Effects of intense noise exposure on fetal sheep auditory brain stem response and inner ear histology.

OBJECTIVE: To evaluate, in two separate experiments, the effects of intense noise exposures delivered to fetal sheep in utero during a time of rapid auditory development. DESIGN: In the first experiment, auditory brain stem response (ABR) thresholds to clicks and tone bursts were recorded from chronically instrumented fetal sheep in utero before and after exposure of pregnant ewes to intense broadband noise. A single 16 hr exposure was delivered at 113 days gestational age, a time when the ABR is just emerging. Thresholds were compared with an age-matched, nonexposed control group. In the second experiment, fetal sheep at the same gestational age were exposed four times to broadband noise and their cochleae were harvested 20 days later for histological analysis by the use of scanning electron microscopy. Comparisons were made with an age-matched, nonexposed control group. RESULTS: Experiment One: ABR thresholds recorded between 10 to 20 days after the exposure were not as sensitive as thresholds obtained from control fetuses. There was a tendency for thresholds to 0.5 kHz tone bursts to be more affected than thresholds to clicks. Experiment Two: Scanning electron microscopy of the organ of Corti from fetuses exposed to noise from 111 to 114 days gestational age revealed significant damage to inner and outer hair cells in the middle and apical turns of cochleae. Similar hair cell damage was not present in control fetuses. CONCLUSIONS: Intense exogenous noise penetrated the uterus of pregnant sheep and resulted in elevations in ABR thresholds 2 to 3 wk after exposure. In fetuses repeatedly exposed to noise, the middle and apical turns of the cochlea showed greater hair cell damage than found at the same locations in control cochlea. The basal turn of the cochlea was not damaged.

Distribution of Na,K-ATPase is normal in the inner ear of a mouse with a null mutation of the glucocorticoid receptor.

This study was performed in order to test the hypothesis that the glucocorticoid hormone stimulates the formation of Na,K-ATPase in the inner ear of the mouse. An immunohistochemical study with respect to the presence and distribution of glucocorticoid receptors (GR) and Na,K-ATPase in the vestibular and cochlear regions of the inner ear was performed on a C57BL mouse with a null mutation of the glucocorticoid receptor (GR mutant mouse). The wild type C57BL mouse and the CBA mouse served as normal controls. As expected, the homozygous GR mutant mouse showed no specific staining for GR in the inner ear. The heterozygous GR mutant mouse showed faint staining of GR in the spiral limbus, the spiral ganglion, the organ of Corti and the utricle. This staining was markedly less than in the wild type C57BL mouse. Antibody labelling of Na,K-ATPase in the inner ear showed no significant difference between the homozygous and the heterozygous GR mutant mouse as compared to the control wild type C57BL mouse or the CBA mouse. Although earlier studies have shown a positive correlation between levels of glucocorticoid hormone in serum and the concentration of Na,K-ATPase in the inner ear, the hypothesis that glucocorticoid hormones alone stimulate the formation of Na,K-ATPase in the inner ear could not be confirmed by this study. Thus other regulating substances must be considered.

Interaction of methylprednisolone and transient asphyxia on the inner ear of the adrenalectomized rat.

Methylprednisolone has been shown clinically to have beneficial effects on certain types of hearing loss. In the current study, compound action potential (CAP) thresholds, endocochlear potentials (EPs), and potassium concentration (CK+) values in the endolymph were determined under conditions of transient asphyxia (45 seconds) and methylprednisolone treatment (24 hours) in bilateral adrenalectomized rats. Treatment with methylprednisolone significantly reduced the effect of transient asphyxia on CAP thresholds as compared with nontreated animals. Methylprednisolone did not alter the dramatic short-term reduction in the EPs produced by anoxia. Potassium concentrations in treated adrenalectomized rats were significantly lower before transient asphyxia than in nontreated adrenalectomized rats. In the nontreated rats, transient asphyxia induced a reduction in CK+ levels that was not seen in the methylprednisolone-treated animals. The data support the clinical application of methylprednisolone for certain forms of hearing loss and for potassium imbalance in the endolymph.

Ca(2+)-ATPases in the cochlear duct.

Differing levels of the Ca(2+)-ATPase enzymes that reside on the plasma membrane (PM) and on the endoplasmic reticulum (ER) were identified in individual rat cochlear tissues by the use of a semi-quantitative enzyme-linked immunosorbent assay (ELISA). Unlike other studies, a specific antibody to PM Ca(2+)-ATPase was used to detect significantly greater levels (about 2x) of PM Ca(2+)-ATPase in the stria vascularis (SV) than that in the spiral ligament (SL) and organ of Corti (OC) tissues. Similarly, levels of ER Ca(2+)-ATPase were also significantly higher in the SV than in the SL and OC tissues. The presence of ER Ca(2+)-ATPase in the tissues of the SV has not been demonstrated previously. Given the importance of Ca2+ homeostasis in the inner ear, the statistically significantly higher densities of both PM and ER Ca(2+)-ATPase measured in the SV relative to the SL and OC regions would indicate tissue-specific responses to fluctuations in systemic and local Ca2+ concentrations.

Localization of secretory phospholipase A2 in mouse cochlear tissues and cultured cells.

OBJECTIVE: To localize the expression of secretory phospholipase A2 (sPLA2) in the cochlear duct. DESIGN: By means of an immunocytochemistry technique that used rabbit anti-porcine pancreatic phospholipase A2 antiserum, sPLA2 sites were identified in vivo and in vitro in the adult mouse cochlear duct. SUBJECTS: Eight C57BL/6N normal adult male mice (11-12 weeks old) (6 mice for the in vivo study, 2 mice for the in vitro study). RESULTS: Different patterns of immunostaining for sPLA2 were observed in tissues of the cochlear duct. High levels of sPLA2 were detected in the following areas: the basal cells of the stria vascularis, the organ of Corti, the spiral ganglion, and the cochlear nerve; moderate levels of sPLA2 were found in the suprastrial and poststrial portions of the spiral ligament; low levels of sPLA2 were observed in the marginal and intermediate cells of the stria vascularis and the infrastrial portion of the spiral ligament. Immunoreactivity for sPLA2 also was detected in cultured marginal cells of the stria vascularis and in fibrocytes of the spiral ligament. CONCLUSIONS: Secretory phospholipase A2 is present in mouse cochlear tissues and individual cultured cells. These results suggest that the levels of sPLA2 expression in the cochlear duct are cell specific.

Altered calcium homeostasis in the rat cochlear duct and endogenous corticosteroid insufficiency.

Free calcium concentration (CCa2+) profiles were evaluated in perilymph, endolymph, marginal cells, spiral ligament and blood serum of adrenalectomized (ADX) rats. Free CCa2+ was significantly greater in perilymph and significantly reduced in the serum of the ADX animals as compared to sham-operated animals. In addition, higher levels of free CCa2+ were found in the spiral ligament in ADX animals. Free CCa2+ did not appear to be affected by ADX in marginal cells and endolymph. These data suggest that marked reductions in endogenous levels of corticosteroids may have a systematic effect on free CCa2+ that is detectable in blood serum as well as cochlear fluids and tissues.

Structural maturation of the interface region between the stria vascularis and spiral ligament in the neonatal rat cochlea.

The ultrastructural morphology of the interface region between the stria vascularis (SV) and spiral ligament (SL) was examined in the neonatal rat cochlea via transmission electron microscopy. At postnatal day (PND) 3, morphology of both basal cells and fibrocytes was simple and immature. Only a small number of fibrocytes was observed in the SL. Intercellular junctions between basal cells and fibrocytes, and between adjacent fibrocytes, were few. At PND 7, the number of fibrocytes increased, and more organelles appeared within their cytoplasm. From PND 11 to 14, nuclei of the basal cells appeared to be more spindle-shaped and contained more heterochromatin. The cytoplasm of the fibrocytes was pale, and a greater number of cytoplasmic vesicles and mitochondria emerged. More intercellular junctions were observed between basal cells and fibrocytes at the interface region and between fibrocytes in the SL. By PND 21, the morphology of basal cells and fibrocytes and their intercellular junctions appeared to be adult-like. These morphological observations correlate with previous reports on the functional maturation of the developing rat cochlea.

Status of cochlear Na,K-ATPase in the aged SHR rat and its possible role in hearing loss.

The relationship between levels of Na,K-ATPase isoforms was studied in the lateral walls of the cochlea in aged spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Dense staining for the enzyme's alpha 1 subunit was found especially in the basal turns of the cochlea, while external sulcal cells were more intensely stained in the more apical turns in both SHR and WKY rats. In contrast, staining for beta 1 Na,K-ATPase was demonstrable in significant levels in the stria vascularis and suprastrial regions of the SHR rat, with involvement of the basilar suprastrial region pronounced in both strains of animals. Findings suggest a spatially defined, age-induced alteration in cochlear homeostasis with a possible consequent effect on sound perception.

Development of 11 beta-hydroxysteroid dehydrogenase expression in the rat cochlea.

11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) catalyzes the transformation of active glucocorticoid (GC) steroids to inactive 11-oxosteroids, as well as the reverse reaction. 11 beta-HSD was previously demonstrated specifically in the spiral ligament of the lateral cochlear wall where it was co-localized with adrenal steroid receptors. These findings imply that 11 beta-HSD regulates binding of corticoids to their inner ear receptors. The GC receptor expression initially occurs around the critical maturation period of the cochlear duct. 11 beta-HSD, which is an integral part of the cochlear steroid receptor system, could indirectly affect glucocorticoid-mediated induction processes. In this study the expression of 11 beta-HSD was studied in the postnatal rat cochlea from the 3rd to 30th postnatal day. Bouin's fixed, paraffin-embedded cochlear sections were processed for immunocytochemical detection of 11 beta-HSD using polyclonal antibodies against 11 beta-HSD. 11 beta-HSD expression appeared at the 12th postnatal day at low levels in spiral ligament tissues. From the 15th postnatal day, 11 beta-HSD expression was stronger and similar to that of the adult cochlea. No additional inner ear tissue region expressed 11 beta-HSD enzyme during the observed period. 11 beta-HSD expression coincides with the onset of functional maturity of the rat cochlear duct. The expression of 11 beta-HSD is preceded by the expression of GC receptors which appeared at the 7th postnatal day in the rat cochlea. These results further suggest an integrative role of the cochlear steroid receptor system in the homeostasis and functional maturation of the cochlea.

Correlative evidence of hypertension and altered cochlear microhomeostasis: electrophysiological changes in the spontaneously hypertensive rat.

The spontaneously hypertensive rat model has been used to show that hypertension is an important pathophysiological risk factor in age-related hearing loss. In the present study, compound action potential (CAP), electrochemical potential (ECP), and potassium concentration (CK+) measurements were taken from the cochlea of genetically predisposed, spontaneously hypertensive rats (SHR) and from normotensive Wistar-Kyoto (WKY) rats. In the SHR model, as the duration of hypertension increased with the animal's age (from 3 to 8 months), CAP thresholds increased, ECP increased in marginal cells only, and CK+ increased in both endolymph and marginal cells. Collectively, the data suggest that ionic alternations of cellular potentials are involved in hearing changes in the hypertensive state. Ultimately, such data may assist in understanding hearing loss in individuals who are diagnosed with hypertension.

Localization of Cu/Zn-SOD and Mn-SOD in the rat cochlea.

Distributions of the copper/zinc-superoxide dismutase (Cu/Zn-SOD) and the manganese superoxide dismutase (Mn-SOD) in the rat cochlea were studied by immunohistochemistry with specific polyclonal antibodies to Mn-SOD and Cu/Zn-SOD. Coexpression of Mn-SOD and Cu/Zn-SOD was observed in the cochlear tissues with differing immunoactivity intensities. The presence of Cu/Zn- and Mn-SOD implies the existence of a defense system against free oxygen radical (FOR) injury in cochlear tissues.

Electrochemical potentials and potassium concentration profiles recorded from perilymph, endolymph and associated inner ear tissues in adrenalectomized rats.

This study evaluated the electrochemical potentials and potassium concentration (Ck+) profiles in the perilymph, endolymph, marginal cells, and spiral ligament of adrenalectomized rats in which endogenous corticosteroids had been removed. Electrochemical potentials recorded at the four cochlear sites were not affected by adrenalectomy (ADX). Ck+ was greater in the endolymph of the ADX animals as compared to control animals. Additionally, there was an increase of Ck+ in the marginal cells, perilymph, and spiral ligament tissues of the ADX animals as compared to control animals, although the observed increases were not statistically significant. In a previous study (Ma et al., 1995a), it was found that potassium levels in the blood plasma of ADX animals were higher than those identified in normal rats; thus, ADX may have a systemic effect on Ck+ that is detectable in both tissues and fluids within the cochlea. Even though Ck+ was elevated within the cochlea in the ADX model, the functional response of the inner ear, as assessed electrophysiologically, was not altered.

Detection and regulation of Cu/Zn-SOD and Mn-SOD in rat cochlear tissues.

Relative levels of copper/zinc-superoxide dismutase (Cu/Zn-SOD) and manganese-superoxide dismutase (Mn-SOD) in individual cochlear tissues were detected by the use of an enzyme-linked immunosorbent assay (ELISA). A heterogeneous distribution of Cu/Zn-SOD was observed in the individual tissues of control animals: high levels were measured in the stria vascularis (SV), intermediate levels of enzyme were measured in the spiral ligament (SL), and low levels were measured in the organ of Corti region (OC); collectively, these levels were not statistically significant (P = 0.0645). Levels of Mn-SOD in individual tissues of the control group were statistically significant (P < 0.05): high levels were measured in the SV, medium levels were detected in the SL, and low levels were identified in the OC. Following the administration of methylprednisolone (MP), a significant reduction of Cu/Zn-SOD in the SV (P < 0.05) and a non-significant, but noticeable, increase (> 30%) of Mn-SOD in the OC were observed. These results indicate that levels of SOD are tissue specific and that SOD is subject to glucorticoid regulation.

Receptors for glucocorticoids in the human inner ear.

Glucocorticoid receptors were detected in the human inner ear. The highest concentration of glucocorticoid receptor protein was measured by enzyme-linked immunosorbent assay in the spiral ligament tissues; the lowest concentration of glucocorticoid receptors was measured in the macula of the saccule. The demonstration of the presence of glucocorticoid receptors in human Inner ear tissues provides a basis to consider the direct effects of glucocorticoid action on select inner ear cells, rather than assuming a systemic antiinflammatory or immunosuppressive effect during the therapeutic treatment of patients with given inner ear disorders.

Responsiveness of alpha 1 and beta 1 cochlear Na, K-ATPase isoforms to thyroid hormone.

The effects of thyroid hormone on Na, K-ATPase subunit isoforms under euthyroid (EUTH), hypothyroid (HYPO) and hyperthyroid (HYPER) states were investigated via immunocytochemistry and the use of polyclonal antibodies specific to each isoform (alpha 1, alpha 2, alpha 3 and beta 1, beta 2). In HYPO animals, there was a distinct decrease in Na, K-ATPase alpha 1 isoform immunoreactivity in the stria vascularis (SV), spiral ganglion (SG) cells, spiral limbus (SLi) and cochlear nerve (CN) as compared with that in EUTH animals by the 17th day of the experiment. Immunostaining of the alpha 1 isoform increased in HYPER animals as compared with that in HYPO animals, and reached a level comparable to that in EUTH animals after 2 days of triiodothyronine (T3) treatment. Levels of alpha 2, alpha 3 and beta 2 isoforms did not appear to be affected by T3 administration. By the 19th day of a low I2 diet, the immunoreactive intensity of the beta 1 isoform was reduced in cochlear tissues of HYPO animals as compared with that in EUTH animals. The immunoreactivity of the beta 1 isoform increased after treatment with T3 for 4 days and was comparable with levels in EUTH animals. These data indicate that alpha 1 and beta 1 isoforms within specific cochlear regions of the adult rat are responsive to thyroid hormone.

Localization of the mineralocorticoid receptor in rat cochlear tissue.

Distribution of the mineralocorticoid receptor within rat cochlear tissue was studied by immunohistochemistry. Intense immunoreactions of mineralocorticoid receptor antibodies were observed in outer and inner hair cells of the organ of Corti, stria vascularis, spiral ligament and spiral ganglion cells. Moderate to weak immunostaining was observed in the spiral limbus and cochlear nerve. Results of this study suggest that cochlear tissue is a mineralocorticoid hormone target tissue.

In situ real-time sequential potentiometric determinations of potassium concentrations from three cochlear regions in noise-exposed rats.

Double-barrelled potassium selective microelectrodes (K-ISME) were used in situ for real-time sequential determinations of potassium concentrations (CK+) in endolymph, marginal cells and the spiral ligaments of rats exposed to moderate noise at 100 dB for 30 min (NE) and control (CTL) animals. CK+ in NE animals at these sites did not differ significantly when compared to CK+ in CTL animals. However, there was a slight decrease in CK+ in marginal cells in the noise-exposed animals.

Effect of stress on cochlear glucocorticoid protein. II. Restraint.

The effect of restraint stress via immobilization on rat cochlear glucocorticoid receptor (GR) levels was determined using an enzyme-linked immunosorbent assay (ELISA). Results demonstrated that GR levels in cochlear tissues exhibited tissue-specific and time-dependent responses to immobilization (6 hours daily). Similar responses of the GR were observed in rats restrained during two different times of the day. A significant quadratic trend (P = 0.019, R2 = 0.58) was observed in levels of GR in spiral ligament tissues of rats restrained from 10:00 to 16:00 h; levels of GR were elevated by day 2, and by day 21 GR levels had returned to near normal levels. GR levels in the spiral ligament tissues also were found to increase significantly after 2 days in response to repeated restraint stress administered from 06:00 to 12:00 h (P = 0.017, R2 = 0.34). Interestingly, a subtle, but statistically significant, decreasing trend in the organ of Corti's GR levels was detected when the daily restraint stress was applied from 06:00 to 12:00 h for up to 7 days. No significant trends (P > 0.05) were observed in GR levels of stria vascularis tissues regardless of the time of day of the restraint protocol. Stress has been implicated as an etiological factor in Ménière's disease and other ear pathologies. The data presented here indicate that the effect of stress is specific to tissue region and that, as in tissues of other systems, the GR of cochlear tissues are responsive to stress.

Clinical viral infections and temporal bone histologic studies of patients with AIDS.

The brain, eye, and inner ear are each protected from blood-borne infectious agents by a barrier that has some anatomic and functional differences. In patients with AIDS, opportunistic infections of the central nervous system and eye are frequent. Little is known about the incidence of middle and inner ear infections in patients with AIDS, but deafness and severe vertigo are uncommon. We studied 14 homosexual men with AIDS, aged 28 to 55 years, for 1 to 2 years until death. No patient had deafness, but one had vertigo. Adenovirus type 6 and cytomegalovirus were isolated from the middle ear cavity in four patients. Temporal bone histology demonstrated acute otitis media in four, chronic otitis media in two, and serous otitis media in three. Adenovirus type 6 and cytomegalovirus, either alone or with herpes simplex virus type 1, were isolated from inner fluids of three patients. Histologic inner ear findings were abnormal in only one patient. Viruses were isolated or histologically identified in the brains of four patients and in the eyes of five patients. In our patients viral infections were nearly as common in the inner ears as in the brain and eye, suggesting that protection from the blood-labyrinth barrier was similar to that from the other barriers. Because the inner ear viral infections were asymptomatic and there was an absence of pathologic damage and inflammation, we suggest that some viral inner ear infections in patients with AIDS are nonpathogenic and elicit no inflammation or that the viral infections occur terminally and elicit no inflammation because of immunosuppression from the AIDS.

Combined effects of adrenalectomy and noise exposure on compound action potentials, endocochlear potentials and endolymphatic potassium concentrations.

The effects of removal of endogenous corticosteroids via bilateral adrenalectomy in combination with noise exposure (30 min at 100 dB) were determined by recording compound action potential (CAP) and endocochlear potentials (EP), and by measuring potassium concentrations (K+e) within the endolymph. Thirty-eight Long-Evans rats were divided into groups according to experimental treatments: adrenalectomy (ADX) or non-ADX and noise exposure or non-noise exposure. CAP thresholds, EP and K+e values were subjected to repeated-measures analysis of variance with group and time as factors classifying the measurements. Noise exposure resulted in significant elevations of CAP thresholds in both the ADX and non-ADX animals, but had no effect on either EP or endolymphatic K+e. Recovery was noted during all post-exposure measurement periods and was significantly faster for ADX animals. EP and K+e did not change during or after noise exposure. ADX animals showed a non-significant reduction of EP and a statistically significant increase of K+e during all measurement periods as compared to non-ADX animals.