Presented at the Meeting of the Association for Research in Otolaryngology, St. Petersburg Beach, Florida, February 23, 2000.
Aldosterone (Mineralocorticoid) Equivalent to Prednisolone (Glucocorticoid) in Reversing Hearing Loss in MRL/MpJ-Faslpr Autoimmune Mice†
Article first published online: 2 JAN 2009
Copyright © 2000 The Triological Society
Volume 110, Issue 11, pages 1902–1906, November 2000
How to Cite
Trune, D. R., Beth Kempton, J. and Kessi, M. (2000), Aldosterone (Mineralocorticoid) Equivalent to Prednisolone (Glucocorticoid) in Reversing Hearing Loss in MRL/MpJ-Faslpr Autoimmune Mice. The Laryngoscope, 110: 1902–1906. doi: 10.1097/00005537-200011000-00025
Research supported by Grant Nos. DC03573 and DC03955 from the National Institutes of Health—National Institute on Deafness and Other Communication Disorders; and Grant No. RCTR 597-0160 to the National Center for Rehabilitative Auditory Research (Portland, OR) from the Department of Veteran's Affairs, Division of Rehabilitation Research & Development.
- Issue published online: 2 JAN 2009
- Article first published online: 2 JAN 2009
- Manuscript Accepted: 28 JUN 2000
- Autoimmune hearing loss;
- MRL/MpJ-Faslpr autoimmune mice;
Hypothesis Although the glucocorticoid prednisone is the standard therapy for autoimmune sensorineural hearing loss, what this hormone does in the ear to restore hearing is not known. MRL/MpJ-Faslpr autoimmune mice consistently have shown only stria vascularis disease, implying that abnormal ion balances in the endolymph underlie cochlear dysfunction. Previously we have shown that hearing loss in these mice is reversed with prednisolone treatment. This, coupled with the complete lack of cochlear inflammation, suggests that the restoration of hearing with prednisolone is due to its sodium transport function and not to its anti-inflammatory or immune suppression effects. Therefore the hypothesis of this study was that the mineralocorticoid aldosterone, which only increases sodium transport, would be as effective as prednisolone in reversing autoimmune hearing loss.
Study Design MRL/MpJ-Faslpr autoimmune mice were treated with either prednisolone or aldosterone to compare steroid effects on auditory brainstem response (ABR) thresholds and stria morphology.
Methods After baseline ABR audiometry, autoimmune mice were given prednisolone (5 mg/kg per day), aldosterone (15 μg/kg per day), or water in their drinking bottles. After 2 months of treatment the ABR thresholds were remeasured, and ears collected for histological examination.
Results The untreated controls showed continued elevation of ABR thresholds and edematous stria. However, thresholds in most steroid mice were improved or unchanged and their stria morphology improved, particularly with aldosterone treatment.
Conclusions Restoration of hearing with steroid treatment is due to increased sodium transport to re-establish cochlear ionic balances. Aldosterone therapy may offer advantages over prednisone for long-term management of not only autoimmune hearing loss, but also other forms of nonimmune-related deafness for which steroids are currently prescribed.
The glucocorticoids (dexamethasone, prednisone, methylprednisolone) have been the standard therapy for autoimmune and idiopathic hearing loss for several years. 1 However, we have very little understanding of the actual abnormal cellular processes in the ear that cause these forms of hearing loss, and even less knowledge of how these are reversed by steroid treatment. Most glucocorticoids have three functions—immune suppression, anti-inflammation, and increase in sodium transport. The rationale for glucocorticoid therapy in hearing loss has been based on the first two of these functions, immune suppression and anti-inflammation, to suppress hyperactive systemic autoimmune functions and counter presumed inflammatory processes in the ear. However, there is little evidence that direct inflammatory processes in the ear are responsible for the hearing loss, particularly in autoimmune disease.
Numerous autoimmune mouse studies have demonstrated that the major disease in the ear is disruption of the stria vascularis and its blood-labyrinth barrier. 2 This breakdown in sodium and potassium ion transport mechanisms, coupled with the complete lack of inflammation within these autoimmune ears, implies that ion imbalance is the disease underlying autoimmune hearing loss. Also, steroid therapy is effective in forms of idiopathic hearing loss that have no immune origin, suggesting that there is a common cochlear pathological process or condition in the many different types of steroid-responsive hearing loss. These various observations have caused us to question the traditional view that cochlear inflammation underlies autoimmune hearing loss.
The observed stria vascularis disease in autoimmune mice suggests that endolymph sodium-potassium imbalance is the underlying cause of autoimmune hearing loss. This has recently been confirmed by Ruckenstein et al. 3 with their measurement of depression of the endocochlear resting potential in autoimmune mice. Because a third function of most glucocorticoids (except dexamethasone) is to increase sodium transport, 4,5 this stria function may be the actual cochlear cellular activity targeted by this steroid group. Therefore a possible mechanism for glucocorticoid reversal of autoimmune hearing loss is restoration of proper stria ion balances that are compromised by disruption of the blood-labyrinth barrier. 2 This also would explain why glucocorticoids effectively reverse sudden and idiopathic hearing loss that has no immunological or inflammatory correlates.
Increasing sodium transport is the primary role of the mineralocorticoids, such as aldosterone. 4,6 This is accomplished by upregulating the number of sodium channels and increasing cellular Na+,K+-adenosine triphosphatase (Na+,K+-ATPase). If the underlying disease in many of these forms of hearing loss is abnormal ionic balances in the endolymph, aldosterone may be an equally effective treatment. Therefore the aim of the present study was to test the hypothesis that abnormalities in cochlear ion transport underlie autoimmune hearing loss. If so, remediation of the hearing loss would be accomplished by mineralocorticoid treatment because this group of steroids has no effect on inflammation or the immune system. This laboratory has recently shown that hearing dysfunction in MRL/MpJ-Faslpr autoimmune mice can be prevented or reversed by treatment with the glucocorticoid prednisolone. 7–9 Therefore these mice were used to compare the relative therapeutic effects of the mineralocorticoid aldosterone and the glucocorticoid prednisolone. If aldosterone is equally effective in reversing autoimmune hearing loss, it would offer significant new evidence that sodium and potassium imbalances in the endolymph are the underlying cochlear problem in autoimmune hearing loss, as well as other forms of steroid-responsive ear disorders.
MATERIALS AND METHODS
The MRL/MpJ-Faslpr mouse (formerly, MRL/ lpr) is an established model of autoimmune sensorineural hearing loss. 10 Spontaneous systemic autoimmune disease develops in the MRL/MpJ-Faslpr mice, resulting in increased serum immune complexes, anti-DNA antibodies, lymphadenopathy, elevated auditory thresholds, and pathological changes in the stria vascularis. 2,3,10 The hearing loss has been shown to be reversible with prednisolone, 7–9 making these mice useful in the study of the cellular mechanisms of steroid-responsive hearing loss.
Thirty MRL/MpJ-Faslpr autoimmune mice were purchased from Jackson Laboratories (Bar Harbor, ME) at 2 months of age. Onset of systemic autoimmune disease occurs at 2 to 3 months of age, and cochlear thresholds rise shortly thereafter. 10 Therefore the mice were tested with auditory brainstem response (ABR) audiometry at 3 to 4 months of age to establish pretreatment baseline auditory thresholds. Then mice were assigned to aldosterone, prednisolone, or water treatment groups for 2 months, at which time the ABR audiometry was repeated.
Mice were randomly assigned to one of the two steroid groups (n = 10 each) or the untreated control group (n = 10). The respective treatments were continued throughout the 2-month testing period.
The glucocorticoid group was given daily oral doses (5 mg/kg per day) of prednisolone sodium phosphate (Spectrum Quality Products, Inc., Gardena, CA). The steroid was delivered by dissolving 15 mg in the standard 500-mL drinking-water bottle, which has been shown to maintain elevated systemic levels. 11,12 The advantage of this oral method is that it provides a constant source of steroid, parallels oral administration in patients, and avoids the trauma of daily injections. Mice drink 3 to 5 mL of water daily, so the effective dose was approximately 0.15 mg per day.
The mineralocorticoid group was given a daily oral dose of 15 μg/kg per day of aldosterone (d-aldosterone, Sigma, St. Louis, MO). Aldosterone drinking water was prepared by dissolving 50 μg of aldosterone in 50 μL of 100% ethyl alcohol (ETOH) and diluting it in the 500-mL drinking-water bottle. 13 The final effective dose was approximately 0.5 mg per day. The final ETOH concentration was 0.01% and was considered negligible.
Untreated control autoimmune mice were given regular tap water in their bottles to assess the normal progression of auditory dysfunction in autoimmune disease.
Auditory brainstem response audiometry to pure tones was used to evaluate cochlear function and followed our standard protocol. 10 Animals were anesthetized, and the individual ears of each mouse were stimulated with a closed-tube sound delivery system sealed into the ear canal. The ABR to tone-burst stimuli at 4, 8, 16, and 32 kHz was recorded, and thresholds obtained for each ear. The steroid and control mice always were tested on the same day to eliminate any potential instrumentation differences. Each ear's pretreatment and post-treatment ABR threshold shift at the four frequencies was calculated and the total shift for the four frequencies was obtained. The steroid groups were compared with controls receiving water, by means of the χ2 statistic, to determine whether either steroid treatment significantly altered the progression of hearing loss.
Stria Vascularis Morphology
The inner ears of all surviving mice were removed, perilymphatically perfused with fixative (3% paraformaldehyde in 0.1 mol/L phosphate buffer), and immersion-fixed overnight. After decalcification in ethylenediamine tetra-acetic acid (EDTA), the ears were cryostat sectioned according to the protocol of Whitlon et al. 14 Tissues were taken through serial dilutions of 30% sucrose, placed in degassed Optimum Cutting Temperature (OCT) compound, lightly vacuumed, and sectioned at 10 μm for qualitative evaluation of the effects of steroid treatment on the stria vascularis.
The use of the animals reported in this study was approved by the Oregon Health Sciences University Institutional Animal Care and Use Committee to ensure compliance with federal animal welfare guidelines.
The mice tolerated the steroid in the drinking water and showed no adverse affects, such as dehydration, avoidance of drinking, and the like. By the end of the 2-month treatment period, half of the untreated control mice had died of disease, which is typical for this strain. Survival was significantly higher for mice receiving the steroids; seven prednisolone-treated mice and nine aldosterone-treated mice were still alive at the post-treatment ABR test (χ2 = 7.2;P < .01).
Each ear's pretreatment and post-treatment ABR thresholds at the four frequencies were compared to establish any change attributable to treatment. The shifts at each frequency within an ear were added to derive a total shift in threshold per ear. If the ear's combined threshold shifts were lower by 20 dB or more, the ear was considered to be improved (average, 5 dB per frequency). The ear was considered unchanged if the combined threshold shifts were ±15 dB and were rated as worse if the combined threshold shifts were higher by 20 dB or more. The χ2 analyses compared the number of ears within each outcome category for each steroid relative to controls receiving water.
The ABR threshold analysis revealed that thresholds in untreated mice continued to rise with advancing systemic disease, because 80% of the ears were worse (Table I). Only one control ear (10%) improved, and one ear was unchanged. The steroid-treated ears were significantly better than controls after 2 months of treatment. Six of the prednisolone-treated ears were improved, six were unchanged, and only two were worse (P < .00232). The aldosterone-treated ears also were significantly better, with 7 improved, 11 unchanged, and none worse (P < .00001). These results suggest that both steroids had an equivalent effect of significantly improving hearing thresholds or preventing them from declining further.
χ2 results show comparison of each steroid group with the water controls.
Stria Vascularis Morphology
The stria vascularis in untreated control mice showed the typical edema and degeneration seen with advancing autoimmune disease and hearing loss (Fig. 1A and B). Dilated stria vessels and enlarged intercellular spaces were found throughout all turns of the cochlea. There was thinning of the stria in older mice, demonstrating the degeneration of stria structure and function with advancing disease. The prednisolone-treated mice showed some restoration of stria integrity and thickening of the epithelium (Fig. 1C), although dilated vessels and edematous spaces were still seen. The aldosterone-treated mice showed marked improvement in stria morphology, returning to virtually normal appearance (Fig. 1D). The stria vessels in aldosterone-treated mice were normal in size, showing neither dilation nor degeneration. Furthermore, no intercellular edema was seen in any of the aldosterone-treated mice.
The present results for prednisolone treatment parallel earlier studies that showed either hearing improvement or no change in most of the treated autoimmune mice. 7–9 This reinforces the validity of the MRL/MpJ-Faslpr autoimmune mice as models for investigation of the otopathological mechanisms of spontaneous autoimmune disease and the cochlear cellular mechanisms by which steroids reverse hearing loss. The most significant new finding of the present study was that aldosterone treatment resulted in hearing improvement comparable to prednisolone. Aldosterone also restored the stria vascularis epithelium to a qualitatively normal appearance. These two findings have tremendous implications regarding the underlying cochlear disease associated with autoimmune hearing loss. The fact that one can merely increase sodium transport to compensate for the underlying cochlear problem is a major advancement in our understanding of potential autoimmune disease processes of the ear.
However, as provocative as these results are, they are not entirely surprising. Aldosterone therapy was investigated because of numerous observations by this laboratory regarding the autoimmune disease processes in the cochlea. The stria consistently has been identified as the only site of disease in mice with autoimmune hearing loss, and no hair cell disease has ever been observed. 10 Furthermore, breakdown of the stria blood-labyrinth barrier occurs with the systemic autoimmune disease, 2 but inflammation is never observed, despite leakage of immunoglobulin into the stria pericapillary spaces. 15 The fact that autoimmune hearing loss in patients and mice is reversible with steroids 7–9 suggests that the cellular processes which control cochlear potentials are compromised, but can regenerate to normal function. Thus the underlying pathological condition appears to be in the stria because Ruckenstein et al. 3 have shown that the endocochlear potential is reduced in the autoimmune mice and Lin and Trune 2 have shown that the stria blood-labyrinth barrier is compromised. The observed reversal of hearing loss also implies stria disease, because it can recover after trauma 16 and hair cells cannot. Further, because any disruption of the stria is ultimately going to impair its function of sodium and potassium ion transport, it is a logical conclusion that restoring sodium and potassium balances with a mineralocorticoid also will restore normal hearing. These preliminary findings of the present study have been confirmed in extensive dose-response comparisons of these two steroids and the systemic immunological changes that parallel hearing improvement. 17
Recently, animal studies have shown both mineralocorticoid and glucocorticoid receptors in the normal ear that respond to systemic administration of both steroid groups. 18 Although it is not yet clear which of the steroid responsive cellular functions within the ear explain the present results, recovery probably is due to mineralocorticoid receptor upregulation of sodium transport. It is well known that glucocorticoids have a higher affinity for the mineralocorticoid receptor than they do for their own. 19 Thus both aldosterone and prednisolone treatment would lead to upregulation of mineralocorticoid receptor processes (i.e., increased sodium channels and elevation of Na+,K+-ATPase levels). The implication of hearing improvement and stria regeneration being related to mineralocorticoid function parallels recent findings by Ruckenstein et al. 20 They treated autoimmune mice with dexamethasone, another common glucocorticoid therapy for autoimmune hearing loss, and observed suppressed systemic autoimmune disease but no improvement in stria morphology. This would be expected because dexamethasone is one of the few glucocorticoids that has no sodium transport function, which, based on the present study, would be required to fully restore stria morphology and physiology.
Although the present findings represent a potentially exciting advancement in our understanding of cochlear disease mechanisms, one must carefully evaluate alternative explanations for the findings. For example, the improvement in hearing may be due indirectly to improved systemic ion levels from increased kidney sodium transport function, upregulation of systemic Na+,K+-ATPase levels or other enzymes that impact ion transport, suppression of the immune system by the mineralocorticoid, or upregulation of other systemic hormones that can affect the immune system, such as estrogen. However, the improvement in stria morphology is strong support for the conclusion that the aldosterone directly impacts stria cellular function.
Certainly, more studies will be necessary to identify the exact cellular mechanisms of steroid-responsive ear disease. Notwithstanding, the results of the present study provide a provocative challenge to traditional theories on cochlear autoimmune disease mechanisms and treatment. The fact that numerous clinical hearing disorders (autoimmune, idiopathic, and sudden sensorineural hearing loss) respond to prednisone suggests that they have a final common pathway of disruption of stria ion transport. Mineralocorticoids may have application to these hearing disorders with depressed endocochlear potentials and may provide an alternative for long-term management of hearing loss in patients who cannot tolerate prolonged glucocorticoid therapy.
Aldosterone was equivalent to prednisolone in reversing autoimmune hearing loss. This finding offers significant new evidence that the restoration of hearing with steroid treatment is due to increased stria sodium-potassium transport to re-establish normal ionic balances in the endolymph. Aldosterone may have application in the long-term management of autoimmune hearing loss, as well as other forms of sudden or idiopathic deafness that have no underlying immunological or inflammatory cause.
- 14.Cryoembedding and sectioning of cochleas for immunocytochemistry and in situ hybridization. Brain Res Protocols. Accepted for publication., , .
- 17.Aldosterone and prednisolone control of cochlear function in MRL/MpJ-Faslpr autoimmune mice. Hear Res. Submitted for review., , .