Abstract Research has indicated that general anesthesia may cause neuroapoptosis and long-term cognitive dysfunction in developing animals, however, the precise mechanisms orchestrating these outcomes remain inadequately elucidated within scholarly discourse. The purpose of this study was to investigate the impact of sevoflurane on the hippocampus of developing rats by analyzing the changes in microRNA and mRNA and their interactions. Rats were exposed to sevoflurane for 4 h on their seventh day after birth, and the hippocampus was collected for analysis of neuroapoptosis by Western blot and immunohistochemistry. High-throughput sequencing was conducted to analyze the variances in miRNA and mRNA expression levels, and the Morris water maze was employed to assess long-term memory in rats exposed to sevoflurane after 8 weeks. The results showed that sevoflurane exposure led to dysregulation of 5 miRNAs and 306 mRNAs in the hippocampus. Bioinformatic analysis revealed that these dysregulated miRNA-mRNA target pairs were associated with pathological neurodevelopment and developmental disorders, such as regulation of axonogenesis, regulation of neuron projection development, regulation of neuron differentiation, transmission of nerve impulse, and neuronal cell body. Further analysis showed that these miRNAs formed potential network interactions with 44 mRNAs, and two important nodes were identified, miR-130b-5p and miR-449c-5p. Overall, this study suggests that the dysregulation of the miRNA-mRNA signaling network induced by sevoflurane may contribute to neurodevelopmental toxicity in the hippocampus of rats and be associated with long-term cognitive dysfunction. Keywords: Sevoflurane, Neurotoxicity, miRNA-mRNA network, Neuroapoptosis 1. Introduction An increasing amount of evidence suggests that various anesthetics agents may have deleterious impacts on developing neural cells, thereby leading to persistent cognitive impairment [[35]1,[36]2]. The phenomenon of anesthetic-induced developmental neurotoxicity encompasses a spectrum of pathological events, including neuronal apoptosis, neurodegenerative processes, changes in neurogenesis and synaptogenesis, as well as damage to brain circuits [[37]3]. Sevoflurane is an inhaled anesthetic, and is widely used for both adult and pediatric patients in clinical practice due to its desirable properties, such as lack of pungent odor, low blood-gas partition coefficient, rapid onset and offset, and limited cardiopulmonary depression. However, some studies suggest that sevoflurane may have deleterious impacts on developing nervous system, particularly in infants and young children [[38]4]. This toxicity could potentially affect the cognitive and behavioral development of children, which has raised concerns [[39]5]. Nevertheless, the mechanism underlying sevoflurane-induced neurotoxicity remains incompletely understood. MicroRNAs (miRNAs) are small (approximately 21 nucleotides), non-coding RNA molecules that are highly conserved throughout evolution and are encoded in the genomes of nearly all eukaryotes, from plants to mammals [[40]6]. In mammals, predicted miRNAs control the activity of more than 60 % of the entire protein-coding genes and engage in the modulation of all cellular processes thus far [[41]7]. Generally, miRNAs modulate the expression of genes through binding to partially complementary regions in the mRNA 3′ UTR, and by promoting degradation or inhibiting translation of the target mRNA. Studies have shown that individual genes can be subject to regulation by multiple miRNAs, and a single miRNA can also regulate multiple mRNA targets simultaneously [[42]8,[43]9]. As the field of miRNA research has developed, it has become clear that miRNA function is extremely complex, with a complex network of factors strictly controlling miRNA processing and biological activity. Dysregulated miRNA expression has been found to play a critical role in various pathological processes, including neurological disorders, cardiovascular disease, cancer and viral infections [[44]10]. The multifunctionality and potential of miRNAs make them a valuable resource for both basic research and clinical applications. MiRNAs and their associated regulatory networks are becoming increasingly recognized for their roles in regulating complex neurobiological functions. Research has shown that the spatiotemporal expression patterns of miRNAs play crucial roles in mammalian neural development. Dysregulated miRNA/mRNA interactions can form extensive molecular networks, potentially affecting diverse neural functional pathways in the hippocampus, thereby lead to neuronal cell damage and harm long-term memory [[45]11,[46]12]. Numerous studies have highlighted the pivotal role of microRNAs in sevoflurane-induced developmental neurotoxicity, with specific miRNA/mRNA axes such as miR-330-3p/ULK1, lncRNA Neat1/miR-298-5p/SRPK1, lncRNA PEG13/miR-128-3p/SOX13, and Rik-203/miR-466l-3p/BDNF being implicated in the pathogenesis of sevoflurane-induced neurotoxicity during neurodevelopmental stages [[47][13], [48][14], [49][15], [50][16]]. Therefore, the primary objective of this inquiry is to scrutinize the impact of sevoflurane exposure on the expression profiles of miRNAs and mRNAs in the developing rat hippocampus, employing bioinformatics methodologies to prognosticate disease pathways, delineate miRNA-mRNA interactions, and unravel pertinent cellular pathways. These endeavors aim to provide further insights into the diverse mechanisms underlying the neurotoxic effects of sevoflurane exposure during development. 2. Materials and methods 2.1. Animal studies Approval for this investigation was obtained from the Animal Research Committee of Zhejiang Provincial People's Hospital (No. 2021082), with adherence to the “Guidelines for the Care and Use of Laboratory Animals” in all experimental procedures. All animals used in the experiment were postnatal day 7 (P7) SD rat pups from GemPharmatech (Nanjing, China). The rat pups were exposed to 3.4 % sevoflurane or only fresh gas for a duration of 4 h within a chamber (RWD Life Science) at room temperature [[51]17]. To sustain a consistent concentration of 3.4 % sevoflurane throughout the trials, a Capnomac gas monitor (Datex-Ohmeda) was employed. After the experiment, all animals were euthanized for isolation of hippocampal tissues in accordance with the “AVMA Guidelines for the Euthanasia of Animals”. This process involved placing the animals in a carbon dioxide environment for a duration of 5 min until cessation of respiration and cardiac activity was confirmed. 2.2. Western blot analysis Hippocampal tissues underwent lysis in RIPA buffer supplemented with protease inhibitor, followed by quantification of protein concentration utilizing a BCA kit. Subsequent to lysis, the protein extracts were subjected to separation via SDS-PAGE and transferred onto a PVDF membrane. To mitigate non-specific binding, the membrane was treated with a blocking solution comprising non-fat milk, followed by incubation with primary antibodies alongside a loading control such as GAPDH. Following thorough washing steps, the membrane was exposed to HRP-conjugated secondary antibodies. Visualization and quantification of protein bands were achieved utilizing an ECL kit in conjunction with ImageJ software. 2.3. Immunohistochemistry Immunohistochemical analysis was conducted on formalin-fixed, paraffin-embedded tissue sections. Following deparaffinization and rehydration procedures, antigen retrieval was facilitated by subjecting the sections to boiling in citrate buffer (pH 6.0) for a duration of 20 min. To mitigate endogenous peroxidase activity, treatment with 3 % H[2]O[2] in methanol was administered for a duration of 15 min. Subsequently, incubation with primary antibodies targeting the proteins of interest was carried out overnight at 4 °C. Following thorough washing with PBS, the sections underwent incubation with HRP-conjugated secondary antibodies for 30 min at ambient temperature. Signal visualization was accomplished through the utilization of DAB substrate, accompanied by counterstaining with hematoxylin. Subsequent to dehydration, the slides were mounted and subjected to microscopic imaging. Negative controls were integrated by excluding the primary antibody from the staining protocol. 2.4. Morris water maze (MWM) test Eight weeks post-exposure to sevoflurane, spatial learning and memory in rats were assessed through the Morris water maze. The maze setup included a circular pool with water rendered opaque by the addition of non-toxic white paint. A platform, 10 cm in diameter, was submerged 1 cm beneath the water surface within one of the four quadrants. Training for each rat spanned five days, during which they were taught to locate the platform using visual cues present in the room. On the sixth day, a probe trial was conducted where the platform was removed, allowing the rats to swim for 120 s. Measurements were taken of the time spent in each quadrant and the frequency of platform crossings. The test was performed blind to the experimental group. 2.5. Expression profile analysis of miRNA and mRNA Total RNA extraction from tissue samples was carried out utilizing a commercial kit, adhering strictly to the provided protocol. The quality and quantity of the extracted RNA were evaluated with a NanoDrop spectrophotometer. The RNA was then sent to a commercial sequencing company for library preparation and sequencing. miRNA uses Bowtie-1.0.0 for read alignment and ACGT101-miR (v4.2) software for miRNA identification. Expression levels are normalized using norm values, and target gene prediction is performed using a combination of TargetScan v5.0 and Miranda v3.3 software [[52]18]. For RNA-seq, reads of all samples were aligned to the reference genome of the research species using HISAT2 [[53]19]. StringTie and Ballgown were used to estimate transcript expression levels and calculate FPKM values for mRNAs. The R package EdgeR was employed to process both miRNA-seq and mRNA-seq data between two conditions utilizing a negative binomial generalized linear model with a likelihood ratio test (glmLRT). To visualize the abundance of miRNAs/genes, perform heatmap clustering analysis, and investigate miRNA-mRNA correlations, the count-per-million values were converted to log 2 scale (LogCPM). The results of miRNA and mRNA expression profiling were integrated to identify potential miRNA-mRNA regulatory networks. 2.6. RT-qPCR Total RNA was isolated from tissue samples following the manufacturer's protocol using a commercial extraction kit. The RNA's quality and concentration were determined with a NanoDrop spectrophotometer. Subsequently, cDNA was synthesized from 1 μg of total RNA utilizing a reverse transcription kit. Real-time PCR was carried out with a SYBR Green master mix and gene-specific primers. The thermal cycling parameters were set as follows: an initial denaturation at 95 °C for 10 min, then 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The relative expression levels of target genes were quantified using the 2^−ΔΔCt method, with normalization to an endogenous reference gene. 2.7. Statistical analysis Statistical analyses were conducted utilizing GraphPad Software and SPSS 24.0. Data are expressed as mean ± standard deviation. Comparisons between two groups were made using a Student's t-test, while comparisons among more than two groups were performed using a one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test. A P-value of less than 0.05 was deemed statistically significant. Animal allocation was randomized according to weight, and all animals were included in the study without any exclusions. 3. Result 3.1. Sevoflurane exposure has no effect on physiological conditions in rat During sevoflurane anesthesia, all rat pups survived, and their body temperature was consistently maintained at 37 ± 0.5 °C. Additionally, arterial blood gas parameters ([54]Table 1) showed no significant differences between the control group and the sevoflurane-exposed group, indicating that sevoflurane anesthesia did not have a significant impact on the physiological condition of the rat pups. Table 1. Arterial blood pressure and arterial blood gas analysis. Time pH PaCO[2], mmHg PaO[2], mmHg SaO[2] % MAP, mmHg Con (n = 6) 0 h 6.87 ± 0.03 39.4 ± 3.8 98.2 ± 6.3 99.5 ± 0.3 50 ± 4 6 h 6.79 ± 0.04 40.3 ± 4.2 99.1 ± 7.2 99.3 ± 0.5 48 ± 3 3.4 % SEVO (n = 6) 0 h 6.82 ± 0.06 39.8 ± 5.3 98.7 ± 7.1 99.1 ± 0.4 49 ± 6 6 h 6.78 ± 0.04 41.3 ± 4.3 99.2 ± 6.6 98.5 ± 0.6 48 ± 5 [55]Open in a new tab PaCO[2] = arterial carbon dioxide tension; PaO[2] = arterial oxygen tension; SaO[2] = arterial oxygen saturation. 3.2. Sevoflurane can cause developmental neurotoxic effects Exposure to sevoflurane for 4 h increased the expression of Bax and cleaved caspase 3 proteins, while decreasing the expression of BcL-2 protein in the hippocampus of day 7 rat pups, indicating induction of apoptosis ([56]Fig. 1A). The TUNEL assay was utilized to identify apoptotic cells within the hippocampal tissue samples. Following TUNEL and hematoxylin staining, apoptotic cell nuclei exhibited a brown stain, while normal nuclei appeared blue. Analysis revealed a noteworthy elevation in apoptotic cell count within the sevoflurane-exposed group compared to the control group (P < 0.05) ([57]Fig. 1B). Additionally, to further investigate the impact of sevoflurane on learning and memory ability in neonatal rat, the Morris water maze (MWM) test were carried out in rat 8 weeks after Sevoflurane exposure ([58]Fig. 1C). Our findings indicate a significant increase in swim latencies and a notable decrease in crossing numbers during the MWM in rats following sevoflurane exposure ([59]Fig. 1D).These findings suggest that sevoflurane exposure may impair long-term memory function. Fig. 1. [60]Fig. 1 [61]Open in a new tab Sevoflurane exposure causes hippocampal cell apoptosis and impairs long-term memory in rat pups. (A) Apoptosis in the hippocampus of rats was evaluated by detecting the expression of apoptosis-related proteins Bax, Bcl-2, and cleaved caspase 3 using Western blotting. The unedited images are referenced in [62]Fig. S2 (B) TUNEL staining was used to identify cell apoptosis by immunohistochemistry. (C) The escape latencies were measured over five consecutive training days to assess learning and memory in the two groups. (D) The average number of crossings over the platform-site during the probe trial was calculated as an indicator of spatial memory. 3.3. Sevoflurane exposure alters mRNA profiles in the hippocampus of rat pup To investigate the transcriptional changes induced by sevoflurane exposure, we conducted a high-throughput RNA-sequencing (RNA-seq) analysis aiming to identify similarities and differences in gene expression profiles. Utilizing violin plot analysis and Pearson's correlation, we observed that intergroup correlations between sevoflurane and control mRNA expression profile on Day 7 rats were high similarity ([63]Fig. 2A and B). Out of the 28,970 mRNA transcripts that were analyzed, 306 were found to be dysregulated (168 upregulated and 138 downregulated) in the hippocampi of rats exposed to sevoflurane at Day 7 (|logFC| > 2, P < 0.05). The volcano plot depicted the differential abundance of mRNAs within the hippocampi between the control and sevoflurane groups ([64]Fig. 2C, [65]Table S1). Furthermore, heatmaps were created using the most significant differentially expressed genes in each group, revealing a notably distinct pattern associated with sevoflurane exposure in contrast to the control group ([66]Fig. 2D). Fig. 2. [67]Fig. 2 [68]Open in a new tab Sevoflurane induces differential mRNA expression profiles in the hippocampus of rats. (A, B) Violin plots and Pearson correlation coefficients heatmap show the similar distribution of normalized mRNA signal intensities in the hippocampus of six rats. (C) The volcano plot displays the differentially expressed mRNAs between the control and sevoflurane groups. Red dots indicate upregulated mRNAs, while blue dots indicate downregulated mRNAs, with p < 0.05 and fold change ≥2 compared to the control. (D) The heatmap clustering analysis shows the expression profiles of differentially expressed mRNAs induced by sevoflurane in the hippocampus of rats (p < 0.05). Each row represents the relative expression level of an mRNA gene. (For interpretation of the references to colour in this figure legend, the reader is referred