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Deafness is the most common sensory impairment, affecting approximately 5% or 430 million people worldwide as per the World Health Organization

This research is the first to produce induced pluripotent stem cell-derived inner ear sensory neurons in the Neurog1+/− heterozygote mouse using blastocyst complementation. Additionally, this approach corrected non-sensory deficits associated with Neurog1 heterozygosity, indicating that complementation is specific to endogenous Neurog1 function. This work validates the use of blastocyst complementation as a tool to create novel insight into the function of developmental genes and highlights blastocyst complementation as a potential platform for generating chimeric inner ear cell types that can be transplanted into damaged inner ears to improve hearing.

Postmortem examination of the cochlea with a cochlear implant in the scala tympani presents several challenges. It is technologically difficult to section a cochlea with an implant due to the presence of its wires and metal components that are adjacent to the membranous and bony tissues of the cochlea. These metal components damage traditional steel blades of a microtome in celloidin, paraffin or frozen embedded tissues. However, plastic embedded implanted cochleas have been successfully sectioned using specialized methods (Irving et al., 2013). An alternative non-destructive method is to optically section a chemically cleared cochlea using light-sheet microscopy, which we will describe in this publication. However, since this method uses a light-sheet to section the cochlea the opaque and reflective metal components of the implant results in some artifacts in the 2D optical sections. The best image quality using light-sheet fluorescent microscopy is when the implant is removed prior to imaging.

This research is the first to produce induced pluripotent stem cell-derived inner ear sensory neurons in the Neurog1 +/- heterozygote mouse using blastocyst complementation. Additionally, this approach corrected non-sensory deficits associated with Neurog1 heterozygosity, indicating that complementation is specific to endogenous Neurog1 function. This work validates the use of blastocyst complementation as a tool to create novel insight into the function of developmental genes and highlights blastocyst complementation as a potential platform for generating chimeric inner ear cell types that can be transplanted into damaged inner ears to improve hearing.

This research is the first to produce induced pluripotent stem cell-derived inner ear sensory neurons in the Neurog1+/− heterozygote mouse using blastocyst complementation. Additionally, this approach corrected non-sensory deficits associated with Neurog1 heterozygosity, indicating that complementation is specific to endogenous Neurog1 function. This work validates the use of blastocyst complementation as a tool to create novel insight into the function of developmental genes and highlights blastocyst complementation as a potential platform for generating chimeric inner ear cell types that can be transplanted into damaged inner ears to improve hearing. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-021-02184-1.

Passive neutron coincidence/multiplicity counters are subject to non-ideal behavior, such as double pulsing and dead time. It has been shown in the past that double-pulsing exhibits a distinct signature in a Rossi-alpha distribution, which is not readily noticed using traditional Multiplicity Shift Register analysis. However, it has been assumed that the use of a pre-delay in shift register analysis removes any effects of double pulsing. In this work, we use high-fidelity simulations accompanied by experimental measurements to study the effects of double pulsing on multiplicity rates. By exploiting the information from the double pulsing signature peak observable in the Rossi-alpha distribution, the double pulsing fraction can be determined. Algebraic correction factors for the multiplicity rates in terms of the double pulsing fraction have been developed. We discuss the role of these corrections across a range of scenarios.

The transcription factor sex determining region Y-box 2 (SOX2) is required for the formation of hair cells and supporting cells in the inner ear and is a widely used sensory marker. Paradoxically, we demonstrate via fate mapping that, initially, SOX2 primarily marks nonsensory progenitors in the mouse cochlea, and is not specific to all sensory regions until late otic vesicle stages. SOX2 fate mapping reveals an apical-to-basal gradient of SOX2 expression in the sensory region of the cochlea, reflecting the pattern of cell cycle exit. To understand SOX2 function, we undertook a timed-deletion approach, revealing that early loss of SOX2 severely impaired morphological development of the ear, whereas later deletions resulted in sensory disruptions. During otocyst stages, SOX2 shifted dramatically from a lateral to medial domain over 24-48 h, reflecting the nonsensory-to-sensory switch observed by fate mapping. Early loss or gain of SOX2 function led to changes in otic epithelial volume and progenitor proliferation, impacting growth and morphological development of the ear. Our study demonstrates a novel role for SOX2 in early otic morphological development, and provides insights into the temporal and spatial patterns of sensory specification in the inner ear.

Globally 360 million people have disabling hearing loss and, of these, 32 million are children. Human hearing relies on 15,000 hair cells that transduce mechanical vibrations to electrical signals in the auditory nerve. The process is powered by the endo-cochlear potential, which is produced by a vascularized epithelium that actively transports ions in conjunction with a gap junction (GJ) system. This "battery" is located "off-site" in the lateral wall of the cochlea. The GJ syncytium contains the GJ protein genes beta 2 (GJB2/connexin26 (Cx26)) and 6 (GJB6/connexin30 (Cx30)), which are commonly involved in hereditary deafness. Because the molecular arrangement of these proteins is obscure, we analyze GJ protein expression (Cx26/30) in human cochleae by using super-resolution structured illumination microscopy. At this resolution, the Cx26 and Cx30 proteins were visible as separate plaques, rather than being co-localized in heterotypic channels, as previously suggested. The Cx26 and Cx30 proteins thus seem not to be co-expressed but to form closely associated assemblies of GJ plaques. These results could assist in the development of strategies to treat genetic hearing loss in the future.

In the field of nuclear safeguards, passive neutron multiplicity counting (PNMC) is a method typically employed in non-destructive assay (NDA) of special nuclear material (SNM) for nonproliferation, verification and accountability purposes. PNMC is generally performed using a well-type thermal neutron counter and relies on the detection of correlated pairs or higher order multiplets of neutrons emitted by an assayed item. To assay SNM, a set of parameters for a given well-counter is required to link the measured multiplicity rates to the assayed item properties. Detection efficiency, die-away time, gate utilization factors (tightly connected to die-away time) as well as optimum gate width setting are among the key parameters. These parameters along with the underlying model assumptions directly affect the accuracy of the SNM assay. In this paper we examine the role of gate utilization factors and the single exponential die-away time assumption and their impact on the measurements for a range of plutonium materials. In addition, we examine the importance of item-optimized coincidence gate width setting as opposed to using a universal gate width value. Finally, the traditional PNMC based on multiplicity shift register electronics is extended to Feynman-type analysis and application of this approach to Pu mass assay ismore » demonstrated.« less

Various methods of autocorrelation neutron analysis may be used to extract information about a measurement item containing spontaneously fissioning material. The two predominant approaches being the time correlation analysis (that make use of a coincidence gate) methods of multiplicity shift register logic and Feynman sampling. The common feature is that the correlated nature of the pulse train can be described by a vector of reduced factorial multiplet rates. We call these singlets, doublets, triplets etc. Within the point reactor model the multiplet rates may be related to the properties of the item, the parameters of the detector, and basic nuclear data constants by a series of coupled algebraic equations – the so called point model equations. Solving, or inverting, the point model equations using experimental calibration model parameters is how assays of unknown items is performed. Currently only the first three multiplets are routinely used. In this work we develop the point model equations to higher order multiplets using the probability generating functions approach combined with the general derivative chain rule, the so called Faa di Bruno Formula. Explicit expression up to 5th order are provided, as well the general iterative formula to calculate any order. This work represents the first necessary step towards determining if higher order multiplets can add value to nondestructive measurement practice for nuclear materials control and accountancy.

TMPRSS3 (Trans-membrane Serine Protease 3) is a type II trans-membrane serine protease that has proteolytic activity essential for hearing. Mutations in the gene cause non-syndromic autosomal recessive deafness (DFNB8/10) in humans. Knowledge about its cellular distribution in the human inner ear may increase our understanding of its physiological role and involvement in deafness, ultimately leading to therapeutic interventions. In this study, we used super-resolution structured illumination microscopy for the first time together with transmission electron microscopy to localize the TMPRSS3 protein in the human organ of Corti. Archival human cochleae were dissected out during petroclival meningioma surgery. Microscopy with Zeiss LSM710 microscope achieved a lateral resolution of approximately 80 nm. TMPRSS3 was found to be associated with actin in both inner and outer hair cells. TMPRSS3 was located in cell surface-associated cytoskeletal bodies (surfoskelosomes) in inner and outer pillar cells and Deiters cells and in subcuticular organelles in outer hair cells. Our results suggest that TMPRSS3 proteolysis is linked to hair cell sterociliary mechanics and to the actin/microtubule networks that support cell motility and integrity. Electronic supplementary material The online version of this article (10.1007/s00441-018-2793-2) contains supplementary material, which is available to authorized users.

Introduction Cochlear micromechanics and frequency tuning depend on the macromolecular organization of the basilar membrane (BM), which is still unclear in man. Novel techniques in cochlear implantation (CI) motivate further analyses of the BM. Materials and methods Normal cochleae from patients undergoing removal of life-threatening petro-clival meningioma and an autopsy specimen from a normal human were used. Laser-confocal microscopy, high resolution scanning (SEM) and transmission electron microscopy (TEM) were carried out in combination. In addition, one human temporal bone was decellularized and investigated by SEM. Results The human BM consisted in four separate layers: (1) epithelial basement membrane positive for laminin-β2 and collagen IV, (2) BM “proper” composed of radial fibers expressing collagen II and XI, (3) layer of collagen IV and (4) tympanic covering layer (TCL) expressing collagen IV, fibronectin and integrin. BM thickness varied both radially and longitudinally (mean 0.55–1.16 μm). BM was thinnest near the OHC region and laterally. Conclusions There are several important similarities and differences between the morphology of the BM in humans and animals. Unlike in animals, it does not contain a distinct pars tecta (arcuate) and pectinata. Its width increases and thickness decreases as it travels apically in the cochlea. Findings show that the human BM is thinnest and probably most vibration-sensitive at the outer pillar feet/Deiter cells at the OHCs. The inner pillar and IHCs seem situated on a fairly rigid part of the BM. The gradient design of the BM suggests that its vulnerability increases apical wards when performing hearing preservation CI surgery. Electronic supplementary material The online version of this article (doi:10.1007/s00441-014-2098-z) contains supplementary material, which is available to authorized users.

In the measurement field of international nuclear safeguards, passive neutron coincidence counting is used to quantify the spontaneous fission rate of certain special nuclear materials. The shift register autocorrelation analysis method is the most commonly used approach. However, the Feynman-Y technique, which is more commonly applied in reactor noise analysis, provides an alternative means to extract the correlation information from a pulse train. In this work we consider how to select the optimum gate width for each of these two time-correlation analysis techniques. The optimum is considered to be that which gives the lowest fractional precision on the net doublets rate. Our theoretical approach is approximate but is instructional in terms of revealing the key functional dependence. We show that in both cases the same performance figure of merit applies so that common design criteria apply to the neutron detector head. Our prediction is that near optimal results, suitable for most practical applications, can be obtained from both techniques using a common gate width setting. The estimated precision is also comparable in the two cases. The theoretical expressions are tested experimentally using 252Cf spontaneous fission sources measured in two thermal well counters representative of the type in common use by international inspectorates. Fast accidental sampling was the favored method of acquiring the Feynman-Y data. Our experimental study confirmed the basic functional dependences predicted although experimental results when available are preferred. With an appropriate gate setting Feynman-Y analysis provides an alternative to shift register analysis for safeguards applications which is opening up new avenues of data collection and data reduction to explore.

The blood vessels that supply the inner ear form a barrier between the blood and the inner ear fluids to control the exchange of solutes, protein, and water. This barrier, called the blood-labyrinth barrier (BLB) is analogous to the blood-brain barrier (BBB), which plays a critical role in limiting the entry of inflammatory and infectious agents into the central nervous system. We have developed an in vivo method to assess the functional integrity of the BLB by injecting sodium fluorescein into the systemic circulation of mice and measuring the amount of fluorescein that enters perilymph in live animals. In these experiments, perilymph was collected from control and experimental mice in sequential samples taken from the posterior semicircular canal approximately 30 min after systemic fluorescein administration. Perilymph fluorescein concentrations in control mice were compared with perilymph fluorescein concentrations after lipopolysaccharide (LPS) treatment (1 mg/kg IP daily for 2 days). The concentration of perilymphatic fluorescein, normalized to serum fluorescein, was significantly higher in LPS-treated mice compared to controls. In order to assess the contributions of perilymph and endolymph in our inner ear fluid samples, sodium ion concentration of the inner ear fluid was measured using ion-selective electrodes. The sampled fluid from the posterior semicircular canal demonstrated an average sodium concentration of 145 mM, consistent with perilymph. These experiments establish a novel technique to assess the functional integrity of the BLB using quantitative methods and to provide a comparison of the BLB to the BBB.

Administration of an aminoglycoside antibiotic and loop diuretic causes damage to hair cells in the organ of Corti, resulting in their death and the death of their corresponding spiral ganglion neurons. While this phenomenon has been studied previously, analysis of its effects in the whole cochlea has not been reported. The authors sought to evaluate the effects of a combination dose of kanamycin and furosemide in mice cochlea using an imaging system and computer analysis that allowed for nondestructive, whole-cochlea visualization.Study using an animal model.Cochlear analysis laboratory.Five mice received kanamycin and furosemide and 3 mice received saline. Cochleas were harvested and imaged with scanning thin-sheet laser imaging microscopy (sTSLIM) to analyze sensory cells and cochlea structures.The drug-treated animals showed substantial loss of inner hair cells and complete outer hair cell loss. All treated mice showed spiral ganglion neuron loss with fewer neurons than control animals and decreased cell density in the middle turn of the cochlea. The spiral ligament and spiral limbus in the treated animals also showed a decrease in fibrocyte cell density in the middle to apical portion of the cochlea. The stria vascularis appeared normal in all animals.Imaging methods that allow for whole-cochlea analysis provide insight into changes that occur in the cochlea after ototoxic insult. Trends that may not be apparent in cross-section samples of the cochlea can be observed. Computer analysis of these trends allows them to be assessed accurately.

Neutron emissions from fissioning nuclear material are temporally correlated. The detection of these correlated neutrons is frequently used to quantify plutonium (Pu) and other fissile materials for international nuclear safeguards and related activities. However, detector dead time affects the observed rates of correlated neutrons in a non-trivial manner, and must be accounted for to obtain accurate results. A major simplification made in the most widely used dead time corrections is that the neutron detections are occurring randomly in time. A few previous attempts at providing a dead time model for correlated neutrons have been limited in scope, have made simplifying assumptions early in the derivation, and have, in general, not been implemented in the broader safeguards community. This paper provides an exact dead time model for correlated neutron detections in a single channel system assuming an updating dead time, and therefore a paralyzable system. This dead time model includes the assumption that a single exponential, with one characteristic decay constant, can describe the system neutron die-away profile. An exact model for the effects of dead time on measured gate moments is derived which is extendable to an arbitrary order of neutron correlation. This dead time model predicts the measured gate moments based on the dead time and underlying detection rates, including the effects from detection rates with an arbitrarily high order of correlation. The effects of dead time on the apparent singles, doubles, triples and quadruples rates using either event triggered, random or mixed gate structure is also derived. Either the equations for the measured gate moments or the apparent multiplicity rates can be numerically inverted to find the dead time corrected multiplicity rates. Although the model has been explicitly solved for rates up to and including quadruples, it is directly extendable to any order of correlation. This model is presented from the perspective of neutron counting. However, the model is mathematically applicable to any series of events which are temporally correlated through an exponential probability distribution and which are subject to a dead time-like filter.

Passive Neutron Coincidence Counting (PNCC) based on shift register autocorrelation time analysis of the detected neutron pulse train is an important Nondestructive Assay (NDA) method. It is used extensively in the quantification of plutonium and other spontaneously fissile materials for purposes of nuclear materials accountancy. In addition to the totals count rate, which is also referred to as the singles, gross or trigger rate, a quantity known as the reals coincidence rate, also called the pairs or doubles, is obtained from the difference between the measured neutron multiplicities in two measurement gates triggered by the incoming events on the pulse train. The reals rate is a measure of the number of time correlated pairs present on the pulse train and this can be related to the fission rates (and hence material mass) since fissions emit neutrons in bursts which are also detected in characteristic clusters. A closely related measurement objective is the determination of the reactivity of systems as they approach criticality. In this field an alternative autocorrelation signature is popular, the so called Feynman variance-to-mean technique which makes use of the multiplicity histogram formed the periodic, or clock-triggered opening of a coincidence gate. Workers in these two application areas share common challenges and improvement opportunities but are often separated by tradition, problem focus and technical language. The purpose of this paper is to recognize the close link between the Feynman variance-to-mean metric and traditional PNCC using shift register logic applied to correlated pulse trains. We, show using relationships for the late-gate (or accidentals) histogram recorded using a multiplicity shift register, how the Feynman Y -statistic, defined as the excess variance-to-mean ratio, can be expressed in terms of the singles and doubles rates familiar to the safeguards and waste assay communities. These two specialisms now have a direct bridge between them and we anticipate fruitful cross fertilization, for example on assay algorithms, including corrections for measurement item perturbation factors, and on data acquisition systems.

In this article we extend the neutron multiplicity counting dead time correction scheme devised by Dytlewski to include Singles. In this way a self consistent, item specific, Singles dead time correction is obtained under the same physical assumptions and approximations as are used for the Doubles and Triples corrections. This correction for the Singles count rate also affects the Doubles and Triples dead time correction because it is also the correction needed for the number of coincidence gates opened. We develop the new dead time formula, which we call the Croft–Dytlewski formula, as an action on the Accidentals histogram, and apply it to simulated pulse train data for which the correct results are known. The new formula was found to perform better than the standard ad-hoc Dytlewski Singles dead time correction formula, particularly for highly correlated high count rate items.

A standard nondestructive assay technique for determining the mass of plutonium in an item is passive neutron coincidence counting. In passive neutron coincidence counting, the dead time or rate loss corrections for the singles and doubles neutron counting rates are routinely made using empirical relationships that are based on the design and performance of the individual counter used to make the measurements. The empirical methods were developed ahead of any supporting theory for dead time losses and have worked well to date for the majority of safeguards measurement scenarios. Modern applications using highly efficient systems with short neutron lifetimes together with the challenges posed by highly multiplying items mean dead time corrections of higher fidelity are needed. While many attempts have been made to develop dead time corrections that are based on the physical principles of the measurements being performed, these corrections have often been found to be difficult to implement in a real system. For instance, Matthes and Haas developed an approach in 1985 which did not gain favor largely because the form of the doubles correction apparently required numerical integration which was difficult to implement with the computer technology of that time. A recent review into the approach that was developed by Matthes and Haas has determined that a straightforward analytical expression can be derived for the doubles correction that is similar to the singles rate correction that was developed in their original work. An analysis of the expressions is presented here to show how they relate to the traditional empirical methods. Further, we illustrate their implications and limitations. For instance the empirical methods do not address within burst losses i.e. rate related losses due to the correlated neutron bursts from fission, whereas the Matthes and Hass expression for singles counting does exhibit such an effect.

Recently two papers on how to perform passive neutron auto-correlation analysis on time gated histograms formed from pulse train data, generically called time correlation analysis (TCA), have appeared in this journal Dubi et al. Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment (673) (2012) 111; Croft et al. Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment (691) (2012) 152. For those of us working in international nuclear safeguards these treatments are of particular interest because passive neutron multiplicity counting is a widely deployed technique for the quantification of plutonium. The purpose of this letter is to show that the skewness-variance-mean (SVM) approach developed in Dubi et al. (Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment (673) (2012) 111) is equivalent in terms of assay capability to the random trigger interval (RTI) analysis laid out in Croft et al. Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment (691) (2012) 152. Mathematically we could also use other numerical ways to extract the time correlated information from the histogram data including, for example, what we might call the mean, mean square, and mean cube approach. The important feature however, from the perspective of real world applications, is that the correlated information extracted is the same, and subsequently gets interpreted in the same way based on the same underlying physics model.

We report replacement of one side of a static illumination, dual sided, thin-sheet laser imaging microscope (TSLIM) with an intensity modulated laser scanner in order to implement structured illumination (SI) and HiLo image demodulation techniques for background rejection. The new system is equipped with one static and one scanned light-sheet and is called a scanning thin-sheet laser imaging microscope (sTSLIM). It is an optimized version of a light-sheet fluorescent microscope that is designed to image large specimens (

Light sheet fluorescence microscopy (LSFM) functions as a non-destructive microtome and microscope that uses a plane of light to optically section and view tissues with subcellular resolution. This method is well suited for imaging deep within transparent tissues or within whole organisms, and because tissues are exposed to only a thin plane of light, specimen photobleaching and phototoxicity are minimized compared to wide-field fluorescence, confocal, or multiphoton microscopy. LSFMs produce well-registered serial sections that are suitable for three-dimensional reconstruction of tissue structures. Because of a lack of a commercial LSFM microscope, numerous versions of light sheet microscopes have been constructed by different investigators. This review describes development of the technology, reviews existing devices, provides details of one LSFM device, and shows examples of images and three-dimensional reconstructions of tissues that were produced by LSFM.

We describe 2 cases of bilateral Ménière disease with features resembling autoimmune inner ear disease in patients who were found to be carriers of human leukocyte antigen (HLA) B27. For immunohistochemical analysis, mouse inner ear sections were used as the tissue substrate for reaction with serum. Both patients demonstrated an increased immunofluorescence reaction compared with a normal control. We suggest that an antibody-mediated mechanism may be responsible for HLA-B27–associated Ménière disease.

The mouse cochlea database (MCD) provides an interactive, image database of the mouse cochlea for learning its anatomy and data mining of its resources. The MCD website is hosted on a centrally maintained, high-speed server at the following URL: (http://mousecochlea.umn.edu). The MCD contains two types of image resources, serial 2D image stacks and 3D reconstructions of cochlear structures. Complete image stacks of the cochlea from two different mouse strains were obtained using orthogonal plane fluorescence optical microscopy (OPFOS). 2D images of the cochlea are presented on the MCD website as: viewable images within a stack, 2D atlas of the cochlea, orthogonal sections, and direct volume renderings combined with isosurface reconstructions. In order to assess cochlear structures quantitatively, "true" cross-sections of the scala media along the length of the basilar membrane were generated by virtual resectioning of a cochlea orthogonal to a cochlear structure, such as the centroid of the basilar membrane or the scala media. 3D images are presented on the MCD website as: direct volume renderings, movies, interactive QuickTime VRs, flythrough, and isosurface 3D reconstructions of different cochlear structures. 3D computer models can also be used for solid model fabrication by rapid prototyping and models from different cochleas can be combined to produce an average 3D model. The MCD is the first comprehensive image resource on the mouse cochlea and is a new paradigm for understanding the anatomy of the cochlea, and establishing morphometric parameters of cochlear structures in normal and mutant mice.

A new neutron multiplicity counter is being developed that utilizes the fast response of liquid scintillator detectors. The ability to detect fast (vs. moderated) fission neutrons makes possible a coincidence gate of the order of tens of nanoseconds (vs. tens of microseconds). A neutron counter with such a narrow gate will be virtually insensitive to accidental coincidences, making it possible to measure items with a high single neutron background to greater accuracy in less time. This includes impure Pu items with high (α, n) rates as well as items of low-mass HEU where a strong active interrogation source is needed. Liquid scintillator detectors also allow for energy discrimination between interrogation source neutrons and fission neutrons, allowing for even greater assay sensitivity. Designing and building a liquid scintillator multiplicity counter (LSMC) requires a symbiotic effort of simulation and experiment to optimize performance and mitigate hardware costs in the final product. We present preliminary Monte-Carlo studies using the GEANT toolkit.

A novel class of scintillating materials, which are nanocomposites of known scintillators, is described. These nanocomposite materials are expected to have improved properties with respect to the properties of the bulk scintillators from which they are derived. Improvements include enhanced light output, reduced cost and greater size scalability. Optical properties of the nanocomposite phosphor components are described, with emphasis on the origin of the enhanced light yield. Finally, results from our first nanocomposite scintillator, including photopeak measurements, are described. The photopeak measurements demonstrate the proof-of-principle of the nanocomposite scintillator concept.

Decellularized tissues have been used to investigate the extracellular matrix (ECM) in a number of different tissues and species. Santi and Johnson JARO 14:3-15 (2013) first described the decellularized inner ear in the mouse, rat, and human using scanning thin-sheet laser imaging microscopy (sTSLIM). The purpose of the present investigation is to examine decellularized cochleas in the mouse and human at higher resolution using scanning electron microscopy (SEM). Fresh cochleas were harvested and decellularized using detergent extraction methods. Following decellularization, the ECM of the bone, basilar membrane, spiral limbus, and ligament remained, and all of the cells were removed from the cochlea. A number of similarities and differences in the ECM of the mouse and human were observed. A novel, spirally directed structure was present on the basilar membrane and is located at the border between Hensen and Boettcher cells. These septa-like structures formed a single row in the mouse and multiple rows in the human. The basal lamina of the stria vascularis capillaries was present and appeared thicker in the human compared with the mouse. In the mouse, numerous openings beneath the spiral prominence that previously housed the root processes of the external sulcus cells were observed but in the human there was only a single row of openings. These and other anatomical differences in the ECM between the mouse and human may reflect functional differences and/or be due to aging; however, decellularized cochleas provide a new way to examine the cochlear ECM and reveal new observations.

In order to improve the accuracy and capabilities of neutron multiplicity counting, additional quantifiable information is needed in order to address the assumptions that are present in the point model. Extracting and utilizing higher order moments (Quads and Pents) from the neutron pulse train represents the most direct way of extracting additional information from the measurement data to allow for an improved determination of the physical properties of the item of interest. The extraction of higher order moments from a neutron pulse train required the development of advanced dead time correction algorithms which could correct for dead time effects in all of the measurement moments in a self-consistent manner. In addition, advanced analysis algorithms have been developed to address specific assumptions that are made within the current analysis model, namely that all neutrons are created at a single point within the item of interest, and that all neutrons that are produced within an item are created with the same energy distribution. This report will discuss the current status of implementation and initial testing of the advanced dead time correction and analysis algorithms that have been developed in an attempt to utilize higher order moments to improve the capabilities of correlatedmore » neutron measurement techniques.« less

A number of technical and practical limitations exist within the neutron correlated counting techniques used in safeguards, especially within the algorithms that are used to process and analyze the detected neutron signals. A multi-laboratory effort is underway to develop new and improved analysis and data processing algorithms based on fundamental physics principles to extract additional or more accurate information about nuclear material bearing items.

Auditory sensitivity and frequency resolution depend on the physical properties of the basilar membrane in combination with outer hair cell-based amplification in the cochlea. The physiological role of the tectorial membrane (TM) in hair cell transduction has been controversial for decades. New insights into the TM structure and function have been gained from studies of targeted gene disruption. Several missense mutations in genes regulating the human TM structure have been described with phenotypic expressions. Here, we portray the remarkable gradient structure and molecular organization of the human TM. Ultrastructural analysis and confocal immunohistochemistry were performed in freshly fixed human cochleae obtained during surgery. Based on these findings and recent literature, we discuss the role of human TMs in hair cell activation. Moreover, the outcome proposes that the α-tectorin-positive amorphous layer of the human TM is replenished and partly undergoes regeneration during life.

Hypothesis Thin-sheet laser imaging microscopy (TSLIM) optical sectioning can be used to assess temporal bone soft tissue morphology before celloidin sectioning. Background Traditional human temporal bone (TB) celloidin embedding and sectioning is a lengthy and involved process. Although bone morphology can be assessed with microCT before traditional histology, soft tissue structures are difficult to resolve until after celloidin sectioning. A potential solution is TSLIM, a high-resolution, nondestructive optical sectioning technique first developed to image bone and soft tissue in animal cochleae. Methods Two temporal bones from 1 individual were used to evaluate TSLIM's capacity to image human temporal bones (bone and soft tissue) before traditional histology. The right TB was trimmed to the cochlea, prepared for and imaged with TSLIM, then processed for celloidin sectioning. The left TB, serving as a control, was directly prepared for traditional celloidin sectioning. Results TSLIM imaging of the right TB showed adequate resolution of all major tissue structures but barely resolved cells. Celloidin sections produced from the TSLIM-imaged right TB were equivalent in cytologic detail to those from the traditionally prepared left TB. TSLIM 3-dimensional (3D) reconstructions were superior to those obtained from celloidin sections because TSLIM produced many more sections that were without mechanical sectioning artifacts or alignment issues. Conclusion TSLIM processing disturbs neither gross nor detailed morphology and integrates well with celloidin histology, making it an ideal method to image soft tissue before celloidin sectioning.

The structure of side, tip, and “attachment” links of chinchilla outer hair cell (OHC) stereocilia was studied by transmission and scanning electron microscopy using tannic acid and Cuprolinic blue histochemical procedures. Tannic acid, which interacts with many different types of proteins and glycoproteins irrespective of their electrical charge, showed strong reactivity for the central area of the side links and weak reactivity for the marginal area of these links adjacent to the stereocilia membrane. Tannic acid treatment revealed the tip links as thin strands, about 5 nm thick. Attachment links were poorly visualized after tannic acid treatment and appeared as sparse filamentous strands at tips of the tallest OHC stereocilia. Cuprolinic blue, at a high critical electrolyte concentration, reacted with strongly negative, primarily sulfated, carbohydrate residues of glycoconjugate macromolecules. In contrast to the tannic acid treatment, the central portions of the OHC stereocilia side links were unstained after Cuprolinic blue treatment; however, membrane-associated ends of these links were darkly stained. The tip links showed a similar appearance as after tannic acid treatment; however, Cuprolinic blue revealed an electron-dense substructure at both ends of its insertion into the stereocilia. Cuprolinic blue reactive structures were also observed as attachment links only at the tips of the OHC stereocilia of the tallest row in each bundle. These structures formed a crown-like array around the tip of each stereocilium. Their primary function appears to be attachment of type B fibrils of the tectorial membrane to the tallest OHC stereocilia. Cuprolinic blue reactive structures of the side, tip, and attachment links appear to contain acidic, sulfated residues of proteoglycans or glycoproteins. These structures may function as connective elements between the stereocilia links and the hair cell cytoskeleton.

Francisco Aboitiz*, F. Xavier Castellanos and Ximena Carrasco Departamento de Psiquiatria, Facultad de Medicina, and Centro Interdisciplinario de Neurociencia, Pontificia Universidad Catolica de Chile, Santiago, Chile New York University Child Study Center, Nathan Kline Institute for Psychiatric Research, New York, NY, USA Servicio de Neurologia y Psiquiatria infantil, Hospital Luis Calvo Mackenna Facultad de Medicina, Universidad de Chile, Santiago, Chile

The molecular and supramolecular structure of the tectorial membrane (TM) was studied by transmission electron microscopy (TEM). Collagen (type A) fibrils in the TM were found associated with proteoglycans (PGs) and type B fibrils. Most PGs were orthogonally oriented and attached D-periodically to collagen fibrils. Computer averaged projections of PG particles and linear aggregates of PGs in crystalline arrays, stained with Cuprolinic blue, showed an elongated, electron-dense structure 50-65 nm in length and 10 nm in width. Image analysis of type B fibrils showed that they are constructed of globular domains arranged with a periodicity of 12-14 nm. Each globular domain contains two thin 'arms', extended in opposite directions, which contact the 'arms' of adjacent fibrils. Numerous type B fibrils were found between collagen fibrils. They are attached to adjacent collagen fibrils by the 'arms' of their globular domains. An association of type B fibrils and PGs with collagen seems to result in the local ordered arrangement of the TM matrix. A hypothetical model of the TM matrix supramolecular structure is presented.

Keratan sulfate (KS) was immunolocalized in the chinchilla cochlea and vestibular system using indirect immunohistochemistry and a monoclonal antibody (clone 5-D-4) directed against a proteoglycan core antigen. As a positive control, anti-KS Mab reactivity was found in the pericellular matrix and lacuna walls of temporal bone osteocytes. In the cochlea, anti-KS Mab reactivity was abundant in the basal cell layer of the stria vascularis and in the marginal band and Hensen's stripe of the tectorial membrane. Less anti-KS Mab reactivity was present in the cover net, Hardesty's membrane and the upper fibrous zone of the limbal layer of the tectorial membrane. In the vestibular system, anti-KS Mab reactivity was immunolocalized to a portion of the epithelium overlying the cupula of the crista ampullaris, in the apical surface of crista ampullaris epithelium, crista ampullaris stereocilia and in the otoconia. Elucidating the distribution of KS in the cochlea will improve our understanding of cochlear anatomy and is a first step toward understanding the etiology of hearing loss observed in diseases involving KS metabolism, namely, mucopolysaccharidosis type IV (Morquio's syndrome).