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Steroid Control of Acute Middle Ear Inflammation in a Mouse Model
Carol J. MacArthur, MD; Jacqueline M. DeGagne, BS; J. Beth Kempton, BS; Dennis R. Trune, PhD

Objective: To investigate steroids for their potential for therapeutic approaches to control otitis media. Glucocorticoids and mineralocorticoids have differential effects on inflammation and fluid absorption, but little is known of their control of middle and inner ear manifestations of acute otitis media. Design: Both glucocorticoid (prednisolone and dexamethasone) and mineralocorticoid (aldosterone and fludrocortisone) steroids were investigated for their ability to reduce inflammatory symptoms in a mouse otitis media model. Setting: Academic medical center. Subjects: Acute inflammation was induced by transtympanic injection of heat killed Streptococcus pneumoniae to 100 BALB/c mice. Interventions: Twenty mice in each experimental group (prednisolone, dexamethasone, aldosterone, and fludrocortisone) were given a steroid in their drinking water

the day before inoculation, and these treatments were continued until the mice were killed for histologic examination. Twenty control mice were treated with water only.
Main Outcome Measures: Histologic measure of inflammation: middle ear fluid, inflammatory cell number, and tympanic membrane thickness. Results: Histologic middle ear morphometrics showed significant steroid effects at both 3 and 5 days in reduction of fluid area, cell number, and tympanic membrane thickness. Conclusions: Glucocorticoids were most effective in controlling inflammation. Interestingly, the mineralocorticoids were also effective in reducing the inflammatory response at 5 days, suggesting that their fluid transport function helped clear disease. Thus, steroid control of middle ear disease may be useful in alleviating symptoms faster and reducing the risk to the inner ear.

Arch Otolaryngol Head Neck Surg. 2009;135(5):453-457 strategies are needed for alleviating the pain and hearing loss that accompany AOM. Inflammation results from the innate immune response to bacteria present in the ME. Thus, strategies to decrease fluid and inflammation would be potentially beneficial in the treatment of AOM. The lack of therapeutic options beyond antibiotics or tympanostomy tubes has led to considerable animal research efforts to better understand the mechanisms of AOM and develop new strategies for its prevention or treatment. Glucocorticoids lessen inflammation by suppressing the immune response, but they also bind to the mineralocorticoid receptor to enhance Na (sodium) transport and fluid absorption. Mineralocorticoids, such as aldosterone, only act on fluid homeostasis by increasing production of Na , K (potassium)–adenosine triphosphatase and the epithelial sodium channel. It has been reported in both in vivo and in vitro studies that the ME epithelium is involved in acWWW.ARCHOTO.COM

Author Affiliations: Department of Otolaryngology–Head & Neck Surgery and Oregon Hearing Research Center, Oregon Health & Science University, Portland.

TITIS MEDIA (OM) IS ONE of the most prevalent inflammatory diseases in the pediatric population.1 The only effective primary treatment for acute OM (AOM) is antibiotics. However, a consequence of antibiotic therapy is bacterial death and release of bacterial inflammatory products (lipopolysaccharide, peptidoglycan, and DNA), which can exacerbate and prolong inflammation in the middle ear (ME).2-4 Alleviation of symptoms can be accomplished by oral anti-inflammatory agents, oral antibiotics, or the infection can resolve on its own up to 70% of the time.5 Recent efforts to withhold the use of antibiotics for this disease entity have been put forward to decrease the number of antibiotic prescriptions written every year for this common disorder.6 Decreasing the use of antibiotics should decrease bacterial resistance to antibiotics, an emerging problem in many parts of the world. Therefore, new



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increasing to 3158 µm2 by day 5. measurements were made at days 3 and 5 because these postinoculation times would provide the maximum inflammation for the demonstration of any potential steroid effects. The ANOVA results showed significant overall group differences with regard to steroid effects at both 3 and 5 days (Figure 2). The steroid treatments served to reduce this inflammation. WWW. The inflammatory cells were primarily neutrophils (acute phase inflammatory cells). These MEs were often clear of fluid and inflammatory cells. RESULTS HISTOPATHOLOGIC ANALYSIS Control (bacteria only) ears often had considerable inflammation of the ME at both days 3 and 5 on histologic analysis. INFLAMMATION MEASURES To determine if there were quantitative differences in the extent of inflammation.05). embedded in glycol methacrylate plastic. Institutional guidelines regarding animal experimentation were followed. MAY 2009 454 ©2009 American Medical Association. and coverslipped. Occasionally. fixative was intracardially perfused (1.8 This fluid homeostasis function of the ME epithelium is likely to have a major role in removing fluid that accumulates during inflammation such as seen with acute and chronic OM.tive fluid transport via the epithelial sodium channel. such as fluid or inflammatory cells. and thickening of the round window membrane and TM (Figure 1A-C). The prednisolone-treated mice had 10 MEs without symptoms. 4 showed MEs completely free of inflammation (Table). fluid homeostasis (mineralocorticoids) or immune suppression (glucocorticoids). would experimentally provide the control of ME fluid and cellular infiltration in this model of AOM. the total number of ears showing inflammation at the 2 ages was evaluated to determine if the steroid treatments altered the normal pathologic progression. stained with basic fuchsin and methylene blue. These parameters were previously determined to best assess acute inflammation. Animals drank the medication-containing water ad libitum. The fluid area was measured with a calibrated micrometer grid in the eyepiece and the cells within the fluid area were counted. Illinois) were then performed on data from the treatment groups and controls to determine if steroid treatments suppressed ME inflammation. serially mounted on glass slides. Tympanic membrane thickness was measured with a micrometer scale within the opposite eyepiece. An assessment was made of the number of ears with any degree of inflammation. Oral steroid treatments were given in the drinking water per our previous protocol10 and consisted of either prednisolone (5 mg/kg/d). with significant inflammation first occurring at day 3. or slower inflammation development. an ear will not manifest an inflammatory response in spite of bacterial inoculation. Features of inflammation commonly seen were accumulation of inflammatory exudate. increasing at day 5. sectioned in the horizontal plane at 5 µm. This significant steroid effect was an overall reduction in all histologic measures at both 3 and 5 days. and the 3 individual measures for each ME parameter were averaged to derive 1 mean value for each parameter for each ear. and tympanic membrane (TM) thickness. The middle and inner ears were left intact and connected to each other by the skull base so both ears were processed together for histologic analysis and sectioning.ARCHOTO. number of inflammatory cells. By contrast. If the fluid were controlled. Many ears appeared normal. All rights reserved. fludrocortisone (10 µg/kg/d). ANOVA was conducted on the measures of fluid area. and TM thickness. with the exception of fluid area at day 3 (Figure 2).or 5-day treatment end point by an overdose of anesthetic (ketamine and xylazine).9 (Figure 2). and the dissected skulls were immersed in fixative overnight. Dexamethasone was given as a subcutaneous injection daily (0.5% glutaraldehyde–3% paraformaldehyde in 0. Portland. Control mice (n=20) received no steroid treatment and 10 were killed at each time point. They were then bilaterally inoculated transtympanically with 3. Often. However. Results from 2 analyses showed that both of these were statistically significantly better than controls (Table). steroid-treated ears had less inflammation (Figure 1D).COM (REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 135 (NO. The remaining mice (n=80) were given steroid treatments that began the day before inoculation and continued until they were killed.9 The goal of the study was to differentiate which steroid roles. hearing would be restored and pain from inflammation would be alleviated. while 11 dexamethasone-treated ears were clear. number of inflammatory cells. a particular animal being more resistant to bacterial stimulation.7. leaving 19 ears in this group for analysis. glucocorticoid-treated ears showed a significantly higher incidence of inflammation-free ears.5 to 167. Ten mice were examined at each time point for each treatment. Statistical analyses (analysis of variance [ANOVA]. Control mice without steroid treatment showed the normal progression of inflammation measures that paralleled the qualitative changes described herein. Three sections were measured. and the mucosal epithelium was seldom hypertrophied. or aldosterone (15 µg/kg/d). Therefore. hypertrophy of the ME mucosal on 02/12/2013 . The possibility that both glucocorticoids and mineralocorticoids could affect ME inflammation in OM was investigated using a previously developed animal model of AOM. One ear from a 5-day aldosterone–treated mouse was not available for evaluation.75 mg/kg/d).9 Therefore. suggesting that these steroids did not reduce the overall incidence of inflammation.5 µL of heat-killed Streptococcus pneumoniae (109 organisms per milliliter) and their MEs examined after 3 days or 5 days of treatment. Chicago. All animal procedures were approved by the Institutional Animal Care and Use Committee of Oregon Health & Science University. Extensive ME fluid and inflammatory cells were present as well. This could be due to the inoculate draining before an innate immune response is initiated.1M phosphate buffer). Middle ears were assessed at a 10 magnification of a standard ME section (level of the stapedial artery) for fluid area.jamanetwork. mineralocorticoid-treated ears were no different than the bacteriaonly controls (P . Tissues were microwave decalcified in EDTA. The animals were killed at either the 3. The mean fluid area was 1902 µm2 at 3 days. fluid filled the entire ME space. In comparison. Of the 40 inoculated ears in the control mice. 5). while the number of inflammatory cells within the fluid increased proportionately from 106. and resolving by day 7. SPSS Inc. METHODS BALB/c mice (N = 100) were examined with the otomicroscope to establish absence of infection in the external ear or ME. Downloaded From: http://archotol.

00 13. There is considerable inflammation in the ME space. Table. The reduction in TM thickness measured in the mineralocorticoid-treated mice was not different from controls.002 .jamanetwork. MAY 2009 455 ©2009 American Medical Association.A B Co RW SA ME C D Co Co SA SA RW ME RW ME Figure 1. with mean values being reduced in all steroid-treated groups. By day 5. of Ears Treatment Control Prednisolone Dexamethasone Aldosterone Fludrocortisone a The 2 Clear 4 10 11 4 5 Inflamed 36 30 29 35 35 2 Value a P Value . 5). Overall treatment effects were seen with regard to fluid area.61 0. essentially filling the cavity with fluid and inflammatory cells. The impact of bacterial inoculation on middle ear (ME) inflammation.60 10. A.COM (REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 135 (NO.001 . All rights reserved. note the hypertrophy of the ME mucosal epithelium and inflammatory infiltrate nearly filling the middle ear space.278 values were derived from the steroid treatment groups compared with controls. The appearance of a similar section in a control ear taken at 5 days (original magnification 20). Downloaded From: http://archotol. The thickness of the TM is measured away from the injection site to reflect changes due to the bacterial stimulation and not due to needle trauma. WWW. A prednisolone-treated ear at 5 days is completely clear of the typical inflammatory changes seen in untreated mice (original magnification 20). but the post hoc test results showed statistical significance only between the prednisolone and fludrocortisone results. B. Both glucocorticoid- treated groups were significantly different from the controls for TM thickness at day 3 (Figure 2). Prednisolone and dexamethasone both appeared to reduce cell number. although the cochlea (Co) is on 02/12/2013 . Post hoc comparisons showed that the steroids had an impact as early as day 3 on the number of cells and TM thickness (Figure 2).ARCHOTO.028 0. Middle ear inflammatory changes seen at 3 days (original magnification 10) in a control mouse.87 . despite a lower mean value. 2 Analysis of Inflammation-Free Ears at 3 and 5 Days No. D. Again. C. The fluid opposes the round window membrane (RW). all of the steroid treatments had some impact when compared with controls (Figure 2). Middle ear mucosal epithelium thickening and hypertrophy seen at a higher magnification (original magnification 20). SA indicates stapedial artery.

on fluid area (A). compared with controls. the dexamethasone-treated mice were statistically significantly different from the controls. Downloaded From: http://archotol. Presumably. Aldosterone is basically a pure mineralocorticoid (10 000:1 mineralocorticoid vs glucocorticoid action). µm2 3 Days 5 Days B 220 200 180 F = 3.ARCHOTO. all steroids were effective in reducing the number of inflammatory cells by day 5. Error bars indicate SE. MAY 2009 456 ©2009 American Medical Association.01 F = 6. while the role of the eustachian tube is more to allow gas pressure equilibration. Specifically. the mineralocorticoids were also moderately effective at WWW.85 P = . prednisolone and on 02/12/2013 . generally showing only one-third of the cells seen in control mice. an investigation of the use of steroids to clear the ME fluid that accompanies OM has not been explored. Middle ear fluid present during OM causes conductive hearing loss by virtue of decreasing the transmission of sound through the ME.001 No. There was an overall group effect for the steroids. Currently. 5). number of inflammatory cells (B). of Inflammatory Cells 160 140 120 100 80 60 40 20 0 3 Days 5 Days ∗ ∗ ∗ ∗ C 8 7 6 F = 3. Thus. but only at the 5-day end point. no good immediate therapy exists to clear fluid with the exception of myringotomy or tympanostomy tube placement. for the dexamethasone-treated mice.69 P = .03 3000 Control Pred Dex Aldo Fludro 2500 2000 ∗ 1500 1000 500 0 trols.14 Interestingly. were the most effective in our study in reducing ME inflammation in response to bacterial challenge to the ME at both 3 and 5 days.74 P = . the mean fluid area was statistically significantly less compared with the control mice. and they did not differ from each other at day 5. there is now evidence that the ME epithelium plays a major role in fluid absorption.007 F = 2.89 P < .72 P = . Asterisks signify which treatments were statistically significant from controls at the P . One could speculate that the mineralocorticoid action of prednisolone could also be acting in a positive way to decrease ME fluid. Post hoc analysis compared treatment groups with controls. while the glucocorticoid agents were effective in decreasing fluid area and cell count in the ME. All rights reserved. While clearance of ME fluid has long been ascribed to the function of the eustachian tube.11-13 to our knowledge. The glucocorticoids. and fludrocortisone has a much stronger mineralocorticoid than glucocorticoid effect (12. However. µm (REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 135 (NO.7 Other laboratories have reported that fluid regulation in the ME is via the Na channel–dependant process in the ME mucosal epithelium.jamanetwork.A 3500 F = 1. animal research on the use of various steroids for OM has not been conducted. prednisolone also has a modest mineralocorticoid effect.05 level. Interestingly. earlier resolution of this fluid would alleviate troubling sequelae of OM such as pain and decreased hearing. Morphometric results from analysis of variance (F and P values) for overall treatment effects. and tympanic membrane (TM) thickness. The use of mineralocorticoids offers some appeal. COMMENT Fluid Area. the results of the steroid treatments were generally similar in reducing ME inflammation during the observation period. The TM healing that occurs between days 3 and 5 is reflected by the reduction in TM thickness during this period. The number of inflammatory cells was significantly reduced in all steroid groups compared with con- While systemic steroids have been used extensively for ear diseases such as sudden sensorineural hearing loss and in earlier trials for the treatment of OM with effusion. the mineralocorticoids were also found to reduce ME inflammatory response to bacterial injections. However.COM TM Thickness. this is due to the strong immunosuppressive and anti-inflammatory action of these steroids. Findings from the post hoc tests showed that all steroid results were not different from each other.04 5 4 3 2 1 0 ∗ ∗ ∗ 3 Days 5 Days Days of Treatment Figure 2. suggesting that some aspects of inflammation were sensitive to both steroid groups. one-fifth that of the glucocorticoid effect. Therefore.15 F = 2. Thus.5:1 mineralocorticoid vs glucocorticoid action). however. Post hoc comparisons generally showed that 1 or both of the glucocorticoids were better at reducing inflammation than the mineralocorticoids.44 P = . since the use of such an agent would avoid the systemic morbid sequelae of glucocorticoids.

London. Mucin production in the middle ear in response to lipopolysaccharides.COM ©2009 American Medical Association. Hunter SE. both steroid groups were equally effective at day 5. 3. accepted August 7. Correspondence: Carol J. Randell SH. 2004. (REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 135 (NO. This would indicate that fluid area was affected by both types of steroids and affected equally well by the mineralocorticoids. Lecain E. Submitted for Publication: April 7. Otolaryngol Head Neck Surg. Otitis Media in Infants and Children. Oral or topical nasal steroids for hearing loss associated with otitis media with effusion in children. Otitis Media With Effusion in Young Children: Clinical Practice Guideline. Acquisition of data: MacArthur and Kempton. Rosenfeld RM. Drafting of the manuscript: MacArthur and Trune. 9. Thus. While we did not see a significant effect on fluid area at the 3-day end point for the mineralocorticoids compared with the glucocorticoids. Systemic steroids for otitis media with effusion in children. At the 5-day end point.the low therapeutic doses used in this study. Klein JO. MD: Agency for Health Care Policy and Research. Schimmer BP. 9th ed. Prazma JP. July the mineralocorticoids may have been more effective at the 5-day end point because their actions (clearance of fluid) would be expected to take longer than the stronger pure immune suppressive glucocorticoids. 1996:1459-1475.jamanetwork. 1999. Cochrane Database Syst Rev. 11. MacArthur CJ. Hunter SE. 2008. nor an overall effect. Number 12. PA: WB Saunders. Son EJ. final revision received July 14. Trune DR. 2001. Study supervision: MacArthur and Trune. 2. Bluestone CD. 4. inhibitors of the synthesis and actions of adrenocortical hormones. 2001. ENaC. Arch Otolaryngol Head Neck Surg. 12. Ruddon RW. Kania R. Downloaded From: http://archotol.ARCHOTO. Prazma J. Jewett BS.58(1):1-8. 2006. 5. Li J-P. National Institutes of Health. MacArthur.121(1):7-12. 14. Melhus A. 10. Parrish SK. 2008. Nevertheless.210(1-2):1-8. R21 DC007443. 2002. Oregon Health & Science University. New York. Systemic reactivation of otitis media with effusion in a rat model. Jewett BS. Hear Res. MD.48(3):397-402. Bluestone CD. and Trune.and CFTR-dependent ion and fluid transport in human middle ear epithelial cells. Funding/Support: This study was supported by grants R01 DC05593 and DC005593-S1. 2008. Statistical analysis: MacArthur. Kempton JB. Gilman AG. In: Goodman LS. All rights reserved. Department of Otolaryngology–Head & Neck Surgery and Oregon Hearing Research Center. Hear Res. et al. mineralocorticoids were also moderately effective in reducing the inflammatory response at 5 days. Otolaryngol Head Neck Surg. dexamethasone was the only treatment significantly better at decreasing fluid area compared with the other steroids. 5). Berman S. 2006. 7. 1999. Goodman & Gilman’s The Pharmacological Basis of Therapeutics. 1991. Limbird LE. MAY 2009 457 WWW. 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Also. Choi JY.(4):CD001935. Hefeneider SH. Further investigations are ongoing into the relative efficacy of steroids and antibiotics for the suppression of inflammatory response to OM in the mouse model. Berg AO. Author Contributions: Dr MacArthur had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. England: BC Decker Inc.219(1-2): 12-23. Hear Res. Rosenfeld RM. OR 97239 (macarthc@ohsu. Stool SE. and Trune.120(6):884-888. 2006. US Dept of Health and Human Services. 2005. McCoy SL. Kempton JB. 2nd ed. Portland. Gross ND. Singla AK. Obtained funding: MacArthur and Trune. and material support: MacArthur.211(1-2):26-32. Philadelphia. et al. Observation option toolkit for acute otitis media. on 02/12/2013 . Kempton. Mandel EM. Rosenfeld RM. Mineralocorticoid receptor mediates glucocorticoid treatment effects in the autoimmune mouse ear.117(9):984-989. 13. Kim JL. Pillsbury HC III. and Trune. 212(1-2):22-32. This lends support to the concept that sodium transport of ME fluid may be an important function in clearing fluid and thus in controlling inflammation in the ME. 3rd ed. These studies offer insight into the potential steroid control of middle and inner ear disease during AOM. Kempton. van der Voort JH. et al. J Antimicrob Chemother. Evidence-Based Otitis Media. Int J Pediatr Otorhinolaryngol. and Trune. The immune-suppressive actions of glucocorticoids were most effective in reducing the severity of the ME innate immune response. eds. steroid control of ME disease may be useful in alleviating symptoms faster and reducing risk to the inner ear. Rockville.