Multiple exposures to sevoflurane during the neonatal period impair hippocampus development and cognitive function in young mice

Sevoflurane is a commonly used anesthesia for infants and young children. Sevoflurane has potential neurotoxicity in immature brains; however, its specific mechanism has not been fully elucidated. Therefore, we used an established sevoflurane anesthesia model and evaluated hippocampal synaptic function using transcriptome sequencing, biochemical analyses, and animal behavior to investigate the effect of multiple neonatal sevoflurane exposures on the hippocampus in mice. C57BL/6J mice were randomly divided into sevoflurane and control groups. All experimental conditions were identical in the two groups except for the anesthetization procedure. Mice in the sevoflurane group were anesthetized with 2.5% sevoflurane for 2 h daily for 3 consecutive days (postnatal days 6−8). Mice in the control group did not receive sevoflurane anesthesia. During anesthesia, mice were administered 50% oxygen, and the respiratory rate and skin color were monitored. On day 3 of modeling, half of the mice were randomly selected to undergo harvesting of the hippocampus. RNA sequencing (RNA-seq) of RNA extracted from the hippocampus identified 736 differentially expressed genes (DEGs), including 433 upregulated and 303 downregulated DEGs, after multiple sevoflurane exposures. Gene ontology term enrichment analysis results suggested that sevoflurane exposure altered the expression of neurodevelopment-related genes in neonatal mice. Several enriched biological processes involved in brain development (axon/forebrain development) and adenosine monophosphate-activated protein kinase signaling pathways were highlighted. Comparison with RNA-seq database information showed that DEGs of the neonatal hippocampus after multiple exposures to sevoflurane were specific to neonatal mice. Furthermore, Morris water maze testing confirmed that sevoflurane anesthesia induced learning and memory impairments in young mice. Additionally, Western blot and immunofluorescence analyses showed that sevoflurane treatment decreased synaptic protein levels, such as postsynaptic density protein 95, synaptosomal-associated protein, 25 kDa, and B-cell lymphoma 2-associated athanogene 3, in the hippocampus, which induced synaptic dysfunction, resulting in impaired nervous system development in young mice.


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There is a growing need for infants and young children to undergo general anesthesia 57 for surgery or clinical examination. However, the effects of general anesthetic agents on the 58 function and structure of the brain during early-stage development are unclear. The New 59 York Medical Database suggests that general anesthesia may be toxic to the 60 neurodevelopment of infants 0−3 years of age [1]. The United States Food and Drug 61 Administration issued a drug safety statement concerning the use of 11 commonly prescribed 62 sedative and anesthetic drugs, including sevoflurane, with potential neurotoxic effects on 63 children younger than 3 years of age [2]. A previous study also indicated that clinicians  Sevoflurane is neurotoxic to the developing brain when used repeatedly or for long 71 periods. Chen et al. found that the offspring of female rats exposed to anesthesia with 72 sevoflurane during pregnancy had social interaction deficits [5]. In mice, sevoflurane may 73 increase the risk of cognitive dysfunction and induce impulsive behavior in adulthood, 74 similar to that associated with attention-deficit/hyperactivity disorder [6]. Various studies 75 have demonstrated that multiple exposures to sevoflurane in neonatal mice significantly 4 76 altered the genome-wide expression profile of different brain regions in young mice [7], rats 77 [8], and monkeys [9]. However, these studies only investigated the expression of 78 differentially expressed genes (DEGs) in young animals. No study has identified neonatal 79 hippocampal transcriptome alterations through high-throughput RNA sequencing (RNA-seq). 80 Therefore, we investigated the mechanism of sevoflurane-induced neurotoxicity in the 81 newborn hippocampus using a well-established model of multiple exposures to sevoflurane 82 during the neonatal period. Furthermore, we evaluated the influence of sevoflurane on 83 cognitive function behaviors in young mice. The main objective of this study was to 84 demonstrate the genome-wide expression profile and DEGs of the neonatal hippocampus of 85 sevoflurane-treated and control mice using RNA-seq. Therefore, enrichment of biological 86 processes and signaling pathways of DEGs were analyzed. Western blotting and 87 immunostaining were performed to further elucidate the molecular mechanism by which 88 sevoflurane induces abnormal brain development and cognitive dysfunction.

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This study was designed to investigate the effects of sevoflurane on the hippocampus 92 of neonatal mice and brain function ( Fig 1A). Neonatal mice were exposed to sevoflurane for

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There were 15 and 14 mice in the control and sevoflurane groups, respectively. All 133 mice grew normally, and there was no significant difference in body weight between groups.

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The circular pool of the experiment was divided into four equal 90° quadrants (southwest,  Specifically, two water entry points were randomly selected from the four quadrants to begin 139 training. If the mice did not reach the target platform within 1 min, they were guided 140 manually and kept on the platform for an additional 10 s. Escape latency was calculated as 141 the time from when the mice entered the water to the time when they reached the platform.   Sucrose solutions (20%, 25%, and 30%) were sequentially replaced every 24 h for gradient 161 dehydration. After the cerebrum was removed, it was embedded in an optimal cutting 162 temperature compound, and the slices of the classic hippocampal structure were obtained 163 using a freezing microtome. The slides were removed, rinsed four times with 1× phosphate-164 buffered saline, and permeabilized with 0.3% TritonX-100 for 15 min. Antigen retrieval was 165 performed with 1× citric acid retrieval solution, and cells were blocked at room temperature 166 for 1 h. Bag3 antibody (1:1000; Abcam, UK) was incubated at 4°C overnight, and the 167 secondary antibody was incubated at room temperature for 1 h. Thereafter, 4',6-diamidino-2-    trial, mice previously exposed to sevoflurane spent significantly less time in the target area 257 than the control group ( Fig 5A). Additionally, the sevoflurane group had longer first-time 258 target latency than the control group (Fig 5B). Although there was no significant difference in 259 the frequency of crossing the target area between the sevoflurane and control groups, there 260 was a tendency for reduced frequency in the sevoflurane group. During the training trial, 261 mice exposed to sevoflurane required more time to find the platform compared to that for the 262 control mice when they were trained on days 4 and 5 (Fig 5D). These results indicate that 263 exposure to sevoflurane impaired the ability of mice to learn and memorize. After discovering deficits in learning and memory after sevoflurane treatment, we 274 attempted to elucidate the molecular mechanism underlying these occurrences. We analyzed 275 the protein expression levels of synaptic-related genes. Our results showed that the 276 expression levels of PSD95, SNAP25, and Bag3 were significantly decreased after 277 sevoflurane exposure. The phosphorylation level of Bag3 increased after sevoflurane 278 treatment (Fig 6A, B). In agreement with the Western blotting results, immunofluorescence 279 staining confirmed that the expression of Bag3 in the hippocampus and cortex was 280 significantly decreased with sevoflurane exposure (Fig 6C). hippocampus. This is the first study to assay the neonatal hippocampus transcriptome and 293 investigate the molecular mechanism of memory deficits of neonatal mice exposed to and neuroendocrine effects [13]. Synaptic plasticity also has an essential role in the 14 320 development of neural circuits, and impaired synaptic plasticity can lead to major 321 neuropsychiatric disorders, especially hippocampal-dependent memory impairment [14]. The

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Morris water maze test has been widely used to investigate hippocampal-dependent cognitive 323 functions in young mice. The present results showed that the escape latency and platform-324 seeking time were significantly prolonged, the residence time in the target area was 325 shortened, and the number of crossings was reduced in mice with sevoflurane exposure. 326 These results demonstrated that sevoflurane exposure in neonatal mice causes memory 327 deficits in young mice. 328 We evaluated synapse-related protein levels in the hippocampus after sevoflurane 329 modeling and found that SNAP25 protein levels were significantly decreased after  Recently, several studies have suggested that Bag3 plays a vital role in synaptic function by 341 regulating synaptic protein degradation [20]. Furthermore, sevoflurane can abnormally plasticity and long-term potentiation in neurons. We found that the expression of Bag3 was 346 decreased and that of p-Bag3 was increased in the sevoflurane group compared to that in the 347 control group, suggesting that CDK5 phosphorylated and promoted Bag3 degradation, which 348 may be involved in sevoflurane-induced nervous system dysfunction.