Elsevier

NeuroToxicology

Volume 81, December 2020, Pages 172-179
NeuroToxicology

Application of microRNA profiling to understand sevoflurane-induced adverse effects on developing monkey brain

https://doi.org/10.1016/j.neuro.2020.10.002Get rights and content

Abstract

We have described that prolonged sevoflurane exposure at a clinically-relevant concentration of 2.5 % caused neuronal cell death in the developing monkey brain. Postnatal day 5 or 6 rhesus monkeys (n = 3) were exposed to 2.5 % sevoflurane for 8 h. Monkeys kept at environmental conditions in the procedure room served as controls (n = 3). Brain tissues were harvested four hours after sevoflurane exposure for histological analysis, and RNA or protein extraction. MicroRNA (miRNA) profiling on the frontal cortex of monkey brains was performed using next-generation sequencing. 417 miRNAs were identified in the frontal cortex, where most neuronal cell death was observed. 7 miRNAs were differentially expressed in frontal cortex after sevoflurane exposure. Five of these were expressed at significantly lower levels than controls and the other two miRNAs were expressed significantly higher. These differentially expressed miRNAs (DEMs) were then loaded to the Ingenuity Pathway Analysis database for pathway analysis, in which targeting information was available for 5 DEMs. The 5 DEMs target 2,919 mRNAs which are involved in pathways that contribute to various cellular functions. Of note, 78 genes that are related to axon guidance signaling were targeted, suggesting that development of the neural circuit may be affected after sevoflurane exposure. Such changes may have long-term effects on brain development and function. These findings are supplementary to our previous observations and provide more evidence for better understanding the adverse effects of sevoflurane on the developing brain after an 8 -h exposure.

Introduction

Sevoflurane is frequently used in pediatric anesthesia. Our previous work has demonstrated that a 9 -h sevoflurane exposure caused neuronal cell death in the developing monkey brain (Liu et al., 2015), which is consistent with the findings of other anesthetics studied by our own and other groups (Kodama et al., 2011; Liu et al., 2015, 2013; Perez-Zoghbi et al., 2017; Zhang et al., 2016). There has been public concern over the safe use of anesthetics in young children. To provide sound guidance on the safe use of pediatric anesthetics, it is urgent to understand the detailed effects of those anesthetics, to decipher the significant mechanisms by which anesthetics cause any adverse effects, and to find biomarkers that may indicate adverse effects. A comprehensive understanding of these anesthetics is critical for the development of protective strategies against the adverse effects and to recommend a safe anesthesia regimen for clinical practice.

Analysis of gene expression profiles to understand the mechanisms by which chemicals/agents induce toxic, including neurotoxic, effects has been widely explored. In addition, application of toxicogenomics to neurotoxicity studies is occurring more frequently. MicroRNAs (miRNAs) have been recognized as an essential regulator in brain function, ranging from neural development, neurodegenerative diseases, responses to hypoxia and ischemia, to neurotoxicity. Approximately 70 % of identified miRNAs are expressed in brain (Fineberg et al., 2009), some of which are expressed in brain abundantly or exclusively (Nowak and Michlewski, 2013). In recent years emerging studies on miRNA expression profiles in the brain have advanced our knowledge on the roles of miRNAs in regulating central nervous system (CNS) functions under physiological and pathological conditions. Moreover, changes in miRNA expression have shown to be promising biomarkers for diseases and toxicity (Cogswell et al., 2008; Deng et al., 2014; Sheinerman and Umansky, 2013; Siddeek et al., 2014). In the present study, we examined the miRNA expression profile in the infant monkey brain after an 8 -h sevoflurane exposure using next-generation sequencing (NGS). The comparison of miRNA expression levels in sevoflurane-exposed animals with those of controls generated differentially expressed miRNAs (DEMs) and their regulatory roles in sevoflurane-induced adverse effects in the infant monkey brain were analyzed. Although various methods have been applied to investigate the effects of anesthetics on the developing brain, analysis of miRNA expression profiles of the anesthetic-exposed brain is still rare. The present NGS analysis not only confirmed some of our previous findings, but also extended our knowledge on understanding the mechanisms related to sevoflurane-induced adverse effects. The functions associated with the DEM-targets suggest new pathways leading to the adverse effects that sevoflurane may have on the developing brain.

Section snippets

Animals

All animal procedures were approved by the FDA National Center for Toxicological Research (NCTR)’s Institutional Animal Care and Use Committee and performed in full accordance with the Public Health Service Policy on the Humane Care and Use of Laboratory Animals. Postnatal day (PND) 5 or 6 rhesus monkeys (Macaca mulatta) (n = 6) were obtained from the NCTR nonhuman primate breeding colony and handled as previously described (Liu et al., 2015). The 6 animals were randomly assigned to control (3

Statistical analysis

Statistical analyses on physiological parameters and Western blots data between control and sevoflurane-exposed monkeys were performed and graphs were produced using GraphPad Prism. Data are expressed as mean ± SD. Parameter comparisons between the control and treated group was performed using Student t-test. All analyses were considered statistically different with a p value < 0.05.

Physiological parameters of experimental animals

During the experimental procedures, stable physiological conditions of control and sevoflurane-exposed animals were observed. Table 1 summarizes the critical physiological measurements for both groups, which were maintained within normal ranges with no significant differences between the groups.

Neuronal cell death in frontal cortex

An 8 -h exposure to sevoflurane caused increased Fluoro-Jade C-positive cells in frontal cortex compared to the control group, indicating more neuronal cell death. The representative pictures show the

Discussion

Sevoflurane-induced developmental neurotoxicity has been observed in pre-clinical models. However, it is still a goal for researchers to understand how such anesthetics cause the histological, cellular and molecular changes to the brain and to identify biomarkers to predict neurotoxicity in pediatric patients. In the present study we analyzed the miRNA profiles in monkey frontal cortex and found that some miRNAs (i.e., DEMs) changed the expression levels after sevoflurane exposure. The DEMs may

Disclaimer

The information in these materials is not a formal dissemination of information by the FDA and does not represent agency position or policy.

CRediT authorship contribution statement

Fang Liu: Conceptualization, Funding acquisition, Methodology, Project administration, Writing - original draft. Binsheng Gong: Data curation. Qiang Gu: Investigation, Data curation. Shuliang Liu: Investigation, Data curation. Charles Matthew Fogle: Investigation. Tucker A. Patterson: Validation, Writing - review & editing. Joseph P. Hanig: Validation, Writing - review & editing. William Slikker: Supervision, Methodology, Writing - review & editing. Cheng Wang: Conceptualization, Data curation,

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgement

We would like to thank Dong Wang for his suggestion on the statistical analyses. The study was supported by NCTR/US Food and Drug Administration (Protocol ID: E07285.21).

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