Functional and physical outcomes following use of a flexible CO2 laser fiber and bipolar electrocautery in close proximity to the rat sciatic nerve with correlation to an in vitro thermal profile model.

This study compared functional and physical collateral damage to a nerve when operating a Codman MALIS Bipolar Electrosurgical System CMC-III or a CO2 laser coupled to a laser, with correlation to an in vitro model of heating profiles created by the devices in thermochromic ink agarose. Functional damage of the rat sciatic nerve after operating the MALIS or CO2 laser at various power settings and proximities to the nerve was measured by electrically evoked nerve action potentials, and histology of the nerve was used to assess physical damage. Thermochromic ink dissolved in agarose was used to model the spatial and temporal profile of the collateral heating zone of the electrosurgical system and the laser ablation cone. We found that this laser can be operated at 2 W directly above the nerve with minimal damage, while power settings of 5 W and 10 W resulted in acute functional and physical nerve damage, correlating with the maximal heating cone in the thermochromic ink model. MALIS settings up to 40 (11 W) did not result in major functional or physical nerve damage until the nerve was between the forceps tips, correlating with the hottest zone, localized discretely between the tips.

Basilar membrane and tectorial membrane stiffness in the CBA/CaJ mouse.

The mouse has become an important animal model in understanding cochlear function. Structures, such as the tectorial membrane or hair cells, have been changed by gene manipulation, and the resulting effect on cochlear function has been studied. To contrast those findings, physical properties of the basilar membrane (BM) and tectorial membrane (TM) in mice without gene mutation are of great importance. Using the hemicochlea of CBA/CaJ mice, we have demonstrated that tectorial membrane (TM) and basilar membrane (BM) revealed a stiffness gradient along the cochlea. While a simple spring mass resonator predicts the change in the characteristic frequency of the BM, the spring mass model does not predict the frequency change along the TM. Plateau stiffness values of the TM were 0.6 ± 0.5, 0.2 ± 0.1, and 0.09 ± 0.09 N/m for the basal, middle, and upper turns, respectively. The BM plateau stiffness values were 3.7 ± 2.2, 1.2 ± 1.2, and 0.5 ± 0.5 N/m for the basal, middle, and upper turns, respectively. Estimations of the TM Young's modulus (in kPa) revealed 24.3 ± 25.2 for the basal turns, 5.1 ± 4.5 for the middle turns, and 1.9 ± 1.6 for the apical turns. Young's modulus determined at the BM pectinate zone was 76.8 ± 72, 23.9 ± 30.6, and 9.4 ± 6.2 kPa for the basal, middle, and apical turns, respectively. The reported stiffness values of the CBA/CaJ mouse TM and BM provide basic data for the physical properties of its organ of Corti.

Spread of cochlear excitation during stimulation with pulsed infrared radiation: inferior colliculus measurements.

Infrared neural stimulation (INS) has received considerable attention over the last few years. It provides an alternative method to artificially stimulate neurons without electrical current or the introduction of exogenous chromophores. One of the primary benefits of INS could be the improved spatial selectivity when compared with electrical stimulation. In the present study, we have evaluated the spatial selectivity of INS in the acutely damaged cochlea of guinea pigs and compared it to stimulation with acoustic tone pips in normal-hearing animals. The radiation was delivered via a 200 µm diameter optical fiber, which was inserted through a cochleostomy into the scala tympani of the basal cochlear turn. The stimulated section along the cochlear spiral ganglion was estimated from the neural responses recorded from the central nucleus of the inferior colliculus (ICC). ICC responses were recorded in response to cochlear INS using a multichannel penetrating electrode array. Spatial tuning curves (STCs) were constructed from the responses. For INS, approximately 55% of the activation profiles showed a single maximum, ∼22% had two maxima and ∼13% had multiple maxima. The remaining 10% of the profiles occurred at the limits of the electrode array and could not be classified. The majority of ICC STCs indicated that the spread of activation evoked by optical stimuli is comparable to that produced by acoustic tone pips.

Unbiased counting of neurons in the cochlea of developing gerbils.

Accurate counting of neurons in the cochlea has a significant impact on the interpretation of research and clinically relevant data. However, reports of numbers of neurons in the spiral ganglion are widely variable across studies, even within the same species. We suggest that the implementation of a more standardized, unbiased counting method will improve the consistency and accuracy of neuron counts and will impact scientific interpretations. To test this view, we compared, in different ways, the numbers of neurons in the spiral ganglia of developing gerbils, previously reported to decrease by 22-27% between birth and age 7 days. Cochleae from gerbils, aged newborn, 7 days, 20 days, 1.5 years and 2.5 years were embedded in Araldite and serially sectioned at 5 µm. A computer based stereological method was used to unambiguously count every neuron in serial sections, either throughout the entire cochlea, or in a 100-µm segment of the cochlea. No significant changes in neuron numbers during cochlear maturation were found. We demonstrate that in methods using sampling of sections, the identity of the starting section and the interval between sections impacts the variability of the estimate of neuron numbers. In addition, we show that packing density differs between the newborn and seven-day old animals. The data demonstrate that variability in counting methods and the comparison of non-uniform samples can lead to neuron number estimates that show differences where none exist. We propose that a standardized counting protocol be implemented across studies and suggest possible approaches to different types of comparisons between neurons of spiral ganglia from different sources.

Visualizing soft tissue in the mammalian cochlea with coherent hard X-rays.

This paper concerns an important aspect of current developments in medical and biological imaging: the possibility for imaging soft tissue at relatively high resolution in the micrometer range or better, without tedious and/or entirely destructive sample preparation. Structures with low absorption contrast have been visualized using in-line phase contrast imaging. The experiments have been performed at the Advanced Photon Source, a third generation source of synchrotron radiation. The source provides highly coherent X-ray radiation with high photon flux (>10(14) photons/s) at high photon energies (5-70 keV). Thick gerbil cochlear slices have been imaged and were compared with those obtained by light microscopy. Furthermore, intact gerbil cochleae have been imaged to identify the soft tissue structures involved in the hearing process. The present experimental approach was essential for visualizing the inner ear structures involved in the hearing process in an intact cochlea.

Survival and morphology of auditory neurons in dissociated cultures of newborn mouse spiral ganglion.

We have systematically characterized neuronal survival and growth in cultures derived from newborn/postnatal day 1 mouse cochlea. Dissociated cultures of the cochlear spiral ganglion provide an experimental environment in which to examine molecular mechanisms of survival, development and physiology of auditory neurons. To relate survival to the total number of neurons present in the source tissue, three cochleas from different newborn CD-1 mice were embedded in Araldite resin and serially sectioned at 5 mum thickness. All neurons were counted. To avoid overcounting, each section served as a lookup section for the next, giving 8240+/-423 (S.D.) neurons per ganglion. Cultures maintained in the presence of adjacent non-neural tissue, brain-derived neurotrophic factor, neurotrophin 3, leukemia inhibitory factor (LIF) and 10% fetal bovine serum returned the best overall survival (30%) at 42 h post-plating. Best overall survival required the continuous presence of a serum component(s) larger than 100,000 MW. Plating efficiency (number of neurons that attach to the well after 4 h) was similar in the presence or absence of LIF. Inclusion of LIF maintained 100% survival of plated neurons over 42 h of culture; without LIF, a large fraction of the neurons did not survive. LIF appeared to maintain survival by preferentially preserving a population of bipolar neurons, while having little effect on the number of monopolar neurons. This work provides quantitative measures of survival and morphology of auditory neurons in vitro. The results support the idea that survival of spiral ganglion neurons in vivo may depend on interactions with adjacent, non-neural tissue and raise the possibility that maintenance of bipolar morphology after hair cell damage may require biochemical mechanisms in addition to those induced by neurotrophins.

Cochlear dimensions obtained in hemicochleae of four different strains of mice: CBA/CaJ, 129/CD1, 129/SvEv and C57BL/6J.

Because homologies between mice and human genomes are well established and hereditary abnormalities are similar in both, mice present a valuable animal model to study hereditary hearing disorders in humans. One of the manifestations of hereditary hearing disorders might be in the structure of cochlear elements, such as the gross morphology of the cochlea. Cochlear dimensions, however, are one factor that determines inner ear mechanics and thus hearing function. Therefore, gross cochlear dimension might be important when different strains of mice are compared regarding their hearing. Although several studies have examined mouse inner ear structures on a sub-cellular level, only few have studied cochlear gross morphology. Moreover, the sparse data available were acquired from fixed and dehydrated tissue. Dehydration, however, produces severe distortion of gel-like cochlear structures such as the tectorial membrane and the basilar membrane hyaline matrix. In this study, the hemicochlea technique, which allows fresh mouse cochlear material to be viewed from a radial perspective, was used to provide an itemized study of the dimensions of gross cochlear structures in four mouse strains (CBA/CaJ, 129/SvEv, 129/CD1 and C57BL/6J). Except for the CBA/CaJ, these strains are known to possess genes for age-related hearing loss. The measurements showed no major differences among the four strains. However, when compared with previous data, the thickness measures of the basilar membrane were up to 10 times larger. Such differences are likely to result from the different techniques used to process the material. The hemicochlea technique eliminates much of the distortion caused by dehydration, which was present in previous experiments.

Use of the pinna reflex as a test of hearing in mutant mice.

Although it is a gross measure, the pinna reflex test is easily administered and is, therefore, incorporated as a general screening tool in mutagenesis programs. Our recent application of this approach indicated that mutant mice lacking one of the small Maf proteins, in this case MafG, failed to exhibit a pinna reflex. In contrast, littermate controls, with the same mixed 129/CD1 background, and including both wild type and heterozygous mutant animals, passed the test. Because previous studies indicate that mafG is expressed in both cochlear and vestibular parts of the mouse inner ear, the source of this 'presumed deafness' was further assessed by making round window recordings to determine compound action potential thresholds. Auditory brainstem responses were also acquired to assess function along portions of the central auditory pathway. In all cases, responses in homozygous mutants (-/-) were comparable to those obtained from littermate controls, either wild type (+/+) or heterozygous mutants (+/-). Gross anatomy of the organ of Corti was also found to be similar in all three groups of mice. Hence, the lack of a pinna reflex must relate to nonauditory causes.

A reconsideration of sound calibration in the mouse.

Although it is traditional to perform sound calibrations in anesthetized animals by placing a probe-tube microphone near the tympanic membrane, these measurements are inaccurate at high stimulus frequencies where hearing must be quantified in the mouse. Hence, our motivation to develop another approach using the mouse ear canal as a coupler. Results of real-ear-canal calibrations indicate that an average calibration can be used to estimate sound pressure levels in the three mouse strains tested. Similar estimates were also obtained using a tubing coupler, whose volume was comparable to that of the ear canal, thereby offering a simpler alternative. In addition, real-head calibrations were also performed to provide a procedure that can be used in situations where the ear is not dissected, as in measurements of the auditory brainstem response. Calibrations for open, rather than closed, sound-delivery systems were also evaluated using a modified method of substitution.

Development of the gerbil inner ear observed in the hemicochlea.

A frequency-dependent change in hearing sensitivity occurs during maturation in the basal gerbil cochlea. This change takes place during the first week after the onset of hearing. It has been argued that the mass of a given cochlear segment decreases during development and thus increases the best frequency. Changes in mass during cochlear maturation have been estimated previously by measuring the changes in cochlear dimensions. Fixed, dehydrated, embedded, or sputter-coated tissues were used in such work. However, dehydration of the tissue, a part of most histological techniques, results in severe distortion of some aspects of cochlear morphology. The present experiments, using a novel preparation, the hemicochlea, show that hydrated structures, such as the tectorial membrane and the basilar membrane hyaline matrix, are up to 100% larger than estimated previous studies. Therefore, the hemicochlea was used to study the development of cochlear morphology in the gerbil between the day of birth and postnatal day 19. We used no protocols that would have resulted in severe distortion of cochlear elements. Consequently, a detailed study of cochlear morphology yields several measures that differ from previously published data. Our experiments confirm growth patterns of the cochlea that include a period of remarkably rapid change between postnatal day 6 and 8. The accelerated growth starts in the middle of the cochlea and progresses toward the base and the apex. In particular, the increase in height of Deiters' cells dominated the change, "pushing" the tectorial membrane toward scala vestibuli. This resulted in a shape change of the tectorial membrane and the organ of Corti. The tectorial membrane was properly extended above the outer hair cells by postnatal day 12. This time coincides with the onset of hearing. The basilar membrane hyaline matrix increased in thickness, whereas the multilayered tympanic cover layer cells decreased to a single band of cells by postnatal day 19. Before and after the period of rapid growth, the observed gross morphological changes are rather small. It is unlikely that dimensional changes of cochlear structures between postnatal days 12 and 19 contribute significantly in the remapping of the frequency-place code in the base of the cochlea. Instead, structural changes affecting the stiffness of the cochlear partition might be responsible for the shift in best frequency.

Sodium, potassium, chloride and calcium concentrations measured in pigeon perilymph and endolymph.

According to Davis' (1965) model of the inner ear, a potential difference between the endocochlear potential and the hair cell resting potential drives the transduction current across the apical hair cell membrane. It is assumed that the endocochlear potential (EP) consists of two components. The first is a diffusion potential, which depends on the ionic composition of endolymph and perilymph and on the permeability of the perilymph-endolymph barrier. The second is an electrogenic component which is determined by active ion transport across the perilymph-endolymph barrier. In birds, the EP is between +8 and +20 mV. Little is known about the underlying mechanisms responsible for the measured EP in birds. The present paper studies whether ionic compositions of endo- and perilymph might explain the EP in birds. Concentrations of Na+, K+, Ca2+ and Cl- in pigeon scala vestibuli, scala tympani and scala media were determined with ion-selective microelectrodes. Na+, K+, Ca2+ and Cl- were 150.0, 4.2, 1.4 and 117.0 mM in perilymph (scala tympani and scala vestibuli). In scala media, the concentrations of K+, Ca2+ and Cl- were 140.6, 0.23 and 142.1 mM.

Evidence for an anion exchange mechanism for uptake of conjugated bile acid from the rat jejunum.

Absorption of conjugated bile acids from the small intestine is very efficient. The mechanisms of jejunal absorption are not very well understood. The aim of this study was to clarify the mechanism of absorption of conjugated bile acid at the apical membrane of jejunal epithelial cells. Brush-border membrane vesicles from intestinal epithelial cells of the rat were prepared. Absorption of two taurine-conjugated bile acids that are representative of endogenous bile acids in many variate vertebrate species were studied. In ileal, but not jejunal brush-border membrane vesicles, transport of conjugated bile acids was cis-stimulated by sodium. Transport of conjugated bile acids was trans-stimulated by bicarbonate in the jejunum. Absorption of conjugated dihydroxy-bile acids was almost twice as fast as of trihydroxy-bile acids. Coincubation with other conjugated bile acids, bromosulfophthalein, and DIDS, as well as by incubation in the cold inhibited the transport rate effectively. Absorption of conjugated bile acids in the jejunum from the rat is driven by anion exchange and is most likely an antiport transport.

Morphology of the unfixed cochlea.

Our knowledge of cochlear geometry is based largely upon anatomical observations derived from fixed, dehydrated, embedded and/or sputter-coated material. We have now developed a novel preparation, the hemicochlea, where for the first time living cochlear structures can be observed in situ and from a radial perspective. The experiments were performed on the Mongolian gerbil. Ion substitution experiments suggest that no significant swelling or shrinkage occurs when the preparation is bathed in normal culture medium, so long as calcium concentration is kept at endolymph-like (20 microM) levels. The tectorial membrane-reticular lamina relationship appears to remain well preserved. Hensen's stripe maintains a close relationship with the inner hair cell stereociliary bundle, unless the mechanical coupling becomes disturbed. In addition, standard fixation and/or dehydration procedures are used to quantify changes due to shrinkage artifacts. Various morphometric gradients are examined in unfixed specimens from apical, middle, and basal turns.

Basilar membrane vibration in the gerbil hemicochlea.

Excised gerbil cochleae were cut along the mid-modiolar plane (hemicochlea). Along one-half turn of this preparation, fluorescent microbeads were placed on the basilar membrane (BM). The BM was vibrated with click stimuli (50 micros) produced mechanically by a piezo pusher. The stimulus delivery probe could be positioned either more apical or more basal from the beads. Vibration patterns were measured with a wide bandwidth photomultiplier from the movements of the beads. When the probe was positioned more basal, the responses to click stimuli were brief, damped sinusoids. According to the fast Fourier transforms (FFTs) of the averaged time wave forms, the best frequency between successive beads decreased toward the apex (0.8 octave/mm). Sharpness of tuning of the normalized FFT spectra (NQ10dB) on average was 1.5. Response amplitude at a fixed input level, measured at different beads away from the stimulation site, dropped exponentially (58 dB/mm). In addition, for each individual bead, amplitude dropped linearly with decreasing stimulus intensity. In experiments where the stimulating probe was placed more apical, two major properties were observed: first, beads revealed only the spectral components present in the motion of the probe. Second, magnitude reduction of the displacement of the cochlear partition was greater, on average 155 dB/mm, indicating a lack of significant propagation in the reverse direction.

Modeling stochastic spike train responses of neurons: an extended Wiener series analysis of pigeon auditory nerve fibers.

A general method for developing data-based, stochastic nonlinear models of neurons by means of extended functional series expansions was applied to neural activities of pigeon auditory nerve fibers responding to Gaussaian white noise stimuli. To determine Wiener series representations of the investigated neurons the fast orthogonal search algorithm was used. The results suggest that nonlinearities are only instantaneous and that the signal transduction of the investigated sensory system can be described by cascades of dynamic linear and static nonlinear devices. However, only slight improvements result from the nonlinear terms. Considerable improvements are, nevertheless, possible by generalizing the ordinary Wiener series, so that prior neural activity can be taken into account. These extended series were used to develop stochastic models of spiking neurons. The models are able to generate realistic interspike interval distributions and rate-intensity functions. Finally, it will be shown that the irregularity in real and modeled action potential trains has advantages concerning the decoding of neural responses.

Development of activity patterns in auditory nerve fibres of pigeons.

Little is known about inner ear development in pigeons. This paper addresses the question of maturation in activity patterns of pigeon auditory nerve fibres, Pigeons that were 1, 2 and 4 weeks and 1, 2, 3 and 4 years old were investigated. Adult-like activity patterns are found 4 weeks post-hatching. Spontaneous activities of fibres in immature animals (about 40 spikes/s) are half that found in adults. Spontaneous discharge rate does not increase with decreasing characteristic frequency (CF) of the fibre if the animals are immature. Rate threshold are less sensitive in immature animals. Differences between the age groups are generally significant if the CFs of the fibres are below 1.3 kHz. Sharpness of tuning is already adult-like in l-week-old animals. Inter-spike time interval histograms (ISTH) of auditory fibres recorded in animals of all age groups often show Poisson-like distributions. Preferred intervals are found in 10% of the ISTHs of fibres in immature animals but in 30% of adults. Cross-correlations between heart beats of the animal and spontaneous activities show good correlation for about 70% of the fibres in immature animals. With the growth of the animals, the number of fibres showing correlation of spontaneous activities and heart beats decreases to about 40%. The basilar papilla of a 1-week-old animal is smaller than in an adult animal (by 10% in length and by 10% in width), judge by scanning electron microscopy (SEM), Changes of activity patterns in this study are likely to be a result of maturation of the middle ear. In addition to the latter, development of the inner ear is conceivable.

Rate-intensity-functions of pigeon auditory primary afferents.

Rate-intensity-functions (RI-functions) were determined in 150 primary auditory afferents in anaesthetized pigeon. Acoustic stimulation was either at characteristic frequency (CF) or half an octave below or above CF. Stimulated at CF, 37% of the fibres showed saturating RI-functions, whereas 50% showed sloping and 13% straight RI-functions. In the sloping RI-functions, a bend was found about 20 dB above the fibres' thresholds. For non-CF stimuli, the general shape of the RI-functions remained constant. However, the maximum evoked discharge rates were lower for frequencies below CF and higher for frequencies above CF. The data show that a population of neurones, the sloping and straight ones, code stimulus intensities over a wide intensity range. In combination with the scatter of the thresholds, intensity ranges greater than 100 dB are conceivable. It was concluded that the nonlinearities found in pigeon are not caused by basilar membrane (BM) mechanics, rather an origin at the hair cell-afferent nerve fibre system has to be considered.

Preferred intervals in birds and mammals: a filter response to noise?

Quasi-periodic spontaneous activity (preferred intervals, PIs) has been reported from avian primary auditory afferents. In mammals, PIs have not been reported, as yet. As the length of PIs is close to 1/characteristic frequency, it has been suggested that this type of spontaneous activity indicates particular mechanisms in avian inner ear transduction. However, the present paper shows that pigeon auditory fibres possessing preferred intervals in their spontaneous activity always belong to the most sensitive and the most sharply-tuned fibres recorded. This leads to the assumption that preferred intervals are the response of narrow-band filters to noise. This view is supported by three additional findings: (i) Near-threshold noise provokes PIs in avian fibres that show no spontaneous PIs. (ii) Similarly, PIs can also be evoked in mammalian (gerbil) auditory afferents by low level noise. (iii) Phase-locking of auditory afferents can be achieved by sound stimuli 10-20 dB below rate threshold. It is argued that no conclusions may be drawn from the presence of PIs about the nature of the underlying filter.

Effects of endolymphatic and perilymphatic application of salicylate in the pigeon. I: Single fiber activity and cochlear potentials.

The effects of salicylate on the mammalian cochlea function are well documented. However, there is a lack of reports on salicylate effects on the avian auditory periphery and it might well be that salicylate is not ototoxic at all in submammalian vertebrates. We therefore recorded single fiber activities, compound action potential (CAP) and endocochlear potential (EP) during application of salicylate (calculated final concentration of about 2-18 mmol/l) into the scala media of pigeons. We furthermore recorded CAP and EP during perilymphatic perfusion of salicylate (2-20 mmol/l). Salicylate applied into the scala media led to an elevation of tip threshold in single fibers ranging from 5 to 35 dB. The characteristic frequencies of the fibers were not changed. This effect on auditory nerve fibers was reflected in an elevation of CAP thresholds. The mean spontaneous discharge rate was either slightly increased or remained unchanged in the majority of fibers. Perilymphatic salicylate perfusion also led to an elevation of CAP thresholds that was reversible following subsequent perfusion with artificial perilymph. The EP remained unchanged in both application modes. The effects of salicylate were dose dependent and more pronounced in the mid- to high-frequency range. These results are consistent with an action of salicylate on the process (electrical or mechanical, or both) responsible for the sensitivity and frequency selectivity in the avian peripheral hearing organ.

Effects of endolymphatic and perilymphatic application of salicylate in the pigeon. II: Fine structure of auditory hair cells.

Large doses of salicylate are known to cause reversible ototoxic effects including fine structural alterations of the auditory hair cells in mammals. To investigate possible fine structural correlates of salicylate effects on pigeon auditory hair cells, the basilar papillae following perilymphatic or endolymphatic application of salicylate were fixed and processed for transmission electron microscopy. The pigeon auditory hair cells possessed organelles typically described in avians. A single or multi-layered array of cisternae along the cytoplasmic side of the lateral plasma membrane, i.e. subsurface cisternae that are characteristic for mammalian outer hair cells, was not seen. The most prominent fine structural alterations of hair cells after salicylate application were an increase in the luminal width of smooth and rough endoplasmic reticulum as well as the frequent occurrence of prominent single-membrane-bound vesicles filled with electron-dense bodies. Based on the assumption that subsurface cisternae represent a specialized form of endoplasmic reticulum, the present findings indicate that the structural correlates of salicylate toxicity are similar in mammalian and avian auditory hair cells.