Compensation between FOXP transcription factors maintains proper striatal function.

Spiny projection neurons (SPNs) of the striatum are critical in integrating neurochemical information to coordinate motor and reward-based behavior. Mutations in the regulatory transcription factors expressed in SPNs can result in neurodevelopmental disorders (NDDs). Paralogous transcription factors Foxp1 and Foxp2, which are both expressed in the dopamine receptor 1 (D1) expressing SPNs, are known to have variants implicated in NDDs. Utilizing mice with a D1-SPN-specific loss of Foxp1, Foxp2, or both and a combination of behavior, electrophysiology, and cell-type-specific genomic analysis, loss of both genes results in impaired motor and social behavior as well as increased firing of the D1-SPNs. Differential gene expression analysis implicates genes involved in autism risk, electrophysiological properties, and neuronal development and function. Viral-mediated re-expression of Foxp1 into the double knockouts is sufficient to restore electrophysiological and behavioral deficits. These data indicate complementary roles between Foxp1 and Foxp2 in the D1-SPNs.

Compensation between FOXP transcription factors maintains proper striatal function.

Spiny projection neurons (SPNs) of the striatum are critical in integrating neurochemical information to coordinate motor and reward-based behavior. Mutations in the regulatory transcription factors expressed in SPNs can result in neurodevelopmental disorders (NDDs). Paralogous transcription factors Foxp1 and Foxp2, which are both expressed in the dopamine receptor 1 (D1) expressing SPNs, are known to have variants implicated in NDDs. Utilizing mice with a D1-SPN specific loss of Foxp1, Foxp2, or both and a combination of behavior, electrophysiology, and cell-type specific genomic analysis, loss of both genes results in impaired motor and social behavior as well as increased firing of the D1-SPNs. Differential gene expression analysis implicates genes involved in autism risk, electrophysiological properties, and neuronal development and function. Viral mediated re-expression of Foxp1 into the double knockouts was sufficient to restore electrophysiological and behavioral deficits. These data indicate complementary roles between Foxp1 and Foxp2 in the D1-SPNs.

A single-cell trajectory atlas of striatal development.

The striatum integrates dense neuromodulatory inputs from many brain regions to coordinate complex behaviors. This integration relies on the coordinated responses from distinct striatal cell types. While previous studies have characterized the cellular and molecular composition of the striatum using single-cell RNA-sequencing at distinct developmental timepoints, the molecular changes spanning embryonic through postnatal development at the single-cell level have not been examined. Here, we combine published mouse striatal single-cell datasets from both embryonic and postnatal timepoints to analyze the developmental trajectory patterns and transcription factor regulatory networks within striatal cell types. Using this integrated dataset, we found that dopamine receptor-1 expressing spiny projection neurons have an extended period of transcriptional dynamics and greater transcriptional complexity over postnatal development compared to dopamine receptor-2 expressing neurons. Moreover, we found the transcription factor, FOXP1, exerts indirect changes to oligodendrocytes. These data can be accessed and further analyzed through an interactive website ( https://mouse-striatal-dev.cells.ucsc.edu ).

MeCP2 regulates Gdf11, a dosage-sensitive gene critical for neurological function.

Loss- and gain-of-function of MeCP2 causes Rett syndrome (RTT) and MECP2 duplication syndrome (MDS), respectively. MeCP2 binds methyl-cytosines to finely tune gene expression in the brain, but identifying genes robustly regulated by MeCP2 has been difficult. By integrating multiple transcriptomics datasets, we revealed that MeCP2 finely regulates growth differentiation factor 11 (Gdf11). Gdf11 is down-regulated in RTT mouse models and, conversely, up-regulated in MDS mouse models. Strikingly, genetically normalizing Gdf11 dosage levels improved several behavioral deficits in a mouse model of MDS. Next, we discovered that losing one copy of Gdf11 alone was sufficient to cause multiple neurobehavioral deficits in mice, most notably hyperactivity and decreased learning and memory. This decrease in learning and memory was not due to changes in proliferation or numbers of progenitor cells in the hippocampus. Lastly, loss of one copy of Gdf11 decreased survival in mice, corroborating its putative role in aging. Our data demonstrate that Gdf11 dosage is important for brain function.

FOXP1 negatively regulates intrinsic excitability in D2 striatal projection neurons by promoting inwardly rectifying and leak potassium currents.

Heterozygous loss-of-function mutations in the transcription factor FOXP1 are strongly associated with autism. Dopamine receptor 2 expressing (D2) striatal projection neurons (SPNs) in heterozygous Foxp1 (Foxp1+/-) mice have higher intrinsic excitability. To understand the mechanisms underlying this alteration, we examined SPNs with cell-type specific homozygous Foxp1 deletion to study cell-autonomous regulation by Foxp1. As in Foxp1+/- mice, D2 SPNs had increased intrinsic excitability with homozygous Foxp1 deletion. This effect involved postnatal mechanisms. The hyperexcitability was mainly due to down-regulation of two classes of potassium currents: inwardly rectifying (KIR) and leak (KLeak). Single-cell RNA sequencing data from D2 SPNs with Foxp1 deletion indicated the down-regulation of transcripts of candidate ion channels that may underlie these currents: Kcnj2 and Kcnj4 for KIR and Kcnk2 for KLeak. This Foxp1-dependent regulation was neuron-type specific since these same currents and transcripts were either unchanged, or very little changed, in D1 SPNs with cell-specific Foxp1 deletion. Our data are consistent with a model where FOXP1 negatively regulates the excitability of D2 SPNs through KIR and KLeak by transcriptionally activating their corresponding transcripts. This, in turn, provides a novel example of how a transcription factor may regulate multiple genes to impact neuronal electrophysiological function that depends on the integration of multiple current types - and do this in a cell-specific fashion. Our findings provide initial clues to altered neuronal function and possible therapeutic strategies not only for FOXP1-associated autism but also for other autism forms associated with transcription factor dysfunction.

Single-Cell Analysis of Foxp1-Driven Mechanisms Essential for Striatal Development.

The striatum is a critical forebrain structure integrating cognitive, sensory, and motor information from diverse brain regions into meaningful behavioral output. However, the transcriptional mechanisms underlying striatal development at single-cell resolution remain unknown. Using single-cell RNA sequencing (RNA-seq), we examine the cellular diversity of the early postnatal striatum and show that Foxp1, a transcription factor strongly linked to autism and intellectual disability, regulates the cellular composition, neurochemical architecture, and connectivity of the striatum in a cell-type-dependent fashion. We also identify Foxp1-regulated target genes within distinct cell types and connect these molecular changes to functional and behavioral deficits relevant to phenotypes described in patients with FOXP1 loss-of-function mutations. Using this approach, we could also examine the non-cell-autonomous effects produced by disrupting one cell type and the molecular compensation that occurs in other populations. These data reveal the cell-type-specific transcriptional mechanisms regulated by Foxp1 that underlie distinct features of striatal circuitry.

FOXP transcription factors in vertebrate brain development, function, and disorders.

FOXP transcription factors are an evolutionarily ancient protein subfamily coordinating the development of several organ systems in the vertebrate body. Association of their genes with neurodevelopmental disorders has sparked particular interest in their expression patterns and functions in the brain. Here, FOXP1, FOXP2, and FOXP4 are expressed in distinct cell type-specific spatiotemporal patterns in multiple regions, including the cortex, hippocampus, amygdala, basal ganglia, thalamus, and cerebellum. These varied sites and timepoints of expression have complicated efforts to link FOXP1 and FOXP2 mutations to their respective developmental disorders, the former affecting global neural functions and the latter specifically affecting speech and language. However, the use of animal models, particularly those with brain region- and cell type-specific manipulations, has greatly advanced our understanding of how FOXP expression patterns could underlie disorder-related phenotypes. While many questions remain regarding FOXP expression and function in the brain, studies to date have illuminated the roles of these transcription factors in vertebrate brain development and have greatly informed our understanding of human development and disorders. This article is categorized under: Nervous System Development > Vertebrates: General Principles Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics Nervous System Development > Vertebrates: Regional Development.

Controlled aliasing for improved parallel imaging with a 3D spiral staircase trajectory.

PURPOSE: To introduce a modified 3D stack-of-spirals trajectory and efficient SENSE reconstruction for improved through-plane undersampling, while maintaining SNR efficiency and other benefits of spiral acquisitions. METHODS: A novel spiral staircase trajectory is introduced. This trajectory is a modified stack of spirals, in which spiral arms are distributed between partitions along kz . The trajectory maintains the efficient separable reconstruction with a Cartesian fast Fourier transform along the kz direction, followed by a 2D slice-by-slice gridding reconstruction. An additional intermediate step introduces a phase correction to collapse the spiral arms into the prescribed slice planes. For data undersampled through plane, this produces aliasing with reduced coherence, controlled by the arm-ordering. Undersampled data can then be reconstructed with reduced g-factor using a conjugate gradient-based iterative SENSE algorithm. RESULTS: The trajectory significantly improves g-factor for through-plane accelerated acquisitions. Improvement manifests through both reduced overall g-factor and reduced structure in the g-factor maps. In the presented experiments, the mean g-factor decreased from 1.26 to 0.93 and the maximum g-factor decreased from 3.89 to 1.15 for R = 2 spiral staircase when compared with stack of spirals, and the mean g-factor decreased from 2.51 to 0.94 and the maximum g-factor decreased from 8.26 to 1.35 for R = 3 spiral staircase when compared with stack of spirals. CONCLUSION: The novel spiral staircase trajectory offers improved aliasing characteristics for through-plane parallel imaging acceleration in 3D spiral acquisitions.

Beyond bulk: a review of single cell transcriptomics methodologies and applications.

Single-cell RNA sequencing (scRNA-seq) is a promising approach to study the transcriptomes of individual cells in the brain and the central nervous system (CNS). This technology acts as a bridge between neuroscience, computational biology, and systems biology, enabling an unbiased and novel understanding of the cellular composition of the brain and CNS. Gene expression at the single cell resolution is often noisy, sparse, and high-dimensional, creating challenges for computational analysis of such data. In this review, we overview fundamental sample preparation and data analysis processes of scRNA-seq and provide a comparative perspective for analyzing and visualizing these data.

Derepression of sonic hedgehog signaling upon Gpr161 deletion unravels forebrain and ventricular abnormalities.

Inverse gradients of transcriptional repressors antagonize the transcriptional effector response to morphogens. However, the role of such inverse regulation might not manifest solely from lack of repressors. Sonic hedgehog (Shh) patterns the forebrain by being expressed ventrally; however, absence of antagonizing Gli3 repressor paradoxically cause insufficient pathway activation. Interestingly, lack of the primary cilia-localized G-protein-coupled receptor, Gpr161 increases Shh signaling in the mouse neural tube from coordinated lack of Gli3 repressor and Smoothened-independent activation. Here, by deleting Gpr161 in mouse neuroepithelial cells and radial glia at early mid-gestation we detected derepression of Shh signaling throughout forebrain, allowing determination of the pathophysiological consequences. Accumulation of cerebrospinal fluid (hydrocephalus) was apparent by birth, although usual causative defects in multiciliated ependymal cells or aqueduct were not seen. Rather, the ventricular surface was expanded (ventriculomegaly) during embryogenesis from radial glial overproliferation. Cortical phenotypes included polymicrogyria in the medial cingulate cortex, increased proliferation of intermediate progenitors and basal radial glia, and altered neocortical cytoarchitectonic structure with increased upper layer and decreased deep layer neurons. Finally, periventricular nodular heterotopia resulted from disrupted neuronal migration, while the radial glial scaffold was unaffected. Overall, suppression of Shh pathway during early mid-gestation prevents ventricular overgrowth, and regulates cortical gyration and neocortical/periventricular cytoarchitecture.

Foxp1 in Forebrain Pyramidal Neurons Controls Gene Expression Required for Spatial Learning and Synaptic Plasticity.

Genetic perturbations of the transcription factor Forkhead Box P1 (FOXP1) are causative for severe forms of autism spectrum disorder that are often comorbid with intellectual disability. Recent work has begun to reveal an important role for FoxP1 in brain development, but the brain-region-specific contributions of Foxp1 to autism and intellectual disability phenotypes have yet to be determined fully. Here, we describe Foxp1 conditional knock-out (Foxp1cKO) male and female mice with loss of Foxp1 in the pyramidal neurons of the neocortex and the CA1/CA2 subfields of the hippocampus. Foxp1cKO mice exhibit behavioral phenotypes that are of potential relevance to autism spectrum disorder, including hyperactivity, increased anxiety, communication impairments, and decreased sociability. In addition, Foxp1cKO mice have gross deficits in learning and memory tasks of relevance to intellectual disability. Using a genome-wide approach, we identified differentially expressed genes in the hippocampus of Foxp1cKO mice associated with synaptic function and development. Furthermore, using magnetic resonance imaging, we uncovered a significant reduction in the volumes of both the entire hippocampus as well as individual hippocampal subfields of Foxp1cKO mice. Finally, we observed reduced maintenance of LTP in area CA1 of the hippocampus in these mutant mice. Together, these data suggest that proper expression of Foxp1 in the pyramidal neurons of the forebrain is important for regulating gene expression pathways that contribute to specific behaviors reminiscent of those seen in autism and intellectual disability. In particular, Foxp1 regulation of gene expression appears to be crucial for normal hippocampal development, CA1 plasticity, and spatial learning.SIGNIFICANCE STATEMENT Loss-of-function mutations in the transcription factor Forkhead Box P1 (FOXP1) lead to autism spectrum disorder and intellectual disability. Understanding the potential brain-region-specific contributions of FOXP1 to disease-relevant phenotypes could be a critical first step in the management of patients with these mutations. Here, we report that Foxp1 conditional knock-out (Foxp1cKO) mice with loss of Foxp1 in the neocortex and hippocampus display autism and intellectual-disability-relevant behaviors. We also show that these phenotypes correlate with changes in both the genomic and physiological profiles of the hippocampus in Foxp1cKO mice. Our work demonstrates that brain-region-specific FOXP1 expression may relate to distinct, clinically relevant phenotypes.

Three-dimensional ultrashort echo-time imaging using a FLORET trajectory.

PURPOSE: Three-dimensional ultrashort echo-time (UTE) imaging commonly makes use of an isotropic 3D radial projection acquisition. The FLORET sequence is proposed and evaluated as a more efficient alternative. METHODS: The properties of the FLORET trajectory are contrasted with those of a 3D radial projection trajectory. The theoretical advantages of FLORET, including greater sampling and SNR efficiency, are evaluated based upon experimental data. The effect of T2* decay on FLORET is analyzed in comparison to the 3D radial, Cones, and Density Adapted Radial trajectories. FLORET UTE image quality is compared with 3D radial UTE image quality. RESULTS: FLORET is shown to have several advantages over 3D radial acquisitions with respect to image quality, scan time, signal-to-noise, and off-resonance blurring for UTE data. The signal and resolution losses from T2* decay for a FLORET acquisition are shown to be comparable to those of Density Adapted Radial and Density Compensated Cones trajectories. CONCLUSION: The FLORET sequence is recommended as an alternative to 3D radial projection sequences for musculoskeletal UTE imaging as well as other UTE applications that accommodate modest to long per shot sampling times. FLORET is not recommended for imaging extremely short T2 species such as dentin. Magn Reson Med 78:1038-1049, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

FoxP1 orchestration of ASD-relevant signaling pathways in the striatum.

Mutations in the transcription factor Forkhead box p1 (FOXP1) are causative for neurodevelopmental disorders such as autism. However, the function of FOXP1 within the brain remains largely uncharacterized. Here, we identify the gene expression program regulated by FoxP1 in both human neural cells and patient-relevant heterozygous Foxp1 mouse brains. We demonstrate a role for FoxP1 in the transcriptional regulation of autism-related pathways as well as genes involved in neuronal activity. We show that Foxp1 regulates the excitability of striatal medium spiny neurons and that reduction of Foxp1 correlates with defects in ultrasonic vocalizations. Finally, we demonstrate that FoxP1 has an evolutionarily conserved role in regulating pathways involved in striatal neuron identity through gene expression studies in human neural progenitors with altered FOXP1 levels. These data support an integral role for FoxP1 in regulating signaling pathways vulnerable in autism and the specific regulation of striatal pathways important for vocal communication.

Respiratory-induced venous blood flow effects using flexible retrospective double-gating.

BACKGROUND: To demonstrate a novel velocity sensitive acquisition and retrospective cardiorespiratory double-gated reconstruction scheme to examine respiratory effect on venous blood flow in healthy volunteers. METHODS: Radial two dimensional (2D) phase contrast MR is performed at 3 Tesla in the internal jugular vein (IJV) of healthy volunteers (n = 6). Data are retrospectively partitioned based on respiratory waveforms using three schemes: moving average for respiration plateaus, gradient for active respiration, and ten respiratory phases that are cardiac time-averaged. A single 4D flow MR scan is performed in the neck of a healthy volunteer. After gradient operation, blood velocity measurements are made along the IJV length. Percent changes from expiration to inspiration for moving average and gradient techniques are statistically compared with paired t-tests. RESULTS: Percent change increase in summed IJV mean and peak blood flow during active inspiration versus active expiration in 2D was significant (mean flow: 11.5 ± 8.0%, peak flow: 11.9 ± 5.9%, P < 0.01). Smallest cross-sectional area and largest blood velocity are seen during inspiration phases (phase number: area-6.5 ± 3.6, velocity-6.2 ± 3.2). Significant increase in mean velocity along the length of the IJV was observed in 3D, with increasing percent changes more proximal to the chest (mean, 39 ± 30%; range, 0-93%, P = 0.001). CONCLUSION: With a radial acquisition, this pilot study demonstrates feasibility of simultaneous retrospective cardiorespiratory gating in IJV flow. Greatest differences in flow occur between active respiration phases, increasing in magnitude more proximal to the chest.

Adaptive retrospective correction of motion artifacts in cranial MRI with multicoil three-dimensional radial acquisitions.

Despite reduction in imaging times through improved hardware and rapid acquisition schemes, motion artifacts can compromise image quality in magnetic resonance imaging, especially in three-dimensional imaging with its prolonged scan durations. Direct extension of most state-of-the-art two-dimensional rigid body motion compensation techniques to the three-dimensional case is often challenging or impractical due to a significant increase in sampling requirements. This article introduces a novel motion correction technique that is capable of restoring image quality in motion corrupted two-dimensional and three-dimensional radial acquisitions without a priori assumptions about when motion occurs. The navigating properties of radial acquisitions-corroborated by multiple receiver coils-are exploited to detect actual instances of motion. Pseudorandom projection ordering provides flexibility of reconstructing navigator images from the obtained motion-free variable-width subsets for subsequent estimation of rigid body motion parameters by coregistration. The proposed approach does not require any additional navigators or external motion estimation schemes. The capabilities and limitations of the method are described and demonstrated through simulations and representative volunteer cranial acquisitions.

High levels of retinal docosahexaenoic acid do not protect photoreceptor degeneration in VPP transgenic mice.

PURPOSE: To determine whether docosahexaenoic acid can protect against hereditary retinal degenerations in transgenic mice expressing the V20G, P23H, and P27L (VPP) rhodopsin mutations. METHODS: Female transgenic mice expressing the VPP rhodopsin mutation, known to cause a retinal degeneration, were bred to male transgenic mice expressing the fat-1 gene, which can convert n6 to n3 polyunsaturated fatty acids (PUFA). Several weeks before breeding, the female mice were fed a standard diet containing 10% safflower oil (SFO), which is high in n6 and low in n3 PUFA (n6/n3=273). Offspring were genotyped and four groups identified: Fat1(+)/VPP(+), Fat1(-)/VPP(+), Fat1(+)/VPP(-), and Fat1(-)/VPP(-). Dams were maintained on the SFO diet during the nursing period and offspring were kept on the SFO diet after weaning. At 4, 16, and 28 weeks of age, retinal function was evaluated by electroretinography (ERG), photoreceptor cell loss was quantified by measuring outer nuclear layer thickness, and rhodopsin levels were determined. Fatty acid profiles were analyzed in whole retina, plasma, and liver at 4 and 28 weeks of age. RESULTS: Expression of fat-1 in the absence of dietary n3 PUFA led to the generation of two groups of mice with distinctly different levels of n3 and n6 PUFA in the three tissues that were analyzed. Already at four weeks of age, the retinas of fat-1 positive animals had higher levels of n3 PUFA than their wild-type counterparts (23%-29% versus 6.4%-6.5%). In addition, by four weeks of age, there was a significant loss of rod photoreceptor cells in the VPP mice. Progression of retinal degeneration occurred with increasing age in VPP positive mice, with photoreceptor cell death occurring in both inferior and superior regions. Amplitudes of the a- and b-waves of the ERG were significantly reduced with age, with VPP positive mice showing the greatest change. Rhodopsin content was lower in the VPP transgenic mice. There were no significant differences in outer nuclear layer thickness or ERG amplitudes between Fat1(+)/VPP(+) and Fat1(-)/VPP(+), or between Fat1(+)/VPP(-)and Fat1(-)/VPP(-) mice at any of the three ages. CONCLUSIONS: High levels of retinal docosahexaenoic acid do not protect mice expressing the VPP rhodopsin mutation from retinal degeneration.

DHA does not protect ELOVL4 transgenic mice from retinal degeneration.

PURPOSE: Dominant Stargardt macular dystrophy (STGD3) is caused by several different mutations in a gene named ELOVL4, which shares sequence homologies with a family of genes that encode proteins involved in the ELOngation of Very Long chain fatty acids. Studies have suggested that patients with STGD3 have aberrant metabolism of docosahexaenoic acid (DHA, 22:6n3), the major polyunsaturated fatty acid (PUFA) in retinal rod outer segment membranes. We tested the effect of DHA on the progression of retinal degeneration in transgenic mice that express one of the mutations identified in STGD3. METHODS: Transgenic mice expressing mutant human ELOVL4 (TG2) were bred to mice expressing the fat-1 protein, which can convert n6 to n3 PUFA. Mice were maintained on an n3-deficient diet containing 10% safflower oil (SFO, enriched in n6 PUFA; n6/n3=273) so that four experimental groups were produced that differed only in levels of n3 PUFA and expression of the hELOVL4 transgene. These groups were identified by genotyping and named Fat1+/TG2+, Fat1(-)/TG2+, Fat1+/TG2(-), and Fat1(-)/TG2(-). All were continued on the SFO diet for 4 to 16 weeks such that those not expressing Fat1 would be deficient in n3 fatty acids. At both time points, animals were analyzed for retinal function by electroretinography (ERG), photoreceptor cell viability by outer nuclear layer (ONL) thickness measurements, fatty acid profiles in several tissues, and rhodopsin levels. RESULTS: Mice expressing the fat-1 transgene had significantly higher levels of n3 PUFA, primarily DHA, in retina, liver, and plasma lipids at 4 and 16 weeks of age. Retinal DHA levels in fat-1 mice were twice those of controls. By 16 weeks of age, mice expressing the mutant hELOVL4 transgene had a significantly greater loss of photoreceptor cells, reduced ERG amplitudes, and lower rhodopsin levels than control mice. There was no effect of retinal fatty acids on the rate of degeneration of retinas expressing the ELOVL4 transgene. CONCLUSIONS: We found no evidence that high levels of DHA in retinal membranes protected photoreceptor cells expressing mutant ELOVL4 from retinal degeneration. We conclude that DHA is not beneficial for the treatment of retinal degeneration in this animal model of human STGD3 macular dystrophy.

Microbiologic assessment of disposable sterile endoscopic sheaths to replace high-level disinfection in reprocessing: a prospective clinical trial with nasopharygoscopes.

BACKGROUND: Conventional reprocessing of endoscopes with high-level disinfection is labor intensive, expensive, delays the turnover of instruments, and involves potential exposure of personnel to toxic chemicals. We report a prospective clinical trial with rigorous microbiologic assessment of a novel disposable, sterile, polyurethane sheath, which can be easily and snugly applied over a nasopharyngoscope before performing the endoscopic procedure, with enzymatic cleansing and disinfection of the instrument with 70% ethanol following the procedure to determine whether the use of the novel sheath can provide reliable protection against bacterial contamination and obviate the need for routine high-level disinfection in reprocessing. METHODS: Baseline cultures were obtained at 3 time periods from the control heads and insertion shafts of nasopharyngoscopes used in 100 clinical examinations: before application of the protective sheath and execution of the procedure; immediately after the procedure and removal of the sheath; and after enzymatic cleaning, disinfection with 70% ethanol, and drying. All 100 used sheaths and 20 unused sheaths were subjected to high-pressure leak testing to assess barrier integrity. RESULTS: Bacteria were detected on 16 control heads and 6 shafts prior to the procedure; from 13 heads and 1 shaft immediately following the nasopharyngoscopic procedure and sterile sheath removal; and none of the instruments following cleaning, ethanol disinfection, and drying. No sheath showed loss of barrier integrity on leak testing. CONCLUSION: Use of a high-quality, snugly fitting, sterile, disposable polyurethane sheath on a nasopharyngoscope during a clinical examination, combined with enzymatic detergent cleaning and disinfection with 70% ethanol, can provide a reliably decontaminated, patient-ready instrument, eliminating the need for high-level disinfection of endoscopes.