Three-dimensional reconstruction of the guinea pig inner ear, comparison of OPFOS and light microscopy, applications of 3D reconstruction.

Three-dimensional (3D) reconstruction of anatomical structures can give additional insight into the morphology and function of these structures. We compare 3D reconstructions of the guinea pig inner ear, using light microscopy and orthogonal plane fluorescence optical sectioning microscopy. Applications of 3D reconstruction of the inner ear are further explored. For each method two bullas were prepared for 3D reconstruction. Both methods are explained. In general, the 3D reconstructions using orthogonal plane fluorescence optical sectioning microscopy are superior to light microscopy. The exact spiral shape of the cochlea could be reconstructed using orthogonal plane fluorescence optical sectioning microscopy and the length of the basilar membrane measured. When a resolution of 20 microm is sufficient, orthogonal plane fluorescence optical sectioning microscopy is a superior technique for 3D reconstruction of inner ear structures in animals.

A Bast-like valve in the pigeon?

The first description of the presence of a utriculo-endolymphatic valve in human fetuses was given by Bast in 1928. Since then this valve-like structure is called Bast's valve. Its exact function has not yet been established. The general opinion is that it has a protective function by having the possibility to separate the superior endolymphatic compartments of the labyrinth from the inferior compartment. Phylogenetically seen birds are the first vertebrates with a cochlear duct and a distinct inferior and superior part of the labyrinth. A structure in the pigeon inner ear, resembling Bast's valve in mammals, is described.

The relationship of the round window membrane to the cochlear aqueduct shown in three-dimensional imaging.

The round window membrane and cochlear aqueduct complex in the guinea pig are reconstructed with 3D-imaging, using orthogonal plane fluorescence optical sectioning (OPFOS). The 3D-images show that the periotic duct and the aqueduct are connected to a pouch-like extension of the round window. The function of this may be regulation of aqueduct flow resistance under the influence of a pressure difference between inner ear fluid and middle ear.

Morphology of the endolymphatic sac in the guinea pig after an acute endolymphatic hydrops.

The role of the endolymphatic sac (ES) in endolymph volume homeostasis is speculative. The present study investigates changes of the ES's epithelia and luminal filling after induction of an acute endolymphatic hydrops. After microinjection of 1.1 mul artificial endolymph into scala media of the cochlea, guinea pigs were terminated immediately (n = 6) or after different time intervals ; 1/2 h (n = 3), 1 h (n = 4) and 2 h (n = 4). Inner ear specimens were processed for light and/or transmission electron microscopy. The non-injected contralateral ear served as a histological control. Correct injection was confirmed by detection of microspheres in the endolymphatic compartment after the same microinjection procedure. In all specimens, ribosome rich cells and intraluminal macrophages appeared to be actively involved in degradation of homogeneous substance (HS) by secreting lytic enzymes and digestion, respectively. Amazingly, in our study no ES differences were found between injected and non-injected ears and no distinct changes were observed in guinea pigs terminated after different time intervals. The ES's luminal HS was always present and often to a large extent. This is in contrast with [Hear. Res. 138, 81] dramatic changes were observed. Endolymph volume homeostasis is a complex mechanism, in which the role of HS remains obscure.

Cochlear aqueduct flow resistance depends on round window membrane position in guinea pigs.

The resistance for fluid flow of the cochlear aqueduct was measured in guinea pigs for different positions of the round window membrane. These different positions were obtained by applying different constant pressures to the middle ear cavity. Fluid flow through the aqueduct was induced by small pressure steps superimposed on these constant pressures. It was found that the resistance for fluid flow through the aqueduct depended on the round window position but not on flow direction. The results can be explained by special fibrous structures that connect the round window with the entrance of the aqueduct. It was also found that the equilibrium inner ear pressure depends on middle ear pressure, indicating that the aqueduct does not connect the inner ear with a cavity with constant pressure.

Spontaneous otoacoustic emissions in the European edible frog (Rana esculenta): spectral details and temperature dependence.

Spontaneous otoacoustic emissions were recorded in 41 ears of 29 European edible frogs (Rana esculenta). Emission frequencies ranged from 450 to 1350 Hz. The distribution of frequencies shows two distinct populations: one above and one below 1 kHz. With one exception, a maximum number of two emissions were recorded per ear, each in a different population. An amplitude distribution of a frog emission was sampled, from which it was concluded that the emission is generated by an active oscillator. The spectral width of an emission ranged from 1 to 200 Hz (average 38 Hz). There was negative correlation between sound pressure level of an emission and spectral width. In 4 frogs the dependence of emission power and frequency on temperature was investigated. An emission could be 'switched on and off' within a few degrees centigrade. At temperatures below the switching interval no emission was recorded; for higher temperatures emission power showed no dependence on temperature. Frequency increased with temperature (Q10 = 1.1 to 1.3). This yields a mismatch with temperature dependence of best frequencies of auditory fibers. The consequences of this mismatch are discussed.

Dissecting the frog inner ear with Gaussian noise. I. Application of high-order Wiener-kernel analysis.

Wiener kernel analysis was used to characterize the auditory pathway from tympanic membrane to single primary auditory nerve fibers in the European edible frog, Rana esculenta. Nerve fiber signals were recorded in response to white Gaussian noise. By cross-correlating the noise stimulus and the nerve fiber response, we computed (1) the full second-order Wiener kernel, and (2) the diagonals of the zeroth- to fourth-order Wiener kernels. These diagonals are usually referred to as polynomial correlation functions. The measured Wiener kernels were fitted with a 'sandwich' model. A new fitting procedure was used to compute the response characteristics of (1) the first filter, (2) the static nonlinearity, and (3) the second filter, which form the functional components of the model. The first filter is a bandpass filter. In the majority of low frequency fibers, with best excitatory frequency (BEF) < 800 Hz, this filter was tuned to two frequencies. This dual tuning mechanism gives rise to 'off-diagonal' components in the second-order Wiener kernel. The static nonlinearity resembles a rectifier, and is dominated by second-order (quadratic) nonlinearity. As a function of BEF, the shape of the nonlinearity changes systematically. Finally, the last filter in the model was a low pass filter. Across fibers, its cutoff frequency f-3dB ranged from 106 to 434 Hz.

Dissecting the frog inner ear with Gaussian noise. II. Temperature dependence of inner ear function.

The temperature dependence of the response of single primary auditory nerve fibers (n = 31) was investigated in the European edible frog, Rana esculenta (seven ears). Nerve fiber responses were analyzed with Wiener kernel analysis and polynomial correlation. The responses were described with a cascade model, consisting of a linear bandpass filter, a static nonlinearity, and a linear lowpass filter. From the computed Wiener kernels and the polynomial correlation functions, the characteristics of the three model components were obtained. With increasing temperature (1) tuning of the first filter increased in the majority (n = 16) of amphibian papilla fibers (best excitatory frequency, BEF < 1 kHz, n = 21) but remained unchanged in the majority (n = 10) of basilar papilla fibers (BEF > 1 kHz, n = 11), (2) the gain of the first filter remained unchanged, (3) the shape of nonlinear IO function remained unchanged, (4) the combined gain of the static nonlinearity and the second filter usually increased, but displayed considerable scatter across fibers (from -0.7 dB/degrees C to 3 dB/degrees C), and (5) the cutoff frequency of the second lowpass filter increases, with average 0.13 oct/degrees C. The immunity of the shape of the nonlinearity is considered evidence of a temperature independent gating mechanism in the transduction channels. The temperature dependence of the second filter may have resulted from a decrease of the hair cell membrane resistance, but may also reflect changes in subsequent staging of nerve fiber excitation.

DC injection alters spontaneous otoacoustic emission frequency in the frog.

A permanent wire electrode was implanted in the inner ear of the green frog (Rana esculenta). Through this electrode direct current was delivered to the inner ear fluid of the frog, during recording of a spontaneous otoacoustic emission (SOAE). Both SOAE frequency and amplitude turned out to depend on DC level and polarity. Possible explanations for the observed phenomena are given, based on a dissipative limit-cycle oscillator model for SOAE generation, and the assumption that SOAE frequency depends on hair cell ion channel inductance.

Low-voltage field-emission scanning electron microscopy of non-coated guinea-pig hair cell stereocilia.

The stereociliar structures of the guinea-pig cochlear organ of Corti were studied at low-voltage (1-5 kV) with field-emission scanning electron microscope (SEM) using various pre- and post-fixation methods, such as OTOTO (OsO4/thiocrbohydrazide/OsO4/thiocarbohydrazide/OsO4) and TAO (tannic acid/arginine/OsO4), and different dissection procedures of the cochlea. A perfusion and immersion pre-fixation with glutaraldehyde, in combination with removal of the bony wall and stria vascularis from the cochlea, followed by the TAO non-coating treatment gave the best result at 2 kV acceleration voltage. Due to these new technique, several interesting delicate structures of the stereocilia, in particular fine surface structures, were detected for the first time using SEM. These findings include the different types of cross-links and tip links, i.e., the fine surface morphology of the stereocilia and their attachments and imprints in the tectorial membrane (TM). One of the most interesting findings in this study is a network of long filamentous structures, which has been identified mainly at the top of the longest stereocilia and the undersurface of the TM and which may represent the glycocalyx. These findings and their possible implications in the process of mechanoelectrical transduction will be discussed.

Sensory cell damage in two-phase endolymphatic hydrops: a morphologic evaluation of a new experimental model by low-voltage scanning techniques.

HYPOTHESIS: The aim of this study was to create a more dynamic animal model of Ménière's disease combining multiple causes, such as the role of endocrine factors and endolymphatic sac dysfunction, that may mimic the fluctuant characteristics of Ménière's disease. BACKGROUND: Endolymphatic hydrops remains to be considered a pathologic substrate in the etiology of Ménière's disease. The classic guinea pig model of inducing hydrops by total destruction of the endolymphatic sac is a nonphysiologic rigid model of Ménière's disease. METHODS: The authors developed the two-phase endolymphatic hydrops model by inducing hydrops by mild chronic endolymphatic sac dysfunction, in combination with increased endolymph production by aldosterone. Sensory cell damage was evaluated by low-voltage field emission scanning microscopy. RESULTS: This study describes a wide spectrum of morphologic effects of the outer hair cells in radial gradients, in which most effects were observed in the third to second row of outer hair cells, and longitudinal gradients in which the most severe effects were observed in the apical turns. Most affected were the ears that underwent distal endolymphatic sac dissection followed by the administration of aldosterone. Damaging effects proceeded from degeneration and absence of short stereocilia of outer hair cells and even some inner hair cells in the apical turns, to stereociliary disarrangement and atrophy, followed by degeneration and absence of outer hair cells, which were replaced by supporting cells. CONCLUSION: The two-phase endolymphatic hydrops model seems to represent a functional model that may mimic the fluctuant characteristics of Ménière's disease and emphasizes the influence of multiple and coexisting hydrops-inducing influences.

Caloric stimulation of the vestibular system of the pigeon under minimal influence of gravity.

Vestibular microphonics in response to acoustical stimulation were recorded in cochlea-deprived pigeons. Endolymph flow in the semicircular canals alters this response. A caloric stimulus was applied to the horizontal canal or the posterior vertical canal close to the ampulla. To minimize the influence of gravity, the canal being studied was positioned in the horizontal plane. Local temperature changes were generated with an electrically heated miniature copper probe, producing an approximately trapezoidal temperature profile. Because of the opposite hair cell polarization in the horizontal and the vertical ampulla, we could prove that the observed effects were of (hydro-)mechanical origin and were not caused by a direct effect of sensory epithelium temperature change. The first effect of temperature increase was an utriculopetal endolymph flow, as predicted by the 'expansion theory'.

Bárány's theory is right, but incomplete. An experimental study in pigeons.

The horizontal semicircular canal in pigeons was thermally stimulated with a miniature Peltier-element device. Sound evoked electric responses from the vestibular organ were modulated by the caloric stimulus. Experiments were performed under minimal influence of gravity, (horizontal canals in horizontal plane), and with the pigeons head tilted forwards or backwards. On the basis of the results it was concluded that the influence of gravity can be described by Bárány's convection theory. Extra effects are, however, present and they are most probably partly of mechanical origin (fluid expansion) and partly due to a direct influence of temperature on the vestibular receptors. Further experiments are needed to investigate the origin of these extra effects in more detail.

Two-phase endolymphatic hydrops: a new dynamic guinea pig model.

The classical guinea pig model for Meniere's disease, in which endolymphatic hydrops was achieved by destruction of the endolymphatic sac and obliteration of the endolymphatic duct, is a non-physiological profound model with shortcomings in relation to Meniere's disease as seen in patients. We developed a more subtle animal model; the two-phase endolymphatic hydrops. This model is based on a combination of chronic endolymphatic sac dysfunction, induced by slight destruction of the most distal part of the endolymphatic sac, and acute stress-induced endolymph production by stimulation of the Na/K-ATPase in the stria vascularis with aldosterone. Light microscopy of the fluid compartments of four groups of cochleas was used to examine them for the presence of endolymphatic hydrops: i) Normal (control) cochleas showed no hydrops; ii) some of the non-operated (no destruction) aldosterone-treated cochleas showed small degrees of hydrops mainly present in the basal turns; iii) mild dissection of the endolymphatic sac without administration of aldosterone produced a hydrops which was mainly present in the cochlear apex; iv) combination of chronic endolymphatic sac dysfunction and acute attacks of endolymph production by aldosterone administration revealed the most severe degrees of hydrops in all cochlear windings, damage to cochlear structures, and cellular disturbances of the epithelial lining of the endolymphatic sac. This new model may represent a more physiologic and dynamic approach to Meniere's disease and may explain the etiology of many symptoms in patients such as the fluctuant nature and the types of sensoneuronal hearing losses.

Change of guinea pig inner ear pressure by square wave middle ear cavity pressure variation.

The inner ear fluid pressure of guinea pigs was measured during square wave middle ear cavity pressure variation. Time constants were derived for the slopes of the inner ear pressure recovery curves after middle ear pressure change. A "single exponential" function did not fit well and therefore more complicated functions were used for this purpose. For middle ear pressure increasing from zero to a few centimetres of water, returning to zero again, decreasing from zero to minus a few centimetres of water and then returning to zero again, time constants for the inner ear pressure recovery curves were on average 15.0, 8.6, 2.5 and 2.5 s, respectively. The results could not be described using a linear model with constant window membrane compliance and cochlear aqueduct flow resistance. A possible explanation for the large difference in time constants for positive or negative middle ear pressure changes is a dependence on aqueduct flow resistance or round window membrane position.

The vestibular evoked response to linear, alternating, acceleration pulses without acoustic masking as a parameter of vestibular function.

In this study, short latency vestibular evoked potentials (VsEPs) were recorded in five guinea pigs in response to alternating linear acceleration pulses with and without acoustic masking. A steel bolt was implanted in the skull and coupled to a shaker. Linear acceleration pulses (n = 400) in upward, downward or alternating directions were given, with a peak acceleration of 4g after 0.5 msec. Tests were repeated with acoustic masking, after modiolus destruction and after application of KCl in the vestibule. Stimuli of the vestibular nerve were recorded with a platinum electrode in the bony facial nerve canal in the bulla. Unilateral linear acceleration showed a shallow plateau at 0.5 msec, which disappeared with alternating acceleration impulses and after modiolus destruction. Therefore all further tests were done with alternating impulses. After a latency time of 0.8 msec a multiwave response was seen, with a first positive peak P1 at 1.16 ms. These were followed by other positive and negative peaks (N1, P2, N2, P3, N3). With the elimination of cochlear influences by using acoustic masking, P1 remained stable, while subsequent peaks were altered or eliminated. After modiolus destruction, the P1 peak remained, although with a smaller amplitude due to vestibular damage. After application of a saturated KCl solution in the vestibule all responses, including P1, disappeared, thus confirming the vestibular origin of these responses. We conclude that the onset latency of the VsEP and the peak latency and level of the first positive peak P1 in response to alternating linear acceleration pulses without acoustic masking, measured in the facial canal, are good and stable parameters of vestibular function in guinea pigs.

Monitoring inner ear pressure changes in normal guinea pigs induced by the Meniett20.

The inner ear fluid pressure of guinea pigs was measured during a series of complex oscillating middle ear pressure changes induced by the Meniett20 (Pascal Medical, Sweden), a possible therapeutic pressure generator to be used by patients with Meniere's disease. Middle ear pressure changes were transferred instantly to the inner ear, although inner ear pressure declined while middle ear pressure stayed relatively stable. An average undershoot of -1.0 cm H2O with respect to the steady-state pressure was seen after application of a pressure pulse, which was released in a few seconds. The results did not fully comply with a simple linear model in which a constant flow resistance between the inner ear and cerebrospinal space was assumed.

Dynamics of inner ear pressure change caused by intracranial pressure manipulation in the guinea pig.

Previous studies have shown that pressure changes in the cerebrospinal fluid compartment are transmitted to the inner ear. The main route for pressure transfer is the cochlear aqueduct. about which little is known with regard to its dynamic properties. In the present study, sudden intracranial pressure changes (square waves and short pulses) were created in guinea pigs by means of an electronically controlled infusion system. Simultaneously with pressure manipulation, hydrostatic pressure was monitored in both the peridural space and the perilymphatic compartment of the inner ear. The onset of an inner ear pressure change following manipulation of intracranial pressure was immediate. Inner ear pressure increased or decreased without a measurable time lag, and equalized within a few seconds. During square wave intracranial pressure manipulation, inner ear pressure equalized somewhat more slowly after pressure increase than after pressure decrease. To a first approximation, the pressure equalization curves for the inner ear could be fitted with a single exponential function, rising or falling with a time constant in the range 1-3 s, and the system can be described as a low-pass filter composed of a constant compliance and a constant flow resistance. Detailed analysis, however, showed small deviations from a purely exponential recovery process. With a more complicated (non-linear) model, almost perfect fits to the inner ear pressure equalization curves could be obtained. This non-linearity may be a consequence of the dependence of the compliance and, or flow resistance on pressure.

Ultrastructure of the endolymphatic sac in two-phase endolymphatic hydrops in the guinea pig.

Two-phase endolymphatic hydrops is a subtle experimental model for Meniere's disease. Chronic dysfunction of the endolymphatic sac, induced by dissection of the most distal part without causing damage to the intermediate part, is combined with increased endolymph production induced by administration of aldosterone which stimulates the N/K-ATPase in the stria vascularis. A transmission electron microscopic study was performed on the endolymphatic sacs of four groups of guinea pig cochleas: controls: non-operated aldosterone-treated cochleas; operated (dissection of the endolymphatic sac) cochleas; operated and aldosterone-treated cochleas. Light and electron microscopy showed a normal morphology in the controls. Aldosterone treatment had no visible effect. Dissected ears revealed severe deviations. The epithelium of the intermediate sac was low, showed dilated lateral intercellular spaces indicating elevated fluid transport and displayed serious degenerative processes. Distally, the endolymphatic sac was completely blocked by newly formed bone. Additional aldosterone treatment had no cumulative effect on the dissected ears.

Three-dimensional reconstruction of the pigeon inner ear.

Three-dimensional reconstructions of the inner ear of the pigeon (Columba livia domestica), from two-dimensional images, obtained with (conventional) light microscopy or orthogonal-plane fluorescence optical sectioning (OPFOS), are presented. The results are compared with available information on the dimensions and orientation of the semicircular canals.