BACKGROUND:Inhalational anesthetics are known to disrupt PDZ2 domain-mediated protein-protein interactions of the postsynaptic density (PSD)-95 protein. The aim of this study is to investigate the underlying mechanisms in response to early isoflurane exposure on synaptic PSD-95 PDZ2 domain disruption that altered spine densities and cognitive function. The authors hypothesized that activation of protein kinase-G by the components of nitric oxide (NO) signaling pathway constitutes a mechanism that prevents loss of early dendritic spines and synapse in neurons and cognitive impairment in mice in response to disruption of PDZ2 domain of the PSD-95 protein. METHODS:Postnatal day 7 mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active PSD-95 wild-type PDZ2 peptide or soluble guanylyl cyclase activator YC-1 along with their respective controls. Primary neurons at 7 days in vitro were exposed to isoflurane or PSD-95 wild-type PDZ2 peptide for 4 h. Coimmunoprecipitation, spine density, synapses, cyclic guanosine monophosphate-dependent protein kinase activity, and novel object recognition memory were assessed. RESULTS:Exposure of isoflurane or PSD-95 wild-type PDZ2 peptide relative to controls causes the following. First, there is a decrease in PSD-95 coimmunoprecipitate relative to N-methyl-d-aspartate receptor subunits NR2A and NR2B precipitate (mean ± SD [in percentage of control]: isoflurane, 54.73 ± 16.52, P = 0.001; and PSD-95 wild-type PDZ2 peptide, 51.32 ± 12.93, P = 0.001). Second, there is a loss in spine density (mean ± SD [spine density per 10 µm]: control, 5.28 ± 0.56 vs. isoflurane, 2.23 ± 0.67, P < 0.0001; and PSD-95 mutant PDZ2 peptide, 4.74 ± 0.94 vs. PSD-95 wild-type PDZ2 peptide, 1.47 ± 0.87, P < 0.001) and a decrease in synaptic puncta (mean ± SD [in percentage of control]: isoflurane, 41.1 ± 14.38, P = 0.001; and PSD-95 wild-type PDZ2 peptide, 50.49 ± 14.31, P < 0.001). NO donor or cyclic guanosine monophosphate analog prevents the spines and synapse loss and decline in the cyclic guanosine monophosphate-dependent protein kinase activity, but this prevention was blocked by soluble guanylyl cyclase or protein kinase-G inhibitors in primary neurons. Third, there were deficits in object recognition at 5 weeks (mean ± SD [recognition index]: male, control, 64.08 ± 10.57 vs. isoflurane, 48.49 ± 13.41, P = 0.001, n = 60; and female, control, 67.13 ± 11.17 vs. isoflurane, 53.76 ± 6.64, P = 0.003, n = 58). Isoflurane-induced impairment in recognition memory was preventable by the introduction of YC-1. CONCLUSIONS:Activation of soluble guanylyl cyclase or protein kinase-G prevents isoflurane or PSD-95 wild-type PDZ2 peptide-induced loss of dendritic spines and synapse. Prevention of recognition memory with YC-1, a NO-independent activator of guanylyl cyclase, supports a role for the soluble guanylyl cyclase mediated protein kinase-G signaling in countering the effects of isoflurane-induced cognitive impairment. EDITOR’S PERSPECTIVE:

BACKGROUND:The volatile anaesthetic sevoflurane induces time (single or multiple exposures)-dependent effects on tau phosphorylation and cognitive function in young mice. The underlying mechanism for this remains largely undetermined. METHODS:Mice received 3% sevoflurane for 0.5 h or 2 h daily for 3 days on postnatal day (P) 6, 9, and 12. Another group of mice received 3% sevoflurane for 0.5 h or 1.5 h (3 × 0.5) on P6. We investigated effects of sevoflurane anaesthesia on tau phosphorylation on P6 or P12 mice, on cognitive function from P31 to P37, and on protein interactions, using in vivo studies, in vitro phosphorylation assays, and nanobeam single-molecule level interactions in vitro. RESULTS:An initial sevoflurane exposure induced CaMKIIα phosphorylation (132 [11]% vs 100 [6]%, P<0.01), leading to tau phosphorylation at serine 262 (164 [7]% vs 100 [26]%, P<0.01) and tau detachment from microtubules. Subsequent exposures to the sevoflurane induced GSK3β activation, which phosphorylated detached or free tau (tau phosphorylated at serine 262) at serine 202 and threonine 205, resulting in cognitive impairment in young mice. In vitro phosphorylation assays also demonstrated sequential tau phosphorylation. Nanobeam analysis of molecular interactions showed different interactions between tau or free tau and CaMKIIα or GSK3β, and between tau and tubulin at a single-molecule level. CONCLUSIONS:Multiple exposures to sevoflurane can induce sequential tau phosphorylation, leading to cognitive impairment in young mice, highlighting the need to investigate the underlying mechanisms of anaesthesia-induced tau phosphorylation in developing brain.

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.

AIMS:Multiple surgical procedures and anesthesia increase the risk of the development in children. However, the influence of such exposures on the developing childhood immunity organs is rarely reported. MATERIALS AND METHODS:High-throughput sequencing of T-cell receptor (TCR) repertoires (TCRseq) from rhesus monkeys' thymus was performed to investigate whether anesthetics could induce de novo antigen recognition via TCR or TCR development impairments. KEY FINDINGS:No significant difference between sevoflurane and control groups regarding VJ gene combinations and diversity of V and J gene was seen, nor was there an obvious change in similar average number of Complementarity Determining Region 3 (CDR3) aa clonotypes. Our analysis of Rank abundance, Gini coefficient, Simpson index, Normalized Shannon Diversity Entropy (NSDE), Morisita-Horn Similarity Index (MHSI) and Bhattacharyya Distance (BD) indicated there is no difference in TCR diversity and similarity. SIGNIFICANCE:These results suggest early events in thymic T cell development and repertoire generation are not abnormality after multiple sevoflurane exposure during childhood. The stabilization of the immune repertoires suggested the safety of sevoflurane in host immune response in children.

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.

Background:Millions of children experienced surgery procedures requiring general anesthesia (GA). Any potential neurodevelopmental risks of pediatric anesthesia can be a serious public health issue. Various animal studies have provided evidence that commonly used GA induced a variety of morphofunctional alterations in the developing brain of juvenile animals. Methods:We conducted a systematic review to provide a brief overview of preclinical studies and summarize the existing clinical studies. Comprehensive literature searches of PubMed, EMBASE, CINAHL, OVID Medline, Web of Science, and the Cochrane Library were conducted using the relevant search terms "general anesthesia," "neurocognitive outcome," and "children." We included studies investigating children who were exposed to single or multiple GA before 18, with long-term neurodevelopment outcomes evaluated after the exposure(s). Results:Seventy-two clinical studies originating from 18 different countries published from 2000 to 2022 are included in this review, most of which are retrospective studies ( = 58). Two-thirds of studies ( = 48) provide evidence of negative neurocognitive effects after GA exposure in children. Neurodevelopmental outcomes are categorized into six domains: academics/achievement, cognition, development/behavior, diagnosis, brain studies, and others. Most studies focusing on children <7 years detected adverse neurocognitive effects following GA exposure, but not all studies consistently supported the prevailing view that younger children were at greater risk than senior ones. More times and longer duration of exposures to GA, and major surgeries may indicate a higher risk of negative outcomes. Conclusion:Based on current studies, it is necessary to endeavor to limit the duration and numbers of anesthesia and the dose of anesthetic agents. For future studies, we require cohort studies with rich sources of data and appropriate outcome measures, and carefully designed and adequately powered clinical trials testing plausible interventions in relevant patient populations.

Anesthetics and sedatives may cause long-term negative neurocognitive consequences in children. Many clinical reports on this subject have had a profound impact on the field of clinical pediatric anesthesiology. Findings from animal models suggest that early exposure to anesthesia might cause neurocognitive impairment and apoptotic cell death in the brain. Even though the findings from the experimental animals cannot be directly translated to the use of anesthesia in pediatric population due to many variable factors, parents and government regulatory bodies have become sensitive and attentive to the potential adverse effects of anesthesia in children. Multiple epidemiological investigations in human have added to the growing body of evidence showing neurological impairment and cognitive decline after early anesthetic exposure. This is supported by several outcome indicators, including validated neuropsychologic testing, educational interventions for neurodevelopmental or behavioral disorders, and academic performance or school readiness. These outcomes have been evaluated in clinical studies involving children who have been subjected to general anesthesia. The primary goal of this article is to critically review the clinical findings, perform systematic analyses of the evidence, discuss potential underlying mechanisms of neurotoxicity, the pathophysiology of anesthesia-induced developmental neurotoxicity involving mitochondria, endoplasmic reticulum, and lysosomes, and the ethical considerations of pediatric anesthesia. Although detailed well-controlled clinical studies are warranted, the evidence so far support that the potential adverse effects of surgical anesthesia to induce neurotoxicity in pediatric population are not exaggerated.

BACKGROUND:The inhalational anesthetic isoflurane is commonly utilized in clinical practice, particularly in the field of pediatric anesthesia. Research has demonstrated its capacity to induce neuroinflammation and long-term behavioral disorders; however, the underlying mechanism remains unclear [1]. The cation-chloride cotransporters Na-K-2Cl-1 (NKCC1) and K-2Cl-2 (KCC2) play a pivotal role in regulating neuronal responses to gamma-aminobutyric acid (GABA) [2]. Imbalances in NKCC1/KCC2 can disrupt GABA neurotransmission, potentially leading to neural circuit hyperexcitability and reduced inhibition following neonatal exposure to anesthesia [3]. Therefore, this study postulates that anesthetics have the potential to dysregulate NKCC1 and/or KCC2 during brain development. METHODS:We administered 1.5% isoflurane anesthesia to neonatal rats for a duration of 4 h at postnatal day 7 (PND7). Anxiety levels were assessed using the open field test at PND28, while cognitive function was evaluated using the Morris water maze test between PND31 and PND34. Protein levels of NKCC1, KCC2, BDNF, and phosphorylated ERK (P-ERK) in the hippocampus were measured through Western blotting analysis. Pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were quantified using ELISA. RESULTS:We observed a decrease in locomotion trajectories within the central region and a significantly shorter total distance in the ISO group compared to CON pups, indicating that isoflurane induces anxiety-like behavior. In the Morris water maze (MWM) test, rats exposed to isoflurane exhibited prolonged escape latency onto the platform. Additionally, isoflurane administration resulted in reduced time spent crossing in the MWM experiment at PND34, suggesting long-term impairment of memory function. Furthermore, we found that isoflurane triggered activation of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α; downregulated KCC2/BDNF/P-ERK expression; and increased the NKCC1/KCC2 ratio in the hippocampus of PND7 rats. Bumetadine (NKCC1 specific inhibitors) reversed cognitive damage and effective disorder induced by isoflurane in neonatal rats by inhibiting TNF-α activation, normalizing IL-6 and IL-1β levels, restoring KCC2 expression levels as well as BDNF and ERK signaling pathways. Based on these findings, it can be speculated that BDNF, P-ERK, IL-1β, IL-6 and TNF - α may act downstream of the NKCC1/KCC2 pathway. CONCLUSIONS:Our findings provide evidence that isoflurane administration in neonatal rats leads to persistent cognitive deficits through dysregulation of the Cation-Chloride Cotransporters NKCC1 and KCC2, BDNF, p-ERK proteins, as well as neuroinflammatory processes.

As the recent update of General anaesthesia compared to spinal anaesthesia (GAS) studies has been published in 2019, together with other clinical evidence, the human studies provided an overwhelming mixed evidence of an association between anaesthesia exposure in early childhood and later neurodevelopment changes in children. Pre-clinical studies in animals provided strong evidence on how anaesthetic and sedative agents (ASAs) causing neurotoxicity in developing brain and deficits in long-term cognitive functions. However pre-clinical results cannot translate to clinical practice directly. Three well designed large population-based human studies strongly indicated that a single brief exposure to general anesthesia (GAs) is not associated with any long-term neurodevelopment deficits in children's brain. Multiple exposure might cause decrease in processing speed and motor skills of children. However, the association between GAs and neurodevelopment in children is still inconclusive. More clinical studies with larger scale observations, randomized trials with longer duration exposure of GAs and follow-ups, more sensitive outcome measurements, and strict confounder controls are needed in the future to provide more conclusive and informative data. New research area has been developed to contribute in finding solutions for clinical practice as attenuating the neurotoxic effect of ASAs. Xenon and Dexmedetomidine are already used in clinical setting as neuroprotection and anaesthetic sparing-effect, but more research is still needed.