High-resolution in situ imaging of cochlear implant electrode arrays in cat temporal bones using Tuned Aperture Computed Tomography (TACT).

OBJECTIVE: To determine the suitability of Tuned Aperture Computed Tomography (TACT) to generate high-resolution images of intracochlear electrode arrays, in situ, with sufficient anatomic and electrode detail to relate the location of individual electrode contacts to important anatomic landmarks in cat cadaveric temporal bones. The ultimate objective is to develop an imaging technology whereby variations in electrode location, relative to the target neural tissues, can be accurately determined and related to variations in performance with the cochlear implant. DESIGN: Cat temporal bones were implanted with an experimental scala tympani electrode array and an external fiducial landmark. A series of conventional 2D digital radiographs were collected from a variety of x-ray source projection angles and served as for generation of 3D volume renderings using the TACT software toolbox. The 3D renderings were then reoriented and resliced interactively to view the cochlear and electrode features of interest. RESULTS: Significant electrode and anatomical details could be visualized including the course of the electrode wires (<40 microm diameter), the location of all electrode contacts and the outline of the scala tympani. CONCLUSIONS: TACT generates high-resolution 3D images from 2D conventional radiographs. With TACT, the 3D renderings can be interactively reoriented and resectioned to permit visualization of any cochlear or electrode feature. In the present study, this aspect of TACT affords the opportunity to view of the location of each electrode contact relative to the adjacent cochlear features, such as the scalar walls. Because TACT uses conventional radiographic images to generate the volume renderings, the quality and resolution of the resulting 2D images do not suffer from artifacts characteristic of CT. These findings suggest that TACT may be a powerful tool for understanding the contribution of electrode placement to perceptual performance with the cochlear implant.

Optimization of TACT imaging protocols for in situ visualization of cochlear electrode arrays in cat temporal bones.

OBJECTIVE: To explore the effect of the number of two-dimensional (2D) images and x-ray projection angles on the resolution of reconstructed three-dimensional (3D) volumes of intracochlear electrode arrays in cadaveric cat temporal bones using Tuned Aperture Computed Tomography (TACT). DESIGN: Multiple 2D radiographs (basis images (BI)) of implanted cadaveric cat temporal bones were acquired using a range of projection angles, and imported into the TACT workbench. 3D volumes were reconstructed using varying numbers of BIs. Contrast resolution in the image was determined by comparing the contrast ratio (using maximum and minimum grayscale values) in specified anatomic areas of interest. RESULTS: Systematically increasing the number of BIs used in the reconstruction process resulted in a systematic increase in contrast resolution. Likewise, increasing the range of effective projection angles, as also the number of such angles used in the TACT computation also increased the contrast resolution of the resulting images. CONCLUSIONS: Precise determination of the location of cochlear implant electrodes in situ is critical to understanding the factors influencing efficacy of electrical stimulation of the deaf ear. Renderings generated with the TACT algorithm produce 3D images permitting visualization of implant electrode features and anatomic details with resolution sufficient to accurately localize electrode contacts within scala tympani. The quality of resulting images, evaluated as a function of image contrast, improved with a larger number of BIs in the reconstruction. Wider projection angles also improved image detail in addition to generating thinner slices. Any loss in contrast was compensated for by the number of BIs. TACT can thus be optimized to provide useful data to help characterize the location of intracochlear electrode arrays.

Use of Web-based materials to enhance anatomy instruction in the health sciences.

Teaching anatomy by dissection is under considerable pressure to evolve and/or even be eliminated, and curricular hours in the dissection laboratory are decreasing. As a possible means of easing this pressure, an online interactive anatomy program has been created to enhance the dissection experience, observational learning, and three-dimensional comprehension of human anatomy. An assessment was made of the utility of the program in preparing students for dissection laboratories and for examinations. The efficacy of the application was evaluated by first-year students and faculty with pre- and post-use surveys in anatomy courses at three medical schools. It was found that students felt better prepared if they utilized the Web site prior to their dissection laboratory, and faculty reported spending less time explaining basic concepts or techniques. It is concluded that a comprehensive online program significantly enhances the quality and efficiency of instruction in human anatomy in the dissection laboratory and could prove to be a useful tool at other institutions.

Smooth muscle in the annulus fibrosus of the tympanic membrane in bats, rodents, insectivores, and humans.

The annulus fibrosus and its attachment to the bony tympanic ring were studied in a series of mammals. In the pallid bat, Antrozous pallidus, there is an extensive plexus of large interconnected blood sinuses in the part of the annulus that borders the tympanic bone. The spaces between the sinuses are packed with smooth muscle cells. Most of the cells have a predominately radial orientation; they extend from the bony tympanic sulcus to a dense collagenous matrix (apical zone) where radially oriented fibers of the pars tensa are confluent with the annulus. The muscles and vessels constitute a myovascular zone. A structurally similar myovascular zone is also present in the European hedgehog. In rodents, the annulus lacks the large interconnected blood sinuses but many small vessels are present. Smooth muscle is concentrated in the broad area of attachment of the annulus to the tympanic bone. In the gerbil, smooth muscle seems to be concentrated in the central part of the width of the annulus where it is attached to bone and radiates toward the tympanic membrane. In humans collections of radially oriented smooth muscle cells were found in several locations. The smooth muscle in all species studied appears to form a rim of contractile elements for the pars tensa. This arrangement suggests a role in controlling blood flow and/or creating and maintaining tension on the tympanic membrane.

Quantitative anatomy of the guinea pig endolymphatic sac.

The endolymphatic sac is believed to represent one of the primary loci for endolymph volume regulation in the inner ear. Quantitative analysis of physiologic measurements from the endolymphatic sac requires knowledge of the anatomy of the structure, specifically the luminal volume and the variation of cross-sectional area with distance along the sac. Recently techniques have become available to make these measurements. In the present study, fixed, isolated specimens of the guinea pig endolymphatic sac were imaged by high-resolution magnetic resonance microscopy (MRM) or by histological serial sections. Structures were reconstructed and quantified using image analysis software. In specimens imaged by MRM the endolymphatic sac volume, including tissue and lumen, was 359 nl for the intraosseous region and 106 nl for the extraosseous region, totaling 465 nl for the entire structure. The luminal volumes were 131 nl for the intraosseous region and 13 nl for the extraosseous region, totaling 144 nl. In histological specimens the volume, including tissue and lumen, was 414 nl for the intraosseous region and 121 nl for the extraosseous region, totaling 535 nl for the entire structure. The luminal volumes were 152 nl for the intraosseous region and 26 nl for the extraosseous region, totaling 179 nl. Differences in volume estimates obtained by the two methods were not statistically significant and variation was dominated by inter-specimen variation. Pooling the data, the total volume of the endolymphatic sac in the guinea pig including tissue and lumen was 506 nl (S.D. 100, n=17) and the volume of the lumen was 169 nl (S.D. 48, n=14).

The presence and arrangement of type II collagen in the basilar membrane.

Previous studies demonstrating the presence of collagen II in the basilar membrane have used a biochemical approach or have used immunohistochemistry at the light microscopic level. In this investigation both the presence and arrangement of collagen II were demonstrated at the ultrastructural level using pre- and post-embedding immunoelectron microscopy. Labeling was dependent on the development of protocols to expose epitopes while maintaining identifiable ultrastructure. Both positive and negative controls indicate that the labeling was specific for collagen II. Collagen II was detected in the fibrous sheet of the pars tecta and in the two fibrous layers of the pars pectinata. It was detected in situ and on isolated individual 10-12 nm fibrils. The presence of collagen II in all the fibrous layers of the basilar membrane places constraints on the biomechanical properties of this important structure.

Immunolabeling type II collagen in the basilar membrane, a pre-embedding approach.

This paper describes the development of a protocol that can be used to detect collagen II in the healthy adult basilar membrane (BM) at the electron microscopic level. This protocol required aggressive epitope exposure techniques to break the crosslinks that bind the collagen molecules tightly into fibrils and to remove a dense mat of ground substance that surrounds the fibrils. On the other hand, the steps had to be carefully controlled to preserve BM ultrastructure and the collagen II epitopes that are typically labile. These requirements were satisfied by introducing a targeted crosslink breakage method and by regulating the duration of epitope exposure based on changes in tissue appearance observed with differential interference contrast microscopy. High levels of immunolabeling were achieved by substituting tissue preservation techniques for most or all of fixation; this was important because fixation reduces antigenicity directly and impedes epitope exposure. When these techniques were combined with more traditional trypsin and pepsin treatments, the result was dense immunolabeling and preservation of ultrastructure that allowed accurate localization of the immunolabeling. This pre-embedding immunoelectron microscopic method is the first to be carried out on the BM and may be adaptable to future studies of the BM as well as other tissues with similar molecular composition.

Smooth muscle in the annulus fibrosus of the tympanic membrane: physiological effects on sound transmission in the gerbil.

In a wide variety of mammals, the rim of the tympanic membrane (annulus fibrosus) has an array of contractile elements, either smooth muscle [Henson and Henson, J. Assoc. Res. Otolaryngol. 1 (2000) 25-32] or myofibroblasts [Kuijpers et al., Hear. Res. 128 (1999) 80-88]. These elements are anchored peripherally to the bony tympanic ring and centrally to incoming fibers of the pars tensa. Their arrangement suggests that they are involved in the control of tympanic membrane tension. In this study, cochlear microphonic (CM) threshold changes were recorded in gerbils to study the physiological effects of these contractile elements. It was demonstrated that the application of substances known to make smooth muscle contract (vanadate and norepinephrine) caused concentration-dependent elevations in CM thresholds. Maximum changes of 8-9 dB occurred with the lowest frequency tested (2.16 kHz). The application of muscle-relaxing drugs reversed these effects. Controls showed that the threshold changes were not induced by effects on middle or inner ear structures. These results add to emerging evidence that the tympanic membrane has intrinsic control of tension and is potentially able to have some control over energy levels reaching the cochlea.

Quantitative anatomy of the round window and cochlear aqueduct in guinea pigs.

In order to analyze the entry of solutes through the round window membrane, a quantitative description of round window anatomy in relationship to scala tympani is required. High-resolution magnetic resonance microscopy was used to visualize the fluid spaces and tissues of the inner ear in three dimensions in isolated, fixed specimens from guinea pigs. Each specimen was represented as consecutive serial slices, with a voxel size of approximately 25 microm(3). The round window membrane, and its relationship to the terminal portion of scala tympani in the basal turn, was quantified in six specimens. In each image slice, the round window membrane and scala tympani were identified and segmented. The total surface area of the round window membrane averaged 1.18 mm(2) (S.D. 0.08, n=6). The length and variation of cross-sectional area as a function of distance for the cochlear aqueduct was determined in five specimens. The cochlear aqueduct was shown to enter scala tympani at the medial limit of the round window membrane, which corresponded to a distance of approximately 1 mm from the end of the scala when measured along its mid-point. These data are of value in simulating drug and other solute movements in the cochlear fluids and have been incorporated into a public-domain simulation program available at http://oto.wustl.edu/cochlea/.

Ultrastructure of the inner ear of NKCC1-deficient mice.

The transduction of the auditory signal is dependent on the flow of ions within the inner ear. We have generated mice deficient in NKCC1, an ion cotransporter that is thought to be involved in the secretion of K+ by the strial marginal cells. Inner ear histology revealed partial to almost total absence of the scala media and collapse of Reissner's membrane. Ultrastructural analysis showed that Reissner's membrane consists of 3-4 cell layers instead of the usual two, and a substance of unknown composition is present between Reissner's membrane and underlying structures. Within the tunnel of Corti, hair cells and supporting cells were difficult to identify. The location of the tectorial membrane was altered, and a precipitate was observed surrounding it. Severe structural defects were noted in the interdental cells and Boettcher cells, and mild defects were observed in the stria vascularis and in type II and type IV fibrocytes. The finding that major defects occur predominantly in cells that are not known to express NKCC1 suggests that loss of NKCC1 results in functional defects in cells expressing NKCC1 and a morphological effect on cell populations downstream in the proposed K+ recycling pathway.

The tympanic membrane: highly developed smooth muscle arrays in the annulus fibrosus of mustached bats.

The annulus fibrosus tympanicus is the thickened peripheral rim of the pars tensa of the tympanic membrane. It is an area into which the connective tissue matrix of the membrane extends to attach to the tympanic bone (ring). Light microscopy, SEM, TEM and confocal microscopy were used to study the area in mustached bats Pteronotus p. parnellii, P. p. portoricensis and P. quadridens. In cross sections of the annulus morphologically distinct apical and basal or myovascular zones could be recognized. The apical zone had a collagenous matrix that was continuous with the pars tensa. The myovascular zone contained an extensive network of thin walled endothelial tubes and a well-developed array of radially arranged smooth muscle cells that filled the interval between the vessels. The muscle tissue occupied the interval between the densely collagenous "apical zone" and the bony tympanic ring and was closely associated with bundles of unmyelinated nerve fibers. The structure and arrangement of the tissue suggests a highly developed specialization for the tonic control of tension of the pars tensa and a system that potentially regulates sound transmission to the middle and inner ear.

Cochlear fluid space dimensions for six species derived from reconstructions of three-dimensional magnetic resonance images.

OBJECTIVES: To establish the dimensions and volumes of the cochlear fluid spaces. STUDY DESIGN: Fluid space volumes, lengths, and cross-sectional areas were derived for the cochleas from six species: human, guinea pig, bat, rat, mouse, and gerbil. METHODS: Three-dimensional reconstructions of the fluid spaces were made from magnetic resonance microscopy (MRM) images. Consecutive serial slices composed of isotropic voxels (25 microm3) representing the entire volume of fixed, isolated cochleas were obtained. The boundaries delineating the fluid spaces, including Reissner's membrane, were resolved for all specimens, except for the human, in which Reissner's membrane was not consistently resolved. Three-dimensional reconstructions of the endolymphatic and perilymphatic fluid spaces were generated. Fluid space length and variation of cross-sectional area with distance were derived by an algorithm that followed the midpoint of the space along the length of the spiral. The total volume of each fluid space was derived from a voxel count for each specimen. RESULTS: Length, volume, and cross-sectional areas are provided for six species. In all cases, the length of the endolymphatic fluid space was consistently longer than that of either perilymphatic scala, primarily as a result of a greater radius of curvature. For guinea pig specimens, the measured volumes of the fluid spaces were considerably lower than those suggested by previous reports based on histological data. CONCLUSIONS: The quantification of cochlear fluid spaces provided by this study will enable the more accurate calculation of drug and other solute movements in fluids of the inner ear during experimental or clinical manipulations.

The innervation of outer hair cells: 3D reconstruction from TEM serial sections in the Japanese macaque.

Transmission electron micrographs from serial sections were obtained from the neural pole of outer hair cells (OHCs) in the Japanese macaque (Macaca fuscata) and reconstructions of nerve terminals were made using computer software. Data are based on observations of six cells in the basal turn, eight in the middle turn and four in the apex. In general, the number of afferent (type II) terminals on each OHC increased from base to apex, and for a given turn, the numbers appeared unrelated to OHC row. On the other hand, the number of efferent terminals was greater in the middle turn than in other areas, and the number decreased from row 1 to row 3. Reciprocal synapses increased in frequency from the upper basal turn apicalward. The total number of terminals synapsing on an individual OHC increased from base to apex by nearly 100%. Three-dimensional reconstructions showed that nerve fibers terminating on basal and middle turn OHCs ascended directly from sub-OHC regions to synapse on the subnuclear regions of the OHC. In contrast, apical turn fibers ran horizontally at the level of the subnuclear region and the terminals appeared as en passant swellings along a single fiber. Although physiological data are wanting for the macaque, the anatomical findings suggest that functional differences may exist along the length of the cochlea.

Tonic efferent-induced cochlear damping in roosting and echolocating mustached bats.

The activity of the medial olivocochlear (MOC) efferent system in mustached bats, Pteronotus p. parnellii, was studied by monitoring changes in the mechanical properties of the cochlea. The changing properties were expressed by the decay time (DT) of cochlear microphonic potentials produced by transient-induced ringing (Henson et al., 1995). Tape-recorded roost noise (biosonar and communication sounds) produced sudden, marked decreases in DT when presented to the contralateral ear of animals adapted to the quiet. When the animals were first removed from their roosts the DT was relatively short (1.2-1.5 ms) but this gradually lengthened by about 0.5-1.0 ms as they rested in a quiet chamber. The time required to reach a stable, quiet-adapted state after noise exposure varied with SPL and exposure time; in many experiments recovery was in the range of 90-120 min. When quiet-adapted bats were isolated and allowed to fly and echolocate for 20 min, the DTs measured within a few minutes after the end of the flight were also short and only slowly returned to longer preflight values. The administration of a single dose of gentamicin, which blocks MOC effects, greatly reduced the amount of suppression (damping) observed after periods of noise and echolocation sound exposure. We conclude that tonic MOC activity is induced by the natural vocalizations and roost noise and this activity probably regulates and protects the highly resonant cochlear partition.

Changes in cochlear mechanics during vocalization: evidence for a phasic medial efferent effect.

The mustached bat, Pteronotus p. parnellii, has a finely tuned cochlea that rings at its resonant frequency in response to an acoustic tone pip. The decay time (DT) and frequency of these damped oscillations can be measured from the cochlear microphonic potential (CM) to study changes in cochlear mechanics. In this report, we describe phasic changes that occur in synchrony with communication sound vocalizations of the bat. Three animals with chronically implanted electrodes were studied. During the experiments, 1-2 ms tone pips were emitted from a speaker every 200 ms. This triggered a computer analysis of the resulting CM to determine the DT and cochlear resonance frequency (CRF) of the ringing. The time relative to vocalizations was determined by monitoring the output of a microphone placed near a bat's mouth. Similar results were obtained from all three bats tested. In a representative case, the average DT was 2.33 +/- 0.25 ms while the bat was quiet, but it decreased by 46% to 1.26 +/- 0.75 during vocalizations, which indicates a greater damping of the cochlear partition. Sometimes, DT started decreasing immediately before the bat vocalized. After the end of a vocalization, the return to baseline values varied from rapid (milliseconds) to gradual (1-2 seconds). The CRF also changed from baseline values during vocalization, although the amount and direction of change were not predictable. When gentamicin was administered to block the action of medial olivocochlear (MOC) efferents, DT reduction was still evident during vocalization but less pronounced. We conclude that phasic changes in damping occur in synchrony with vocalization, and that the MOC system plays a role in causing suppression. Since suppression can begin prior to vocalization, this may be a synkinetic effect, mediated by neural outflow to the ear in synchrony with neural outflow to the middle ear muscles and the muscles used for vocalization.

The course and distribution of medial efferent fibers in the cochlea of the mustached bat.

The course and distribution of medial olivocochlear (MOC) nerve fibers were studied in the cochlea of the mustached bat. This animal is of interest because of the very sharp tuning of the ear and fine frequency resolution in small frequency bands near 60 and 90 kHz. The MOC fibers arise from about 400 cells in the dorsomedial periolivary (DMPO) nucleus and they are distributed to approximately 4500 outer hair cells (OHCs), resulting in an average OHC unit size of 11.25. Individual fibers appear to have a small number of branches and each branch entering the tunnel of Corti terminates on a patch of OHCs. The patch size is typically 1-3 OHCs with the smallest average patch sizes in the regions tuned to 60 and 90 kHz. The majority of the MOC terminals are derived from the contralateral DMPO. Contralateral vs. ipsilateral projecting fibers are not preferentially distributed within any of the three rows of OHCs or within specific regions throughout most of the cochlea. It can be concluded that the main differences between the mustached bat's MOC system and that of most other mammals are: (1) origin from a single nucleus; (2) relatively small sizes of the patches; (3) a single terminal on each OHC; (4) a gradient in the size of the terminals but not in the number of terminals from row to row or from base to apex.

Detection and quantification of endolymphatic hydrops in the guinea pig cochlea by magnetic resonance microscopy.

Three-dimensional magnetic resonance microscopy (MRM) was used to study normal and hydropic cochleae of the guinea pig. With this technique consecutive serial slices representing the entire volume of isolated, fixed cochleae were obtained. The voxels (volume elements) making up the contiguous slices were isotropic (25 microns 3) and in each slice the boundaries of scala media, including the position of Reissner's membrane, were clearly delineated. Three-dimensional reconstructions of the endolymphatic and perilymphatic scale were generated. Custom software was developed to quantify cross-sectional area (CSA) of all scalae. In the normal cochlea all 3 scalae, including scala media, showed a gradual decrease in CSA from base to apex. Marked differences existed between our findings and previously reported cochlear dimensions, especially for the perilymphatic scalae in the basal turn. In hydropic cochleae the scala media was enlarged to a varying extent in different turns and marked changes in the degree of distension of Reissner's membrane occurred along the cochlea. MRM and subsequent computer analysis of the isotropic data provide excellent methods for imaging and quantifying the fluid spaces of normal and hydropic cochleae.

The effect of contralateral stimulation on cochlear resonance and damping in the mustached bat: the role of the medial efferent system.

In the unanesthetized mustached bat, stimulation of the ear with an acoustic transient produces damped oscillations which are evident in the cochlear microphonic potential. In this report we demonstrate how the decay time of these oscillations is affected by broadband noise presented to the contralateral ear (CLN). In the absence of CLN, the mean decay time was 1.94 +/- 0.23 ms, but during the presentation of CLN the decay time consistently decreased. The changes were finely graded, the higher the CLN, the greater the change. The effect could be maintained at a constant level for extended periods of time and this was evident when the CLN exceeded 40 dB SPL. The latency of the reflex for 64 dB noise was about 11 ms and near maximum changes occurred within 15 ms of CLN onset. Sectioning medial efferent nerve fibers in the floor of the fourth ventricle or the administration of a single dose of gentamicin eliminated changes produced by CLN. The prominence of CM responses to damped oscillations and the robust changes in response to CLN make the mustached bat an excellent model for studying the influence of the medial efferent system on cochlear mechanics.

Respiratory muscle activity in relation to vocalization in flying bats.

The structure of the thoracic and abdominal walls of Pteronotus parnellii (Microchiroptera: Mormoopidae) was described with respect to their function in respiration and vocalization. We monitored electromyographic activity of respiratory and flight muscles in relation to echolocative vocalization. In flight, signals were telemetered with a small FM transmitter modified to summate the low-frequency myopotentials with biosonar signals from a ceramic-crystal microphone. Recordings were also made from the same bats confined to a small cage. Vocalizations were used as the parameter by which all muscle activities were correlated. A discrete burst of activity in the lateral abdominal wall muscles accompanied each vocalization. Diaphragmatic myopotentials occurred between groups of calls and did not coincide with activity of the abdominal wall or with vocalizations. Flight muscles were not active in resting bats. During flight, vocalizations and the abdominal muscle activity that accompanied them coincided with myopotentials of the pectoralis and serratus ventralis muscles. We propose that contractions of the lateral abdominal wall provide the primary power for the production of intense biosonar vocalization in flying and in stationary bats. In flight, synchronization of vocalization with activity of the pectoralis and serratus ventralis jointly contribute to the pressurization of the thoraco-abdominal cavity. This utilization of pressure that is normally generated in flight facilitates respiration and allows for the production of intense vocalizations with little additional energetic expenditure.

Morphology of the abdominal wall in the bat, Pteronotus parnellii (Microchiroptera: Mormoopidae): implications for biosonar vocalization.

We investigated the structure of the abdominal wall of Pteronotus parnellii and made comparisons with eight other species of Microchiroptera and one megachiropteran. Similar to other mammals, the abdominal wall of bats consists of the three flank muscles laterally and the m. rectus abdominis ventrally. In Microchiroptera, flank muscles are mostly confined to dorsal portions of the wall. The mm. transversus abdominis and obliquus internus abdominis form the bulk of the wall; the m. obliquus externus is poorly developed. Ventrolaterally, a large portion of the wall is a dense, bilaminar aponeurosis, composed of collagen, elastin, and fibroblasts. The thicker, superficial lamina derives from the mm. obliquus internus and transversus abdominis. The deep lamina is a continuation of the transversalis fascia. Collagen fibers of the two fused laminae are oriented orthogonally, resulting in a resilient, composite fabric. Fascicles of the flank muscles are oriented along the margins of the aponeurosis so that their forces appear to be concentrated onto the aponeurosis. We suggest that this system is adapted for the regulation and generation of intra-abdominal pressure. The abdominal wall of Pteropus, the one megachiropteran examined, lacks the derived aponeurosis and is similar to other mammals. We consider the abdominal wall of Microchiroptera to be analogous to the diaphragma, in that it functions in the regulation of pressure within body cavities and facilitates biosonar vocalization.