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.

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.

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/.

A comparison study on the effects of prewarming patients in the outpatient surgery setting.

Maintenance of core body temperature in surgical patients presents a challenge to perioperative nurses. Core temperatures less than 36 degrees C are associated with multiple adverse outcomes postoperatively. Internal redistribution of heat from the body core to the colder periphery results in core temperature decreases of 0.5 degrees C to 1.5 degrees C in the first 30 minutes after induction of anesthesia. The purpose of this study was to determine if there was a difference in arrival temperatures to the PACU between surgical patients who had been warmed preoperatively with a forced warm air blanket and those patients warmed with cotton blankets. One hundred patients were randomly assigned to receive prewarming by using a forced-air warm blanket (n = 50) or a cotton blanket (n = 50). Temperatures were monitored every 15 minutes throughout the preoperative and postoperative periods. Patients in the forced warm air group had significantly higher temperatures on arrival to the PACU from the OR than did patients in the warm blanket group (P =.000). Patients in the forced warm air group exhibited a change in temperature of 0.0067 degrees C (+/-.52) compared with a decrease of 0.22 degrees C (+/-.48) for patients in the control group.

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.

Psychophysical correlates of contralateral efferent suppression. I. The role of the medial olivocochlear system in "central masking" in nonhuman primates.

An extensive physiological literature, including experimental and clinical studies in humans, demonstrates that activation of the medial olivocochlear (MOC) efferent system, by either contralateral sound or electrical stimulation, can produce significant alterations in cochlear function and suggests a role for the MOC system in influencing the auditory behavior of binaural hearing. The present data are from psychophysical studies in nonhuman primates which seek to determine if the noted physiological changes in response to contralateral acoustic stimulation have a perceptual counterpart. Four juvenile Japanese macaques were trained to respond to the presence of 1-s sinusoids, presented to the test ear, in an operant reinforcement paradigm. Thresholds were compared for frequencies ranging from 1.0 to 4.0 kHz in quiet, with thresholds measured when continuous, two octave-band noise, centered on the test tone frequency, was presented in the contralateral ear. Contralateral noise was presented at levels of 10-60 dB above detection threshold for the test-tone frequency. While some variability was evident across subjects, both in the frequency distribution and magnitude (as a function of contralateral noise level), all subjects exhibited an increase, or suppression of thresholds in the presence of contralateral noise. On average, thresholds increased systematically with contralateral noise level, to a peak of 7 dB. In one subject, the threshold increase seen with contralateral noise was significantly reduced when the MOC was surgically sectioned on the floor of the IVth ventricle. The characteristics of the measured shifts in behavioral thresholds, in the presence of contralateral noise reported here, are qualitatively and quantitatively similar to both efferent physiological suppression effects and psychophysical central masking threshold shifts which have been reported previously. These data suggest that at least some aspects of "central masking" are efferent-mediated peripheral processes, and that the term "central masking" may be incorrect.

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.

Rapid decalcification of temporal bones with preservation of ultrastructure.

Decalcification of temporal bones, especially from primates, has routinely required long periods of time and has been a major deterrent to many types of morphological studies. In this investigation, temporal bones from the monkey, Macaca fuscata, were decalcified with ethylene diamine tetraacetic acid (EDTA) in a microwave oven. To isolate effects of microwaves on decalcification, tissue was fixed and embedded using routine methods; only decalcification was carried out in the microwave oven. The procedure is described in detail. Instead of months, decalcification was complete in two working days. Control procedures included decalcification at room temperature and use of a regular oven at a temperature equal to that reached in the microwave. The ultrastructure of cochlear tissue was equal to or better than that obtained with routine decalcification.

Synaptic specializations associated with the outer hair cells of the Japanese macaque.

Across species the innervation of outer hair cells (OHCs) shows a remarkable similarity. There are, however, notable differences in fine structure. The present work describes the normal synaptic morphology of OHCs in the Japanese macaque (Macaca fuscata), as determined by examination of serial sections with transmission electron microscopy. The nerve endings at the base of OHCs were divided primarily into two groups: vesiculated (efferent) and non-vesiculated (afferent). In addition, we found supranuclear efferent nerve endings and reciprocal synapses in all three cochlear turns. We also found presynaptic bodies in OHCs at the afferent synapse, the branching of afferent nerve fibers and axodendritic synapses between afferent and efferent fibers in the outer spiral bundle and just beneath OHCs. In terms of synaptic structure, the data indicate that that the Japanese macaque is more similar to that of the human than other species examined to date.

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.

Reconstructions and cross-sectional area measurements from magnetic resonance microscopic images of the cochlea.

In this study, magnetic resonance (MR) microscopy was used to obtain serial sections through the cochleae of mustached bats. As previously reported, 25 microns isotropic voxels can be obtained. Specific areas in each slice were segmented and then three-dimensional (3-D) reconstructions of the perilymphatic and endolymphatic spaces and spiral ligament were obtained. Quantitative measurements of the cross-sectional areas were made with customized macros written for the public-domain software, NIH Image. Results of this study revealed enlargements of the scalae and spiral ligament in areas known to be involved with processing of the animal's biosonar and fine-frequency analysis.

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.

Imaging the cochlea by magnetic resonance microscopy.

The isolated, fixed cochlea of the mustached bat was studied with three dimensional magnetic resonance (MR) microscopy. The cochlea of this animal is about 4 mm in diameter and its entire volume was imaged. With the field of view and matrix size used, the volume elements (voxels) making up the volume data set were isotropic 25 x 25 x 25 micron cubes. Three dimensional (3D) MR microscopy based on isotropic voxels has many advantages over commonly used light microscopy: 1) it is non destructive; 2) it is much less time consuming; 3) no dehydration is required and shrinkage is minimized; 4) the data set can be used to create sections in any desired plane; 5) the proper alignment of sections is inherent in the 3D acquisition so that no reference points are required; 6) the entire data set can be viewed from any point of view in a volume rendered image; 7) the data is digital and features can be enhanced by computer image processing; and 8) the isotropic dimensions of the voxels make the data well-suited for structural reconstructions and measurements. Good images of the osseous spiral lamina, spiral ligament, scala tympani, scala vestibuli, and nerve bundles were obtained. The vestibular (Reissner's) membrane was easily identified in the mustached bat and it appears to bulge into the scala vestibuli. The visibility of this structure suggests that MR microscopy would be well-suited for studies of endolymphatic hydrops.

AChE-staining of type II ganglion cells, processes and terminals in the cochlea of the mustached bat.

There have been a number of reports showing that ganglion cells of sensory neurons may be stained by traditional acetylcholinesterase (AChE) histochemical techniques commonly used to demonstrate efferent nerve fibers and terminals. AChE-staining has been described for cell bodies in the vestibular and spiral ganglia; staining of peripheral and central processes, however, is rare and the presence of reaction product in afferent terminals has not been reported. The outer hair cells of mustached bats, Pteronotus parnellii, differ from those of most mammals in that they typically have a single, large efferent terminal surrounded by 5-7 small, afferent terminals. In this animal an AChE-positive reaction was found not only in efferent fibers and terminals but also in type II ganglion cells, their peripheral and central processes and in outer hair cell terminals. The stained cell bodies were smaller than the unstained type I ganglion cells and they were much fewer in number. The processes of the stained cells could be followed from the soma. The central processes were dispersed throughout the VIIIth nerve trunk. Stained peripheral processes were evident in the osseous spiral lamina, floor of the tunnel of Corti and first space of Nuel and in the outer spiral plexus along the sides of the outer phalangeal (Deiters') cells. AChE-stained afferent terminals were easy to identify after transection of the crossed olivocochlear bundle (COCB) and subsequent degeneration of large efferent terminals. These results are of interest in that assessments of efferent nerve histochemistry after COCB transection need to recognize the potential contribution of AChE reaction product in afferent terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

Doppler-shift compensation by the mustached bat: quantitative data.

Quantitative data for Doppler-shift compensation by Pteronotus parnellii parnellii were obtained with a device which propelled the bats at constant velocities over a distance of 12 m. The bats compensated for Doppler shifts at all velocities tested (0.1-5.0 ms-1). The main findings were (1) that compensation was usually accomplished by a progressive lowering of the approximately 61 kHz second harmonic constant-frequency component of emitted sounds in small frequency steps (93 +/- 72 Hz); (2) that the time needed to reach a steady compensation level averaged 514 +/- 230 ms and the number of pulses required to reach full compensation averaged 10.78 +/- 5.16; (3) that the animals compensated to hold the echo (reference) frequency at a value that was slightly higher than the resting frequency and slightly lower than the cochlear resonance frequency; (4) that reference frequency varied as a function of velocity, the higher the velocity of the animal, the higher was the reference frequency (slope 55 Hz m-1s-2); and (5) that the mean reference frequency was always an undercompensation. The average amount of undercompensation was 15.8%. There was a significant difference (P < or = 0.005) in Doppler-shift compensation data collected at velocities that differed by 0.1 ms-1. A velocity difference of 0.1 ms-1 corresponds to a Doppler-shift difference of about 35 Hz in the approximately 61 kHz signals reaching the ear.

Efferent terminals in the cochlea of the mustached bat: quantitative data.

Efferent terminals in the cochlea of the mustached bat were stained for acetylcholinesterase (AChE) and quantitative data were obtained for the number and size of the endings on the outer hair cells (OHCs) in each row, from base to apex. From TEM micrographs and AChE-stained, surface preparations it was determined that every OHC had a single, large terminal. The mean size of the terminals was significantly different in each row, with the largest occurring in the first row (7.1 microns 2); the mean size in the second and third rows was 5.7 and 5.0 microns 2 respectively. In specific frequency processing regions, the largest mean size (8.4 microns 2) for first row OHCs was consistently found in the distal densely innervated (DDI) area. This region has afferent neurons that are sharply tuned to the second harmonic, constant frequency component of the bat's biosonar signals. Sudden changes in the size of the terminals were observed exactly at the boundaries of the DDI with adjacent sparsely innervated regions. Similar, but less striking, size changes also occurred in and adjacent to the proximal densely innervated (PDI) region, a harmonically related, sharply tuned region, which processes the bat's 91.5 kHz, third harmonic, constant frequency signals. The region of the cochlea with the smallest first row terminals (mean 5.3 microns 2) was the large, sparsely innervated region of the basal turn, a region that does not appear to process biosonar signals. Although the significance of differences in efferent terminal size is not known, the data suggest a possible correlation between OHC stimulation and sharp tuning. The potentially greater influence of the efferent fibers on the first row of OHCs, compared to other rows, is consistent with observations made on other mammals; in the latter, however, the greater influence has been suggested more by number than size. Unlike other mammals, the OHC efferents in the mustached bat have no clear base-to-apex gradient in the number or size of the efferent terminals. It is suggested that this might reflect the high frequency nature of the ear (6-120 kHz) and absence of low frequency hearing.