Early Isoflurane Exposure Impairs Synaptic Development in Fmr1 KO Mice via the mTOR Pathway.

General anesthetics (GAs) may cause disruptions in brain development, and the effect of GA exposure in the setting of pre-existing neurodevelopmental disease is unknown. We tested the hypothesis that synaptic development is more vulnerable to GA-induced deficits in a mouse model of fragile X syndrome than in WT mice and asked whether they were related to the mTOR pathway, a signaling system implicated in both anesthesia toxicity and fragile X syndrome. Early postnatal WT and Fmr1-KO mice were exposed to isoflurane and brain slices were collected in adulthood. Primary neuron cultures isolated from WT and Fmr1-KO mice were exposed to isoflurane during development, in some cases treated with rapamycin, and processed for immunohistochemistry at maturity. Quantitative immunofluorescence microscopy was conducted for synaptic markers and markers of mTOR pathway activity. Isoflurane exposure caused reduction in Synpasin-1, PSD-95, and Gephyrin puncta that was significantly lower in Fmr1-KO mice than in WT mice. Similar results were found in cell culture, where synapse loss was ameliorated with rapamycin treatment. Early developmental exposure to isoflurane causes more profound synapse loss in Fmr1- KO than WT mice, and this effect is mediated by a pathologic increase in mTOR pathway activity.

Early postnatal exposure to isoflurane causes cognitive deficits and disrupts development of newborn hippocampal neurons via activation of the mTOR pathway.

Clinical and preclinical studies indicate that early postnatal exposure to anesthetics can lead to lasting deficits in learning and other cognitive processes. The mechanism underlying this phenomenon has not been clarified and there is no treatment currently available. Recent evidence suggests that anesthetics might cause persistent deficits in cognitive function by disrupting key events in brain development. The hippocampus, a brain region that is critical for learning and memory, contains a large number of neurons that develop in the early postnatal period, which are thus vulnerable to perturbation by anesthetic exposure. Using an in vivo mouse model we demonstrate abnormal development of dendrite arbors and dendritic spines in newly generated dentate gyrus granule cell neurons of the hippocampus after a clinically relevant isoflurane anesthesia exposure conducted at an early postnatal age. Furthermore, we find that isoflurane causes a sustained increase in activity in the mechanistic target of rapamycin pathway, and that inhibition of this pathway with rapamycin not only reverses the observed changes in neuronal development, but also substantially improves performance on behavioral tasks of spatial learning and memory that are impaired by isoflurane exposure. We conclude that isoflurane disrupts the development of hippocampal neurons generated in the early postnatal period by activating a well-defined neurodevelopmental disease pathway and that this phenotype can be reversed by pharmacologic inhibition.

Effects of Early Exposure of Isoflurane on Chronic Pain via the Mammalian Target of Rapamycin Signal Pathway.

Persistent post-surgical pain (PPSP) is a chronic pain condition, often with neuropathic features, that occurs in approximately 20% of children who undergo surgery. The biological basis of PPSP has not been elucidated. Anesthetic drugs can have lasting effects on the developing nervous system, although the clinical impact of this phenomenon is unknown. Here, we used a mouse model to test the hypothesis that early developmental exposure to isoflurane causes cellular and molecular alteration in the pain perception circuitry that causes a predisposition to chronic, neuropathic pain via a pathologic upregulation of the mammalian target of the rapamycin (mTOR) signaling pathway. Mice were exposed to isoflurane at postnatal day 7 and select cohorts were treated with rapamycin, an mTOR pathway inhibitor. Behavioral tests conducted 2 months later showed increased evidence of neuropathic pain, which did not occur in rapamycin-treated animals. Immunohistochemistry showed neuronal activity was chronically increased in the insular cortex, anterior cingulate cortex, and spinal dorsal horn, and activity was attenuated by rapamycin. Immunohistochemistry and western blotting (WB) showed a co-incident chronic, abnormal upregulation in mTOR activity. We conclude that early isoflurane exposure alters the development of pain circuits and has the potential to contribute to PPSP and/or other pain syndromes.

Early Developmental Exposure to General Anesthetic Agents in Primary Neuron Culture Disrupts Synapse Formation via Actions on the mTOR Pathway.

Human epidemiologic studies and laboratory investigations in animal models suggest that exposure to general anesthetic agents (GAs) have harmful effects on brain development. The mechanism underlying this putative iatrogenic condition is not clear and there are currently no accepted strategies for prophylaxis or treatment. Recent evidence suggests that anesthetics might cause persistent deficits in synaptogenesis by disrupting key events in neurodevelopment. Using an in vitro model consisting of dissociated primary cultured mouse neurons, we demonstrate abnormal pre- and post-synaptic marker expression after a clinically-relevant isoflurane anesthesia exposure is conducted during neuron development. We find that pharmacologic inhibition of the mechanistic target of rapamycin (mTOR) pathway can reverse the observed changes. Isoflurane exposure increases expression of phospho-S6, a marker of mTOR pathway activity, in a concentration-dependent fashion and this effect occurs throughout neuronal development. The mTOR 1 complex (mTORC1) and the mTOR 2 complex (mTORC2) branches of the pathway are both activated by isoflurane exposure and this is reversible with branch-specific inhibitors. Upregulation of mTOR is also seen with sevoflurane and propofol exposure, suggesting that this mechanism of developmental anesthetic neurotoxicity may occur with all the commonly used GAs in pediatric practice. We conclude that GAs disrupt the development of neurons during development by activating a well-defined neurodevelopmental disease pathway and that this phenotype can be reversed by pharmacologic inhibition.

Effects of Neonatal Hypoxic-Ischemic Injury and Hypothermic Neuroprotection on Neural Progenitor Cells in the Mouse Hippocampus.

Neonatal hypoxic-ischemic injury (HI) results in widespread cerebral encephalopathy and affects structures that are essential for neurocognitive function, such as the hippocampus. The dentate gyrus contains a reservoir of neural stem and progenitor cells (NSPCs) that are critical for postnatal development and normal adult function of the hippocampus, and may also facilitate the recovery of function after injury. Using a neonatal mouse model of mild-to-moderate HI and immunohistochemical analysis of NSPC development markers, we asked whether these cells are vulnerable to HI and how they respond to both injury and hypothermic therapy. We found that cleaved caspase-3 labeling in the subgranular zone, where NSPCs are located, is increased by more than 30-fold after HI. The population of cells positive for both proliferating cell nuclear antigen and nestin (PCNA+Nes+), which represent primarily actively proliferating NSPCs, are acutely decreased by 68% after HI. The NSPC population expressing NeuroD1, a marker for NSPCs transitioning to become fate-committed neural progenitors, was decreased by 47%. One week after HI, there was a decrease in neuroblasts and immature neurons in the dentate gyrus, as measured by doublecortin (DCX) immunolabeling, and at the same time PCNA+Nes+ cell density was increased by 71%. NSPCs expressing Tbr2, which identifies a highly proliferative intermediate neural progenitor population, increased by 107%. Hypothermia treatment after HI partially rescues both the acute decrease in PCNA+Nes+ cell density at 1 day after injury and the chronic loss of DCX immunoreactivity and reduction in NeuroD1 cell density measured at 1 week after injury. Thus, we conclude that HI causes an acute loss of dentate gyrus NSPCs, and that hypothermia partially protects NSPCs from HI.

Anesthetics interfere with axon guidance in developing mouse neocortical neurons in vitro via a γ-aminobutyric acid type A receptor mechanism.

BACKGROUND: The finding that exposure to general anesthetics (GAs) in childhood may increase rates of learning disabilities has raised a concern that anesthetics may interfere with brain development. The generation of neuronal circuits, a complex process in which axons follow guidance cues to dendritic targets, is an unexplored potential target for this type of toxicity. METHODS: GA exposures were conducted in developing neocortical neurons in culture and in early postnatal neocortical slices overlaid with fluorescently labeled neurons. Axon targeting, growth cone collapse, and axon branching were measured using quantitative fluorescence microscopy. RESULTS: Isoflurane exposure causes errors in Semaphorin-3A-dependent axon targeting (n = 77 axons) and a disruption of the response of axonal growth cones to Semaphorin-3A (n = 2,358 growth cones). This effect occurs at clinically relevant anesthetic doses of numerous GAs with allosteric activity at γ-aminobutyric acid type A receptors, and it was reproduced with a selective agonist. Isoflurane also inhibits growth cone collapse induced by Netrin-1, but does not interfere branch induction by Netrin-1. Insensitivity to guidance cues caused by isoflurane is seen acutely in growth cones in dissociated culture, and errors in axon targeting in brain slice culture occur at the earliest point at which correct targeting is observed in controls. CONCLUSIONS: These results demonstrate a generalized inhibitory effect of GAs on repulsive growth cone guidance in the developing neocortex that may occur via a γ-aminobutyric acid type A receptor mechanism. The finding that GAs interfere with axon guidance, and thus potentially with circuit formation, represents a novel form of anesthesia neurotoxicity in brain development.

Pediatric Intensive Care Unit Patients: Sedation, Monitoring, and Neurodevelopmental Outcomes.

The design and conduct of pediatric sedation studies in critically ill patients have historically been challenging due to the complexity of the pediatric intensive care unit (PICU) environment and the difficulty of establishing equipoise. Clinical trials, for instance, represent 1 important means of advancing our knowledge in this field, but there is a paucity of such studies in the literature. Accounting for ground-level factors in planning for each trial phase (eg, enrollment, intervention, assessment, and follow-up) and the presence of broader system limitations is of key importance. In addition, there is a need for early planning, coordination, and obtaining buy-in from individual study sites and staff to ensure success, particularly for multicenter studies. This review synthesizes the current state of pediatric sedation research and the myriad of challenges in designing and conducting successful trials in this particular area. The review poses consideration for future research directions, including novel study designs, and discusses electroencephalography monitoring and neurodevelopmental outcomes of PICU survivors.

Functional binding interaction identified between the axonal CAM L1 and members of the ERM family.

A yeast two-hybrid library was screened using the cytoplasmic domain of the axonal cell adhesion molecule L1 to identify binding partners that may be involved in the regulation of L1 function. The intracellular domain of L1 bound to ezrin, a member of the ezrin, radixin, and moesin (ERM) family of membrane-cytoskeleton linking proteins, at a site overlapping that for AP2, a clathrin adaptor. Binding of bacterial fusion proteins confirmed this interaction. To determine whether ERM proteins interact with L1 in vivo, extracellular antibodies to L1 were used to force cluster the protein on cultured hippocampal neurons and PC12 cells, which were then immunolabeled for ERM proteins. Confocal analysis revealed a precise pattern of codistribution between ERMs and L1 clusters in axons and PC12 neurites, whereas ERMs in dendrites and spectrin labeling remained evenly distributed. Transfection of hippocampal neurons grown on an L1 substrate with a dominant negative ERM construct resulted in extensive and abnormal elaboration of membrane protrusions and an increase in axon branching, highlighting the importance of the ERM-actin interaction in axon development. Together, our data indicate that L1 binds directly to members of the ERM family and suggest this association may coordinate aspects of axonal morphogenesis.

Anesthetics disrupt growth cone guidance cue sensing through actions on the GABAA α2 receptor mediated by the immature chloride gradient.

BACKGROUND: General anesthetics (GAs) may exert harmful effects on the developing brain by disrupting neuronal circuit formation. Anesthetics that act on γ-aminobutyric acid (GABA) receptors can interfere with axonal growth cone guidance, a critical process in the assembly of neuronal circuitry. Here we investigate the mechanism by which isoflurane prevents sensing of the repulsive guidance cue, Semaphorin 3A (Sema3A). METHODS: Growth cone sensing was assayed by measuring growth cone collapse in dissociated neocortical cultures exposed to recombinant Sema3A in the presence or absence of isoflurane and/or a panel of reagents with specific actions on components of the GABA receptor and chloride ion systems. RESULTS: Isoflurane exposure prevents Sema3A induced growth cone collapse. A GABAA α2 specific agonist replicates this effect (36.83 ±â€¯3.417% vs 70.82 ±â€¯2.941%, in the Sema3A induced control group, p < 0.0001), but an α1-specific agonist does not. Both a Na-K-Cl cotransporter 1 antagonism (bumetanide, BUM) and a chloride ionophore (IONO) prevent isoflurane from disrupting growth cone sensing of Sema3A. (65.67 ±â€¯3.775% in Iso + BUM group vs 67.45 ±â€¯3.624% in Sema3A induced control group, 65.34 ±â€¯1.678% in Iso + IONO group vs 68.71 ±â€¯2.071% in Sema3A induced control group, no significant difference) (n = 96 growth cones per group). CONCLUSION: Our data suggest that the effects of isoflurane on growth cone sensing are mediated by the α2 subunit of the GABAA receptor and also that they are dependent on the developmental chloride gradient, in which Cl- exhibits a depolarizing effect. These findings provide a rationale for why immature neurons are particularly susceptible to anesthetic toxicity.

Current State of and Future Opportunities for Prediction in Microbiome Research: Report from the Mid-Atlantic Microbiome Meet-up in Baltimore on 9 January 2019.

Accurate predictions across multiple fields of microbiome research have far-reaching benefits to society, but there are few widely accepted quantitative tools to make accurate predictions about microbial communities and their functions. More discussion is needed about the current state of microbiome analysis and the tools required to overcome the hurdles preventing development and implementation of predictive analyses. We summarize the ideas generated by participants of the Mid-Atlantic Microbiome Meet-up in January 2019. While it was clear from the presentations that most fields have advanced beyond simple associative and descriptive analyses, most fields lack essential elements needed for the development and application of accurate microbiome predictions. Participants stressed the need for standardization, reproducibility, and accessibility of quantitative tools as key to advancing predictions in microbiome analysis. We highlight hurdles that participants identified and propose directions for future efforts that will advance the use of prediction in microbiome research.

Anesthetics disrupt brain development via actions on the mTOR pathway.

Experiments conducted in non-human primates have recently provided new evidence supporting a longstanding concern that exposure to general anesthesia during late intrauterine life or early childhood can cause lasting cognitive deficits through harmful effects on brain development. The mammalian target of rapamycin (mTOR) signaling system plays a key role in both normal brain development and in a wide range of developmental disorders that are characterized by cognitive deficits. Intriguingly, our recently published work shows that anesthetics can chronically alter mTOR signaling in the hippocampal dentate gyrus and that normalization of mTOR signaling can prevent anesthesia-induced perturbation of structure and function. In this addendum, we briefly discuss the putative role of mTOR in developmental anesthetic neurotoxicity.

Sensitivity to isoflurane anesthesia increases in autism spectrum disorder Shank3+/∆c mutant mouse model.

Autism is a heterogeneous developmental disorder characterized by impaired social interaction, impaired communication skills, and restricted and repetitive behavior. The abnormal behaviors of these patients can make their anesthetic and perioperative management difficult. Evidence in the literature suggests that some patients with autism or specific autism spectrum disorders (ASD) exhibit altered responses to pain and to anesthesia or sedation. A genetic mouse model of one particular ASD, Phelan McDermid Syndrome, has been developed that has a Shank3 haplotype truncation (Shank3+/Δc). These mice exhibit important characteristics of autism that mimic human autistic behavior. Our study demonstrates that a Shank3+/ΔC mutation in mice is associated with a reduction in both the MAC and RREC50 of isoflurane and down regulation of NR1 in vestibular nuclei and PSD95 in spinal cord. Decreased expression of NR1 and PSD95 in the central nervous system of Shank3+/ΔC mice could help reduce the MAC and RREC50 of isoflurane, which would warrant confirmation in a clinical study. If Shank3 mutations are found to affect anesthetic sensitivity in patients with ASD, better communication and stricter monitoring of anesthetic depth may be necessary.

Neurogenesis and developmental anesthetic neurotoxicity.

The mechanism by which anesthetics might act on the developing brain in order to cause long term deficits remains incompletely understood. The hippocampus has been identified as a structure that is likely to be involved, as rodent models show numerous deficits in behavioral tasks of learning that are hippocampal-dependent. The hippocampus is an unusual structure in that it is the site of large amounts of neurogenesis postnatally, particularly in the first year of life in humans, and these newly generated neurons are critical to the function of this structure. Intriguingly, neurogenesis is a major developmental event that occurs during postulated windows of vulnerability to developmental anesthetic neurotoxicity across the different species in which it has been studied. In this review, we examine the evidence for anesthetic effects on neurogenesis in the early postnatal period and ask whether neurogenesis should be studied further as a putative mechanism of injury. Multiple anesthetics are considered, and both in vivo and in vitro work is presented. While there is abundant evidence that anesthetics act to suppress neurogenesis at several different phases, evidence of a causal link between these effects and any change in learning behavior remains elusive.

ERMs colocalize transiently with L1 during neocortical axon outgrowth.

L1 is a member of the Ig superfamily of cell adhesion molecules (CAMs) that functions in many aspects of neuronal development including axonal outgrowth and neuronal migration. These functions require coordination between L1 and the actin cytoskeleton. Because CAMs and the cytoskeleton do not bind directly, membrane-cytoskeletal linkers (MCLs) such as ankyrin are thought to be crucial to their interactions, but data from a knockout mouse suggest that ankyrin is not necessary for the earliest events attributed to L1 function. Recent findings in hippocampal cell culture show that members of the ERM family of proteins (ezrin, radixin, and moesin) can also serve as MCLs between L1 and actin in neurons. Here, we demonstrate that ERM proteins are expressed in extending neuronal processes in the intermediate zone of the developing cortex, a region that is densely packed with migrating neurons and growing axons. ERMs and L1 are codistributed extensively over a transient time course that coincides with rapid axon growth and cortical expansion. This codistribution is strong at embryonic day 17 and 19 but diminishes by postnatal day 0, at which time ankyrin-L1 codistribution increases dramatically. These findings suggest that in the developing neocortex, ERMs are the predominant MCL for L1 during migration and axon extension, neither of which requires ankyrin function. Furthermore, these data suggest that there is a developmentally regulated switch in MCL function in the developing brain.

Conversion of hemiblock to complete heart block by intraoperative motor-evoked potential monitoring.

Intraoperative monitoring of nervous system pathways, including assessing the integrity of descending motor pathways with motor-evoked potentials, is often performed in intracranial and spine operations to reduce the risk of iatrogenic neurological impairment. We present a case in which intraoperative monitoring with motor-evoked potentials resulted in complete heart block in a patient with a history of hemiblock. Neuromonitoring has been associated with arrhythmias in patients with ostensibly normal conduction systems, and we propose that monitoring personnel, anesthesiologists, and surgeons need to be aware of this risk and exercise caution when monitoring motor-evoked potentials in patients with known conduction deficits.

ERM proteins regulate growth cone responses to Sema3A.

Axonal growth cones initiate and sustain directed growth in response to cues in their environment. A variety of events such as receptor internalization, kinase activation, and actin rearrangement can be stimulated by guidance cues and are essential for mediating targeted growth cone behavior. Surprisingly little is known about how such disparate actions are coordinated. Our data suggest that ezrin, radixin, and moesin (ERMs), a family of highly homologous, multifunctional proteins may be able to coordinate growth cone responses to the guidance cue Semaphorin 3A (Sema3A). We show that active ERMs concentrate asymmetrically in neocortical growth cones, are rapidly and transiently inactivated by Sema3A, and are required for Sema3A-mediated growth cone collapse and guidance. The FERM domain of active ERMs regulates internalization of the Sema3A receptor, Npn1, and its coreceptor, L1CAM, while the ERM C-terminal domain binds and caps F-actin. Our data support a model in which ERMs can coordinate membrane and actin dynamics in response to Sema3A.