Hyoepiglottic Ligament Collagen and Elastin Fiber Composition and Changes Associated With Aging

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2 The Laryngoscope VC 2018 The American Laryngological, Rhinological and Otological Society, Inc. Hyoepiglottic Ligament Collagen and Elastin Fiber Composition and Changes Associated With Aging Leslie E. Irvine, MD; Zhi Yang, MD; Eric J. Kezirian, MD, MPH ; Marcel E. Nimni, PhD; Bo Han, PhD Objective: The epiglottis may contribute to upper airway obstruction in approximately 10% of patients with obstructive sleep apnea. Clinical experience indicates that older patients may be more likely to have epiglottis-related obstruction. This study was designed to examine tissue characteristics of the hyoepiglottic ligament as a possible factor in epiglottis-related obstruction based on previous research suggesting that older adults have fewer collagen, elastin, and muscle fibers in the hyoepiglottic ligament. Methods: This is a cross-sectional study of 25 human cadaver hyoepiglottic ligaments. Specimens were stained using Masson s trichrome and Picrosirius red for collagen fibers and with Verhoeff-Van Gieson for elastin fibers. Percentage of collagen and elastin fiber staining for each specimen was calculated and averaged over three regions of each ligament section. Regression analysis was used to determine the association between age, smoking history, and collagen and elastin composition of the hyoepiglottic ligament. Results: The average age of the specimens was years (range years). Increasing age was associated with a lower percentage of collagen and elastin fibers. When accounting for tobacco use, each 1-year increase in age was associated with a 0.53% decrease in Masson s trichrome staining (P ), a 0.35% decrease in Picrosirius red staining (P ), and a 0.33% decrease in Verhoeff-Van Gieson staining (P ). Conclusion: Increasing age is associated with decreases in the collagen and elastin content of the hyoepiglottic ligament. Key Words: Hyoepiglottic ligament, epiglottis, collagen, elastin. Level of Evidence: NA. Laryngoscope, 128: , 2018 INTRODUCTION Epiglottic collapse can contribute to obstructive sleep apnea (OSA). Epiglottis-related obstruction may occur with an epiglottis that displaces posteriorly during sleep (in some cases, there is posterior displacement during wakefulness) or with folding of the epiglottis to obstruct the airway (often with decreased structural integrity of the epiglottis). Both mechanisms of From the Santa Barbara Plastic Surgery Center (L.E.I.), Santa Barbara, California, USA; Department of Surgery (Z.Y., B.H.); the Department of Otolaryngology Head and Neck Surgery (E.J.K.), University of Southern California Keck School of Medicine; Department of Biochemistry and Molecular Biology (M.E.N.); and the Department of Biomedical Engineering (M.E.N., B.H.), University of Southern California, Los Angeles, California, U.S.A Editor s Note: This Manuscript was accepted for publication December 18, This work was conducted at the Keck School of Medicine of the University of Southern California. Presented at the Combined Otolaryngology Sections Meeting in Coronado, California, U.S.A, January 22 24, E.J.K.: Inspire Medical Systems (research funding); advisory board for Nyxoah, ReVENT Medical, Pillar Palatal, Split Rock Scientific, Cognition Life Science, Berendo Scientific; intellectual property ownership in Magnap. Funding was provided by the USC Caruso Department of Otolaryngology Head & Neck Surgery and the Department of Surgery, USC Keck School of Medicine. The authors have no other funding, financial relationships, or conflicts of interest to disclose. Send correspondence to Eric J. Kezirian, MD, MPH, 1450 San Pablo Street, Suite 5100, Los Angeles, CA eric.kezirian@ med.usc.edu DOI: /lary epiglottis-related obstruction have been seen during drug-induced sleep endoscopy. 1 3 Epiglottis-related obstruction may occur in isolation but may also contribute specifically to difficulty tolerating positive airway pressure therapy. The latter may occur if positive pressure pushes the epiglottis in an unfavorable direction to obstruct, rather than open, the airway. 4,5 Epiglottis-related airway obstruction has been associated with neurological disorders, 5 neurologic injury, 6 neck surgery, 7 and aging. 8 The hyoepiglottic ligament connects the hyoid bone and the epiglottis, and changes associated with this ligament may contribute to epiglottis-related airway obstruction. Differences in collagen, elastin, and muscle fiber content of the hyoepiglottic ligament have previously been described in relatively young versus elderly adult human cadavers. 9 The objective of this study was to examine the collagen and elastin content of the hyoepiglottic ligament across a more diverse age range, incorporating stains specific for collagen. MATERIALS AND METHODS The study protocol was confirmed as exempt from review by the institutional review board of the University of Southern California (Los Angeles, CA). Fresh cadaveric laryngeal tissue was obtained from the University of Southern California tissue lab. Tissue was fixed in 10% formalin. After fixation, specimens were bisected in the midline of the epiglottis, cut into sagittal sections, and embedded in paraffin. Specimens consisted of a 1245

3 stained with Masson s trichrome, Picrosirius red for collagen, and Verhoeff-Van Gieson for elastin fibers. Images were taken using a Nikon camera and light microscope at 4 3 magnification. The portion of the median hyoepiglottic ligament between the base of the epiglottis and the center of the ligament was captured (Fig. 1). Three pixel regions from the 4x images were cropped using ImageJ software version 1.49o (National Institutes of Health, Bethesda, MD). Each cropped region image was analyzed for collagen and elastin content using color threshold settings. Density values of each stain were measured and divided by the total area of the image (Fig. 2). The staining density was averaged across the three cropped region images for each specimen. Mean and standard deviations were calculated for age and the percentages of tissue stained, both of which were treated as continuous variables. The Shapiro-Wilk test was used to evaluate the normality of these variables. Smoking history was considered as a dichotomous variable. Linear regression examined the association between percentage of tissue stained (for each stain) and age, adjusted for history of tobacco smoking. Fig. 1. Sagittal section through the hyoepiglottic ligament of a 75- year-old man, stained with Verhoeff Van-Gieson. Box overlies the hyoepiglottic ligament and represents the region where the cropped images were taken. short arrow: epiglottis; long arrow: base of tongue muscle; arrowhead: hyoid bone location (truncated). portion of the epiglottis, tongue base musculature, the median and lateral hyoepiglottic ligament, and the preepiglottic fat inferior to the ligament. Five micron-thick sagittal sections of the median hyoepiglottic ligament were taken. A representative section was identified from each cadaver specimen for staining. Sections were RESULTS Twenty-five cadaver larynges were examined (19 males and 6 females). Age was years, with a range of 30 to 90 years. The majority (72%, 18 of 25) of the specimens were from individuals with a smoking history. The percentages stained are shown in Table I. The Shapiro-Wilk test demonstrated that age and the percentages of staining for Masson s trichrome and Picrosirius red had a normal distribution, although the percentage for Verhoeff-Van Gieson staining did not (P ). Greater age was associated with a decrease in the percentages of Masson s trichrome, Picrosirius red, and Verhoeff-Van Gieson staining before (Table II) and after (Table III) adjustment for a history of tobacco smoking. DISCUSSION This study confirms that increasing age is associated with decreased collagen and elastin content in the hyoepiglottic ligament. The present study builds on the one previous study in this area 9 in three meaningful ways: The present study used stains specific for collagen (Masson s trichrome and Picrosirius red) instead of Fig. 2. Cropped sections of the hyoepiglottic ligament from a 68-year-old specimen (43). Verhoeff-Van Gieson stain (A) shows distribution of elastin fibers. Masson s trichrome (B) and Picrosirius red (C) show distribution of collagen fibers. 1246

4 TABLE I. Percentage of Images Stained. n Mean SD Minimum Maximum Masson s trichrome Picrosirius red Verhoeff-Van Gieson SD 5 standard deviation. generalized hematoxylin-eosin staining. Also, the present study examined cadavers distributed more broadly over a age range of 30 to 90 years, compared to previous work examining cadavers limited to ages 31 to 48 versus 81 to 91 years. The present study also adjusted for a history of tobacco smoking, potentially important due to the association between smoking and both decreased subcutaneous collagen synthesis and increased production of some matrix metalloproteinases associated with collagen and elastin breakdown in skin, as well as altered collagen fiber density and alignment in the vocal ligament. 14 Changes in the hyoepiglottic ligament may affect the position and movement of the epiglottis at rest and during normal functions, such as swallowing. The hyoepiglottic ligament consists of medial and lateral components. The median hyoepiglottic ligament is a fanshaped fibrous structure connecting the medial hyoid bone with the anterior epiglottis. The lateral hyoepiglottic ligaments are two short bands of tissues connecting the lateral epiglottis to the greater cornua of the hyoid bone. 15 This ligament transmits force from the hyoid bone to the epiglottis. Mechanical properties of the hyoepiglottic ligament depend on its composition of collagen and elastic fibers. Although there was a decrease in collagen and elastin fiber content, it was not clear what soft tissue components were increased. We perceived that there was simply a corresponding broad increase in other soft tissue components. The prevalence of OSA increases with patient age, 16 and clinical experience suggests that epiglottis-related obstruction during drug-induced sleep endoscopy also is more common with advanced patient age. If epiglottisrelated obstruction seen during drug-induced sleep endoscopy mirrors events during natural sleep, changes in the hyoepiglottic ligament composition may increase laxity of the ligament and contribute to posterior displacement TABLE II. Percentage of Image Stained Associated With Age (Years), Without Adjusted for Tobacco Smoking Status. Estimate Age P Value Adjusted R 2 Masson s trichrome Picrosirius red Verhoeff-Van Gieson TABLE III. Percentage of Image Stained Associated With Age (Years), Adjusted for Tobacco Smoking Status. (the anteroposterior epiglottis-related obstruction seen during drug-induced sleep endoscopy), 1 potentially explaining at least part of the increased prevalence of OSA. Current approaches for treating epiglottis-related obstruction in adults include partial epiglottis resection, 17 laryngoplasty, 18 and hyoid suspension to advance and stabilize the hyoid bone. 19 The present study suggests that tightening the hyoepiglottic ligament may have therapeutic implications for OSA in selected individuals. This study has limitations. First, there were fewer young cadavers as compared to older cadavers. Information such as history of sleep apnea was also not available. In addition, the study was underpowered to examine gender differences. Future studies may examine collagen subtypes in the hyoepiglottic ligament and changes associated with age, potentially with larger numbers to evaluate possible gender differences. CONCLUSION Increasing age is associated with a lower density of collagen and elastin fibers in the hyoepiglottic ligament. This may have functional and therapeutic implications for epiglottis-related obstruction in OSA. 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