitive impairment in young mice after multiple neonatal exposures to sevoflurane and provide insight into a potential therapeutic strategy for sevoflurane-related neurotoxicity.

Neonatal Sevoflurane Exposure Impairs Learning and Memory by the Hypermethylation of Hippocampal Synaptic Genes.

Sevoflurane anesthesia is widely used in pediatric patients. Clinical studies report memory impairment in those exposed to general anesthesia early in life. DNA methylation is essential for the modulation of synaptic plasticity through regulating the transcription of synaptic genes. Therefore, we tested whether neonatal sevoflurane exposure affects learning and memory underlying the hippocampal DNA methylation of synaptic genes. Male Sprague-Dawley rats were exposed to 3% sevoflurane or air for 2h daily from postnatal day 7 (P7) to P9. 5-aza-2-deoxycytidine (5-AZA), an inhibitor of DNA methyltransferases (DNMTs), was intraperitoneally injected 30min before sevoflurane or air exposure on P7-9. The rats were euthanized 6, 12, 24h, and 28days after the last sevoflurane exposure, followed by the determination of global and gene-specific DNA methylation. The expression of synaptic proteins and synaptic density and the transcription of Dnmts and ten eleven translocations (Tets) in the hippocampus were measured. The ability of learning and memory was assessed using Morris water maze, novel object recognition, and intruder tests. Repeated neonatal sevoflurane exposure impaired cognitive, social, and spatial memory. The memory impairment was associated with the increased Dnmt1, Dnmt3a, and 5-methylcytosine level and the decreased Tet1 and 5-hydromethylcytosine level. Sevoflurane subsequently induced hypermethylation of Shank2, Psd95, Syn1, and Syp gene and down-regulated the expression of synaptic proteins, which finally led to the decrease of synaptic density in a time-dependent manner. Notably, 5-AZA pretreatment ameliorated learning and memory in sevoflurane-treated rats. In conclusion, neonatal exposure to sevoflurane can impair learning and memory through DNA methylation of synaptic genes.

Neonatal sevoflurane exposure induces impulsive behavioral deficit through disrupting excitatory neurons in the medial prefrontal cortex in mice

Sevoflurane, in particular multiple exposures, has been reported to cause the abnormal neurological development including attention-deficit/hyperactivity disorder (ADHD). This study is to investigate ADHD-like impulsivity in adult mice after repeated sevoflurane exposures at the neonatal stage. Six-day-old pups were exposed to 60% oxygen in the presence or absence of 3% sevoflurane for 2 h and the treatment was administrated once daily for three consecutive days. To assess the impulsivity, the cliff avoidance reaction (CAR) was carried out at the 8th week. Our results showed that repeated sevoflurane treatment increased the number of jumps and shortened the jumping latency in the CAR test. The cortices were harvested for immunostaining to detect c-Fos and calmodulin-dependent protein kinase IIα (CaMKIIα) expression in the medial prefrontal cortex (mPFC). We found that mPFC neurons, especially excitatory neurons, were highly activated and related to impulsive behavior. The activation viruses (AAV-CaMKIIα-hM3Dq) were injected to evaluate the effects of specific activation of mPFC excitatory neurons on impulsive behavior in the presence of clozapine-N-oxide (CNO). Likewise, the inhibitory viruses (AAV-CaMKIIα-hM4Di) were injected in the sevoflurane group to explore whether the mPFC excitatory neuronal inhibition reduced the impulsivity. Our results revealed that chemogenetic activation of mPFC excitatory neurons induced impulsive behavior whereas inhibition of mPFC excitatory neurons partially rescued the deficit. These results indicate that repeated sevoflurane exposures at the critical time induce impulsive behavior accompanied with overactivation of mPFC excitatory neurons in adult stages. This work may further extend to understand the ADHD-like impulsive behavior of the anesthetic neurotoxicity.

Involvement of homodomain interacting protein kinase 2-c-Jun N-terminal kinase/c-Jun cascade in the long-term synaptic toxicity and cognition impairment induced by neonatal Sevoflurane exposure.

Sevoflurane is one of the most widely used anesthetics with recent concerns rising about its pediatric application. The synaptic toxicity and mechanisms underlying its long‐term cognition impairment remain unclear. In this study, we investigated the expression and roles of homeodomain interacting protein kinase 2 (HIPK2), a stress activating kinase involved in neuronal survival and synaptic plasticity, and its downstream c‐Jun N‐terminal kinase (JNK)/c‐Jun signaling in the long‐term toxicity of neonatal Sevoflurane exposure. Our data showed that neonatal Sevoflurane exposure results in impairment of memory, enhancement of anxiety, less number of excitatory synapses and lower levels of synaptic proteins in the hippocampus of adult rats without significant changes of hippocampal neuron numbers. Up‐regulation of HIPK2 and JNK/c‐Jun was observed in hippocampal granular neurons shortly after Sevoflurane exposure and persisted to adult. 5‐((6‐Oxo‐5‐(6‐(piperazin‐1‐yl)pyridin‐3‐yl)‐1,6‐dihydropyridin‐3‐yl)methylene)thiazolidine‐2,4‐dione trifluoroacetate, antagonist of HIPK2, could significantly rescue the cognition impairment, decrease in long‐term potentiation, reduction in spine density and activation of JNK/c‐Jun induced by Sevoflurane. JNK antagonist SP600125 partially restored synapse development and cognitive function without affecting the expression of HIPK2. These data, in together, revealed a novel role of HIPK2‐JNK/c‐Jun signaling in the long‐term synaptic toxicity and cognition impairment of neonatal Sevoflurane exposure, indicating HIPK2‐JNK/c‐Jun cascade as a potential target for reducing the synaptic toxicity of Sevoflurane. Cover Image for this issue: doi: 10.1111/jnc.14757.

Cognitive impairment and transcriptomic profile in hippocampus of young mice after multiple neonatal exposures to sevoflurane.

Children with repeated inhalational anesthesia may develop cognitive disorders. This study aimed to investigate the transcriptome-wide response of hippocampus in young mice that had been exposed to multiple sevoflurane in the neonatal period. Mice received 3% sevoflurane for 2 h on postnatal day (PND) 6, 8, and 10, followed by arterial blood gas test on PND 10, behavioral experiments on PND 31–36, and RNA sequencing (RNA-seq) of hippocampus on PND 37. Functional annotation and protein-protein interaction analyses of differentially expressed genes (DEGs) and quantitative reverse transcription polymerase chain reaction (qPCR) were performed. Neonatal sevoflurane exposures induced cognitive and social behavior disorders in young mice. RNA-seq identified a total of 314 DEGs. Several enriched biological processes (ion channels, brain development, learning, and memory) and signaling pathways (oxytocin signaling pathway and glutamatergic, cholinergic, and GABAergic synapses) were highlighted. As hub-proteins, Pten was involved in nervous system development, synapse assembly, learning, memory, and behaviors, Nos3 and Pik3cd in oxytocin signaling pathway, and Cdk16 in exocytosis and phosphorylation. Some top DEGs were validated by qPCR. This study revealed a transcriptome-wide profile in mice hippocampus after multiple neonatal exposures to sevoflurane, promoting better understanding of underlying mechanisms and investigation of preventive strategies.