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Abstract

Objectives/Hypothesis:

Systemic glucocorticoids (GC)s are employed to treat various voice disorders. However, GCs have varying pharmacodynamic properties with adverse effects ranging from changes in epithelial integrity, skeletal muscle catabolism, and altered body weight. We sought to characterize the acute temporal effects of systemic dexamethasone and methylprednisolone on vocal fold (VF) epithelial glucocorticoid receptor (GR) nuclear translocation, epithelial tight junction (ZO-1) expression, thyroarytenoid (TA) muscle fiber morphology, and body weight using an established pre-clinical model. We hypothesized dexamethasone and methylprednisolone will elicit changes in VF epithelial GR nuclear translocation, epithelial ZO-1 expression, TA muscle morphology, and body weight compared to placebo-treated controls.

Methods:

Forty-five New Zealand white rabbits received intramuscular injections of methylprednisolone (4.5 mg; n = 15), dexamethasone (450 µg; n = 15), or volume matched saline (n = 15) into the iliocostalis/longissimus muscle for 6 consecutive days. Vocal folds from 5 rabbits from each treatment group were harvested at 1-, 3-, or 7 days following the final injection and subjected to immunohistochemistry for ZO-1 and GR as well as TA muscle fiber cross-sectional area (CSA) measures.

Results:

Dexamethasone increased epithelial GR nuclear translocation and ZO-1 expression 1-day following injections compared to methylprednisolone (P = .024; P = .012). Dexamethasone and methylprednisolone increased TA CSA 1-day following injections (P = .011). Methylprednisolone decreased body weight 7 days following injections compared to controls (P = .004).

Conclusions:

Systemic dexamethasone may more efficiently activate GR in the VF epithelium with a lower risk of body weight loss, suggesting a role for more refined approaches to GC selection for laryngeal pathology.

Keywords

voice vocal folds glucocorticoids ZO-1 glucocorticoid receptor steroids dexamethasone methylprednisolone

Introduction

Glucocorticoids (GCs) are commonly used to treat a wide variety of voice disorders due to their potent anti-inflammatory properties and hypothesized anti-fibrotic effects.^1-3 Systemic GCs, including oral, intravenous, and/or intramuscular injection (IM), are associated with variable efficacy to treat laryngeal indications.^1,4-11 Oral GCs are most commonly prescribed to treat rheumatologic laryngeal lesions and acute laryngitis.¹ Intravenous GCs are commonly employed to manage vocal fold (VF) and airway angioedema and laryngotracheobronchitis.^1,12 Intramuscular GCs have demonstrated efficacy for laryngotracheobronchitis.^4,5 Systemic GCs have also been reported to manage laryngeal sarcoidosis.^9-11 Dexamethasone and methylprednisolone are the most frequently prescribed GCs in otolaryngology,¹ and drug bioavailability between oral and IM administration of these GCs is comparable.^13-15 The mechanism of action of GCs is well-established in other tissues and disorders, and known to be initiated via intracellular binding of GC to the glucocorticoid receptor (GR).^16-19 The GC-GR complex dissociates from chaperone proteins to regulate gene expression via 3 mechanisms: (1) direct binding to GC responsive elements on genomic DNA to promote anti-inflammatory protein production^16,20; (2) indirectly through interactions between proteins and other transcription factors, mitigating inflammatory protein production^17,20; and (3) via non-genomic pathways in the cytoplasm and at cell and organelle membranes.^18,20-22

Although these immunomodulatory properties make GCs an attractive therapeutic option for treating laryngeal pathologies, it remains unclear how GCs induce physiologic changes within vocal fold (VF) tissue. Glucocorticoids vary in pharmacodynamic properties, including differences in drug potency, half-life, and mineralocorticoid activity. Glucocorticoids are also associated with adverse effects ranging from hypertension, hyperglycemia,²⁰ behavioral/cognitive changes,^2,23 reduced therapeutic effect,²⁴ changes in tight junction/epithelial barrier function,^25-28 and skeletal muscle catabolism.^29,30 Glucocorticoid receptor nuclear translocation is an indicator of genomic steroid activation associated with therapeutic efficacy.^31-34 Tight junction proteins, like ZO-1, contribute to epithelial cell barrier^35,36 and protect the vocal folds from environmental pathogens³⁷ as well as phonotraumatic injury.³⁸ GC-induced skeletal muscle catabolism is also well documented in non-laryngeal skeletal muscle.^30,39,40 Notably, reports of GC-induced VF atrophy are increasing. Mechanistically, GC injection into the VFs decreased hyaluronic acid synthesis in the lamina propria⁴¹ and thyroarytenoid (TA) muscle fiber size.⁴² Clinically, a single triamcinolone injection into the VFs was associated with VF atrophy in some patients.⁴³ Similarly, 5 to 6-weekly subepithelial dexamethasone injections into the VF were associated with VF atrophy in a retrospective case report.⁴⁴ Vocal fold atrophy can have profound detrimental effects on voice production.^44-46

Despite known diversity of drug activity and associated adverse effects across GCs, clinical judgment was reported to drive GC selection rather than evidence of differential efficacy.¹ Characterization of GC-induced tissue changes in the VFs, and in particular, differential effects between GCs, is necessary to better understand the utility of GCs and to accurately customize drug selection for patient-specific needs. In the current study, we sought to characterize the temporal effects of acute systemic GC treatment on healthy VF tissue. Specifically, we investigated epithelial GR activation, tight junction expression, TA muscle morphology, and body weight. We hypothesized methylprednisolone and dexamethasone, 2 of the most prescribed GCs in otolaryngology,¹ will elicit changes in these tissue-level events. Information regarding the effects of GCs on healthy VF tissue will provide foundational insight regarding the utility of these compounds to improve clinical outcomes and mitigate potential adverse effects.

Methods

Preclinical GC Injections

This study was approved by the Vanderbilt Institutional Animal Care and Use Committee. Forty-five New Zealand white rabbits (Oryctolagus cuniculus) were weighed, and then 0.225 mL of 4.5 mg methylprednisolone (n = 15), 450 µg dexamethasone (n = 15), or volume matched saline (n = 15) was injected into the iliocostalis/longissimus muscle. This protocol was repeated for 6 consecutive days at consistent times. Doses were derived from the highest dosing ranges for GC IM injections: 1 to 9 mg for dexamethasone^47-50,10 to 90 mg for methylprednisolone,^51,52 assuming an average weight of 60 kg for humans and 3 kg for rabbits (eg, 4.5 mg methylprednisolone for 3 kg rabbit is analogous to 90 mg methylprednisolone for 60 kg human). Selected doses were based on clinical dosing ranges for IM dexamethasone and methylprednisolone and are not intended to be equivalent between the 2 GCs. Rabbits were randomized to treatment group.

After the 6 days injection protocol, 5 rabbits from each treatment group were weighed and euthanized at 1 day, 3 days, or 7 days. The larynges were harvested and bisected in the sagittal plane; the right half was formalin-fixed and paraffin processed, and the left half was fresh frozen in Optimal Cutting Temperature (OCT) medium. These timepoints were selected based on previous reports regarding GC-induced changes in tight junction expression^26-28 and skeletal muscle morphology^53-55 between 1 and 7 days following treatment.

Body Weight

Body weight was acquired immediately prior to the first injection and immediately prior to euthanasia at each timepoint using a Rice Lake VS-11 scale (Rice Lake, Wisconsin). Percent change from pre-injection weight and post-injection weight was calculated for each animal.

Immunofluorescence Labeling

Immunofluorescence labeling techniques were previously described by our group.⁵⁶ Briefly, OCT blocks were sectioned coronally at 12 µm using a Leica CM1900 cryostat and adhered to positively charged slides. Labeling was performed in triplicate on mid membranous VF sections. Samples were methanol fixed and permeabilized in 0.01% Triton X-100. Slides were antigen blocked in 10% goat serum (1 hour, 20°C) and incubated in mouse monoclonal primary antibodies against tight junction marker ZO-1 (Thermo Fisher Scientific; 1:250) and GR (Abcam; 1:250) in 1% goat serum overnight at 4°C. Secondary antibody detection was performed using Alexa Fluor^® 594 Goat anti-Mouse IgG conjugate (Thermo Fisher Scientific) diluted 1:500 in 1% goat serum for 1 hour at room temperature in a darkened chamber. Samples were mounted with glass coverslips using Vectashield^® Mounting Medium with DAPI (Vector Laboratories).

Hematoxylin and Eosin (H&E) Staining

Paraffin blocks were sectioned coronally at 10 µm using a Leica HistoCore BIOCUT microtome, floated in a Leica HI1210 heated water bath, and adhered to positively charged slides. Once dried, samples were deparaffinized using xylene and dehydrated in descending gradients of ethanol. Slides were stained with hematoxylin and counterstained with eosin performed in triplicate on midmembranous VF sections, followed by rehydration in an ascending gradient of ethanol. Following clarification with xylene, samples were mounted with glass coverslips and SecureMount™ low viscosity mounting medium.

Microscopy

All samples were imaged using a Nikon Eclipse 90i microscope. Immunofluorescence images were captured digitally using a Hamamatsu C10600 black and white camera and H&E images were captured digitally using a Nikon DS-Fi1 color camera. All images were captured at 10× magnification, to include the full depth of the VF including the epithelium, lamina propria (LP), and muscle fibers.

Image Analysis

ImageJ (National Institutes of Health) was employed to calculate epithelial GR nuclear translocation by a blinded rater. A region of interest (ROI) of the VF epithelium was acquired. “Otzu” thresholding, an algorithm to dichotomizes pixels into foreground and background, was used for DAPI (nuclei) labeled images. “Triangle” thresholding was also employed to differentiate foreground and background pixels in GR labeled images. A ratio was derived from positive GR pixels that overlapped with positive DAPI pixels, indicating GR nuclear translocation.

To calculate epithelial ZO-1 expression, images were magnified to 200% at a consistent, fixed region of the epithelium in all samples. A ROI of the epithelium was acquired, and “Moments” thresholding was used to identify ZO-1 positive pixels. This algorithm is better suited for automatic image segmentation based on ZO-1 expression. A ratio was calculated of positive ZO-1 pixels within the total epithelial ROI.

Thyroarytenoid muscle fiber cross-sectional area (CSA) is a standard technique used to assess skeletal muscle atrophy,^39,40,54,57 and has been described previously by our group.⁵⁸ Briefly, a ROI outlining muscle fibers in H&E stained samples was acquired. “Default” thresholding was performed and the particle analysis tool was utilized to calculate total muscle fiber area in each sample. Individual muscle fibers were then manually counted using the multipoint tool and the average individual muscle fiber CSA for each image was calculated.

Statistical Analyses

All statistical analyses were calculated using GraphPad Prism V.7.0 (GraphPad Software, San Diego, California, USA) and employed non-parametric Kruskal-Wallis tests with Dunn’s multiple comparisons tests due to heterogeneity of variance between groups and/or non-normal distribution within conditions.

Results

Dexamethasone Increased Epithelial GR Nuclear Translocation

Dexamethasone significantly increased GR nuclear translocation compared to methylprednisolone 1-day following injections (H(2) = 7.46, P = .024; Figure 1). No differences were observed at the other time points. Intra-rater reliability was established using the correlation coefficient of 10 randomly selected and redundantly analyzed samples; excellent reliability was observed (r = .98; P < .01).

Figure 1.

Epithelial GR nuclear translocation in (A) saline, (B) dexamethasone, and (C) methylprednisolone treated groups 1-day following injections. Graph (D) displays the mean and SEM across treatments.

Methylprednisolone Decreased Epithelial ZO-1 Expression

Methylprednisolone significantly decreased epithelial ZO-1 protein abundance compared to dexamethasone 1-day following injections (H(2) = 8.66, P = .012; Figure 2). No differences were observed at the other time points. Intra-rater reliability was established using the correlation coefficient of 10 randomly selected and redundantly analyzed samples; excellent reliability was observed (r = .97; P < .01).

Figure 2.

Epithelial ZO-1 fluorescence expression in (A) saline, (B) dexamethasone, and (C) methylprednisolone treated groups 1-day following injections. Graph (D) displays the mean and SEM across treatments.

Glucocorticoids Increased TA Muscle Fiber CSA

Dexamethasone and methylprednisolone significantly increased TA CSA compared to the controls 1-day following injections (H(2) = 8.91, P = .026; 0.029; Figure 3). No differences were observed between other time points or treatment conditions. Intra-rater reliability for quantifying muscle CSA was established using the correlation coefficient of 10 randomly selected and redundantly analyzed samples; excellent reliability (r = .96; P < .01) was observed.

Figure 3.

TA muscle fiber CSA in (A) saline, (B) dexamethasone, and (C) methylprednisolone treated groups 1-day following injections. Graph (D) displays the mean and SEM across treatments.

Methylprednisolone Decreased Body Weight

Methylprednisolone significantly decreased body weight 7 days post-injection compared to control (H(2) = 10.5, P = .004; Figure 4). No differences were observed at other time points.

Figure 4.

Changes in body weight in response to saline, dexamethasone, and methylprednisolone treated groups 7 days following injections. Graph displays the mean and SEM across treatments.

Discussion

Systemic GCs are commonly used to treat a wide variety of voice disorders. However, GCs have varying pharmacodynamic properties with adverse effects ranging from changes in epithelial tight junctions, skeletal muscle catabolism, and changes in body weight. It is critical to characterize the effects of GCs in healthy VF tissue to provide foundational insight regarding utility and optimal dosing of these compounds. In the current study, we sought to provide foundational data to, ultimately, improve clinical outcomes and mitigate potential adverse drug effects.

Glucocorticoid receptor nuclear translocation is associated with therapeutic response to GCs.⁵⁹ Dexamethasone significantly increased epithelial GR nuclear translocation compared to methylprednisolone 1-day following injections. However, GR nuclear translocation did not significantly differ between GCs and control group. No differences were observed between treatment conditions at the 3 and 7 days post-injection time points. Dexamethasone (36-72 hours) has a significantly longer biological half-life than methylprednisolone (12-36 hours).⁶⁰ This difference may, at least partially, explain these findings. Dexamethasone may have actively induced epithelial GR nuclear translocation 1 day following treatment, and this effect diminished by day 3. It is also unclear if methylprednisolone induced immediate GR nuclear translocation, but this induction diminished 1 day following injection. Repeated methylprednisolone injections may have decreased GR nuclear translocation due to tolerance and/or excessive stimulation of transcription factors interacting with GR,^61,62 possibly related to the unique pharmacokinetic profile of methylprednisolone.

GR nuclear translocation in the VFs confirmed adequate concentration of systemic dexamethasone was delivered to the vocal folds to elicit a predictable response. Dexamethasone may more efficiently activate GR in the epithelium compared to methylprednisolone. Other extraneous factors such as sleep⁶³ and stress⁶⁴ have been shown to influence endogenous GC secretion. Although attempts were made to control for potential confounding variables, the inherent variability in the dexamethasone and saline conditions precludes meaningful statistical analyses. Further studies regarding GC effects on GR activation in in the context of laryngeal injury are required to interrogate the clinical efficiency of these drugs.

ZO-1, an epithelial tight junction protein, adheres adjacent cells to decrease paracellular permeability and maintain barrier integrity.³⁶ In the current study, GCs did not significantly alter ZO-1 expression compared to controls, and ZO-1 was distributed in the epithelial cell-cell junction across all treatment groups. Dexamethasone increased epithelial ZO-1 expression compared to methylprednisolone 1 day following injections. The translational value of this finding is unclear due to ZO-1 being present in the epithelium in all conditions, and no significant differences between GCs and controls. Dexamethasone has been reported to increase ZO-1 expression in other tissues.^65-67 The effects of methylprednisolone on ZO-1 across tissue types is unknown, but other GCs have variable effects on epithelial permeability.^25-28 Decreased ZO-1 expression is associated with increased endothelial and epithelial paracellular permeability.^68,69 Increased ZO-1 may suggest systemic dexamethasone favorably impacted epithelial barrier function in the vocal folds, an under-investigated and favorable therapeutic effect of steroids given that barrier function is compromised in injury.^70-72 Regardless, these data suggest systemic GCs do not acutely affect epithelial ZO-1 distribution.

Skeletal muscle atrophy an observed adverse effect of GCs. Many catabolic conditions result in muscle atrophy (eg, sepsis, cachexia, starvation, and metabolic acidosis) are associated with increased GC levels.⁷³ Additionally, GC induced skeletal muscle catabolism has been observed in vitro and in vivo.^40,55,74 These catabolic effects are believed to be elicited, in part, via inhibitory effects on the IGF-I/PI3K/Akt/mTOR pathway and stimulatory effects on the Ubiquitin-Proteasome System (UPS).^29,30 Surprisingly, dexamethasone and methylprednisolone increased TA muscle fiber CSA 1-day following systemic injection. Only a small subset of studies utilizing intracordal injections address GC induced atrophy in VF tissue, with little attention to the specific mechanism(s) involved, mucosa/muscle specificity, or dose and GC specificity.^41,43,44 Increased TA muscle fiber CSA may be related to VF tissue specificity and/or influence of variables associated with systemic injections. However, these mechanistic hypotheses are speculative and require further investigation.

Methylprednisolone decreased body weight 7 days following injections. This finding may be due to the inhibitory effects of methylprednisolone on the endocrine system, specifically IGF-1.^55,75 Dose-dependent, decreased body weight was reported in rats after repeated methylprednisolone administration,⁷⁶ which was postulated to result from decreased circulating IGF-I. Decreased body weight may also be related to reduced food intake, as GCs are reported to have mixed effects on appetite. Dose-dependent decrease in food intake and body weight were reported in rats^77,78; contradictory data have also been reported.^79-81 Rabbits in the current study had open access to food and associations between food intake, body weight, and GC treatment were not included in the current study.

Certainly, the uniqueness of rabbit VF structure and function may influence the generalizability of GC effects to human VFs. Although rabbits demonstrate similarities to humans in the context of VF tissue, microarchitectural properties, and extracellular matrix components, the rabbit only has 2 to 4 layers of epithelium and a 2-layered LP ^82-84; humans have 5 to 10 epithelial layers and a tertiary intermediate LP layer.^85,86 Additionally, although humans have both type I and II muscle fibers in the TA muscle, rabbits have been reported to only have type II muscle fibers,⁸⁷ which are believed to be less resistant to skeletal muscle atrophy.⁸⁸ Although no atrophy was observed in the current experiments, it is unclear whether different muscle fiber types have variable resistance to skeletal muscle atrophy induced by GCs. In that regard, “atrophy” in this study was defined as only a muscular finding, but atrophy is a multi-modal phenomenal observed to effect the VF lamina propria and epithelium.^41,42 Additionally, GCs are typically administered for laryngeal indications that frequently involve an inflammatory response in the VFs or surrounding tissue. It is reasonable to believe that this inflammatory response, characterized by an influx of immune cells, the release of inflammatory mediators, and chemoattraction of other cell types, influences how GCs interact with the VF tissue. Uninjured VFs were employed in the current study as a starting point for mechanistic inquiry.

Conclusion

Repeated systemic GC treatment did not adversely affect VF epithelial tight junction ZO-1 or TA muscle morphology compared to control tissue. However, methylprednisolone decreased body weight. Additionally, VF epithelial GR nuclear translocation was less efficient in systemic methylprednisolone treatment than dexamethasone. These results suggest systemic dexamethasone may more efficiently activate GR in the VF epithelium with a lower risk of body weight loss. Although further investigation is needed, these data suggest differential systemic effects of GCs. This finding suggests a role for a refined approach to GC selection and administration for laryngeal disease. Future studies will interrogate similar outcomes in the context of VF injury and additional GCs, doses, duration, and timepoints to elucidate chronic steroid use and mimic clinical scenarios more closely.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by research supported by The University of Pittsburgh Clinical and Translational Science Institute (NIH/NCATS TL1-TR001858) and the NIH/NIDCD (R01DC015405 and R01DC017397).

ORCID iD

Gary Gartling

References

Govil

Rafii

Paul

Ruiz

Amin

Branski

RC.

Glucocorticoids for vocal fold disease: a survey of otolaryngologists. J Voice. 2014;28(1):82-87. doi:10.1016/j.jvoice.2013.04.015

Cope

Bova

Steroids in otolaryngology. Laryngoscope. 2008;118(9):1556-1560. doi:10.1097/MLG.0b013e31817c0b4d

Mortensen

Laryngeal steroid injection for vocal fold scar. Curr Opin Otolaryngol Head Neck Surg. 2010;18(6):487-491. doi:10.1097/MOO.0b013e32833fe112

Cetinkaya

Tüfekçi

Kutluk

A comparison of nebulized budesonide, and intramuscular, and oral dexamethasone for treatment of croup. Int J Pediatr Otorhinolaryngol. 2004;68(4):453-456. doi:10.1016/J.IJPORL.2003.11.017

Cruz

Stewart

Rosenberg

Use of dexamethasone in the outpatient management of acute laryngotracheitis. Pediatrics. 1995;96(2 Pt 1):220-223.

Mishra

Rosen

Murry

Acute management of the performing voice. Otolaryngol Clin North Am. 2000;33(5):957-966.

Sataloff

Shaw

Markiewicz

Acute laryngitis in a professional singer. Ear Nose Throat J. 2001;80(7):436-436.

Watts

Clark

Early

Acoustic measures of phonatory improvement secondary to treatment by oral corticosteroids in a professional singer: a case report. J Voice. 2001;15(1):115-121. doi:10.1016/S0892-1997(01)00011-X

Bower

Belen

Weg

Dantzker

DR.

Manifestations and treatment of laryngeal sarcoidosis. Am Rev Respir Dis. 1980;122(2):325-332.

10.

Neel

McDonald

TJ.

Laryngeal sarcoidosis: report of 13 patients. Ann Otol Rhinol Laryngol. 1982;91(4):359-362.

11.

Weisman

Canalis

Powell

WJ.

Laryngeal sarcoidosis with airway obstruction. Ann Otol Rhinol Laryngol. 1980;89(1):58-61.

12.

François

Bellissant

Gissot

, et al. 12-h pretreatment with methylprednisolone versus placebo for prevention of postextubation laryngeal oedema: a randomised double-blind trial. Lancet. 2007;369:1083-1089. doi:10.1016/S0140-6736(07)60526-1

13.

Daley-Yates

Gregory

Brooks

CD.

Pharmacokinetic and pharmacodynamic assessment of bioavailability for two prodrugs of methylprednisolone. Br J Clin Pharmacol. 1997;43(6):593-601.

14.

Antal

Wright Ce 3rd Gillespie

Albert

KS.

Influence of route of administration on the pharmacokinetics of methylprednisolone. J Pharmacokinet Biopharm. 1983;11(6):561-576. doi:10.1007/BF01059057

15.

Egerman

Pierce

4th Andersen

Umstot

Carr

Sibai

BM.

A comparison of the bioavailability of oral and intramuscular dexamethasone in women in late pregnancy. Obstet Gynecol. 1997;89(2):276-280.

16.

Barnes

PJ.

How corticosteroids control inflammation: Quintiles Prize Lecture 2005. Br J Pharmacol. 2006;148(3):245-254. doi:10.1038/sj.bjp.0706736

17.

Scheinman

Gualberto

Jewell

Cidlowski

Baldwin

Jr.

Characterization of mechanisms involved in transrepression of NF-kappa B by activated glucocorticoid receptors. Mol Cell Biol. 1997;15(2):943-953.

18.

Wang

Hunter

, et al. Dynamic regulation of mitochondrial function by glucocorticoids. Proc Natl Acad Sci USA. 2009;106(9):3543-3548. doi:10.1073/pnas.0812671106

19.

Stahn

Buttgereit

Genomic and nongenomic effects of glucocorticoids. Nat Clin Pract Rheumatol. 2008;4(10):525-533. doi:10.1038/ncprheum0898

20.

Rafii

Sridharan

Taliercio

, et al. Glucocorticoids in laryngology: a review. Laryngoscope. 2014;124(7):1668-1673. doi:10.1002/lary.24556

21.

Higgins

Lees

The acute inflammatory process, arachidonic acid metabolism and the mode of action of anti-inflammatory drugs. Equine Vet J. 1984;16(3):163-175. doi:10.1111/j.2042-3306.1984.tb01893.x

22.

Löwenberg

Verhaar

Bilderbeek

, et al. Glucocorticoids cause rapid dissociation of a T-cell-receptor-associated protein complex containing LCK and FYN. EMBO Rep. 2006;7(10):1023-1029. doi:10.1038/sj.embor.7400775

23.

Kershner

Wang-Cheng

Psychiatric side effects of steroid therapy. Psychosomatics. 1989;30(2):135-139. doi:10.1016/S0033-3182(89)72293-3

24.

Lippman

Clinical implications of glucocorticoid receptors in human leukemia. Am J Physiol Endocrinol Metab. 1982;243(2):E103-E108. doi:10.1152/ajpendo.1982.243.2.E103

25.

Grzanka

Misiołek

Golusiński

Jarząb

Molecular mechanisms of glucocorticoids action: implications for treatment of rhinosinusitis and nasal polyposis. Eur Arch Otorhinolaryngol. 2011;268(2):247-253. doi:10.1007/s00405-010-1330-z

26.

Kao

Fluhr

Man

, et al. Short-term glucocorticoid treatment compromises both permeability barrier homeostasis and stratum corneum integrity: inhibition of epidermal lipid synthesis accounts for functional abnormalities. J Investig Dermatol. 2003;120(3):456-464. doi:10.1046/j.1523-1747.2003.12053.x

27.

Kadmiel

Janoshazi

Cidlowski

JA.

Glucocorticoid action in human corneal epithelial cells establishes roles for corticosteroids in wound healing and barrier function of the eye. Exp Eye Res. 2016;152:10-33.

28.

Chen

Luo

Han

, et al. Corticosteroid regulates epithelial occludin expression in nasal polyps through a MKP-1-dependent pathway. ORL J Otorhinolaryngol Relat Spec. 2014;76(5):248-256. doi:10.1159/000368652

29.

Schakman

Gilson

Thissen

JP.

Mechanisms of glucocorticoid-induced myopathy. J Endocrinol. 2008;197(1):1-10. doi:10.1677/JOE-07-0606

30.

Schakman

Kalista

Barbé

Loumaye

Thissen

JP.

Glucocorticoid-induced skeletal muscle atrophy. Int J Biochem Cell Biol. 2013;45(10):2163-2172. doi:10.1016/j.biocel.2013.05.036

31.

Karin

New twists in gene regulation by glucocorticoid receptor: is DNA binding dispensable?

Cell. 1998;93(4):487-490.

32.

Ito

Barnes

Adcock

IM.

Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits interleukin-1β-induced histone H4 acetylation on lysines 8 and 12. Mol Cell Biol. 2000;20(18):6891-6903.

33.

Greening

Ind

Northfield

Shaw

Added salmeterol versus higher-dose corticosteroid in asthma patients with symptoms on existing inhaled corticosteroid. Lancet. 1994;344(8917):219-224.

34.

Usmani

Ito

Maneechotesuwan

, et al. Glucocorticoid receptor nuclear translocation in airway cells after inhaled combination therapy. Am J Respir Crit Care Med. 2005;172(6):704-712.

35.

Tsukita

Yamazaki

Katsuno

Tamura

Tsukita

Tight junction-based epithelial microenvironment and cell proliferation. Oncogene. 2008;27(55):6930-6938. doi:10.1038/onc.2008.344

36.

Wolburg

Lippoldt

Tight junctions of the blood-brain barrier: development, composition and regulation. Vasc Pharmacol. 2002;38(6):323-337.

37.

Levendoski

Leydon

Thibeault

SL.

Vocal fold epithelial barrier in health and injury: a research review. J Speech Lang Hear Res. 2014;57(5):1679-1691. doi:10.1044/2014_JSLHR-S-13-0283

38.

Kimball

Sayce

Powell

Gartling

Brandley

Rousseau

Different vibratory conditions elicit different structural and biological vocal fold changes in an in-vivo rabbit model of phonation. J Voice. 2021;35:216-225. doi:10.1016/j.jvoice.2019.08.023

39.

Qin

Yang

, et al. Dexamethasone-induced skeletal muscle atrophy was associated with upregulation of myostatin promoter activity. Res Vet Sci. 2013;94(1):84-89. doi:10.1016/j.rvsc.2012.07.018

40.

Mallidis

Bhasin

, et al. Glucocorticoid-induced skeletal muscle atrophy is associated with upregulation of myostatin gene expression. Am J Physiol Endocrinol Metab. 2003;285(2):E363-E371. doi:10.1152/ajpendo.00487.2002

41.

Hashimoto

Kaneko

Kinoshita

, et al. Effects of repeated intracordal glucocorticoid injection on the histology and gene expression of rat vocal folds. J Voice. Published online July 2021. doi:10.1016/j.jvoice.2021.06.013

42.

Jin

Lee

, et al. Morphological and histological changes of rabbit vocal fold after steroid injection. Otolaryngol Head Neck Surg. 2013;149(2):277-283. doi:10.1177/0194599813489657

43.

Lee

Yeo

Choi

, et al. Local steroid injection via the cricothyroid membrane in patients with a vocal nodule. Arch Otolaryngol Head Neck Surg. 2011;137(10):1011-1016. doi:10.1001/archoto.2011.168

44.

Shi

Giraldez-Rodriguez

Johns

3rd . The risk of vocal fold atrophy after serial corticosteroid injections of the vocal fold. J Voice. 2016;30(6):762.e11-762.e13. doi:10.1016/j.jvoice.2015.10.004

45.

Takano

Kimura

Nito

Imagawa

Sakakibara

Tayama

Clinical analysis of presbylarynx-vocal fold atrophy in elderly individuals. Auris Nasus Larynx. 2010;37:461-464. doi:10.1016/j.anl.2009.11.013

46.

Omori

Slavit

Matos

Kojima

Kacker

Blaugrund

SM.

Vocal fold atrophy: quantitative glottic measurement and vocal function. Ann Otol Rhinol Laryngol. 1997;106(7 Pt 1):544-551. doi:10.1177/000348949710600702

47.

Dexamethasone 4 mg tablets - Summary of Product Characteristics (SmPC) - (emc). Accessed December 7, 2020. https://www.medicines.org.uk/emc/product/7395/smpc#gref

48.

Dexamethasone (systemic): drug information - UpToDate. Accessed June 26, 2023. https://www.uptodate.com/contents/dexamethasone-systemic-drug-information

49.

Decadron, Dexamethasone Intensol (dexamethasone) dosing, indications, interactions, adverse effects, and more. Accessed June 26, 2023. https://reference.medscape.com/drug/decadron-dexamethasone-intensol-dexamethasone-342741

50.

Dexamethasone dosage guide + max dose, adjustments - Drugs.com. Accessed June 26, 2023. https://www.drugs.com/dosage/dexamethasone.html#Usual_Adult_Dose_for_Anti_inflammatory

51.

Methylprednisolone dosage guide with precautions - Drugs.com. Accessed December 7, 2020. https://www.drugs.com/dosage/methylprednisolone.html

52.

Methylprednisolone dosage guide + max dose, adjustments - Drugs.com. Accessed June 26, 2023. https://www.drugs.com/dosage/methylprednisolone.html#Usual_Adult_Dose_for_Allergic_Rhinitis

53.

Inder

Jang

Obeyesekere

Alford

FP.

Dexamethasone administration inhibits skeletal muscle expression of the androgen receptor and IGF-1–implications for steroid-induced myopathy. Clin Endocrinol. 2010;73(1):126-132. doi:10.1111/j.1365-2265.2009.03683.x

54.

Gilson

Schakman

Combaret

, et al. Myostatin gene deletion prevents glucocorticoid-induced muscle atrophy. Endocrinology. 2007;148(1):452-460. doi:10.1210/en.2006-0539

55.

Gayan-Ramirez

Vanderhoydonc

Verhoeven

Decramer

Acute treatment with corticosteroids decreases IGF-1 and IGF-2 expression in the rat diaphragm and gastrocnemius. Am J Respir Crit Care Med. 1999;159(1):283-289. doi:10.1164/ajrccm.159.1.9803021

56.

Gartling

Sayce

Kimball

Sueyoshi

Rousseau

A comparison of the localization of integral membrane proteins in human and rabbit vocal folds. Laryngoscope. 2021;131(4):E1265-E1261. doi:10.1002/lary.29243

57.

Ceglia

Niramitmahapanya

Price

Harris

Fielding

Dawson-Hughes

An evaluation of the reliability of muscle fiber cross-sectional area and fiber number measurements in rat skeletal muscle. Biol Proced Online. 2013;15:6. doi:10.1186/1480-9222-15-6

58.

Gartling

Nakamura

Sayce

, et al. Acute in vitro and in vivo effects of dexamethasone in the vocal folds: a pilot study. Laryngoscope. 2022; In press. doi:10.1002/lary.30461

59.

Matthews

Ito

Barnes

Adcock

IM.

Defective glucocorticoid receptor nuclear translocation and altered histone acetylation patterns in glucocorticoid-resistant patients. J Allergy Clin Immunol. 2004;113(6):1100-1108.

60.

Goodman

Gilman

The Pharmacological Basis of Therapeutics. Macmillan; 1955.

61.

Barnes

PJ.

Mechanisms and resistance in glucocorticoid control of inflammation. J Steroid Biochem Mol Biol. 2010;120(2-3):76-85.

62.

Ramamoorthy

Cidlowski

JA.

Exploring the molecular mechanisms of glucocorticoid receptor action from sensitivity to resistance. Endocr Dev. 2013;24:41-56. doi:10.1159/000342502

63.

Chung

Son

Kim

Circadian rhythm of adrenal glucocorticoid: its regulation and clinical implications. Biochim Biophys Acta Mol Basis Dis. 2011;1812(5):581-591.

64.

Hellhammer

Wüst

Kudielka

BM.

Salivary cortisol as a biomarker in stress research. Psychoneuroendocrinology. 2009;34(2):163-171.

65.

Hue

Cho

Cao

Dale Bass

Meaney

Morrison

3rd . Dexamethasone potentiates in vitro blood-brain barrier recovery after primary blast injury by glucocorticoid receptor-mediated upregulation of ZO-1 tight junction protein. J Cereb Blood Flow Metab. 2015;35(7):1191-1198. doi:10.1038/jcbfm.2015.38

66.

Romero

Radewicz

Jubin

, et al. Changes in cytoskeletal and tight junctional proteins correlate with decreased permeability induced by dexamethasone in cultured rat brain endothelial cells. Neurosci Lett. 2003;344(2):112-116. doi:10.1016/S0304-3940(03)00348-3

67.

Singer

Stevenson

Woos

Firestone

. Relationship of serine/threonine phosphorylatiod dephosphorylation signaling to glucocorticoid regulation of tight junction permeability and ZO-1 distribution in nontransformed mammary epithelial cells. J Bio Chem. 1994;269(23):16108-16115.

68.

Scudamore

Jepson

Hirst

Hugh

Miller

The rat mucosal mast cell chymase, RMCP-11, alters epithelial cell monolayer permeability in association with altered distribution of the tight junction proteins ZO-1 and occludin. Eur J Cell Biol. 1998;75(4):321-330. doi:10.1016/S0171-9335(98)80065-4

69.

Wang

Liu

Zhao

Xue

Krüppel-like factor 4 regulates blood-tumor barrier permeability via ZO-1, Occludin and Claudin-5. J Cell Physiol. 2014;229(7):916-926. doi:10.1002/jcp.24523

70.

Shen

Zhang

Song

Zheng

WP.

Bone-marrow mesenchymal stem cells reduce rat intestinal ischemia-reperfusion injury, ZO-1 downregulation and tight junction disruptionviaa TNF-α-regulated mechanism. World J Gastroenterol. 2013;19(23):3583-3595. doi:10.3748/wjg.v19.i23.3583

71.

Rousseau

Suehiro

Echemendia

Sivasankar

Raised intensity phonation compromises vocal fold epithelial barrier integrity. Laryngoscope. 2011;121(2):346-351. doi:10.1002/lary.21364

72.

Kojima

Valenzuela

Novaleski

, et al. Effects of phonation time and magnitude dose on vocal fold epithelial genes, barrier integrity, and function. Laryngoscope. 2014;124(12):2770-2778. doi:10.1002/lary.24827

73.

Lecker

Solomon

Mitch

Goldberg

AL.

Muscle protein breakdown and the critical role of the ubiquitin-proteasome pathway in normal and disease states. J Nutr. 1999;129(1):227S-237S. doi:10.1093/jn/129.1.227S

74.

Mallidis

Artaza

Taylor

Gonzalez-Cadavid

Bhasin

Characterization of 5'-regulatory region of human myostatin gene: regulation by dexamethasone in vitro. Am J Physiol Endocrinol Metab. 2001;281(6):E1128-E1136. doi:10.1152/ajpendo.2001.281.6.E1128

75.

Sassoon

Zhu

Pham

, et al. Acute effects of high-dose methylprednisolone on diaphragm muscle function. Muscle Nerve. 2008;38(3):1161-1172. doi:10.1002/mus.21048

76.

Skjærbæk

Frystyk

Grøfte

, et al. Serum free insulin-like growth factor-I is dose-dependently decreased by methylprednisolone and related to body weight changes in rats. Growth Horm IGF Res. 1999;9(1):74-80. doi:10.1054/ghir.1999.0090

77.

Won Jahng

Kim

Ryu

, et al. Dexamethasone reduces food intake, weight gain and the hypothalamic 5-HT concentration and increases plasma leptin in rats. Eur J Pharmacol. 2008;581(1-2):64-70. doi:10.1016/j.ejphar.2007.11.029

78.

Liu

Shi

, et al. Glucocorticoids decrease body weight and food intake and inhibit appetite regulatory peptide expression in the hypothalamus of rats. Exp Ther Med. 2011;2(5):977-984. doi:10.3892/etm.2011.292

79.

Zakrzewska

Cusin

Stricker-Krongrad

, et al. Induction of obesity and hyperleptinemia by central glucocorticoid infusion in the rat. Diabetes. 1999;48(2):365-370. doi:10.2337/diabetes.48.2.365

80.

Buchbinder

Hoving

Green

Hall

Forbes

Nash

Short course prednisolone for adhesive capsulitis (frozen shoulder or stiff painful shoulder): a randomised, double blind, placebo controlled trial. Ann Rheum Dis. 2004;63(11):1460-1469. doi:10.1136/ard.2003.018218

81.

Halvorsen

Ræder

White

, et al. The effect of dexamethasone on side effects after coronary revascularization procedures. Anesth Analg. 2003;96(6):1578-1583.

82.

Thibeault

Gray

Bless

Chan

Ford

CN.

Histologic and rheologic characterization of vocal fold scarring. J Voice. 2002;16(1):96-104. doi:10.1016/S0892-1997(02)00078-4

83.

Kurita

A comparative study of the layer structure of the vocal fold. Jibi Rinsho Kai. 1981;28(4):3-21.

84.

Gray

Hammond

TH.

Biomechanical and histologic observations of vocal fold fibrous proteins. 2000;109(1):77-85. doi:10.1177/000348940010900115

85.

Hallén

Johansson

Laurent

Dahlqvist

Hyaluronan localization in the rabbit larynx. Anat Rec. 1996;246(4):441-445. doi:10.1002/(SICI)1097-0185(199612)246:4<441::aid-ar3>3.0.CO;2-Y

86.

Arens

Glanz

Wönckhaus

Hersemeyer

Kraft

Histologic assessment of epithelial thickness in early laryngeal cancer or precursor lesions and its impact on endoscopic imaging. Eur Arch Otorhinolaryngol. 2007;264(6):645-649. doi:10.1007/s00405-007-0246-8

87.

Hoh

JF.

Laryngeal muscle fibre types. Acta Physiol Scand. 2005;183(2):133-149. doi:10.1111/j.1365-201X.2004.01402.x

88.

Wang

Pessin

JE.

Mechanisms for fiber-type specificity of skeletal muscle atrophy. Curr Opin Clin Nutr Metab Care. 2013;16(3):243-250. doi:10.1097/MCO.0b013e328360272d

Acute Effects of Systemic Glucocorticoids on the Vocal Folds in a Pre-Clinical Model

Abstract

Objectives/Hypothesis:

Methods:

Results:

Conclusions:

Keywords

Introduction

Methods

Preclinical GC Injections

Body Weight

Immunofluorescence Labeling

Hematoxylin and Eosin (H&E) Staining

Microscopy

Image Analysis

Statistical Analyses

Results

Dexamethasone Increased Epithelial GR Nuclear Translocation

Methylprednisolone Decreased Epithelial ZO-1 Expression

Glucocorticoids Increased TA Muscle Fiber CSA

Methylprednisolone Decreased Body Weight

Discussion

Conclusion

Footnotes

Declaration of Conflicting Interests

Funding

ORCID iD

References