Understanding the diversity and dynamics of in vivo efferocytosis: Insights from the fly embryo.

The clearance of dead and dying cells, termed efferocytosis, is a rapid and efficient process and one that is critical for organismal health. The extraordinary speed and efficiency with which dead cells are detected and engulfed by immune cells within tissues presents a challenge to researchers who wish to unravel this fascinating process, since these fleeting moments of uptake are almost impossible to catch in vivo. In recent years, the fruit fly (Drosophila melanogaster) embryo has emerged as a powerful model to circumvent this problem. With its abundance of dying cells, specialist phagocytes and relative ease of live imaging, the humble fly embryo provides a unique opportunity to catch and study the moment of cell engulfment in real-time within a living animal. In this review, we explore the recent advances that have come from studies in the fly, and how live imaging and genetics have revealed a previously unappreciated level of diversity in the efferocytic program. A variety of efferocytic strategies across the phagocytic cell population ensure efficient and rapid clearance of corpses wherever death is encountered within the varied and complex setting of a multicellular living organism.

Fluorogenic Chemical Probes for Wash-free Imaging of Cell Membrane Damage in Ferroptosis, Necrosis, and Axon Injury.

Selectively labeling cells with damaged membranes is needed not only for identifying dead cells in culture, but also for imaging membrane barrier dysfunction in pathologies in vivo. Most membrane permeability stains are permanently colored or fluorescent dyes that need washing to remove their non-uptaken extracellular background and reach good image contrast. Others are DNA-binding environment-dependent fluorophores, which lack design modularity, have potential toxicity, and can only detect permeabilization of cell volumes containing a nucleus (i.e., cannot delineate damaged volumes in vivo nor image non-nucleated cell types or compartments). Here, we develop modular fluorogenic probes that reveal the whole cytosolic volume of damaged cells, with near-zero background fluorescence so that no washing is needed. We identify a specific disulfonated fluorogenic probe type that only enters cells with damaged membranes, then is enzymatically activated and marks them. The esterase probe MDG1 is a reliable tool to reveal live cells that have been permeabilized by biological, biochemical, or physical membrane damage, and it can be used in multicolor microscopy. We confirm the modularity of this approach by also adapting it for improved hydrolytic stability, as the redox probe MDG2. We conclude by showing the unique performance of MDG probes in revealing axonal membrane damage (which DNA fluorogens cannot achieve) and in discriminating damage on a cell-by-cell basis in embryos in vivo. The MDG design thus provides powerful modular tools for wash-free in vivo imaging of membrane damage, and indicates how designs may be adapted for selective delivery of drug cargoes to these damaged cells: offering an outlook from selective diagnosis toward therapy of membrane-compromised cells in disease.

Changes in the term neonatal electroencephalogram with general anesthesia - a systematic review with narrative synthesis.

While effects of general anesthesia on neuronal activity in the human neonatal brain are incompletely understood, electroencephalography (EEG) provides some insight and may identify age-dependent differences. A systematic search (MEDLINE, Embase, PUBMED, Cochrane Library to November 2023) retrieved English language publications reporting EEG during general anesthesia for cardiac or non-cardiac surgery in term neonates (37 to 44 weeks post-menstrual age). Data were extracted and risk of bias (ROBINS-I Cochrane tool) and quality of evidence (GRADE checklist) assessed. From 1155 abstracts, nine publications (157 neonates; 55.7% male) fulfilled eligibility criteria. Data were limited and study quality was very low. The occurrence of discontinuity, a characteristic pattern of alternating higher and lower amplitude EEG segments, was reported with general anesthesia (94 of 119 neonates, six publications) and with hypothermia (23 of 23 neonates, two publications). Decreased power in the delta (0.5-4Hz) frequency range was also reported with increasing anesthetic dose (39 neonates; three publications). While evidence gaps were identified, both increasing sevoflurane concentration and decreasing temperature are associated with increasing discontinuity.

Topographical Features of Pediatric Electroencephalography during High Initial Concentration Sevoflurane for Inhalational Induction of Anesthesia.

BACKGROUND: High-density electroencephalographic (EEG) monitoring remains underutilized in clinical anesthesia, despite its obvious utility in unraveling the profound physiologic impact of these agents on central nervous system functioning. In school-aged children, the routine practice of rapid induction with high concentrations of inspiratory sevoflurane is commonplace, given its favorable efficacy and tolerance profile. However, few studies investigate topographic EEG during the critical timepoint coinciding with loss of responsiveness-a key moment for anesthesiologists in their everyday practice. The authors hypothesized that high initial sevoflurane inhalation would better precipitate changes in brain regions due to inhomogeneities in maturation across three different age groups compared with gradual stepwise paradigms utilized by other investigators. Knowledge of these changes may inform strategies for agent titration in everyday clinical settings. METHODS: A total of 37 healthy children aged 5 to 10 yr underwent induction with 4% or greater sevoflurane in high-flow oxygen. Perturbations in anesthetic state were investigated in 23 of these children using 64-channel EEG with the Hjorth Laplacian referencing scheme. Topographical maps illustrated absolute, relative, and total band power across three age groups: 5 to 6 yr (n = 7), 7 to 8 yr (n = 8), and 9 to 10 yr (n = 8). RESULTS: Spectral analysis revealed a large shift in total power driven by increased delta oscillations. Well-described topographic patterns of anesthesia, e.g., frontal predominance, paradoxical beta excitation, and increased slow activity, were evident in the topographic maps. However, there were no statistically significant age-related changes in spectral power observed in a midline electrode subset between the groups when responsiveness was lost compared to the resting state. CONCLUSIONS: High initial concentration sevoflurane induction causes large-scale topographic effects on the pediatric EEG. Within the minute after unresponsiveness, this dosage may perturb EEG activity in children to an extent where age-related differences are not discernible.

A systematic review of the prevalence of chronic postsurgical pain in children.

Two prior reviews highlight the scarcity and conflicting nature of available data on chronic postsurgical pain in children, reporting a wide prevalence range of 3.2% to 64% (at ≥3 months). This updated systematic review aimed to consolidate information on the prevalence of pediatric chronic postsurgical pain. A thorough literature search of full English-text publications from April 2014 to August 2021 was conducted using Ovid MEDLINE, PubMed, and Cochrane Database of Systematic Reviews, with search terms: postoperative pain, child, preschool, pediatrics, adolescent, chronic pain. Seventeen relevant studies were identified. Most assessed chronicity once greater than 3 months duration postoperatively (82%), were predominantly prospective (71%) and conducted in inpatient settings (88%). The surgeries examined included orthopedic (scoliosis and limb), urological, laparotomy, inguinal, and cardiothoracic procedures, involving numbers ranging from 36 to 750, totaling 3137 participants/2792 completers. The studies had wide variations in median age at surgery (6 days to 16 years), the percentage of female participants (unspecified or 12.5% to 90%), and follow-up duration (2.5 months to 9 years). Various pain, functional, psychosocial, and health-related quality of life outcomes were documented. Chronic postsurgical pain prevalence varied widely from 2% to 100%. Despite increased data, challenges persist due to heterogeneity in definitions, patient demographics, mixed versus single surgical populations, diverse perioperative analgesic interventions, follow-up durations and reported outcomes. Interpretation is further complicated by limited information on impact, long-term analgesia and healthcare utilization, and relatively small sample sizes, hindering the assessment of reported associations. In some cases, preoperative pain and deformity may not have been addressed by surgery and persisting pain postoperatively may then be inappropriately termed chronic postsurgical pain. Larger-scale, procedure-specific data to better assess current prevalence, impact, and whether modifiable factors link to negative long-term outcomes, would be more useful and allow targeted perioperative interventions for at-risk pediatric surgical patients.

Anesthesia and Developing Brains: Unanswered Questions and Proposed Paths Forward.

Anesthetic agents disrupt neurodevelopment in animal models, but evidence in humans is mixed. The morphologic and behavioral changes observed across many species predicted that deficits should be seen in humans, but identifying a phenotype of injury in children has been challenging. It is increasingly clear that in children, a brief or single early anesthetic exposure is not associated with deficits in a range of neurodevelopmental outcomes including broad measures of intelligence. Deficits in other domains including behavior, however, are more consistently reported in humans and also reflect findings from nonhuman primates. The possibility that behavioral deficits are a phenotype, as well as the entire concept of anesthetic neurotoxicity in children, remains a source of intense debate. The purpose of this report is to describe consensus and disagreement among experts, summarize preclinical and clinical evidence, suggest pathways for future clinical research, and compare studies of anesthetic agents to other suspected neurotoxins.

Ena orchestrates remodelling within the actin cytoskeleton to drive robust Drosophila macrophage chemotaxis.

The actin cytoskeleton is the engine that powers the inflammatory chemotaxis of immune cells to sites of tissue damage or infection. Here, we combine genetics with live in vivo imaging to investigate how cytoskeletal rearrangements drive macrophage recruitment to wounds in Drosophila We find that the actin-regulatory protein Ena is a master regulator of lamellipodial dynamics in migrating macrophages, where it remodels the cytoskeleton to form linear filaments that can then be bundled together by the cross-linker Fascin (also known as Singed in flies). In contrast, the formin Dia generates rare, probing filopods for specialised functions that are not required for migration. The role of Ena in lamellipodial bundling is so fundamental that its overexpression increases bundling even in the absence of Fascin by marshalling the remaining cross-linking proteins to compensate. This reorganisation of the lamellipod generates cytoskeletal struts that push against the membrane to drive leading edge advancement and boost cell speed. Thus, Ena-mediated remodelling extracts the most from the cytoskeleton to power robust macrophage chemotaxis during their inflammatory recruitment to wounds.

Neurodevelopmental outcome at 5 years of age after general anaesthesia or awake-regional anaesthesia in infancy (GAS): an international, multicentre, randomised, controlled equivalence trial.

BACKGROUND: In laboratory animals, exposure to most general anaesthetics leads to neurotoxicity manifested by neuronal cell death and abnormal behaviour and cognition. Some large human cohort studies have shown an association between general anaesthesia at a young age and subsequent neurodevelopmental deficits, but these studies are prone to bias. Others have found no evidence for an association. We aimed to establish whether general anaesthesia in early infancy affects neurodevelopmental outcomes. METHODS: In this international, assessor-masked, equivalence, randomised, controlled trial conducted at 28 hospitals in Australia, Italy, the USA, the UK, Canada, the Netherlands, and New Zealand, we recruited infants of less than 60 weeks' postmenstrual age who were born at more than 26 weeks' gestation and were undergoing inguinal herniorrhaphy, without previous exposure to general anaesthesia or risk factors for neurological injury. Patients were randomly assigned (1:1) by use of a web-based randomisation service to receive either awake-regional anaesthetic or sevoflurane-based general anaesthetic. Anaesthetists were aware of group allocation, but individuals administering the neurodevelopmental assessments were not. Parents were informed of their infants group allocation upon request, but were told to mask this information from assessors. The primary outcome measure was full-scale intelligence quotient (FSIQ) on the Wechsler Preschool and Primary Scale of Intelligence, third edition (WPPSI-III), at 5 years of age. The primary analysis was done on a per-protocol basis, adjusted for gestational age at birth and country, with multiple imputation used to account for missing data. An intention-to-treat analysis was also done. A difference in means of 5 points was predefined as the clinical equivalence margin. This completed trial is registered with ANZCTR, number ACTRN12606000441516, and ClinicalTrials.gov, number NCT00756600. FINDINGS: Between Feb 9, 2007, and Jan 31, 2013, 4023 infants were screened and 722 were randomly allocated: 363 (50%) to the awake-regional anaesthesia group and 359 (50%) to the general anaesthesia group. There were 74 protocol violations in the awake-regional anaesthesia group and two in the general anaesthesia group. Primary outcome data for the per-protocol analysis were obtained from 205 children in the awake-regional anaesthesia group and 242 in the general anaesthesia group. The median duration of general anaesthesia was 54 min (IQR 41-70). The mean FSIQ score was 99·08 (SD 18·35) in the awake-regional anaesthesia group and 98·97 (19·66) in the general anaesthesia group, with a difference in means (awake-regional anaesthesia minus general anaesthesia) of 0·23 (95% CI -2·59 to 3·06), providing strong evidence of equivalence. The results of the intention-to-treat analysis were similar to those of the per-protocol analysis. INTERPRETATION: Slightly less than 1 h of general anaesthesia in early infancy does not alter neurodevelopmental outcome at age 5 years compared with awake-regional anaesthesia in a predominantly male study population. FUNDING: US National Institutes of Health, US Food and Drug Administration, Thrasher Research Fund, Australian National Health and Medical Research Council, Health Technologies Assessment-National Institute for Health Research (UK), Australian and New Zealand College of Anaesthetists, Murdoch Children's Research Institute, Canadian Institutes of Health Research, Canadian Anesthesiologists Society, Pfizer Canada, Italian Ministry of Health, Fonds NutsOhra, UK Clinical Research Network, Perth Children's Hospital Foundation, the Stan Perron Charitable Trust, and the Callahan Estate.

Genetic Analysis of Patients Who Experienced Awareness with Recall while under General Anesthesia.

BACKGROUND: Intraoperative awareness with recall while under apparently adequate general anesthesia is a rare, unexplained, and often very distressing phenomenon. It is possible that a relatively small number of genetic variants might underlie the failure of general anesthetic drugs to adequately suppress explicit memory formation and recall in the presence of apparently adequate anesthesia concentrations. METHODS: The authors recruited 12 adult patients who had experienced an episode of intraoperative awareness with recall (compared with 12 controls), performed whole exome sequencing, and applied filtering to obtain a set of genetic variants that might be associated with intraoperative awareness with recall. The criteria were that the variant (1) had a minor allele frequency less than 0.1% in population databases, (2) was within exonic or splicing regions, (3) caused a nonsynonymous change, (4) was predicted to be functionally damaging, (5) was expressed in the top 50% of genes expressed in the brain, and (6) was within genes in Kyoto Encyclopedia of Genes and Genomes pathways associated with general anesthesia, drug metabolism, arousal, and memory. RESULTS: The authors identified 29 rare genetic variants in 27 genes that were absent in controls and could plausibly be associated with this disorder. One variant in CACNA1A was identified in two patients and two different variants were identified in both CACNA1A and CACNA1S. Of interest was the relative overrepresentation of variants in genes encoding calcium channels and purinergic receptors. CONCLUSIONS: Within the constraints of the filtering process used, the authors did not find any single gene variant or gene that was strongly associated with intraoperative awareness with recall. The authors report 27 candidate genes and associated pathways identified in this pilot project as targets of interest for future larger biologic and epidemiologic studies.

Statistical Analysis Plan for "An international multicenter study of isoelectric electroencephalography events in infants and young children during anesthesia for surgery".

This Statistical Analysis Plan details the statistical procedures to be applied for the analysis of data for the multicenter electroencephalography study. It consists of a basic description of the study in broad terms and separate sections that detail the methods of different aspects of the statistical analysis, summarized under the following headings (a) Background; (b) Definitions of protocol violations; (c) Definitions of objectives and other terms; (d) Variables for analyses; (e) Handling of missing data and study bias; (f) Statistical analysis of the primary and secondary study outcomes; (g) Reporting of study results; and (h) References. It serves as a template for researchers interested in writing a Statistical Analysis Plan.

Enrollment challenges in multicenter, international studies: The example of the GAS trial.

INTRODUCTION: Randomized trials are important for generating high-quality evidence, but are perceived as difficult to perform in the pediatric population. Thus far there has been poor characterization of the barriers to conducting trials involving children, and the variation in these barriers between countries remains undescribed. The General Anesthesia compared to Spinal anesthesia (GAS) trial, conducted in seven countries between 2007 and 2013, provides an opportunity to explore these issues. METHODS: We undertook a descriptive analysis to evaluate the reasons for variation in enrollment between countries in the GAS trial, looking specifically at the number of potential subjects screened, and the subsequent application of four exclusion criteria that were applied in a hierarchical order. RESULTS: A total of 4023 patients were screened by 28 centers in seven countries. Australia and the USA screened the most subjects, accounting for 84% of all potential trial participants. The percentage of subjects eliminated from the screened pool by each exclusion criterion varied between countries. Exclusion due to a predefined condition (H1) eliminated only 5% of potential subjects in Italy and the UK, but 37% in Canada. Exclusions due to a contraindication or a physician's refusal most impacted enrollment in Australia and the USA. The patient being "too large for spinal anesthesia" was the most commonly cited by anesthetists who refused to enroll a patient (64% of anesthetist refusals). The majority of surgeon refusals came from the USA, where surgeons preferred the patient to receive a general anesthetic. The percentage of approached parents refusing to consent ranged from a low of 3% in Italy to a high of 70% in the USA and Netherlands. The most frequently cited reason for parent refusal in all countries was a preference for general anesthesia (median: 43%, range: 32%-67%). However, a sizeable proportion of parents in all countries had a contrasting preference for spinal anesthesia (median: 25%, range: 13%-31%), and 23% of U.S. parents expressed concern about randomization. CONCLUSION: The GAS trial highlights enrollment challenges that can occur when conducting multicenter, international, pediatric studies. Investigators planning future trials should be aware of potential differences in screening processes across countries, and that exclusions by anesthetists and surgeons may vary in reason, in frequency, and by country. Furthermore, investigators should be aware that the U.S. centers encountered particularly high surgeon and parental refusal rates and that U.S. parents were uniquely concerned about randomization. Planning trials that address these difficulties should increase the likelihood of successfully recruiting subjects in pediatric trials.

Clinical Evidence for Any Effect of Anesthesia on the Developing Brain.

A recent U.S. Food and Drug Administration warning advised that prolonged or repeated exposure to general anesthetics may affect neurodevelopment in children. This warning is based on a wealth of preclinical animal studies and relatively few human studies. The human studies include a variety of different populations with several different outcome measures. Interpreting the results requires consideration of the outcome used, the power of the study, the length of exposure and the efforts to reduce the confounding effects of comorbidity and surgery. Most, but not all, of the large population-based studies find evidence for associations between surgery in early childhood and slightly worse subsequent academic achievement or increased risk for later diagnosis of a behavioral disability. In several studies, the amount of added risk is very small; however, there is some evidence for a greater association with multiple exposures. These results may be consistent with the preclinical data, but the possibility of confounding means the positive associations can only be regarded as weak evidence for causation. Finally, there is strong evidence that brief exposure is not associated with any long term risk in humans.

Estimating pediatric general anesthesia exposure: Quantifying duration and risk.

INTRODUCTION: Understanding the duration of pediatric general anesthesia exposure in contemporary practice is important for identifying groups at risk for long general anesthesia exposures and designing trials examining associations between general anesthesia exposure and neurodevelopmental outcomes. METHODS: We performed a retrospective cohort analysis to estimate pediatric general anesthesia exposure duration during 2010-2015 using the National Anesthesia Clinical Outcomes Registry. RESULTS: A total of 1 548 021 pediatric general anesthetics were included. Median general anesthesia duration was 57 minutes (IQR: 28-86) with 90th percentile 145 minutes. Children aged <1 year had the longest median exposure duration (79 minutes, IQR: 39-119) with 90th percentile 210 minutes, and 13.7% of this very young cohort was exposed for >3 hours. High ASA physical status and care at a university hospital were associated with longer exposure times. CONCLUSION: While the vast majority (94%) of children undergoing general anesthesia are exposed for <3 hours, certain groups may be at increased risk for longer exposures. These findings may help guide the design of future trials aimed at understanding neurodevelopmental impact of prolonged exposure in these high-risk groups.

The impact of general anesthesia on child development and school performance: a population-based study.

BACKGROUND: There has been considerable interest in the possible adverse neurocognitive effects of exposure to general anesthesia and surgery in early childhood. AIMS: The aim of this data linkage study was to investigate developmental and school performance outcomes of children undergoing procedures requiring general anesthesia in early childhood. METHODS: We included children born in New South Wales, Australia of 37+ weeks' gestation without major congenital anomalies or neurodevelopmental disability with either a school entry developmental assessment in 2009, 2012, or Grade-3 school test results in 2008-2014. We compared children exposed to general anesthesia aged <48 months to those without any hospitalization. Children with only 1 hospitalization with general anesthesia and no other hospitalization were assessed separately. Outcomes included being classified developmentally high risk at school entry and scoring below national minimum standard in school numeracy and reading tests. RESULTS: Of 211 978 children included, 82 156 had developmental assessment and 153 025 had school test results, with 12 848 (15.7%) and 25 032 (16.4%) exposed to general anesthesia, respectively. Children exposed to general anesthesia had 17%, 34%, and 23% increased odds of being developmentally high risk (adjusted odds ratio [aOR]: 1.17; 95% CI: 1.07-1.29); or scoring below the national minimum standard in numeracy (aOR: 1.34; 95% CI: 1.21-1.48) and reading (aOR: 1.23; 95% CI: 1.12-1.36), respectively. Although the risk for being developmentally high risk and poor reading attenuated for children with only 1 hospitalization and exposure to general anesthesia, the association with poor numeracy results remained. CONCLUSION: Children exposed to general anesthesia before 4 years have poorer development at school entry and school performance. While the association among children with 1 hospitalization with 1 general anesthesia and no other hospitalization was attenuated, poor numeracy outcome remained. Further investigation of the specific effects of general anesthesia and the impact of the underlying health conditions that prompt the need for surgery or diagnostic procedures is required, particularly among children exposed to long duration of general anesthesia or with repeated hospitalizations.

Anesthesia and the developing brain: A way forward for laboratory and clinical research.

All commonly used general anesthetics have been shown to cause neurotoxicity in animal models, including nonhuman primates. Opinion, however, remains divided over how cumulative evidence from preclinical and human studies in this field should be interpreted and its translation to current practices in pediatric anesthesia and surgery. A group of international experts in laboratory and clinical sciences recently convened in Genoa, Italy, to evaluate the current state of both laboratory and clinical research and discuss future directions for basic, translational, and clinical studies in this field. This paper describes those discussions and conclusions. A central goal identified was the importance of continuing to pursue laboratory research efforts to better understand the biological pathways underlying anesthesia neurotoxicity. The distinction between basic and translational experimental designs in this field was highlighted, and it was acknowledged that it will be important for future animal research to try to causally link structural changes with long-term cognitive abnormalities. While inherent limitations will continue to affect the ability of even large observational cohorts to determine if anesthesia impacts neurodevelopment or behavioral outcomes, the importance of conducting further large well-designed cohort studies was also emphasized. Adequately powered cohorts could clarify which populations are at increased risk, provide information on environmental and healthcare-related risk modifiers, and guide future interventional trials. If anesthetics cause structural or functional adverse neurological effects in young children, alternative or mitigating strategies need to be considered. While protective or mitigating strategies have been repeatedly studied in animals, there are currently no human data to support alternative anesthetic strategies in clinical practice. Lastly, it was noted that there is still considerable debate over the clinical relevance of anesthesia neurotoxicity, and the need to evaluate the impact of other aspects of perioperative care on neurodevelopment must also be considered.

Neurodevelopmental outcome at 2 years of age after general anaesthesia and awake-regional anaesthesia in infancy (GAS): an international multicentre, randomised controlled trial.

BACKGROUND: Preclinical data suggest that general anaesthetics affect brain development. There is mixed evidence from cohort studies that young children exposed to anaesthesia can have an increased risk of poor neurodevelopmental outcome. We aimed to establish whether general anaesthesia in infancy has any effect on neurodevelopmental outcome. Here we report the secondary outcome of neurodevelopmental outcome at 2 years of age in the General Anaesthesia compared to Spinal anaesthesia (GAS) trial. METHODS: In this international assessor-masked randomised controlled equivalence trial, we recruited infants younger than 60 weeks postmenstrual age, born at greater than 26 weeks' gestation, and who had inguinal herniorrhaphy, from 28 hospitals in Australia, Italy, the USA, the UK, Canada, the Netherlands, and New Zealand. Infants were randomly assigned (1:1) to receive either awake-regional anaesthesia or sevoflurane-based general anaesthesia. Web-based randomisation was done in blocks of two or four and stratified by site and gestational age at birth. Infants were excluded if they had existing risk factors for neurological injury. The primary outcome of the trial will be the Wechsler Preschool and Primary Scale of Intelligence Third Edition (WPPSI-III) Full Scale Intelligence Quotient score at age 5 years. The secondary outcome, reported here, is the composite cognitive score of the Bayley Scales of Infant and Toddler Development III, assessed at 2 years. The analysis was as per protocol adjusted for gestational age at birth. A difference in means of five points (1/3 SD) was predefined as the clinical equivalence margin. This trial is registered with ANZCTR, number ACTRN12606000441516 and ClinicalTrials.gov, number NCT00756600. FINDINGS: Between Feb 9, 2007, and Jan 31, 2013, 363 infants were randomly assigned to receive awake-regional anaesthesia and 359 to general anaesthesia. Outcome data were available for 238 children in the awake-regional group and 294 in the general anaesthesia group. In the as-per-protocol analysis, the cognitive composite score (mean [SD]) was 98.6 (14.2) in the awake-regional group and 98.2 (14.7) in the general anaesthesia group. There was equivalence in mean between groups (awake-regional minus general anaesthesia 0.169, 95% CI -2.30 to 2.64). The median duration of anaesthesia in the general anaesthesia group was 54 min. INTERPRETATION: For this secondary outcome, we found no evidence that just less than 1 h of sevoflurane anaesthesia in infancy increases the risk of adverse neurodevelopmental outcome at 2 years of age compared with awake-regional anaesthesia. FUNDING: Australia National Health and Medical Research Council (NHMRC), Health Technologies Assessment-National Institute for Health Research UK, National Institutes of Health, Food and Drug Administration, Australian and New Zealand College of Anaesthetists, Murdoch Childrens Research Institute, Canadian Institute of Health Research, Canadian Anesthesiologists' Society, Pfizer Canada, Italian Ministry of Heath, Fonds NutsOhra, and UK Clinical Research Network (UKCRN).