Richard A. Schmiedt

Lateral wall Na, K-ATPase and endocochlear potentials decline with age in quiet-reared gerbils

Changes in the integrity of cochlear ion transport systems with age were examined in gerbils raised for 5-38 months in a quiet environment. Ion transport function was assessed by light microscopic immunohistochemical staining for the enzyme, Na,K-ATPase and by measurement of the endocochlear potential (EP). Small foci of strial atrophy accompanied by loss of immunostaining for Na,K-ATPase were observed in the stria vascularis of the apical and basal turns as early as 5 months of age. Cochleas from 29-38 month-old gerbils showed a loss of immunostaining for Na,K-ATPase in the stria in most of the apical turn with the degeneration extending well into the middle turn in many of the oldest ears. The extent of strial atrophy and loss of immunoreactive Na,K-ATPase in the basal turn varied considerably among the oldest cochleas. Populations of lateral wall fibrocytes (type II fibrocytes) normally rich in Na,K-ATPase exhibited a corresponding decrease in enzyme content in regions of advanced strial atrophy. The volume of immunostained stria vascularis correlated well with the magnitude of the resting EP. The results demonstrate that lateral wall ion transport systems in the gerbil cochlea degenerate as a function of age. The findings also provide good evidence for a functional relationship between the stria vascularis and the Na,K-ATPase-rich type II fibrocytes in generating and maintaining the EP.

Age-related changes in auditory potentials of Mongolian gerbil

The Mongolian gerbil is being evaluated as an animal model of age-related hearing loss (presbyacusis). Part of this evaluation involves estimating auditory thresholds from evoked potentials arising from the auditory nerve and brainstem. The gerbils are born and reared in an environment where the ambient noise level is less than 40 dBA. Some animals are followed longitudinally (8, 19, 23.5 and 36 months), others are studied at 6-8 months (controls), or at 36 months (cross-sectional). Physiological responses are obtained with the animals anesthetized with ketamine and xylazine and transdermal electrodes attached to the head. Auditory signals are tone pips with center frequencies from 1 to 16 kHz in octave steps. Signal levels are varied from 10 to 80 dB SPL in 10 dB steps. For animals (N = 48) in the age range of 6-8 months, mean auditory thresholds were about 20 dB SPL between 2.0 and 8.0 kHz, 25 dB at 16 kHz and 30 dB at 1.0 kHz. By age 22-24 months (N = 15) thresholds had increased by about 10 dB at nearly all frequencies. By age 36 months (N = 37 ears, 32 animals) threshold increases were about 30-35 dB at 8 and 16 kHz, were 25 dB at 4 kHz and 2 kHz, and were 19 dB at 1 kHz. These hearing losses in 36-month gerbil are qualitatively similar to human data for 60-65-year-old males and 70-year-old females. Individual differences in hearing loss were large with the range exceeding 65 dB. While some animals (26/37) had a high-frequency sloping loss, others (11/37) had a bimodal audiometric shape where the hearing loss was smallest at 4 kHz and increased by at least 10 dB at adjacent frequencies.

Spontaneous rates, thresholds and tuning of auditory-nerve fibers in the gerbil: Comparisons to cat data

Characteristics of 245 auditory nerve fibers in eleven Mongolian gerbils are described in terms of spontaneous rates, thresholds, and tuning curves. The animals were reared in a low-noise environment and had similar hearing thresholds across frequency. Tuning curves were obtained with an algorithm developed to characterize the tuning of auditory fibers in cat, thereby allowing direct comparisons to published data from cat. The results demonstrate that basic similarities exist between gerbil and cat data, although some minor differences are also apparent. Tuning curve bandwidths, as measured 10 and 40 dB above the thresholds at the characteristic frequency (CF), follow trends found in cat data. Like cat, auditory nerve fibers in the gerbil have a range of spontaneous rates. In individual gerbils, fibers associated with low spontaneous rates have higher thresholds than do fibers of similar CF with high rates. Five of the eleven gerbils showed profiles of spontaneous rate across frequency reminiscent of those obtained from quiet-raised young cats. The profiles of the remaining gerbils tended to be compressed to a smaller range of spontaneous rates for characteristic frequencies above about five kHz, much like older cats with unknown noise histories. The cause of the spontaneous compression is not obvious. The correspondence between cat and gerbil with regard to spontaneous rate and CF threshold implies the presence of fundamental mechanisms that are common to mammalian auditory systems.

Age-related decreases in endocochlear potential are associated with vascular abnormalities in the stria vascularis.

The density and diameter of strial capillaries were assessed in whole-mount preparations of the cochlear lateral wall from 18 gerbils aged in quiet for at least 36 months. Following morphometric analysis, histopathologic changes in selected regions of the lateral wall were examined by light and transmission electron microscopy. Alterations in strial vasculature were compared with the endocochlear potential (EP) measurements from the same ear. Vascular degeneration occurred in a segmental fashion in that regions of atrophic capillaries were found throughout the cochlea but primarily in the apical and lower basal turns and in the hook. The amount of stria with normal capillaries varied greatly among the aged ears, ranging from 19 to 87%. The resting EP also varied markedly, ranging from 23 to 83 mV. Little correlation was found between vascular alterations and the corresponding EP value from individual cochlear turns. However, significant correlations were found between the total strial area with normal vasculature and both the mean EP value and that recorded at either the round window or first turn in that ear.

Fine structure of the 2f1‐f2 acoustic distortion product: Changes with primary level

The fine frequency structure of the 2f1-f2 acoustic distortion product (ADP fine structure) was examined in ten human subjects with normal hearing. Primary frequencies (f1 and f2) were incremented in steps of 1/32 octave with an f2/f1 ratio of 1.2. The primary levels were kept equal to each other and varied from 45 to 65 dB SPL in 2.5-dB steps. The results show that the ADP fine structure is characterized by a series of peaks and valleys across frequency, with a peak-to-peak frequency spacing of about 3/32 octave and a peak-to-valley amplitude ratio of up to 20 dB. At frequencies below 4000 Hz, as primary level increases, the sharpness of the ADP fine structure is not significantly reduced and the pattern gradually shifts to lower frequencies. At frequencies above 4000 Hz, a flattening of the pattern is sometimes observed at high levels. A consequence of the underlying process responsible for the fine structure is that ADP input/output (I/O) functions can be highly variable in shape. Dramatic shape changes can occur for ADP I/O functions obtained with primary frequency changes of as little as 1/32nd of an octave. The outward cause of I/O function variability is the behavior of the ADP fine structure with level; i.e., it remains robust at high levels and systematically shifts to lower frequencies with level. As a result, ADP peaks can shift to valleys with increasing level and vice versa. Thus, small shifts in primary frequency can result in significant changes in the shape of the ADP I/O function in humans.

Contribution of Bone Marrow Hematopoietic Stem Cells to Adult Mouse Inner Ear: Mesenchymal Cells and Fibrocytes

Bone marrow (BM)‐derived stem cells have shown plasticity with a capacity to differentiate into a variety of specialized cells. To test the hypothesis that some cells in the inner ear are derived from BM, we transplanted either isolated whole BM cells or clonally expanded hematopoietic stem cells (HSCs) prepared from transgenic mice expressing enhanced green fluorescent protein (EGFP) into irradiated adult mice. Isolated GFP+ BM cells were also transplanted into conditioned newborn mice derived from pregnant mice injected with busulfan (which ablates HSCs in the newborns). Quantification of GFP+ cells was performed 3–20 months after transplant. GFP+ cells were found in the inner ear with all transplant conditions. They were most abundant within the spiral ligament but were also found in other locations normally occupied by fibrocytes and mesenchymal cells. No GFP+ neurons or hair cells were observed in inner ears of transplanted mice. Dual immunofluorescence assays demonstrated that most of the GFP+ cells were negative for CD45, a macrophage and hematopoietic cell marker. A portion of the GFP+ cells in the spiral ligament expressed immunoreactive Na, K‐ATPase, or the Na‐K‐Cl transporter (NKCC), proteins used as markers for specialized ion transport fibrocytes. Phenotypic studies indicated that the GFP+ cells did not arise from fusion of donor cells with endogenous cells. This study provides the first evidence for the origin of inner ear cells from BM and more specifically from HSCs. The results suggest that mesenchymal cells, including fibrocytes in the adult inner ear, may be derived continuously from HSCs. J. Comp. Neurol. 496:187–201, 2006.

Ouabain application to the round window of the gerbil cochlea: A model of auditory neuropathy and apoptosis

The physiological and morphological changes resulting from acute and chronic infusion of ouabain onto the intact round-window (RW) membrane were examined in the gerbil cochlea. Osmotic pumps fitted with cannulas allowed chronic (0.5–8 days) infusions of ouabain. Acute and short-term applications of ouabain (1–24 h) induced an increase in auditory-nerve compound action potential (CAP) thresholds at high frequencies with lower frequencies unaffected. The resulting threshold shifts were basically all (no response) or none (normal thresholds), with a sharp demarcation between high and low frequencies. Survival times of 2 days or greater after ouabain exposure resulted in complete auditory neuropathy with no CAP response present at any frequency. Distortion product otoacoustic emissions (DPOAEs) and the endocochlear potential (EP) were largely unaffected by the ouabain indicating normal function of the outer hair cells (OHC) and stria vascularis. One to 3 days after short-term applications, apoptosis was evident among the spiral ganglion neurons assessed both morphologically and with TdT-mediated dUTP-biotin nick end labeling (TUNEL). With 4–8 day survival times, most spiral ganglion cells were absent; however, a few cell bodies remained intact in many ganglia profiles. These surviving neurons had many of the characteristics of type II afferents. Our working hypothesis is that the ouabain induces a spreading depression among the type I ganglion cells by blocking the Na+,K+ ATPase pump. Because of the constant spike activity of these cells, the ouabain rapidly alters potassium concentrations within ([K+]i) and external to ([K+]o) the ganglion cells, thereby initiating an apoptotic cascade.

Boundaries of two-tone rate suppression of cochlear-nerve activity

Two-tone rate suppression was examined in the responses of single cochlear-nerve fibers in Mongolian gerbils. The iso-rate tracking algorithm developed by Kiang and Moxon (Kiang, N.Y.-S. and Moxon, E.C. (1974): J. Acoust Soc. Am. 55, 620-630) for obtaining tuning curves was modified to track iso-rate suppression boundaries as a function of frequency with the excitor tone fixed at the characteristic frequency (CF) of the fiber. Lower threshold boundaries of the areas of suppression flanking the tuning curve above and below CF were outlined for fibers over a large CF range. It was found that the boundaries of rate suppression obtained below CF were very stable in their absolute positions on the intensity-frequency plane. This stability was evident both as a function of fiber CF (0.6-15 kHz) and as a function of the shape of the tuning curve at a given CF. In other words, the suppression boundary obtained below CF was largely independent of the tuning curve. In a second series of experiments tuning curves were taken in the presence of a fixed tone placed in the suppression area located above the fiber CF. The fixed tone by itself was not excitatory. These tuning curves were compared to tuning curves obtained with a single tone. It was found that frequencies around the fiber CF were most affected (suppressed) by the presence of the second tone, and that the low-frequency tail of the tuning curve tended to shift toward the boundary of the suppression area below CF. Because this suppression boundary lies below the threshold of the normal tail of the tuning curve for many mid- and high-CF fibers, these fibers often became hypersensitive at low frequencies in the presence of the second tone above CF.

Age-related changes in cochleas of mongolian gerbils

The effects of aging on the gerbil cochlea were studied in 16 animals raised in a quiet environment. Animals were tested at ages ranging from 33 to 36 months, the approximate average lifespan of gerbils in our colony. Hearing sensitivity was assessed by measures of whole-nerve compound action potential (CAP) thresholds and surface preparations of the organ of Corti were subsequently examined by light microscopy for losses of sensory hair cells. These quiet-aged animals showed a wide range of hair-cell losses and threshold shifts. Outer hair cells often showed significant losses while inner hair cells were rarely absent. All animals had some threshold shift, especially at frequencies above 4 kHz. These shifts ranged from 1 to 68 dB. At high frequencies, threshold shifts often occurred without hair-cell losses at corresponding cochlear locations. At low frequencies, threshold shifts seldom reflected the losses of hair cells commonly found in the cochlear apex. Thus, the correlation of specific hair-cell losses and CAP threshold shifts at corresponding frequencies was poor. On the other hand, the total number of missing hair cells, irrespective of location, was a good, general indicator of the hearing capacity in a given ear. It appears that the factor or factors that makes cochleas susceptible to hair-cell loss with increasing age also affects other cochlear mechanisms that are necessary for normal functioning of the ear.

Nuclear Factor κB Deficiency Is Associated with Auditory Nerve Degeneration and Increased Noise-Induced Hearing Loss

Degeneration of the spiral ganglion neurons (SGNs) of the auditory nerve occurs with age and in response to acoustic injury. Histopathological observations suggest that the neural degeneration often begins with an excitotoxic process affecting the afferent dendrites under the inner hair cells (IHCs), however, little is known about the sequence of cellular or molecular events mediating this excitotoxicity. Nuclear factor κB (NFκB) is a transcription factor involved in regulating inflammatory responses and apoptosis in many cell types. NFκB is also associated with intracellular calcium regulation, an important factor in neuronal excitotoxicity. Here, we provide evidence that NFκB can play a central role in the degeneration of SGNs. Mice lacking the p50 subunit of NFκB (p50−/− mice) showed an accelerated hearing loss with age that was highly associated with an exacerbated excitotoxic-like damage in afferent dendrites under IHCs and an accelerated loss of SGNs. Also, as evidenced by immunostaining intensity, calcium-buffering proteins were significantly elevated in SGNs of the p50−/− mice. Finally, the knock-out mice exhibited an increased sensitivity to low-level noise exposure. The accelerated hearing loss and neural degeneration with age in the p50−/− mice occurred in the absence of concomitant hair cell loss and decline of the endocochlear potential. These results indicate that NFκB activity plays an important role in protecting the primary auditory neurons from excitotoxic damage and age-related degeneration. A possible mechanism underlying this protection is that the NFκB activity may help to maintain calcium homeostasis in SGNs.