Identification of differential m6A RNA methylomes and ALKBH5 as a potential prevention target in the developmental neurotoxicity induced by multiple sevoflurane exposures.

Sevoflurane, as a commonly used inhaled anesthetic for pediatric patients, has been reported that multiple sevoflurane exposures are associated with a greater risk of developing neurocognitive disorder. N6-Methyladenosine (m6A), as the most common mRNA modification in eukaryotes, has emerged as a crucial regulator of brain function in processes involving synaptic plasticity, learning and memory, and neurodevelopment. Nevertheless, the relevance of m6A RNA methylation in the multiple sevoflurane exposure-induced developmental neurotoxicity remains mostly elusive. Herein, we evaluated the genome-wide m6A RNA modification and gene expression in hippocampus of mice that received with multiple sevoflurane exposures using m6A-sequencing (m6A-seq) and RNA-sequencing (RNA-seq). We discovered 19 genes with differences in the m6A methylated modification and differential expression in the hippocampus. Among these genes, we determined that a total of nine differential expressed genes may be closely associated with the occurrence of developmental neurotoxicity induced by multiple sevoflurane exposures. We further found that the alkB homolog 5 (ALKBH5), but not methyltransferase-like 3 (METTL3) and Wilms tumor 1-associated protein (WTAP), were increased in the hippocampus of mice that received with multiple sevoflurane exposures. And the IOX1, as an inhibitor of ALKBH5, significantly improved the learning and memory defects and reduced neuronal damage in the hippocampus of mice induced by multiple sevoflurane exposures. The current study revealed the role of m6A methylated modification and m6A-related regulators in sevoflurane-induced cognitive impairment, which might provide a novel insight into identifying biomarkers and therapeutic strategies for inhaled anesthetic-induced developmental neurotoxicity.

Multiple Exposures to Sevoflurane General Anesthesia During Pregnancy Inhibit CaMKII/CREB Axis by Downregulating HCN2 to Induce an Autism-Like Phenotype in Offspring Mice.

The objective of this investigation was to examine the impact of multiple exposures to general anesthesia (GA) with sevoflurane on the offspring of pregnant mice, as well as to elucidate the underlying mechanism. Neurodevelopmental assessments, including various reflexes and behavioral tests, were conducted on the offspring in the GA group to evaluate neuronal cell development. Furthermore, neonatal mouse neuronal cells were isolated and transfected with a high-expression CREB vector (pcDNA3.1-CREB), followed by treatment with sevoflurane (0.72 mol/L), ZD7288 (50 µmol/L), and KN-62 (10 µmol/L), or a combination of these compounds. The expression of relevant genes was then analyzed using qRT-PCR and western blot techniques. In comparison to the sham group, neonatal mice in the GA group exhibited significantly prolonged latencies in surface righting reflex, geotaxis test, and air righting reflex. Furthermore, there was a notable deceleration in the development of body weight and tail in the GA group. These mice also displayed impairments in social ability, reduced reciprocal social interaction behaviors, diminished learning capacity, and heightened levels of anxious behaviors. Additionally, synaptic trigger malfunction was observed, along with decreased production of c-Fos and neurotrophic factors. Sevoflurane was found to notably decrease cellular c-Fos and neurotrophic factor production, as well as the expression of HCN2 and CaMKII/CREB-related proteins. The inhibitory effects of sevoflurane on HCN2 or CaMKII channels were similar to those observed with ZD7288 or KN-62 inhibition. However, overexpression of CREB mitigated the impact of sevoflurane on neuronal cells. Repetitive exposure to sevoflurane general anesthesia while pregnant suppresses the CaMKII/CREB pathway, leading to the development of autism-like characteristics in offspring mice through the reduction of HCN2 expression.

The effect of N-acetylcysteine on the neurotoxicity of sevoflurane in developing hippocampus cells.

Sevoflurane, a common pediatric anesthetic, has been linked to neurodegeneration, raising safety concerns. This study explored N-acetylcysteine's protective potential against sevoflurane-induced neurotoxicity in rat hippocampi. Four groups were examined: Control: Received 6 hours of 3 l/min gas (air and 30 % O2) and intraperitoneal saline. NAC: Received 6 hours of 3 l/min gas and 150 mg/kg NAC intraperitoneally. Sev: Exposed to 6 hours of 3 l/min gas and 3 % sevoflurane. Sev+NAC: Received 6 hours of 3 l/min gas, 3 % sevoflurane, and 150 mg/kg NAC. Protein levels of NRF-2, NLRP3, IL-1ß, caspase-1, Beclin 1, p62, LC3A, and apoptosis markers were assessed. Sevoflurane and NAC alone reduced autophagy, while Sev+NAC group maintained autophagy levels. Sev group had elevated NRF-2, NLRP3, pNRF2, Caspase-1, and IL-1ß, which were reduced in Sev+NAC. Apoptosis was higher in Sev, but Sev+NAC showed reduced apoptosis compared to the control. In summary, sevoflurane induced neurotoxicity in developing hippocampus, which was mitigated by N-acetylcysteine administration.

The changing of α5-GABAA receptors expression and distribution participate in sevoflurane-induced learning and memory impairment in young mice.

BACKGROUND: Sevoflurane is a superior agent for maintaining anesthesia during surgical procedures. However, the neurotoxic mechanisms of clinical concentration remain poorly understood. Sevoflurane can interfere with the normal function of neurons and synapses and impair cognitive function by acting on α5-GABAAR. METHODS: Using MWM test, we evaluated cognitive abilities in mice following 1 h of anesthesia with 2.7%-3% sevoflurane. Based on hippocampal transcriptome analysis, we analyzed the differential genes and IL-6 24 h post-anesthesia. Western blot and RT-PCR were performed to measure the levels of α5-GABAAR, Radixin, P-ERM, P-Radixin, Gephyrin, IL-6, and ROCK. The spatial distribution and expression of α5-GABAAR on neuronal somata were analyzed using histological and three-dimensional imaging techniques. RESULTS: MWM test indicated that partial long-term learning and memory impairment. Combining molecular biology and histological analysis, our studies have demonstrated that sevoflurane induces immunosuppression, characterized by reduced IL-6 expression levels, and that enhanced Radixin dephosphorylation undermines the microstructural stability of α5-GABAAR, leading to its dissociation from synaptic exterior and resulting in a disordered distribution in α5-GABAAR expression within neuronal cell bodies. On the synaptic cleft, the expression level of α5-GABAAR remained unchanged, the spatial distribution became more compact, with an increased fluorescence intensity per voxel. On the extra-synaptic space, the expression level of α5-GABAAR decreased within unchanged spatial distribution, accompanied by an increased fluorescence intensity per voxel. CONCLUSION: Dysregulated α5-GABAAR expression and distribution contributes to sevoflurane-induced partial long-term learning and memory impairment, which lays the foundation for elucidating the underlying mechanisms in future studies.

Sevoflurane causes neurotoxicity and cognitive impairment by regulating Hippo signaling pathway-mediated ferroptosis via upregulating PRKCD.

BACKGROUND: Sevoflurane (SEV) has been found to induce neurotoxicity and cognitive impairment, leading to the development of degenerative diseases. Protein kinase C delta (PRKCD) is upregulated in the hippocampus of SEV-treated mice and may be related to SEV-related neurotoxicity. However, the underlying molecular mechanisms by which SEV mediates neurotoxicity via PRKCD remain unclear. METHODS: Normal mice and PRKCD knockout (KO) mice were exposed to SEV. Hippocampal neurons were isolated from mice hippocampal tissues. H&E staining was used for pathological morphology of hippocampal tissues, and NISSL staining was used to analyze the number of hippocampal neurons. The mRNA and protein levels were determined using quantitative real-time PCR, western blot, immunofluorescence staining and immunohistochemical staining. The mitochondrial microstructure was observed by transmission electron microscopy. Cell viability was detected by cell counting kit 8 assay, and ferroptosis was assessed by detecting related marker levels. The cognitive ability of mice was assessed by morris water maze test. And the protein levels of PRKCD, ferroptosis-related markers and Hippo pathway-related markers were examined by western bolt. RESULTS: SEV increased PRKCD expression and ferroptosis in hippocampal tissues of mice. Also, SEV promoted mouse hippocampal neuron injury by inducing ferroptosis via upregulating PRKCD expression. Knockout of PRKCD alleviated SEV-induced neurotoxicity and cognitive impairment in mice, and relieved SEV-induced ferroptosis in hippocampal neurons. PRKCD could inhibit the activity of Hippo pathway, and its knockdown also overturned SEV-mediated ferroptosis by activating Hippo pathway. CONCLUSION: SEV could induce neurotoxicity and cognitive impairment by promoting ferroptosis via inactivating Hippo pathway through increasing PRKCD expression.

Mfn2 regulates mitochondria and mitochondria-associated endoplasmic reticulum membrane function in neurodegeneration induced by repeated sevoflurane exposure.

Repeated sevoflurane exposure in neonatal mice can leads to neuronal apoptosis and mitochondrial dysfunction. The mitochondria are responsible for energy production to maintain homeostasis in the central nervous system. The mitochondria-associated endoplasmic reticulum membrane (MAM) is located between the mitochondria and endoplasmic reticulum (ER), and it is critical for mitochondrial function and cell survival. MAM malfunction contributes to neurodegeneration, however, whether it is involved in sevoflurane-induced neurotoxicity remains unknown. Our study demonstrated that repeated sevoflurane exposure induced mitochondrial dysfunction and dampened the MAM structure. The upregulated ER-mitochondria tethering enhanced Ca2+ transition from the cytosol to the mitochondria. Overload of mitochondrial Ca2+ contributed to opening of the mitochondrial permeability transition pore (mPTP), which caused neuronal apoptosis. Mitofusin 2(Mfn2), a key regulator of ER-mitochondria contacts, was found to be suppressed after repeated sevoflurane exposure, while restoration of Mfn2 expression alleviated cognitive dysfunction due to repeated sevoflurane exposure in the adult mice. These evidences suggest that sevoflurane-induced MAM malfunction is vulnerable to Mfn2 suppression, and the enhanced ER-mitochondria contacts promotes mitochondrial Ca2+ overload, contributing to mPTP opening and neuronal apoptosis. This paper sheds light on a novel mechanism of sevoflurane-induced neurotoxicity. Furthermore, targeting Mfn2-mediated regulation of the MAM structure and mitochondrial function may provide a therapeutic advantage in sevoflurane-induced neurodegeneration.

SHARPIN contributes to sevoflurane-induced neonatal neurotoxicity through up-regulating HMGB1 to repress M2 like-macrophage polarization.

Sevoflurane exposure can result in neurotoxicity especially among children, which remains an important complication after surgery. However, its related mechanisms remain unclear. Here, we investigated the biological roles of SHARPIN in sevoflurane-induced neurotoxicity. As detected by qPCR, Western blotting and immunohistochemical staining, SHARPIN and HMGB1 expression was elevated in sevoflurane-stimulated mice as compared with the control mice. SHARPIN depletion attenuated hippocampus injury, repressed the expression of HMGB1 and M1-like macrophage markers (iNOS, TNF-α, IL-1ß, IL-6), but enhanced the expression of M2-like macrophage markers (ARG-1, IL-10). GST pull-down and Co-IP assays demonstrated that SHARPIN directly interacted with HMGB1 to enhance HMGB1 expression in SH-SY5Y cells. The inhibitory effects of SHARPIN silencing on inflammatory reaction and M1-like macrophages were counteracted by HMGB1 overexpression. Finally, SHARPIN-HMGB1 pathway affected neuroinflammation triggered by sevoflurane via modulating macrophage polarization. Collectively, our data suggested that SHARPIN stimulated sevoflurane-induced neurotoxicity via converting M2-like macrophages to M1-like macrophages by enhancing HMGB1 expression. SHARPIN intervention may be a promising therapeutic method to relieve sevoflurane-induced neurotoxicity.

Damage-associated molecular patterns as a mechanism of sevoflurane-induced neuroinflammation in neonatal rodents.

BACKGROUND: General anesthesia is inevitable for pediatric patients undergoing surgery, though volatile anesthetic agents may cause neuroinflammation and neurodevelopmental impairment; however, the underlying pathophysiology remains unclear. We aimed to investigate the neuroinflammation mechanism in developing rat brains associated with sevoflurane exposure time, by identifying the specific damage-associated molecular patterns (DAMPs) pathway and evaluating the effects of non-steroidal anti-inflammatory drugs (NSAIDs) in alleviating neuroinflammation. METHODS: A three-step experiment was conducted to investigate neuroinflammation induced by sevoflurane. First, the exposure time required for sevoflurane to cause neuroinflammation was determined. Next, the specific pathways of DAMPs involved in neuroinflammation by sevoflurane were identified. Finally, the effects of NSAIDs on sevoflurane-induced neuroinflammation were investigated. The expression of various molecules in the rat brain were assessed using immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, western blot analysis, and enzyme-linked immunosorbent assay. RESULTS: In total, 112 rats (aged 7 days) were used, of which six rats expired during the experiment (mortality rate, 5.3%). Expression of CD68, HMGB-1, galectin-3, TLR4, TLR9, and phosphorylated NF-κB was significantly increased upon 6 h of sevoflurane exposure. Conversely, transcriptional levels of TNF-α and IL-6 significantly increased and IFN-γ significantly decreased after 6 h of sevoflurane exposure. Co-administration of NSAIDs with sevoflurane anesthesia significantly attenuated TNF-α and IL-6 levels and restored IFN-γ levels. CONCLUSIONS: In conclusion, 6 h of sevoflurane exposure induces neuroinflammation through the DAMPs pathway, HMGB-1, and galectin-3. Co-administration of ibuprofen reduced sevoflurane-induced neuroinflammation.

Echinatin mitigates sevoflurane-induced neurotoxicity through regulation of ferroptosis and iron homeostasis.

Surgery and anesthesia are vital medical interventions, but concerns over their potential cognitive side effects, particularly with the use of inhalational anesthetics like sevoflurane, have surfaced. This study delves into the neuroprotective potential of Echinatin against sevoflurane-induced neurotoxicity and the underlying mechanisms. Echinatin, a natural compound, has exhibited anti-inflammatory, antioxidant, and anticancer properties. Sevoflurane, while a popular anesthetic, is associated with perioperative neurocognitive disorders (PND) and neurotoxicity. Our investigation began with cellular models, where Echinatin demonstrated a significant reduction in sevoflurane-induced apoptosis. Mechanistically, we identified ferroptosis, a novel form of programmed cell death characterized by iron accumulation and lipid peroxidation, as a key player in sevoflurane-induced neuronal injury. Echinatin notably suppressed ferroptosis in sevoflurane-exposed cells, suggesting a pivotal role in neuroprotection. Expanding our research to a murine model, we observed perturbations in iron homeostasis, inflammatory cytokines, and antioxidants due to sevoflurane exposure. Echinatin treatment effectively restored iron balance, mitigated inflammation, and preserved antioxidant levels in vivo. Behavioral assessments using the Morris water maze further confirmed Echinatin's neuroprotective potential, as it ameliorated sevoflurane-induced spatial learning and memory impairments. In conclusion, our study unveils Echinatin as a promising candidate for mitigating sevoflurane-induced neurotoxicity. Through the regulation of ferroptosis, iron homeostasis, and inflammation, Echinatin demonstrates significant neuroprotection both in vitro and in vivo. These findings illuminate the potential for Echinatin to enhance the safety of surgical procedures involving sevoflurane anesthesia, minimizing the risk of cognitive deficits and neurotoxicity.

Epigenetic Mechanism of SETD1B-mediated Histone Methylation in Cognitive Impairment Induced by Sevoflurane Anesthesia in Neonatal Mice.

Sevoflurane (Sev) anesthesia is associated with cognitive deficits and neurotoxicity. This study explores the epigenetic mechanism of SET domain containing 1B (SETD1B) in Sev-induced cognitive impairment in neonatal mice. Neonatal mice (C57BL/6, n = 72) were exposed to 3% Sev for 2 h per day at P6, 7, and 8, and the control neonatal mice were only separated from the mother for 2 h. The mice were divided into groups of 12 individuals, with an equal number of male and female mice in each group. Mice were intraperitoneally injected with adenovirus-packaged SETD1B overexpression vector. Behavioral tests (Morris water maze, open field test, T-maze, novel object recognition, etc.) were performed at P30. Mouse hippocampal neuronal cells were cultured in vitro. SETD1B, C-X-C motif chemokine receptor 4 (CXCR4), NLR family pyrin domain containing 1 (NLRP1), Cleaved Caspase1, and GSDMD-N expressions in hippocampal tissues or cells were determined by quantitative real-time polymerase chain reaction and Western blot. SETD1B and histone H3 lysine 4 methylation (H3K4me1, H3K4me2, and H3K4me3) enrichment on the CXCR4 promoter was analyzed by ChIP. Sev insulted cognitive impairment and diminished SETD1B expression in mouse hippocampal tissues. SETD1B overexpression mitigated cognitive impairment, enhanced H3K4me3 levels in hippocampal tissues, and restrained hippocampal neuronal pyroptosis. SETD1B increased CXCR4 expression by elevating the H3K4me3 level on the CXCR4 promoter, thereby curbing NLRP1/Caspase1-mediated hippocampal neuronal pyroptosis. To conclude, SETD1B enhances CXCR4 expression by elevating the H3K4me3 level on the CXCR4 promoter, thereby suppressing NLRP1/Caspase1-triggered hippocampal neuronal pyroptosis and alleviating Sev-induced cognitive impairment in neonatal mice.

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

BACKGROUND: Repeated 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. METHODS: From 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). RESULTS: We 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. CONCLUSION: We 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.

Sevoflurane causes cognitive impairment by inducing iron deficiency and inhibiting the proliferation of neural precursor cells in infant mice.

AIMS: Numerous studies on animals have shown that exposure to general anesthetics in infant stage may cause neurocognitive impairment. However, the exact mechanism is not clear. The dysfunction of iron metabolism can cause neurodevelopmental disorders. Therefore, we investigated the effect of iron metabolism disorder induced by sevoflurane (Sev) on cognitive function and the proliferation of neural precursor cells (NPCs) and neural stem cells (NSCs) in infant mice. METHODS: C57BL/6 mice of postnatal day 14 and neural stem cells NE4C were treated with 2% Sev for 6 h. We used the Morris water maze (MWM) to test the cognitive function of infant mice. The proliferation of NPCs was measured using bromodeoxyuridine (BrdU) label and their markers Ki67 and Pax6 in infant brain tissues 12 h after anesthesia. Meanwhile, we used immunohistochemical stain, immunofluorescence assay, western blot, and flow cytometer to evaluate the myelinogenesis, iron levels, and cell proliferation in cortex and hippocampus or in NE4C cells. RESULTS: The results showed that Sev significantly caused cognitive deficiency in infant mice. Further, we found that Sev inhibited oligodendrocytes proliferation and myelinogenesis by decreasing MBP and CC-1 expression and iron levels. Meanwhile, Sev also induced the iron deficiency in neurons and NSCs by downregulating FtH and FtL expression and upregulating the TfR1 expression in the cortex and hippocampus, which dramatically suppressed the proliferation of NSCs and NPCs as indicated by decreasing the colocalization of Pax6+ and BrdU+ cells, and caused the decrease in the number of neurons. Interestingly, iron supplementation before anesthesia significantly improved iron deficiency in cortex and hippocampus and cognitive deficiency induced by Sev in infant mice. Iron therapy inhibited the decrease of MBP expression, iron levels in neurons and oligodendrocytes, and DNA synthesis of Pax6+ cells in hippocampus induced by Sev. Meanwhile, the number of neurons was partially recovered in hippocampus. CONCLUSION: The results from the present study demonstrated that Sev-induced iron deficiency might be a new mechanism of cognitive impairment caused by inhaled anesthetics in infant mice. Iron supplementation before anesthesia is an effective strategy to prevent cognitive impairment caused by Sev in infants.

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.

Neuron-derived exosomes mediate sevoflurane-induced neurotoxicity in neonatal mice via transferring lncRNA Gas5 and promoting M1 polarization of microglia.

Sevoflurane exposure during rapid brain development induces neuronal apoptosis and causes memory and cognitive deficits in neonatal mice. Exosomes that transfer genetic materials including long non-coding RNAs (lncRNAs) between cells play a critical role in intercellular communication. However, the lncRNAs found in exosomes derived from neurons treated with sevoflurane and their potential role in promoting neurotoxicity remain unknown. In this study, we investigated the role of cross-talk of newborn mouse neurons with microglial cells in sevoflurane-induced neurotoxicity. Mouse hippocampal neuronal HT22 cells were exposed to sevoflurane, and then co-cultured with BV2 microglial cells. We showed that sevoflurane treatment markedly increased the expression of the lncRNA growth arrest-specific 5 (Gas5) in neuron-derived extracellular vesicles, which inhibited neuronal proliferation and induced neuronal apoptosis by promoting M1 polarization of microglia and the release of inflammatory cytokines. We further revealed that the exosomal lncRNA Gas5 significantly upregulated Foxo3 as a competitive endogenous RNA of miR-212-3p in BV2 cells, and activated the NF-κB pathway to promote M1 microglial polarization and the secretion of inflammatory cytokines, thereby exacerbating neuronal damage. In neonatal mice, intracranial injection of the exosomes derived from sevoflurane-treated neurons into the bilateral hippocampi significantly increased the proportion of M1 microglia, inhibited neuronal proliferation and promoted apoptosis, ultimately leading to neurotoxicity. Similar results were observed in vitro in BV2 cells treated with the CM from HT22 cells after sevoflurane exposure. We conclude that sevoflurane induces the transfer of lncRNA Gas5-containing exosomes from neurons, which in turn regulates the M1 polarization of microglia and contributes to neurotoxicity. Thus, modulating the expression of lncRNA Gas5 or the secretion of exosomes could be a strategy for addressing sevoflurane-induced neurotoxicity.

Necroptosis involved in sevoflurane-induced cognitive dysfunction in aged mice by activating NMDA receptors increasing intracellular calcium.

Perioperative neurocognitive disorders are a common surgical and postanesthesia complication. Necroptosis contributes to the emergence of various neurological disorders. We conjecture that cognitive impairment is associated with necroptosis of hippocampal neurons, which is mediated by NMDA receptors leading to cytoplasmic calcium imbalance. C57BL/6 J male mice ( 18 months) were randomly divided into the C ( control group), S ( sevoflurane group), S+M ( sevoflurane plus the NMDA receptor antagonist memantine group) and S+N ( sevoflurane plus necrostatin-1) group. We exposed the mice to 3% sevoflurane for 2 h a day for three consecutive days in the S, S+M and S+N groups. Memantine ( 20 mg/kg) or Nec-1 ( 10 mg/kg) was injected intraperitoneally 1 h before sevoflurane anesthesia in the S+M or S+N group. We used the animal behavior tests to evaluate the cognitive function. Pathological damage, the rate of necroptosis, [Ca2+]i, and the expression of necroptosis-related proteins were evaluated. The cognitive function tests, pathological damage, the rate of necroptosis, the expression of necroptosis-related proteins, NMDAR2A and NMDAR2B were significantly different in the S group ( P < 0.05). Alleviated pathological damage, decreased the rate of necroptosis and down-regulated the expression of necroptosis-related proteins occurred in the S+M and S+N group ( P < 0.05). The lower elevated [Ca2+]i, expression of NMDAR2A and NMDAR2B were found in the S+M group. Our findings highlighted sevoflurane-induced cognitive dysfunction is associated with an imbalance in cytoplasmic calcium homeostasis by activating NMDA receptors, which causes hippocampus neurons to undergo necroptosis and ultimately affects cognitive performance in aged mice.

20(S)-Ginsenoside Rh1 alleviates sevoflurane-induced ototoxicity by reducing oxidative stress levels.

CONTEXT: Sevoflurane is an inhalational anesthetic widely used in pediatric surgery. However, animal studies have shown that multiple sevoflurane exposures during the neonatal period led to ototoxicity. 20(S)-Ginsenoside Rh1, a ginsenoside extract, protects against cisplatin-induced ototoxicity by scavenging free radicals. OBJECTIVE: This study aimed to assess the effects of Rh1 on sevoflurane-induced ototoxicity. MATERIALS AND METHODS: Neonatal cochlear explants and House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were cultured and randomly divided into three groups: the control group, the sevoflurane group and the Rh1 pretreatment group. We pretreated cochlear explants or HEI-OC1 cells with 100 µM Rh1 2 hours before performing sevoflurane exposure. Immunofluorescence was used to detect hair cells and spiral ganglion neurons. Cell Counting Kit-8 assay was used to determine cell viability. Annexin V-fluorescein isothiocyanate and propidium iodide were used to evaluate apoptosis. CellROX-Green and MitoSOX-Red probes were used to measure the amount of reactive oxygen species (ROS). Tetramethylrhodamine methyl ester labeling was used to examine mitochondrial membrane potential. RESULTS: Rh1 attenuated spiral ganglion neuron nerve fibers and synapses degeneration in cochlear explants after sevoflurane exposure. Rh1 significantly increased the viability of HEI-OC1 cells, reduced reactive oxygen species accumulation in HEI-OC1 cells, and prevented mitochondrial damage in HEI-OC1 cells after sevoflurane exposure. DISCUSSION AND CONCLUSION: These findings suggest that Rh1 is a promising drug for preventing sevoflurane-induced ototoxicity.

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

AIMS: 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. METHODS: 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 in vivo electrophysiology experiments at age P35. RESULTS: 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. CONCLUSION: Our data suggested that neonatal sevoflurane exposure results in cognitive impairment through decreased synCAM1 expression in PV interneurons in the hippocampus.

Proteomic analysis of gene expression in the prefrontal cortex in infant rhesus macaques after multiple sevoflurane exposures.

PURPOSE: Repeated exposure of infant rhesus macaques to sevoflurane induces neurotoxicity and is associated with neurocognitive impairment in later life. We aimed to investigate the effect of repeated sevoflurane exposure on the expression of proteins in the prefrontal cortex of infant rhesus macaques by proteomics. METHODS: Rhesus macaques were exposed to sevoflurane three times, on postnatal days 7, 21 and 35. Quantitative proteomics employing LC-MS with isobaric labeling (TMT10plex), western blotting, and transmission electron microscopy (TEM) were utilized in the studies. RESULTS: The results of a proteomics investigation of the brain revealed that the proteins that were differentially expressed in rhesus macaques after sevoflurane exposures were associated mainly with mitochondrial respiration. Following multiple sevoflurane exposures, the prefrontal cortices of rhesus macaques exhibited increases in NDUFA8 and COX IV protein levels, while no alterations in mitochondrial morphology were observed through TEM. CONCLUSION: Multiple exposures to sevoflurane increased the mitochondrial protein levels in rhesus macaques.

Tbx2 knockdown alleviated sevoflurane-induced cognitive disorder and neuron damages in aged rats via suppressing oxidative stress and ferroptosis.

Anesthesia with sevoflurane contributes to perioperative neurocognitive disorder (PND), which is characterized by the deficiency in study and memory. T-Box transcription factor 2 (Tbx2), which is involved in the development of hippocampus neurons, was upregulated in the hippocampus of rats exposed to sevoflurane. Our study aimed to explore the role of Tbx2 in sevoflurane-induced cognitive disorder and hippocampus neuron damages. The expression of Tbx2 in hippocampus was upregulated after sevoflurane exposure, which was accompanied by the accumulation of reactive oxygen species and lipid peroxidation, as well as the loss of neurons in hippocampus. In vitro, silencing Tbx2 suppressed oxidative stress and ferroptosis induced by sevoflurane, whereas exogenous overexpression of Tbx2 exacerbated these processes. Importantly, Tbx2 knockdown improved sevoflurane-induced cognitive disorder in aged rats, as evidenced by the increases in behavioral indexes. Mechanistically, the expression of brain-derived neurotrophic factor (BDNF), as well as the downstream nuclear factor erythroid 2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling, was repressed by Tbx2. Mimicking the activation of BDNF with 7,8-dihydroxyflavone rescued the effects of Tbx2 overexpression on oxidative stress and ferroptosis in vitro, indicating that the BDNF/Nrf2/HO-1 signaling may mediate the role of Tbx2 in sevoflurane-induced cognitive disorder and neuron damages. In summary, Tbx2 may contribute to neuronal damages via enhancing the oxidative stress and ferroptosis caused by sevoflurane. BDNF/Nrf2/HO-1 signaling mediates the role of Tbx2 in sevoflurane-induced cognitive disorder. Knockdown of Tbx2 improves sevoflurane-induced cognitive impairment. Our finding provides a novel insight for PND treatment.

Multiple sevoflurane exposures during mid-trimester induce neurotoxicity in the developing brain initiated by 15LO2-Mediated ferroptosis.

AIMS: Mid-gestational sevoflurane exposure may induce notable long-term neurocognitive impairment in offspring. This study was designed to investigate the role and potential mechanism of ferroptosis in developmental neurotoxicity induced by sevoflurane in the second trimester. METHODS: Pregnant rats on day 13 of gestation (G13) were treated with or without 3.0% sevoflurane, Ferrostatin-1 (Fer-1), PD146176, or Ku55933 on three consecutive days. Mitochondrial morphology, ferroptosis-relative proteins, malondialdehyde (MDA) levels, total iron content, and glutathione peroxidase 4 (GPX4) activities were measured. Hippocampal neuronal development in offspring was also examined. Subsequently, 15-lipoxygenase 2 (15LO2)-phosphatidylethanolamine binding protein 1 (PEBP1) interaction and expression of Ataxia telangiectasia mutated (ATM) and its downstream proteins were also detected. Furthermore, Morris water maze (MWM) and Nissl's staining were applied to estimate the long-term neurotoxic effects of sevoflurane. RESULTS: Ferroptosis mitochondria were observed after maternal sevoflurane exposures. Sevoflurane elevated MDA and iron levels while inhibiting GPX4 activity, and resultant long-term learning and memory dysfunction, which were alleviated by Fer-1, PD146176, and Ku55933. Sevoflurane could enhance 15LO2-PEBP1 interaction and activate ATM and its downstream P53/SAT1 pathway, which might be attributed to excessive p-ATM nuclear translocation. CONCLUSION: This study proposes that 15LO2-mediated ferroptosis might contribute to neurotoxicity induced by maternal sevoflurane anesthesia during the mid-trimester in the offspring and its mechanism may be ascribed to hyperactivation of ATM and enhancement of 15LO2-PEBP1 interaction, indicating a potential therapeutic target for ameliorating sevoflurane-induced neurotoxicity.