Changes of Serum Aldosterone Concentration after Noise Exposure in Mice.

Song,  Hyung Min; Lim,  Gil Chai; Lim,  Hyun Woo; Kim,  Min Ju; Choi,  Seung Hyo; Chung,  Jong Woo

Original Article

Noise and hearing conservation, phamacology

Korean Journal of Audiology 2011;15(3):137-140.

Changes of Serum Aldosterone Concentration after Noise Exposure in Mice.

Hyung Min Song, Gil Chai Lim, Hyun Woo Lim, Min Ju Kim, Seung Hyo Choi, Jong Woo Chung

¹Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea. jwchung@amc.seoul.kr
²Department of Otolaryngology, Jeju National University Hospital, Jeju National University College of Medicine, Jeju, Korea.

Abstract

BACKGROUND AND OBJECTIVES
The study was designed to measure the change of serum aldosterone concentration after noise exposure in BALB/c mice.
MATERIALS AND METHODS
BALB/c hybrid mice with < or =25 dB nHL in auditory brainstem responses (ABR) were used. Six mice were exposed to 120 dB SPL broad white band noise for 3 hours per day for 3 consecutive days. ABRs in all mice were examined after noise exposure. Serum aldosterone concentration was checked by radioimmunoassay in three mice without noise exposure, in three mice immediately after noise exposure (0 day after noise group), and in three mice 3 days after noise exposure (3 days after noise group).
RESULTS
Permanent noise exposure-related threshold shift was induced in mice of 0 day after noise group and 3 days after noise group (83.3+/-2.9 and 88.3+/-2.9 dB nHL, respectively). The serum aldosterone concentration of the mice after noise exposure was significantly lower than control mice (p=0.046). The changes of aldosterone concentration were 879+/-137.5 pg/mL without noise exposure, 623+/-75.9 pg/mL immediately after noise exposure, and 683+/-49.2 pg/mL 3 days after noise exposure.
CONCLUSIONS
In BALB/c mice, serum aldosterone concentration is decreased significantly after noise exposure.

Keywords: Aldosterone;Noise-induced;Hearing loss;Mice

Address for correspondence : Jong Woo Chung, MD, Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, Korea
Tel : +82-2-3010-3718, Fax : +82-2-489-2773, E-mail ; jwchung@amc.seoul.kr

Introduction

Aldosterone is a mineralocorticoid (MC) that is released from the zona glomerulosa of the adrenal cortex. Several recent studies have shown the stria vascularis has numerous MC and glucocorticoid receptors.^1,²⁾ The MC receptor of the inner ear controls the homeostasis of the endolymph by means of ion channels and transporters that exist in the cochlear duct cells.^3,⁴⁾ Deprivation or imbalance of such activities affects the morphological functions of the related cells, which leads to hearing loss.^5,^6,⁷⁾
Even though little animal research is available on the relevance of hearing loss and aldosterone, it has been reported that injection of aldosterone to mrl/mpj-fas (lpr) autoimmune mice that have stria vascularis and no hair cell pathology facilitates the regeneration of the stria vascularis to its normal form.⁸⁾ Since stria vascularis abnormality causes abnormal sodium ion (Na+) and potassium ion (K+) transport, it can be suggested that the enhancement of such transport is necessary to fully recover normal hearing level.⁸⁾ Recently, it has also been reported that the difference in serum aldosterone concentration between normal hearing and presbycusic groups is highly significant.⁹⁾ Although there are no explanations available, the decreased serum aldosterone concentration, which affects the expression of Na, K-ATPase and Na-K-Cl cotransporter (NKCC1) on the cell membranes of the inner ear cells, is related to the disrupted homeostasis of endolymph due to abnormal K+ regeneration in the cochlea.⁹⁾
Aldosterone plays a critical role in maintaining the homeostasis of the inner ear. However, previous studies have tended to focus on noise-induced hearing loss and steroid injection.^10,¹¹⁾ Not much is known about the protein changes related to K+ regeneration and the role(s) of aldosterone in noise-induced hearing loss. The aim of this study was to determine whether serum aldosterone is influenced by noise exposure by comparing aldosterone levels of BALB/c mouse before and after noise exposure.

Materials and Methods

Experimental animals
Nine, 6-week-old, male BALB/c mice (Orient Co., Seongnam, Korea) weighing 21-24 g with normal Preyer's reflexes and normal hearing at auditory brain stem response were used. The mice were randomly divided into three groups (n=3 per group; see below), weighed, and bred in a cage. All mice were allowed unrestricted access to food and water.

Organization of experimental groups
Serum aldosterone was measured in each of the three groups. In the control group, mice were not exposed to noise. Another group was noise-exposed for 3 consecutive days prior to measurement of serum aldosterone immediately after termination of the exposure (0 day after noise group). The last group was also noise-exposed for 3 days but serum aldosterone was measured 3 days after termination of the exposure (3 days after group).

Instruments for noise exposure
For noise exposure, a sound-proof noise booth was used. Noise was generated by a speaker with input/output resistance of 8 Ω (290-8L; Altec Lansing, Oklahoma City, OK, USA) that was directly amplified through a model R-399 amplifier (INTER M, Seoul, Korea). The amplifier was placed in the left corner of the room and the speaker was placed above the amplifier, with the speaker horn at a 45° angle.

Noise-induced hearing loss
Noise-exposed mice were rounded up in a cage, placed inside a noise booth, and exposed to white noise of 120 dB peak equivalent SPL for 3 hours per day for 3 days to induce permanent threshold shift (PTS). Noise was generated through Cool Edit 1.52 PC software, and the amplitude of the noise was measured over 120 dB SPL using a sound level meter (B&K, Nærum, Denmark) in the center and each corner of the soundproof room. The amount of noise exposure used in this study to cause PTS has been previously demonstrated to induce continuous threshold change in a study that used the same equipment and conditions.¹²⁾

Measurement of hearing threshold
Traveler Express (Bio-logic Systems, Mundelein, IN, USA) was used to elicit auditory brainstem response (ABR). Alternative click sound of 13 per second was measured as a stimulus by reducing 10 dB per measurement starting from 90 dB nHL and, when an indefinite wave was shown, reducing it in 5 dB steps. Hearing threshold was measured on wave I. Sound frequency filter of the click sound was adjusted between 100-3,000 Hz and the total number of stimulations was 1,024. Mice with normal hearing threshold <20 dB at ABR were selected before the experiment. Threshold was measured before exposure and hearing loss was checked immediately after the termination of the noise exposure of >120 dB SPL for 3 hours for 3 days.

Measurement of serum aldosterone
Three mice in unexposed control group and three mice of the 0 day after noise group undergone blood sampling at an hour after 3 days of noise exposure termination and were immediately euthanized. After injecting intramuscular ketamine hydrochloride (50 mg/kg) and xylazine (2 mg/kg) to induce deep anesthesia, blood was sampled through cardiocentesis using a 21 gauge needle equipped with a 1 cc syringe to collect 0.7-1.0 cc of blood. Serum aldosterone concentration of each mouse was measured by a commonly used radioimmunoassay kit (Aldosterone II RIA Diagnostic kit; Abbott Laboratories, Diagnostic Division, Abbott Park, North Chicago, IL, USA). The same procedure was used to sample blood from the three mice of the 3 days after noise group.

Statistical analyses
SPSS version 12.0 (SPSS, Chicago, IL, USA) was used for statistical analyses. Kruskal Wallis test and Mann-Whitney U-test was used to verify hearing threshold and aldosterone differences between the study groups.

Results

Results of audiologic evaluation
Hearing levels before noise exposure did not show a significant difference between study groups: 16.7±2.9 dB nHL in the control group, 18.3±2.9 dB nHL in the 0 days after noise group, and 16.7±2.9 dB nHL in the 3 days after noise group (p=0.456)(Table 1). Hearing level measured immediately after exposure termination (0 days after noise group) and 3 days after exposure termination were 83.3±2.9 dB nHL and 88.3±2.9 dB nHL, respectively. The difference was insignificant (p=0.368). The hearing threshold measured 3 days after termination of exposure in 3 days after noise group was 46.7±2.9 dB nHL.

Change of serum aldosterone concentration after noise exposure
The mean serum aldosterone concentration of the unexposed control group was 879.3±137.5 pg/mL (Table 2). The mean serum aldosterone concentration of the 0 day after noise group decreased to 623.0±75.9 pg/mL (p=0.046)(Fig. 1), and the mean concentration of the 3 days after noise group (683.3±49.2 pg/mL), while elevated, was still lower than before noise exposure (p=0.046).

Discussion

Unlike corticosterone, which is elevated after noise-exposure,^13,¹⁴⁾ this study demonstrates statistically significant decrease in serum aldosterone concentration after noise-exposure. In a normal hypothalamic-pituitary-adrenal axis reaction, corticosterone down-regulates glucocorticoid receptor (GR) mRNA and protein. As endogenous corticosterone is increased by noise trauma, cochlear GR mRNA expression decreases.¹⁴⁾ However, little is known about the decrease in aldosterone after noise trauma, and the decrease in MC receptor expression.
Aldosterone activates MC receptors in the cytosol, thereby expressing aldosterone-induced proteins, which increase Na+ and K+ active transport.^15,¹⁶⁾ MC receptors of the inner ear regulate the homeostasis of the endolymph through ion channels and transporters that reside inside the cochlear duct cells, including stria vascularis and spiral ligament.^3,⁴⁾ Degeneration of the spiral ligament type 2 fibrocytes, which play a critical role in potassium regeneration of the cochlea, and swelling of the stria vascularis are observed in cochlea with noise trauma.¹⁷⁾ Therefore, considering aldosterone's physiologic function and various findings related to noise-induced hearing loss, it seems likely that aldosterone has a role in either hearing loss after noise trauma or regeneration of hearing.
Yet, presently, a decrease in aldosterone concentration was evident after noise exposure. Considering that stress conditions such as restraint and cold trigger the rise of aldosterone concentration,¹⁸⁾ the fact that aldosterone actually decreased in such stressful condition of noise exposure and isolation in the noise booth is noteworthy. How the structural change inside the cochlear duct and increased threshold due to PTS caused a decrease in aldosterone by affecting the renin-angiotension-aldosterone system was not presently revealed.
Although there was no statistically significant difference between 0 day after noise group (623.0±75.9 pg/mL) and 3 days after noise group (683.3±49.2 pg/mL), the aldosterone concentration showed a tendency to rise in the after 3 days after noise group. It is assumed that after a traumatic noise stimulus is removed, the body recovers to its baseline state, although a longer observation period than used presently will be necessary to confirm this. In addition, this study used a small number of specimens per group. In order to generalize the results, further studies should be carried out with more specimens to collect sufficient data. Moreover, the multiple use of nonparametric comparison raises concern about increasing α error and misunderstanding of results. The application of statistical correction methods was limited due to the small number of experimental animals in this study. So, further experiments will be necessary to maintain statistical validity.
In addition, to clarify the mechanism of aldosterone change after noise exposure, additional studies should compare aldosterone related antibody expression in the cochlea or correlate aldosterone receptor changes evident in Western blotting with serum aldosterone levels. Moreover, the mechanism of disruption of the ion homeostasis inside the cochlea using PTS differs from transient threshold shift (TTS). A study involving chinchillas demonstrated that pillar buckling and uncoupling of the OHC stereocilia from the tectorial membrane occurred in the organ of Corti with TTS.¹⁹⁾ On the other hand, with PTS, loss of adjacent afferent nerve fibers and the hair cells of the organ of Corti has been described.¹⁹⁾ Although this study used PTS induced mice, aldosterone seems relevant to hearing recovery with ion balance of inner ear after noise trauma; further study is needed to observe aldosterone change after only 3 hours of 110 dB SPL exposure of which is to recover to normal.¹³⁾
In this study, the observed decrease in aldosterone concentration after noise exposure shows that noise trauma and hearing changes may influence the systemic electrolyte and water regulation. Further study is needed to determine how the systemic change triggers changes in the cochlea.

Conclusion

Noise exposed BALB/c mice with permanent threshold shift showed significant decrease in serum aldosterone levels in contrast to unexposed groups. This study raised the need for further studies to understand the mechanisms related to the decrease in serum aldosterone levels.

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