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.

Single-nucleus Atlas of Sevoflurane-induced Hippocampal Cell Type- and Sex-specific Effects during Development in Mice.

BACKGROUND: Multiple neonatal exposures to sevoflurane induce neurocognitive dysfunctions in rodents. The lack of cell type-specific information after sevoflurane exposure limits the mechanistic understanding of these effects. In this study, the authors tested the hypothesis that sevoflurane exposures alter the atlas of hippocampal cell clusters and have neuronal and nonneuronal cell type-specific effects in mice of both sexes. METHODS: Neonatal mice were exposed to 3% sevoflurane for 2 h at postnatal days 6, 8, and 10 and analyzed for the exposure effects at postnatal day 37. Single-nucleus RNA sequencing was performed in the hippocampus followed by in situ hybridization to validate the results of RNA sequencing. The Morris Water Maze test was performed to test neurocognitive function. RESULTS: The authors found sex-specific distribution of hippocampal cell types in control mice alongside cell type- and sex-specific effects of sevoflurane exposure on distinct hippocampal cell populations. There were important changes in male but not in female mice after sevoflurane exposure regarding the proportions of cornu ammonis 1 neurons (control vs. sevoflurane, males: 79.9% vs. 32.3%; females: 27.3% vs. 24.3%), dentate gyrus (males: 4.2% vs. 23.4%; females: 36.2% vs. 35.8%), and oligodendrocytes (males: 0.6% vs. 6.9%; females: 5.9% vs. 7.8%). In male but not in female mice, sevoflurane altered the number of significantly enriched ligand-receptor pairs in the cornu ammonis 1, cornu ammonis 3, and dente gyrus trisynaptic circuit (control vs. sevoflurane, cornu ammonis 1-cornu ammonis 3: 18 vs. 42 in males and 15 vs. 21 in females; cornu ammonis 1-dentate gyrus: 21 vs. 35 in males and 12 vs. 20 in females; cornu ammonis 3-dentate gyrus: 25 vs. 45 in males and 17 vs. 20 in females), interfered with dentate gyrus granule cell neurogenesis, hampered microglia differentiation, and decreased cornu ammonis 1 pyramidal cell diversity. Oligodendrocyte differentiation was specifically altered in females with increased expressions of Mbp and Mag. In situ hybridization validated the increased expression of common differentially expressed genes. CONCLUSIONS: This single-nucleus RNA sequencing study reveals the hippocampal atlas of mice, providing a comprehensive resource for the neuronal and nonneuronal cell type- and sex-specific effects of sevoflurane during development.

Disruption of hippocampal P2RX2/CaMKII/NF-κB signaling contributes to learning and memory impairment in C57BL/6 mice induced by surgery plus anesthesia in neonatal period.

A great number of pediatric patients undergoing varied procedures make neonatal surgery plus anesthesia become a matter of great concern owing to underlying neurotoxicity in developing brain. The authors set out to assess long-term effects of surgery plus anesthesia in mouse model. Six-day-old C57BL/6 mice were randomized to receive either anesthesia with 3% sevoflurane, abdominal surgery under the same anesthesia, or the control condition. These mice were examined of learning and memory at juvenile age in Morris water maze test. The brain tissues of mice were harvested for Western blot analysis, including purinergic receptors P2X family, CaMKII and NF-κB. Another battery of mice were administered with inhibitors of P2RX2/3 (e.g., A317491) into hippocampal dentate gyrus before behavioral testing. We found that neonatal surgery plus anesthesia, but not sevoflurane anesthesia alone, impaired the learning and memory of juvenile mice, as evidenced by delayed escape latency and reduced platform-crossing times. Immunoblotting analysis showed that behavioral abnormalities were associated with increased levels of P2RX2, phosphorylated-CaMKIIß and activated NF-κB in mouse hippocampus. Injection of A317491 ameliorated the impaired learning and memory of juvenile mice undergoing neonatal surgery plus anesthesia, and it also mitigated the neonatal surgery-induced signaling enhancement of P2RX2/CaMKII/NF-κB. Together, these results indicate that neonatal surgery plus anesthesia may cause long-term cognitive dysfunction, with potential mechanism of increasing P2RX2 and downstream signaling of phosphorylated-CaMKII and NF-κB. Our findings will promote more studies to assess detrimental effects of surgery and accompanying inflammation, diverse anesthetics and even sleeping deprivation on mouse neurodevelopment and neurobehavioral performance.

Neonatal exposure to sevoflurane impairs preference for social novelty in C57BL/6 female mice at early-adulthood.

Social interaction deficit is core symptom of children with autism, owing to interaction of genetic predisposition and environmental toxins. Sevoflurane could induce neurotoxicity in developing brain in rodent models. This study aims to investigate whether sevoflurane anesthesia in neonatal period could impair social behaviors in male and female mice. Twenty-eight male and thirty-one female mice were randomly assigned to receive 3.0% sevoflurane or 60% oxygen on postnatal day 6. They were tested for social interaction behaviors at one- and two-month-old. In addition, the cortex and hippocampus of neonatal mice undergoing sevoflurane anesthesia were harvested for immunoblotting analysis. As a result, both male and female mice undergoing sevoflurane anesthesia showed strong sociability and weak preference for social novelty at juvenile age. In addition, the male mice developed normal preference for social novelty at early-adulthood; However, the female mice remained weak preference for social novelty. Furthurmore, sevoflurane anesthesia could decrease the levels of PSD95 but not Neuroligin-1 in the hippocampus but not cortex of neonatal mice. In conclusion, sevoflurane anesthesia in neonatal period could disturb development of social memory and impair preference for social novelty in female mice at early-adulthood, with the potential mechanism of decreasing PSD95 expression in the hippocampus of C57BL/6 mice.

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.

Maternal anesthesia with sevoflurane during the mid-gestation induces social interaction deficits in offspring C57BL/6 mice.

Sevoflurane anesthesia in pregnant mice could induce neurotoxicity in the developing brain and disturb learning and memory in the offspring mice. Whether it could impair social behaviors in the offspring mice is uncertain. Therefore, we assessed the neurobehavioral effect of in-utero exposure to sevoflurane on social interaction behaviors in C57BL/6 mice. The pregnant mice were anesthetized with 2.5% sevoflurane in 100% oxygen for 2 h, and their offspring mice were tested in three-chambered social paradigm, which includes three 10-min sessions of habituation, sociability, and preference for social novelty. At the juvenile age, the offspring mice showed abnormal sociability, as proved by not taking more time sniffing at the stranger 1 mouse compared with the empty enclosure (108.5 ± 25.4 vs. 108.2 ± 44.0 s, P = 0.9876). Meanwhile, these mice showed impaired preference for social novelty, as evidenced by not taking more time sniffing at the stranger 2 compared with the stranger 1 mouse (92.1 ± 52.2 vs. 126.7 ± 50.8 s, P = 0.1502). At the early adulthood, the offspring mice retrieved the normal sociability (145.6 ± 33.2 vs. 76.0 ± 31.8 s, P = 0.0001), but remained the impaired preference for social novelty (100.6 ± 29.3 vs. 118.0 ± 47.9 s, P = 0.3269). Collectively, these results suggested maternal anesthesia with sevoflurane could induce social interaction deficits in their offspring mice. Although the disturbance of sociability could be recoverable, the impairment of preference for social novelty could be long-lasting.

Inhibition of cathepsin S attenuates myocardial ischemia/reperfusion injury by suppressing inflammation and apoptosis.

Myocardial ischemia/reperfusion (I/R) injury leads to high mortality and morbidity due to the incomplete understanding of the underlying mechanism and the consequent lack of effective therapy. The present study revealed and validated key candidate genes in relation to inflammation and apoptosis pathways underlying myocardial I/R injury. Cathepsin S was identified as the top hub protein based on the protein-protein interaction analysis, and, thus, its role during myocardial I/R injury was further investigated. Myocardial I/R in mice resulted in significantly increased levels of myocardial injury biomarkers (cardiac troponin I, lactic dehydrogenase, and creatinine kinase-MB) and inflammatory cytokines (interleukin-1ß [IL-1ß], IL-6, and tumor necrosis factor-α), elevated apoptosis rate, and upregulated protein expression of cleaved caspase-8, cleaved caspase-3, and cleaved poly ADP-ribose polymerase. These abovementioned changes were blocked by two different selective cathepsin S inhibitors, LY3000328 or MIV-247. Moreover, Kaplan-Meier survival plot showed that cathepsin S inhibition improved 21-day survival rate following myocardial I/R injury. This study demonstrated that the inhibition of cathepsin S alleviated myocardial I/R-induced injury by suppressing inflammation and apoptosis, which may be used in clinical applications of cardioprotection.

Heat Shock Protein 70 Protects the Heart from Ischemia/Reperfusion Injury through Inhibition of p38 MAPK Signaling.

BACKGROUND: Heat shock protein 70 (Hsp70) has been shown to exert cardioprotection. Intracellular calcium ([Ca2+]i) overload induced by p38 mitogen-activated protein kinase (p38 MAPK) activation contributes to cardiac ischemia/reperfusion (I/R) injury. However, whether Hsp70 interacts with p38 MAPK signaling is unclear. Therefore, this study investigated the regulation of p38 MAPK by Hsp70 in I/R-induced cardiac injury. METHODS: Neonatal rat cardiomyocytes were subjected to oxygen-glucose deprivation for 6 h followed by 2 h reoxygenation (OGD/R), and rats underwent left anterior artery ligation for 30 min followed by 30 min of reperfusion. The p38 MAPK inhibitor (SB203580), Hsp70 inhibitor (Quercetin), and Hsp70 short hairpin RNA (shRNA) were used prior to OGD/R or I/R. Cell viability, lactate dehydrogenase (LDH) release, serum cardiac troponin I (cTnI), [Ca2+]i levels, cell apoptosis, myocardial infarct size, mRNA level of IL-1ß and IL-6, and protein expression of Hsp70, phosphorylated p38 MAPK (p-p38 MAPK), sarcoplasmic/endoplasmic reticulum Ca2+-ATPase2 (SERCA2), phosphorylated signal transducer and activator of transcription3 (p-STAT3), and cleaved caspase3 were assessed. RESULTS: Pretreatment with a p38 MAPK inhibitor, SB203580, significantly attenuated OGD/R-induced cell injury or I/R-induced myocardial injury, as evidenced by improved cell viability and lower LDH release, resulted in lower serum cTnI and myocardial infarct size, alleviation of [Ca2+]i overload and cell apoptosis, inhibition of IL-1ß and IL-6, and modulation of protein expressions of p-p38 MAPK, SERCA2, p-STAT3, and cleaved-caspase3. Knockdown of Hsp70 by shRNA exacerbated OGD/R-induced cell injury, which was effectively abolished by SB203580. Moreover, inhibition of Hsp70 by quercetin enhanced I/R-induced myocardial injury, while SB203580 pretreatment reversed the harmful effects caused by quercetin. CONCLUSIONS: Inhibition of Hsp70 aggravates [Ca2+]i overload, inflammation, and apoptosis through regulating p38 MAPK signaling during cardiac I/R injury, which may help provide novel insight into cardioprotective strategies.

Dexmedetomidine protects H9c2 cardiomyocytes against oxygen-glucose deprivation/reoxygenation-induced intracellular calcium overload and apoptosis through regulating FKBP12.6/RyR2 signaling.

PURPOSE: Intracellular calcium ([Ca2+]i) overload is a major cause of cell injury during myocardial ischemia/reperfusion (I/R). Dexmedetomidine (DEX) has been shown to exert anti-inflammatory and organ protective effects. This study aimed to investigate whether pretreatment with DEX could protect H9c2 cardiomyocytes against oxygen-glucose deprivation/reoxygenation (OGD/R) injury through regulating the Ca2+ signaling. METHODS: H9c2 cardiomyocytes were subjected to OGD for 12 h, followed by 3 h of reoxygenation. DEX was administered 1 h prior to OGD/R. Cell viability, lactate dehydrogenase (LDH) release, level of [Ca2+]i, cell apoptosis, and the expression of 12.6-kd FK506-binding protein/ryanodine receptor 2 (FKBP12.6/RyR2) and caspase-3 were assessed. RESULTS: Cells exposed to OGD/R had decreased cell viability, increased LDH release, elevated [Ca2+]i level and apoptosis rate, down-regulated expression of FKBP12.6, and up-regulated expression of phosphorylated-Ser2814-RyR2 and cleaved caspase-3. Pretreatment with DEX significantly blocked the above-mentioned changes, alleviating the OGD/R-induced injury in H9c2 cells. Moreover, knockdown of FKBP12.6 by small interfering RNA abolished the protective effects of DEX. CONCLUSION: This study indicates that DEX pretreatment protects the cardiomyocytes against OGD/R-induced injury by inhibiting [Ca2+]i overload and cell apoptosis via regulating the FKBP12.6/RyR2 signaling. DEX may be used for preventing cardiac I/R injury in the clinical settings.

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.