Kyle E. Rarey

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.

Localization of glucocorticoid receptors and glucocorticoid receptor mRNAs in the rat cochlea.

The distribution of glucocorticoid (GR) receptor messenger RNAs (mRNAs) and GR receptors was studied by in situ hybridization histochemistry and immunocytochemistry, respectively. In situ hybridization histochemistry was performed with a biotin‐labeled riboprobe complementary to rat GR receptor mRNA. GR receptor mRNAs were demonstrated in spiral ligament cells, spiral limbus cells, and spiral ganglion cells. GR receptor mRNAs were demonstrated neither in cells of the stria vascularis nor in cells of the organ of Corti region. With the use of a monoclonal and a polyclonal antibody, GR receptors were observed in the spiral ligament cells, stria vascularis cells, spiral limbus cells, and spiral ganglion cells by immunocytochemistry. Binding of anti‐GR‐receptor antibodies to a lesser extent was observed in the organ of Corti region; however, cellular distribution of the GR receptors could not be resolved with the applied techniques. These results suggest that the GR receptor is expressed differently in the heterogeneous cochlear tissues.

Presence of type I and type II/IB receptors for adrenocorticosteroid hormones in the inner ear

Aldosterone-Type I and dexamethasone-Type II/IB receptor complexes were identified in cytosol prepared from both cochlear and vestibular tissue samples. The specific binding capacity of Type I receptors in the cochlear tissues was approximately equal to that in the vestibular tissues. Likewise, the binding capacity of Type II/IB receptors in the cochlea was approximately equal to that in the vestibular endorgans. Based on the total specific binding measured with dexamethasone, the Type II/IB receptors appeared to outnumber the Type I receptors in cochlear and vestibular tissues by a factor of approximately 2.6; however, when adjustments were made for the probable cross-binding of dexamethasone to Type I receptors, these ratios were decreased to approximately 1.6. The existence of protein receptors for adrenocorticosteroid hormones demonstrated in the present study clearly suggests a mechanism whereby such hormones may directly regulate fluid and ionic gradients in the inner ear.

Tissue specific levels of glucocorticoid receptor within the rat inner ear

Individual, rat inner ear tissues were isolated and processed for determination of levels of glucocorticoid (GR) receptor by an Enzyme Linked Immuno-Sorbant Assay (ELISA). Differing levels of GR receptor between seven sampled inner ear regions were measured. Levels of GR receptors in the spiral ligament tissues were found to be significantly higher compared to all other tissue samples. GR levels in the tissues of stria vascularis and organ of Corti were different from one another but both were statistically higher than those detected in the vestibular tissue samples (dark cell regions, cristae ampullares and maculae utriculi), which had the lowest GR receptor levels measured. Intermediate levels of GR receptor were found in the endolymphatic sac region. It is suggested that the varying levels of inner ear GR receptors may be indicative of differing biological responses among the given tissues, as well as differences in the magnitudes of such responses to circulating glucocorticoids.

Na,K-ATPase α and β subunit isoform distribution in the rat cochlear and vestibular tissues

The distribution of five Na,K-ATPase subunit isoforms (alpha 1, alpha 2, alpha 3, beta 1 and beta 2) in rat cochlear and vestibular tissues was determined by immunocytochemical techniques using subunit isoform specific polyclonal antibodies. The expression of Na,K-ATPase alpha and beta subunit isoforms varied among different cell regions of the inner ear. The alpha 1 subunit isoform was more extensively distributed in all inner ear tissues than the alpha 2 or alpha 3 subunit isoforms. The beta 1 subunit isoform was distributed primarily in spiral ligament and inner hair cells of the cochlea, and in crista ampullaris and macula of the saccule. The beta 2 subunit isoform was most abundant in the stria vascularis, dark cells of the ampullae and utricle. The alpha 1 beta 1 subunit combination of Na,K-ATPase was most commonly found in the spiral ligament, while the alpha 1 beta 2 combination was most abundant in the stria vascularis. Similarly, alpha 1 beta 2 was confined more to the dark cells of the ampullae and utricle. The alpha 3 beta 1 subunit combination of Na,K-ATPase was identified in the inner hair cells of the cochlea and the sensory regions of the vestibular end organs. These observations may reflect functional diversity of Na,K-ATPase in the individual inner ear regions and may provide insight into the differences between fluid and ion transport in the inner ear and that of other transporting tissues. Overall, the distribution pattern further indicates that the different isoform combinations have specific roles.

Glucocorticoid receptor expression in the postnatal rat cochlea

The glucocorticoid receptor (GR) expression in the neonatal rat cochlea was investigated by utilization of a polyclonal antibody against GR, the immunoreactivity of which exhibited a distinct, age-dependent developmental pattern in tissues of the spiral ligament (SL). Immunostaining of GR appeared initially at the 7th postnatal day (PND), increased rapidly between the 14th and 21st PND, and reached adult-like expression levels by the 21st PND. Less pronounced, developmentally regulated expression patterns of GR were observed in cells of the spiral limbus (SLi), spiral ganglion (SG), organ of Corti (OC), and cochlear nerve (CN). For example, high expression levels of GR were observed in the SLi, SG and OC at 3 PND; subsequently, GR immunoreactivity levels decreased from 7 to 14 PND, and then GR immunoreactivity intensified in these regions by 21 PND. No remarkable changes in GR expression were observed in stria vascularis (SV). These data indicate that GR expression in the inner ear is tissue and age-specific, and that GR expression parallels both Na,K-ATPase expression and endocochlear potential development.

Effect of stress on cochlear glucocorticoid protein: Acoustic stress

Levels of glucocorticoid (GR) receptor protein were determined by a quantitative enzyme-linked immunosorbent assay (ELISA) technique in inner ear tissue of rats exposed daily to 85 dB SPL white noise for 4 hours on 3 consecutive days. GR levels in spiral ligament and organ of Corti tissues were detected using a monoclonal antibody to the GR receptor, BuGR2. A non-significant 13% decrease in GR levels of spiral ligament tissues was observed in the noise exposed animals relative to untreated animals. A statistically significant decrease of 27% in GR protein levels was seen in the organ of Corti region (P < 0.03), however. There was a concomitant increase of serum corticosterone levels (P < 0.03) in noise exposed animals as opposed to those of controls. These results indicate a tissue specific response of GR receptor to acoustic stress. Inner ear GR protein therefore may be a useful marker in determining the effect of stress on the inner ear. Finally, such data may be applicable to support the hypothesis that stress is an etiological agent in Ménières disease.

Ototoxicity of cisplatin vs. platinum analogs CBDCA (JM-8) and CHIP (JM-9).

Cis-diamminedichloroplatinum (cisplatin), a divalent platinum compound and cell-cycle nonspecific chemotherapeutic agent, produces a permanent high-frequency sensorineural hearing loss and a dose-related cumulative renal insufficiency with tubular necrosis and interstitial nephritis. Synthetic platinum analogs are presently being tested to identify an analog with greater antitumor activity, but less ototoxicity and nephrotoxicity than cisplatin. The objectives of this study were to analyze the potential cochlear and nephrotoxic effects of two synthetic platinum analogs presently in phases I and II of clinical trials, CBDCA [JM-8 or cis-diammine, 1,1-cyclobutane dicarboxylato (2)-0,01-platinum (NSC-241240)] and CHIP [JM-9 or cis-dichloro-trans-dihydroxybisisopropylamine platinum IV (NSC-256927)]. Cytocochleography, auditory brain-stem evoked response (ABR), double-blind light microscopy of renal tissues, and gamma emission analysis of 195mpt localization in viscera and inner ear were employed in the evaluation of cisplatin and platinum analogs (JM-8 and JM-9) in adult guinea pigs. Final results indicate that the investigational chemotherapeutic analogs CBDCA (JM-8) and CHIP (JM-9) do not produce the ototoxicity and nephrotoxicity characteristic of cisplatin. Furthermore, these findings demonstrate 195mpt localization in the vestibular labyrinth and confirm previous platinum distribution studies in the organ of Corti and stria vascularis tissues.

Intralabyrinthine Osteogenesis in Cogans's Syndrome

Temporal bones from a 64-year-old man who had Cogans syndrome were examined by light microscopy. Although tissues of the middle ear appeared unremarkable, extensive ectopic bone formation was observed bilaterally in the inner ear membranous labyrinth. Such osteogenesis completely obliterated the cochlear compartments apically. Only the scala tympani compartment of the basal cochlear turn remained patent. Ectopic bone tissue was observed also in the vestibular semicircular canals. Marked degeneration of eighth nerve fibers and associated ganglion cells also was observed, To our knowledge, this report represents only the fourth temporal bone findings of Cogans syndrome and demonstrates a more advanced pathologic state of inner ear pathology than those reported previously.

Na,K-ATPase α- and β-isoforms in the developing cochlea of the mouse

Abstract Immunohistochemistry was used to investigate the presence of Na,K-ATPase α- and β-subunits isoforms (α1, (α2, α3 β1and β2) in the cochlea of the mouse at different ages between embryological day (E) 19 and postnatal day (P)+30. α1 was mainly found in the stria vascularis and in the spiral ligament; it increased steadily from p+4. These data correlates well with the morphological and electrophysiological maturation of the cochlea. α3 predominated in the spiral ganglia and the cochlear nerve. This finding is well in accordance with reports that α3 seems to be associated with the nervous system. The β-subunit was found mainly in those tissues where staining of the α-subunit also was seen. Both subunits were localized in tissue regions where fluid regulation is expected to play an important role. For some isoforms, the expression pattern of Na,K-ATPase during development in the mouse is different from that in the rat. The expression of Na,K-ATPase and that of glucocorticoid receptors during development in the inner ear of the mouse show a similar pattern, which may indicate that glucocorticoid receptors could be involved in regulating the expression of Na,K-ATPase.