Standards for preclinical research and publications in developmental anaesthetic neurotoxicity: expert opinion statement from the SmartTots preclinical working group.

In March 2019, SmartTots, a public-private partnership between the US Food and Drug Administration and the International Anesthesia Research Society, hosted a meeting attended by research experts, anaesthesia journal editors, and government agency representatives to discuss the continued need for rigorous preclinical research and the importance of establishing reporting standards for the field of anaesthetic perinatal neurotoxicity. This group affirmed the importance of preclinical research in the field, and welcomed novel and mechanistic approaches to answer some of the field's largest questions. The attendees concluded that summarising the benefits and disadvantages of specific model systems, and providing guidance for reporting results, would be helpful for designing new experiments and interpreting results across laboratories. This expert opinion report is a summary of these discussions, and includes a focused review of current animal models and reporting standards for the field of perinatal anaesthetic neurotoxicity. This will serve as a practical guide and road map for novel and rigorous experimental work.

Early Postnatal Exposure to Isoflurane Disrupts Oligodendrocyte Development and Myelin Formation in the Mouse Hippocampus.

BACKGROUND: Early postnatal exposure to general anesthetics may interfere with brain development. We tested the hypothesis that isoflurane causes a lasting disruption in myelin development via actions on the mammalian target of rapamycin pathway. METHODS: Mice were exposed to 1.5% isoflurane for 4 h at postnatal day 7. The mammalian target of rapamycin inhibitor, rapamycin, or the promyelination drug, clemastine, were administered on days 21 to 35. Mice underwent Y-maze and novel object position recognition tests (n = 12 per group) on days 56 to 62 or were euthanized for either immunohistochemistry (n = 8 per group) or Western blotting (n = 8 per group) at day 35 or were euthanized for electron microscopy at day 63. RESULTS: Isoflurane exposure increased the percentage of phospho-S6-positive oligodendrocytes in fimbria of hippocampus from 22 ± 7% to 51 ± 6% (P < 0.0001). In Y-maze testing, isoflurane-exposed mice did not discriminate normally between old and novel arms, spending equal time in both (50 ± 5% old:50 ± 5% novel; P = 0.999), indicating impaired spatial learning. Treatment with clemastine restored discrimination, as evidenced by increased time spent in the novel arm (43 ± 6% old:57 ± 6% novel; P < 0.001), and rapamycin had a similar effect (44 ± 8% old:56 ± 8% novel; P < 0.001). Electron microscopy shows a reduction in myelin thickness as measured by an increase in g-ratio from 0.76 ± 0.06 for controls to 0.79 ± 0.06 for the isoflurane group (P < 0.001). Isoflurane exposure followed by rapamycin treatment resulted in a g-ratio (0.75 ± 0.05) that did not differ significantly from the control value (P = 0.426). Immunohistochemistry and Western blotting show that isoflurane acts on oligodendrocyte precursor cells to inhibit both proliferation and differentiation. DNA methylation and expression of a DNA methyl transferase 1 are reduced in oligodendrocyte precursor cells after isoflurane treatment. Effects of isoflurane on oligodendrocyte precursor cells were abolished by treatment with rapamycin. CONCLUSIONS: Early postnatal exposure to isoflurane in mice causes lasting disruptions of oligodendrocyte development in the hippocampus via actions on the mammalian target of rapamycin pathway.

General Anesthesia Alters the Diversity and Composition of the Intestinal Microbiota in Mice.

Dysbiosis of the intestinal microbiota has been shown to result in altered immune responses and increased susceptibility to infection; as such, the state of the intestinal microbiome may have profound implications in the perioperative setting. In this first-in-class study, we used 16s ribosomal RNA sequencing and analysis in a mouse model of general anesthesia to investigate the effects of volatile anesthetics on the diversity and composition of the intestinal microbiome. After 4-hour exposure to isoflurane, we observed a decrease in bacterial diversity. Taxonomic alterations included depletion of several commensal bacteria including Clostridiales. These data identify volatile anesthetics as potential contributors to microbial dysbiosis in the postoperative patient.

Sevoflurane Impairs Growth Cone Motility in Dissociated Murine Neurons.

BACKGROUND: Early postnatal exposure to general anesthetic agents causes a lasting impairment in learning and memory in animal models. One hypothesis to explain this finding is that exposure to anesthetic agents during critical points in neural development disrupts the formation of brain circuitry. Here, we explore the effects of sevoflurane on the neuronal growth cone, a specialization at the growing end of axons and dendrites that is responsible for the targeted growth that underlies connectivity between neurons. METHODS: Dissociated neuronal cultures were prepared from embryonic mouse neocortex. Time-lapse images of live growth cones exposed to anesthetics were taken using differential interference contrast microscopy, and the rate of change of the area of the lamellipodia and the speed of the filopodial tip were quantified as measures of motility. The involvement of the p75 neurotropin receptor (p75NTR) was tested using inhibitors applied to the media and by a coimmunoprecipitation assay. RESULTS: The rate of lamellipodial area change and filopodial tip velocity in both axonal and dendritic growth cones was significantly reduced with sevoflurane exposure between 2% and 6%. Motility could be substantially restored by treatment with Y27632 and TAT-peptide 5, which are inhibitors of Rho Kinase and p75NTR, respectively. Sevoflurane results in reduced coimmunoprecipitation of Rho-Guanosine-5'-diphosphate dissociation inhibitor after pulldown with p75NTR. CONCLUSIONS: Sevoflurane interferes with growth cone motility, which is a critical process in brain circuitry formation. Our data suggest that this may occur through an action on the p75NTR, which promotes growth inhibitory signaling by the Rho pathway.

Molecular Mechanisms of Anesthetic Neurotoxicity: A Review of the Current Literature.

Data from epidemiologic studies and animal models have raised a concern that exposure to anesthetic agents during early postnatal life may cause lasting impairments in cognitive function. It is hypothesized that this is due to disruptions in brain development, but the mechanism underlying this toxic effect remains unknown. Ongoing research, particularly in rodents, has begun to address this question. In this review we examine currently postulated molecular mechanisms of anesthetic toxicity in the developing brain, including effects on cell death pathways, growth factor signaling systems, NMDA and GABA receptors, mitochondria, and epigenetic factors. The level of evidence for each putative mechanism is critically evaluated, and we attempt to draw connections between them where it is possible to do so. Although there are many promising avenues of research, at this time no consensus can be reached as to a definitive mechanism of injury.

Isoflurane impairs the capacity of astrocytes to support neuronal development in a mouse dissociated coculture model.

BACKGROUND: There is growing concern that pediatric exposure to anesthetic agents may cause long-lasting deficits in learning by impairing brain development. Most studies to date on this topic have focused on the direct effects of anesthetics on developing neurons. Relatively little attention has been paid to possible effects of anesthetics on astrocytes, a glial cell type that plays an important supporting role in neuronal development. METHODS: Astrocytes were exposed to isoflurane and then cocultured with unexposed neurons to test for astrocyte-specific toxic effects on neuronal growth. Axon length was measured in the cocultured neurons to assess neuronal growth. RESULTS: We found that neurons cocultured with astrocytes exposed to isoflurane exhibited a 30% reduction in axon outgrowth. Further experimentation showed that this effect is likely due to reduced levels of brain-derived neurotrophic factor in the coculture media. CONCLUSIONS: Isoflurane interferes with the ability of cultured astrocytes to support neuronal growth. This finding represents a potentially novel mechanism through which general anesthetics may interfere with brain development.

Review: effects of anesthetics on brain circuit formation.

The results of several retrospective clinical studies suggest that exposure to anesthetic agents early in life is correlated with subsequent learning and behavioral disorders. Although ongoing prospective clinical trials may help to clarify this association, they remain confounded by numerous factors. Thus, some of the most compelling data supporting the hypothesis that a relatively short anesthetic exposure can lead to a long-lasting change in brain function are derived from animal models. The mechanism by which such changes could occur remains incompletely understood. Early studies identified anesthetic-induced neuronal apoptosis as a possible mechanism of injury, and more recent work suggests that anesthetics may interfere with several critical processes in brain development. The function of the mature brain requires the presence of circuits, established during development, which perform the computations underlying learning and cognition. In this review, we examine the mechanisms by which anesthetics could disrupt brain circuit formation, including effects on neuronal survival and neurogenesis, neurite growth and guidance, formation of synapses, and function of supporting cells. There is evidence that anesthetics can disrupt aspects of all of these processes, and further research is required to elucidate which are most relevant to pediatric anesthetic neurotoxicity.