Implementation of continuous temperature monitoring during perioperative care: a feasibility study.

BACKGROUND: Continuous body temperature monitoring during perioperative care is enabled by using a non-invasive "zero-heat-flux" (ZHF) device. However, rigorous evaluation of whether continuous monitoring capability improves process of care and patient outcomes is lacking. This study assessed the feasibility of a large-scale trial on the impact of continuous ZHF monitoring on perioperative temperature management practices and hypothermia prevention. METHODS: A feasibility study was conducted at a tertiary hospital. Participants included patients undergoing elective surgery under neuraxial or general anesthesia, and perioperative nurses and anesthetists caring for patient participants. Eighty-two patients pre and post introduction of the ZHF device were enrolled. Feasibility outcomes included recruitment and retention, protocol adherence, missing data or device failure, and staff evaluation of intervention feasibility and acceptability. Process of care outcomes included temperature monitoring practices, warming interventions and perioperative hypothermia. RESULTS: There were no adverse events related to the device and feasibility of recruitment was high (60%). Treatment adherence varied across the perioperative pathway (43 to 93%) and missing data due to electronic transfer issues were identified. Provision of ZHF monitoring had most impact on monitoring practices in the Post Anesthetic Care Unit; the impact on intraoperative monitoring practices was minimal. CONCLUSIONS: Enhancements to the design of the ZHF device, particularly for improved data retention and transfer, would be beneficial prior to a large-scale evaluation of whether continuous temperature monitoring will improve patient outcomes. Implementation research designs are needed for future work to improve the complex area of temperature monitoring during surgery.  TRIAL REGISTRATION: Prospective registration prior to patient enrolment was obtained from the Australian and New Zealand Clinical Trials Registry (ANZCTR) on 16th April 2021 (Registration number: ACTRN12621000438853).

Hydrocephalus in Nfix-/- Mice Is Underpinned by Changes in Ependymal Cell Physiology.

Nuclear factor one X (NFIX) is a transcription factor required for normal ependymal development. Constitutive loss of Nfix in mice (Nfix-/-) is associated with hydrocephalus and sloughing of the dorsal ependyma within the lateral ventricles. Previous studies have implicated NFIX in the transcriptional regulation of genes encoding for factors essential to ependymal development. However, the cellular and molecular mechanisms underpinning hydrocephalus in Nfix-/- mice are unknown. To investigate the role of NFIX in hydrocephalus, we examined ependymal cells in brains from postnatal Nfix-/- and control (Nfix+/+) mice using a combination of confocal and electron microscopy. This revealed that the ependymal cells in Nfix-/- mice exhibited abnormal cilia structure and disrupted localisation of adhesion proteins. Furthermore, we modelled ependymal cell adhesion using epithelial cell culture and revealed changes in extracellular matrix and adherens junction gene expression following knockdown of NFIX. Finally, the ablation of Nfix from ependymal cells in the adult brain using a conditional approach culminated in enlarged ventricles, sloughing of ependymal cells from the lateral ventricles and abnormal localisation of adhesion proteins, which are phenotypes observed during development. Collectively, these data demonstrate a pivotal role for NFIX in the regulation of cell adhesion within ependymal cells of the lateral ventricles.

Investigating cortical features of Sotos syndrome using mice heterozygous for Nsd1.

Sotos syndrome is a developmental disorder characterized by a suite of clinical features. In children, the three cardinal features of Sotos syndrome are a characteristic facial appearance, learning disability and overgrowth (height and/or head circumference > 2 SDs above average). These features are also evident in adults with this syndrome. Over 90% of Sotos syndrome patients are haploinsufficient for the gene encoding nuclear receptor-binding Su(var)3-9, Enhancer-of-zesteand Trithorax domain-containing protein 1 (NSD1). NSD1 is a histone methyltransferase that catalyzes the methylation of lysine residue 36 on histone H3. However, although the symptomology of Sotos syndrome is well established, many aspects of NSD1 biology remain unknown. Here, we assessed the expression of Nsd1 within the mouse brain, and showed a predominantly neuronal pattern of expression for this histone-modifying factor. We also generated a mouse strain lacking one allele of Nsd1 and analyzed morphological and behavioral characteristics in these mice, showing behavioral characteristics reminiscent of some of the deficits seen in Sotos syndrome patients.

Partial Loss of USP9X Function Leads to a Male Neurodevelopmental and Behavioral Disorder Converging on Transforming Growth Factor ß Signaling.

BACKGROUND: The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative. METHODS: We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology. RESULTS: Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor ß signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory. CONCLUSIONS: Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor ß signaling and hippocampal function.

NFIX-Mediated Inhibition of Neuroblast Branching Regulates Migration Within the Adult Mouse Ventricular-Subventricular Zone.

Understanding the migration of newborn neurons within the brain presents a major challenge in contemporary biology. Neuronal migration is widespread within the developing brain but is also important within the adult brain. For instance, stem cells within the ventricular-subventricular zone (V-SVZ) and the subgranular zone of dentate gyrus of the adult rodent brain produce neuroblasts that migrate to the olfactory bulb and granule cell layer of the dentate gyrus, respectively, where they regulate key brain functions including innate olfactory responses, learning, and memory. Critically, our understanding of the factors mediating neuroblast migration remains limited. The transcription factor nuclear factor I X (NFIX) has previously been implicated in embryonic cortical development. Here, we employed conditional ablation of Nfix from the adult mouse brain and demonstrated that the removal of this gene from either neural stem and progenitor cells, or neuroblasts, within the V-SVZ culminated in neuroblast migration defects. Mechanistically, we identified aberrant neuroblast branching, due in part to increased expression of the guanylyl cyclase natriuretic peptide receptor 2 (Npr2), as a factor contributing to abnormal migration in Nfix-deficient adult mice. Collectively, these data provide new insights into how neuroblast migration is regulated at a transcriptional level within the adult brain.

Heterozygosity for Nuclear Factor One X in mice models features of Malan syndrome.

BACKGROUND: Nuclear Factor One X (NFIX) haploinsufficiency in humans results in Malan syndrome, a disorder characterized by overgrowth, macrocephaly and intellectual disability. Although clinical assessments have determined the underlying symptomology of Malan syndrome, the fundamental mechanisms contributing to the enlarged head circumference and intellectual disability in these patients remains undefined. METHODS: Here, we used Nfix heterozygous mice as a model to investigate these aspects of Malan syndrome. Volumetric magnetic resonance imaging (MRI) was used to calculate the volumes of 20 brain sub regions. Diffusion tensor MRI was used to perform tractography-based analyses of the corpus callosum, hippocampal commissure, and anterior commissure, as well as structural connectome mapping of the whole brain. Immunohistochemistry examined the neocortical cellular populations. Two behavioral assays were performed, including the active place avoidance task to assess spatial navigation and learning and memory function, and the 3-chambered sociability task to examine social behaviour. FINDINGS: Adult Nfix+/- mice exhibit significantly increased brain volume (megalencephaly) compared to wildtypes, with the cerebral cortex showing the highest increase. Moreover, all three forebrain commissures, in particular the anterior commissure, revealed significantly reduced fractional anisotropy, axial and radial diffusivity, and tract density intensity. Structural connectome analyses revealed aberrant connectivity between many crucial brain regions. Finally, Nfix+/- mice exhibit behavioral deficits that model intellectual disability. INTERPRETATION: Collectively, these data provide a significant conceptual advance in our understanding of Malan syndrome by suggesting that megalencephaly underlies the enlarged head size of these patients, and that disrupted cortical connectivity may contribute to the intellectual disability these patients exhibit. FUND: Australian Research Council (ARC) Discovery Project Grants, ARC Fellowship, NYSTEM and Australian Postgraduate Fellowships.

Neurogenic differentiation by hippocampal neural stem and progenitor cells is biased by NFIX expression.

Our understanding of the transcriptional programme underpinning adult hippocampal neurogenesis is incomplete. In mice, under basal conditions, adult hippocampal neural stem cells (AH-NSCs) generate neurons and astrocytes, but not oligodendrocytes. The factors limiting oligodendrocyte production, however, remain unclear. Here, we reveal that the transcription factor NFIX plays a key role in this process. NFIX is expressed by AH-NSCs, and its expression is sharply upregulated in adult hippocampal neuroblasts. Conditional ablation of Nfix from AH-NSCs, coupled with lineage tracing, transcriptomic sequencing and behavioural studies collectively reveal that NFIX is cell-autonomously required for neuroblast maturation and survival. Moreover, a small number of AH-NSCs also develop into oligodendrocytes following Nfix deletion. Remarkably, when Nfix is deleted specifically from intermediate progenitor cells and neuroblasts using a Dcx-creERT2 driver, these cells also display elevated signatures of oligodendrocyte gene expression. Together, these results demonstrate the central role played by NFIX in neuroblasts within the adult hippocampal stem cell neurogenic niche in promoting the maturation and survival of these cells, while concomitantly repressing oligodendrocyte gene expression signatures.

A morphology independent approach for identifying dividing adult neural stem cells in the mouse hippocampus.

BACKGROUND: Type 1 adult hippocampal neural stem cells (AH-NSCs) continue to generate neurons throughout life, albeit at a very low rate. The relative quiescence of this population of cells has led to many studies investigating factors that may increase their division. Current methods of identifying dividing AH-NSCs in vivo require the identification and tracing of radial processes back to nuclei within the subgranular zone. However, caveats to this approach include the time-intensive nature of identifying AH-NSCs with such a process, as well as the fact that this approach ignores the relatively more active population of horizontally oriented AH-NSCs that also reside in the subgranular zone. RESULTS: Here we describe, and then verify using Hes5::GFP mice, that labeling for the cell cycle marker Ki67 and selection against the intermediate progenitor cell marker TBR2 (Ki67+ve ; TBR2-ve nuclei) is sufficient to identify dividing horizontally and radially oriented AH-NSCs in the adult mouse hippocampus. CONCLUSIONS: These findings provide a simple and accurate way to quantify dividing AH-NSCs in vivo using a morphology-independent approach that will facilitate studies into neurogenesis within the hippocampal stem cell niche of the adult brain. Developmental Dynamics 247:194-200, 2018. © 2017 Wiley Periodicals, Inc.

USP9X deletion elevates the density of oligodendrocytes within the postnatal dentate gyrus.

Neural stem cells (NSCs) within the adult hippocampal dentate gyrus reside in the subgranular zone (SGZ). A dynamic network of signaling mechanisms controls the balance between the maintenance of NSC identity, and their subsequent differentiation into dentate granule neurons. Recently, the ubiquitin-specific protease 9 X-linked (USP9X) was shown to be important for hippocampal morphogenesis, as mice lacking this gene exhibited a higher proportion of proliferating NSCs, yet a decrease in neuronal numbers, within the postnatal dentate gyrus. Here we reveal that Usp9x-deficiency results in the upregulation of numerous oligodendrocytic and myelin-associated genes within the postnatal hippocampus. Moreover, cell counts reveal a significant increase in oligodendrocyte precursor cells and mature oligodendrocytes per unit volume of the mutant dentate gyrus. Collectively, these findings indicate that USP9X may regulate NSC lineage determination within the postnatal SGZ.

Usp9x-deficiency disrupts the morphological development of the postnatal hippocampal dentate gyrus.

Within the adult mammalian brain, neurogenesis persists within two main discrete locations, the subventricular zone lining the lateral ventricles, and the hippocampal dentate gyrus. Neurogenesis within the adult dentate gyrus contributes to learning and memory, and deficiencies in neurogenesis have been linked to cognitive decline. Neural stem cells within the adult dentate gyrus reside within the subgranular zone (SGZ), and proteins intrinsic to stem cells, and factors within the niche microenvironment, are critical determinants for development and maintenance of this structure. Our understanding of the repertoire of these factors, however, remains limited. The deubiquitylating enzyme USP9X has recently emerged as a mediator of neural stem cell identity. Furthermore, mice lacking Usp9x exhibit a striking reduction in the overall size of the adult dentate gyrus. Here we reveal that the development of the postnatal SGZ is abnormal in mice lacking Usp9x. Usp9x conditional knockout mice exhibit a smaller hippocampus and shortened dentate gyrus blades from as early as P7. Moreover, the analysis of cellular populations within the dentate gyrus revealed reduced stem cell, neuroblast and neuronal numbers and abnormal neuroblast morphology. Collectively, these findings highlight the critical role played by USP9X in the normal morphological development of the postnatal dentate gyrus.