The regulatory mechanism of HSP70 in endoplasmic reticulum stress in pepsin-treated laryngeal epithelium cells and laryngeal cancer cells.

BACKGROUNDS: Excessive pepsin can damage both normal laryngeal epithelial cells and laryngeal cancer (LC) cells. Heat shock protein 70 (HSP70) is closely related to pepsin. In this paper, we will explore the different significance of the regulatory role of HSP70 in endoplasmic reticulum stress (ERS) level in pepsin-treated laryngeal epithelial cells and LC cells. METHODS: In cell experiments, laryngeal epithelial cells and LC cells were selected and induced by different concentrations of pepsin. Cell activity was detected by CCK8, cell apoptosis was detected by flow cytometry, and autophagy was detected by autophagy detection kit. The expression of ER)-related proteins was detected by immunofluorescence (IF) and Western blot. Cell transfection was used to inhibit HSP70 expression in both cells, and ERS, apoptosis, and autophagy were measured using related techniques. In animal experiments, a mouse model bearing LC was established. TUNEL assay detected apoptosis, autophagy kit detected autophagy, and ER-related protein expression was detected by Western blot. RESULTS: HSP70 was increased in pepsin-stimulated laryngeal epithelial cells and LC cells, thereby inhibiting ER and ER-induced apoptosis and autophagy. Inhibition of HSP70 reduced the expression of glucose regulated protein 78 (GRP78) in pepsin-stimulated laryngeal epithelial cells and LC cells, and only inhibited downstream apoptosis-related pathways in laryngeal epithelial cells rather than in LC cells. Inhibition of HSP70 and ER could significantly promote apoptosis and inhibit tumor growth in the absence of pepsin stimulation in vivo. CONCLUSION: ER level regulated by HSP70 had different significance in laryngeal epithelial cells and LC cells treated with pepsin.

Changes in the Laryngeal Mucosa After Thyroid Surgery: A Rat Model.

BACKGROUND/AIM: Thyroidectomy can cause various airway symptoms affecting the quality of life. We investigated the changes in extracellular matrix (ECM) composition and markers for inflammation and microcirculation of laryngeal mucosa. MATERIALS AND METHODS: Sixty Sprague-Dawley rats were categorized into control and three surgical groups based on the extent of surgeries, 1) flap elevation (FE) group, 2) thyroid and trachea exposure (TE) group, and 3) thyroid isthmectomy (TI) group. We analyzed the expression of TGF-ß1, VEGFR-3, CD31, and MMP- 9 in relation to the inflammatory and microcirculatory changes in the lamina propria on postoperative days (PODs) 3, 7, and 21. ECM composition of hyaluronic acid (HA) and collagen in the subglottic area (SA) was also evaluated. RESULTS: All parameters increased in surgical groups at each postoperative phase except collagen deposition. On POD 3, TGF-ß1 expression and SA increased in relation to the surgical extent and decreased over time, but more than the control in all surgical groups on POD 21. Surgical groups had more HA and less collagen composition, causing a higher HA to collagen ratio in relation to the surgical extent. VEGFR-3 and CD31 expression increased with time at all postoperative phases according to the surgical extent. Expression of MMP-9 increased in TI groups compared to TE and FE groups on POD 7 and POD 21. CONCLUSION: This study demonstrated that thyroid surgery exposing the thyroid and trachea induces an increase in the SA with a higher HA and lesser collagen composition. Furthermore, the markers for acute inflammation and microcirculation with tissue remodeling increased in the laryngeal mucosa.

Energy metabolism of cells in the macula flava of the newborn vocal fold from the aspect of mitochondrial microstructure.

OBJECTIVE: Cells in the vocal fold of maculae flavae are likely to be tissue stem cells. Energy metabolism of the cells in newborn maculae flavae was investigated from the aspect of mitochondrial microstructure. METHOD: Five normal newborn vocal folds were investigated under transmission electron microscopy. RESULTS: Mitochondria consisted of a double membrane bounded body containing matrices and a system of cristae. However, these membranes were ambiguous. In each mitochondrion, the lamellar cristae were sparse. Intercristal space was occupied by a mitochondrial matrix. Some mitochondria had fused to lipid droplets and rough endoplasmic reticulum, and both the mitochondrial outer and inner membranes had incarcerated and disappeared. CONCLUSION: The features of the mitochondria of the cells in the newborn maculae flavae showed that their metabolic activity and oxidative phosphorylation were low. The metabolism of the cells in the newborn maculae flavae seems to be favourable to maintain the stemness and undifferentiation of the cells.

Mainstream Cigarette Smoke Impacts the Mouse Vocal Fold Epithelium and Mucus Barrier.

OBJECTIVES/HYPOTHESIS: Cigarette smoke (CS) is a primary risk factor for the development of numerous benign and malignant laryngeal diseases. The epithelium and mucus lining the vocal folds (VF) are the first barriers against CS. The primary objective of this study was to investigate epithelial and mucus barrier changes in the mouse laryngeal mucosa upon exposure to subacute CS. The secondary objective was to compare mucus barrier changes in mice and human smokers and nonsmokers. Study Design Animal model. METHODS: Mice were exposed to CS for 4 weeks for 4 hours (N = 12, high dose [HD]) or 1 hour (N = 12, low dose [LD]) per day. Air-exposed mice were used as a control group (N = 10). Larynges were harvested and VF epithelial barrier integrity was evaluated including cellular proliferation and expression of cell junctions. We also investigated mucus production by examining mucus cell area and mucin expression in mice and human smokers and nonsmokers. RESULTS: HD CS increased VF epithelial cellular proliferation but did not alter the expression of cell junctions. HD CS also induced hypertrophy of the mucus-producing submucosal glands. However, only LD CS increased MUC5AC gene expression. MUC5AC staining appeared elevated in laryngeal specimens from smokers, but this was not significant as compared to nonsmokers. CONCLUSIONS: These findings help us identify potential adaptive mechanisms to CS exposure as well as set the foundation for further study of key aspects of epithelial and mucus barrier integrity that may be implicated in laryngeal disease development following prolonged smoking. LEVEL OF EVIDENCE: NA Laryngoscope, 131:2530-2539, 2021.

Lubricin/proteoglycan 4 detected in vocal folds of humans and five other mammals.

OBJECTIVES/HYPOTHESIS: Lubricin/proteoglycan-4 (PRG4) lubricates connective tissues such as joints and tendon sheaths, enabling them to better withstand shearing and frictional forces during motion. We wondered whether PRG4 might play a role in phonation, as normal vocal folds withstand repetitive, high-velocity deformations remarkably well. As a first step, we tested whether PRG4 is expressed in vocal folds. STUDY DESIGN: Laboratory study. METHODS: Anatomical and molecular methods were applied to 47 larynges from humans, macaque (Macaca fascicularis), canines, pigs, calves, and rats. Immunohistochemistry (IHC), Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR) methods were used to test for the presence of PRG4. RESULTS: In all species, the true vocal fold lamina propria (TVF-LP) was positive for PRG4 by IHC, whereas immunoreactivity of the false vocal fold was weak or absent, depending on the species. Human TVF-LP was strongly stained across all layers. Immunoreactivity was seen variably on the vocal fold surface and within the vocal fold epithelium, in the conus elasticus and thyroglottic ligament, and at the tip of vocal process. Western blots of four humans and six pigs demonstrated immunoreactivity at appropriate molecular weight. qRT-PCR of pig tissues confirmed PRG4 mRNA expression, which was highest in the TVF-LP. CONCLUSIONS: PRG4 was found in phonatory tissues of six mammals. We suggest it might act as a lubricant within the lamina propria and possibly on the vocal fold surface, limiting phonation-related damage to vocal fold extracellular matrix and epithelium, and enhancing vocal efficiency by reducing internal friction (viscosity) within the vocal fold. LEVEL OF EVIDENCE: NA Laryngoscope, 129:E229-E237, 2019.

Influence of subglottic secretion drainage on the microorganisms of ventilator associated pneumonia: A meta-analysis for subglottic secretion drainage.

BACKGROUND: The influence of the subglottic secretion drainage (SSD) on the microorganisms of ventilator associated pneumonia (VAP) is still unclear.A meta-analysis focusing on the influence of the SSD on the microorganisms of VAP. METHODS: A comprehensive search was conducted through the online studies of PubMed, Embase, Cochrane Library, Google scholar, CNKI (China National Knowledge Infrastructure), and VIPI (Database for Chinese Technical Periodicals) using specific search terms.Included studies were randomized controlled trials (RCTs) that compare the microorganisms of VAP between SSD and standard endotracheal tube care in mechanically ventilated adults. RESULTS: Nine RCTs were eligible. There was no significant difference in the rate of VAP caused by nonfermentative bacteria and enterobacteria between SSD group and control group (OR = 0.73, 95%CI, 0.53-1.01; P = .06). The episodes of VAP caused by Gram-positive cocci and Haemophilus influenzae organisms were lower in the SSD group (OR = 0.29, 95%CI, 0.18-0.48; P<0.00001). Less mean volume of SSD daily was observed in VAP group (OR = -16.97, 95%CI, -29.87-4.08; P = .010). CONCLUSION: We found SSD to be associated with significant decreases in VAP caused by Gram-positive cocci and H influenzae organisms but no significant differences in VAP caused by nonfermentative bacteria and enterobacteria. Less mean volume of SSD daily was observed in VAP group.

[The effect of miR-497 on laryngeal squamous cell carcinoma invasion through modulating PlexinA4].

Objective: To study the roles of miR-497 and PlexinA4 in the progression of laryngeal squamous cell carcinoma. Methods: The expressions of miR-497 and PlexinA4 in fresh tumor specimens and adjacent normal mucosa tissues as well as in cell lines of laryngeal squamous cell carcinoma (LSCC) were detected with qRT-PCR and immunohistochemistry. The association of miR-497 and PlexinA4 expressions with clinicopathologic factors and their prognostic values in LSCC were evaluated PlexinA4 siRNA and pcDNA3.1 (+ )/PlexinA4 plasmid were transfected into the LSCC and measured by Transwell to evaluate their effect on the invasion of LSCC. Results: miR-497 was low expression in LSCC, which related to pathological differentiation, while PlexinA4 mRNA was high expression in LSCC. Kaplan-Meier method showed that the prognosis of patients with high miR-497 expression was better than that of patients with low miR-497 expression (χ(2)=10.342, P=0.001); . Cox regression analysis showed that miR-497 was an independent prognostic factor for LSCC. The double luciferase reporter gene showed that the variation of the fluorescence activity of wild type PlexinA4 was significantly different from that of the control group (P<0.01). In Hep-2 and TU212 cell line, the number of cells with PlexinA4 siRNA passing through the compartments was 70.00±10.85 and 85.00±6.45, significantly higher than control (F values were 30.251 and 23.936, both P<0.05), the number of cells with pcDNA3.1 (+ ) /PlexinA4 was 170.56±11.95 and 142.00±10.43, also significantly less than control (F values were 35.104 and 29.643, both P<0.05). Conclusion: The expression of miR-497 in LSCC is decreased, indicating poor prognosis, which is as an independent risk factor for prognosis of LSCC. miR-497 may modulate LSCC invasion through PlexinA4.

Fibroblast growth factor receptor 1 and 3 expression is associated with regulatory PI3K/AKT kinase activity, as well as invasion and prognosis, in human laryngeal cancer.

PURPOSE: Aberrant fibroblast growth factor receptor (FGFR) expression is thought to contribute to the development of many types of cancer. As yet, however, their impact on the course and prognosis of head and neck cancer remains to be determined. Here, we aimed to investigate the effects of expression of the FGFR family members FGFR1 and FGFR3, as well as their downstream PI3K/AKT signal-regulated kinases, on the aggressiveness and prognosis of laryngeal cancer. METHODS: In total 137 surgically removed squamous cell laryngeal cancer (SCLC) and 100 matched non-cancerous laryngeal mucosa (NCLM) samples were assessed for mRNA expression using quantitative real-time PCR. The corresponding proteins were analyzed by Western blotting. SLUG expression was assessed by immunohistochemistry. The expression data were subsequently related to tumor front grading (TFG), local/nodal recurrences, prognosis and overall survival. RESULTS: The FGFR1, FGFR3 and PI3K/AKT kinase mRNA and protein levels were found to be significantly higher in the SCLC than the NCLM samples (p < 0.05). A high FGFR1 mRNA/protein expression level was found to be associated with an increased invasion rate, according to TFG scale and SLUG level, a high local/nodal recurrence rate and a poor prognosis (p < 0.05). Similarly, we found that a high FGFR3 mRNA/protein expression level was associated with a shorter survival time (p < 0.05). In addition, we found that high PI3K/AKT kinase mRNA/protein levels were associated with a high TFG (p < 0.05). We also found that FGFR1/3 mRNA and FGFR1 protein levels were inversely associated with overall survival (log-rank test: FGFR1 mRNA p = 0.03, FGFR3 mRNA p = 0.04, FGFR1 protein p = 0.03). Subsequent multivariate analyses revealed that high FGFR3 mRNA expression may serve as an independent poor prognostic factor (HR 2.32, 95% CI 1.03-6.59; p = 0.04). We also found that the p-PI3K regulatory kinase protein level was significantly associated with survival in the cohort studied (HR 1.78, 95% CI 0.64-8.53; p = 0.03). CONCLUSIONS: From our data we conclude that FGFR1 and FGFR3, as well as its downstream regulatory PI3K/AKT kinases, may serve as potential biomarkers for the invasiveness and prognosis of laryngeal cancer. The expression of FGFR1/3-PI3K/AKT regulatory pathway members may be instrumental for the identification of patients at risk for an unfavorable clinical outcome.

The Effect of Human Adipose Tissue-Derived Mesenchymal Stem Cells in Rat's Subglottic Stenosis Model.

OBJECTIVE: Subglottic stenosis remains a clinical challenge. The aim of this study was to evaluate the effect of human adipose tissue-derived mesenchymal stem cells (hAMSCs) in rat model of subglottic stenosis. SUBJECTS AND METHODS: Ninety-six 13-week-old male rats were enrolled in this study. They were divided into 3 groups as normal control (NC) group, a subglottic injury and media injection (SM) group, and a subglottic injury and media-stem cell injection (SMSC) group. The hAMSCs were immediately injected into subglottis after injury. Histologic characteristics of subglottis; the mRNA expressions of interleukin-1ß, cyclooxygenase-2, tumor growth factor-ß and basic fibroblast growth factor; and hAMSCs' survival were evaluated. RESULTS: The hAMSCs survived in the subglottis of the rat until 10 days after implantation. The NC and SMSC groups had a significantly wider subglottic lumen and thinner lamina propria than the SM group at 56 days after injury. Collagen intensity of subglottis was significantly higher in the SM group than in the NC and SMSC groups at 28 days after injury. Gene expression didn't show significant difference between the SM group and the SMSC group. CONCLUSIONS: The hAMSCs injection was found to be helpful for preventing subglottic stenosis in a rat model.

Immunolocalization of CD163+ Tumor-Associated Macrophages and Symmetric Proliferation of Ki-67 as Biomarkers to Differentiate New Different Grades of Laryngeal Dysplasia.

OBJECTIVES: To evaluate CD163+ tumor-associated macrophages (TAMs), Ki-67, and cyclin D1 to differentiate laryngeal dysplasia in the 2017 World Health Organization classification. METHODS: Immunohistochemistry for CD163, Ki-67, and cyclin D1 was performed using paraffin-embedded specimens. CD163+ TAMs infiltrating the epithelium were estimated. Ki-67 and cyclin D1 were evaluated in four parts of the epithelium-basal, parabasal, middle third, and upper third layers. RESULTS: In total, 133 specimens were analyzed, including low-grade dysplasia (n = 31), high-grade dysplasia (n = 49), carcinoma in situ (n = 23), and normal mucosa (n = 30). CD163+ TAMs infiltrating the epithelium were significantly higher in high-grade dysplasia than in low-grade dysplasia. In the basal layer, Ki-67+ and cyclin D1+ cells were overexpressed in high-grade dysplasia (P < .0001). The area under the curve was 0.958 for Ki-67 and 0.909 for CD163+ TAMs (P < .0001). CONCLUSIONS: CD163+ TAMs infiltrating the epithelium and Ki-67 overexpression in the basal layer may serve as biomarkers to differentiate low-grade dysplasia from high-grade dysplasia of the larynx. A symmetric proliferative pattern was observed during laryngeal carcinogenesis following Ki-67 overexpression.

Pepsin as a biomarker for laryngopharyngeal reflux in children with laryngomalacia.

OBJECTIVES/HYPOTHESIS: Laryngomalacia is a common cause of newborn stridor. Laryngopharyngeal reflux (LPR) has been associated with laryngomalacia. Although pepsin, a component of LPR, has been associated with inflammatory diseases of the aerodigestive tract, its presence in the airways of laryngomalacia patients is unknown. STUDY DESIGN: Prospective case-control study comparing patients under age 3 years with laryngomalacia to children without laryngomalacia. METHODS: Children less than 3 years old undergoing supraglottoplasty for laryngomalacia or surgery unrelated to the airway, without a history of laryngomalacia, reflux, or respiratory disease, were offered enrollment. Supraglottic lavage samples (3 mL) were obtained from all subjects. Two-millimeter arytenoid biopsies were collected from laryngomalacia patients. Pepsin Western blot and enzyme-linked immunosorbent assay were performed. RESULTS: Ten laryngomalacia and five control subjects were enrolled. Pepsin was detected in lavages of laryngomalacia patients (8/10) but absent in controls (0/5; P = .007). Pepsin was observed more frequently in lavages (8/10) than biopsies (4/10; P = .046) of laryngomalacia subjects. Higher median pepsin concentration was observed in laryngomalacia than control lavages (P = .025). CONCLUSIONS: Pepsin in supraglottic specimens demonstrated an association with laryngomalacia, supporting a role for refluxed pepsin in laryngomalacia. These data corroborate previous work implicating pepsin in inflammatory diseases of the upper airways. Further studies are warranted to investigate the contribution of pepsin to the pathophysiology of laryngomalacia. LEVEL OF EVIDENCE: 3b. Laryngoscope, 127:2413-2417, 2017.

Effects of Smoke Generated by Electrocautery on the Larynx.

OBJECTIVE: The aim of this study was to investigate effects of smoke produced by electrocautery on the laryngeal mucosa. MATERIALS AND METHODS: We used 16 healthy, adult female Wistar albino rats. We divided the rats into two groups. Eight rats were exposed to smoke for 60 min/d for 4 weeks, and eight rats were not exposed to smoke and served as controls. The experimental group was maintained in a plexiglass cabin during exposure to smoke. At the end of 4 weeks, rats were sacrificed under high-dose ketamine anesthesia. Each vocal fold was removed. An expert pathologist blinded to the experimental group evaluated the tissues for the following: epithelial distribution, inflammation, hyperplasia, and metaplasia. Mucosal cellular activities were assessed by immunohistochemical staining for Ki67. Results taken before and after effect were compared statistically. RESULTS: There was a significant difference in the extent of inflammation between the experimental group and the control group. Squamous metaplasia was detected in each group, but the difference was not significant. None of the larynges in either group developed hyperplasia. CONCLUSIONS: We showed increased tissue inflammation due to irritation by the smoke.

Laryngeal mucus hypersecretion is exacerbated after smoking cessation and ameliorated by glucocorticoid administration.

INTRODUCTION: The mechanisms underlying the effects of cigarette smoke and smoking cessation on respiratory secretion, especially in the larynx, remain unclear. OBJECTIVES: The aims of this study were to determine the effects of cigarette smoke and smoking cessation on laryngeal mucus secretion and inflammation, and to investigate the effects of glucocorticoid administration. METHODS: We administered cigarette smoke solution (CSS) to eight-week-old male Sprague Dawley rats for four weeks, then examined laryngeal mucus secretion and inflammatory cytokine expression on days 1, 28 and 90 after smoking cessation. We also investigated the effects of the glucocorticoid triamcinolone acetonide when administered on day 1 after smoking cessation. RESULTS: Exposure to CSS resulted in an increase in laryngeal mucus secretion that was further excacerbated following smoking cessation. This change coincided with an increase in the expression of mRNA for the inflammatory cytokines tumor necrosis factor and interleukin-6, as well as mRNA for MUC5AC, which is involved in mucin production. Triamcinolone suppressed CSS-induced laryngeal mucus hypersecretion and pro-inflammatory cytokine production. CONCLUSION: Cigarette smoke-associated inflammation may contribute to the exacerbated laryngeal mucus hypersecretion that occurs following smoking cessation. The inflammatory response represents a promising target for the treatment of cigarette smoke-associated mucus hypersecretion.

Gene/protein expression of CAPN1/2-CAST system members is associated with ERK1/2 kinases activity as well as progression and clinical outcome in human laryngeal cancer.

Recent evidence indicates the involvement of calpains (CAPNs), a family of cysteine proteases, in cancer development and progression, as well as the insufficient response to cancer therapies. The contribution of CAPNs and regulatory calpastatin (CAST) and ERK1/2 kinases to aggressiveness, disease course, and outcome in laryngeal cancer remains elusive. This study was aimed to evaluate the CAPN1/2-CAST-ERK1/2 enzyme system mRNA/protein level and to investigate whether they can promote the dynamic of tumor growth and prognosis. The mRNA expression of marker genes was determined in 106 laryngeal cancer (SCLC) cases and 73 non-cancerous adjacent mucosa (NCLM) controls using quantitative real-time PCR. The level of corresponding proteins was analyzed by Western Blot. SLUG expression, as indicator of pathological advancement was determined using IHC staining. Significant increases of CAPN1/2-CAST-ERK1/2 levels of mRNA/protein were noted in SCLC compared to NCLM (p < 0.05). As a result, a higher level of CAPN1 and ERK1 genes was related to larger tumor size, more aggressive and deeper growth according to TFG scale and SLUG level (p < 0.05). There were also relationships of CAPN1/2 and ERK1 with incidences of local/nodal recurrences (p < 0.05). An inverse association for CAPN1/2, CAST, and ERK1/2 transcripts was determined with regard to overall survival (p < 0.05). In addition, a higher CAPN1 and phospho-ERK1 protein level was related to higher grade and stage (p < 0.05) and was found to promote worse prognosis. This is the first study to show that activity of CAPN1/2- CAST-ERK1/2 axis may be an indicator of tumor phenotype and unfavorable outcome in SCLC.

Validation of the secretion severity rating scale.

Accumulation of secretions within the hypopharynx, aditus laryngis, and trachea is one characteristic of severe dysphagia and is of high clinical and therapeutic relevance. For the graduation of the secretion severity level, a secretion scale was provided by Murray et al. in 1996. The purpose of the study presented here is the validation of this scale by analyzing the intra-rater and inter-rater reliability as well as concurrent validity. For examination of reliability and validity, a reference standard was defined by two expert clinicians who reviewed 40 video recordings of fiberendoscopic swallowing evaluations, with 10 videos for each severity grade. These videos were rated and rerated independently and blinded by 4 ENT-residents with an interval of 4 weeks. Both the intra-rater (Kendall's τ > 0.847***) and inter-rater reliability (Kendall's W > 0.951***) were highly significant and can be considered good or very good. Correlation of the median of all ratings with the reference standard was close to the highest possible value 1 (τ = 0.984***). The scale was proved to be a reliable and valid instrument for graduation of one of the principal symptoms of oropharyngeal dysphagia and is recommended as an evidence-based instrument for standardized fiberoptic endoscopic evaluation of swallowing.

[Expression of BTG1 protein in laryngeal squamous cell carcinoma and its clinical significance].

OBJECTIVE: To investigate the expression of B-cell translocation gene 1 (BTG1) and to determine the relationship between BTG1 expression and clinicopathological features, biological behaviors in laryngeal squamous cell carcinoma. METHOD: Immunohistochemistry and Western blot were used to analyze BTG1 protein expression in 70 cases of laryngeal cancer and 35 cases of adjacent corresponding laryngeal mucosal tissues to illuminate the relationship between BTG1 expression and clinical factors. RESULT: The positive rate of BTG1 protein expression was 31.43% in laryngeal carcinoma tissues, significantly lower than 91.43% in the adjacent laryngeal tissues (P < 0.05). Western blot showed the relative expression of BTG1 protein between cancer lesion and adjacent tissue were 0.217 ± 0.032 and 0.918 ± 0.081, showing the difference with statistical significance (P < 0.05). The expression of protein was significantly correlated with the tumor invasion, lymph node metastasis, clinic stage and histological grade (P < 0.05 or P < 0.01), but not with sex, age and tumor location (P > 0.05) of patients with laryngeal cancer. CONCLUSION: The expression of BTG1 protein was decreased in laryngeal squamous cell carcinoma, suggesting that BTG1 gene may be closely associated with the carcinogenesis and the degree of malignancy. Detection of BTG1 expression may be useful in diagnosis, treatment and prognosis of laryngeal carcinoma.

Identification of distinct layers within the stratified squamous epithelium of the adult human true vocal fold.

OBJECTIVES/HYPOTHESIS: A precise molecular schema for classifying the different cell types of the normal human vocal fold epithelium is lacking. We hypothesize that the true vocal fold epithelium has a cellular architecture and organization similar to that of other stratified squamous epithelia including the skin, cornea, oral mucosa, and esophagus. In analogy to disorders of the skin and gastrointestinal tract, a molecular definition of the normal cell types within the human vocal fold epithelium and a description of their geometric relationships should serve as a foundation for characterizing cellular changes associated with metaplasia, dysplasia, and cancer. STUDY DESIGN: Qualitative study with adult human larynges. METHODS: Histologic sections of normal human laryngeal tissue were analyzed for morphology (hematoxylin and eosin) and immunohistochemical protein expression profile, including cytokeratins (CK13 and CK14), cornified envelope proteins (involucrin), basal cells (NGFR/p75), and proliferation markers (Ki67). RESULTS: We demonstrated that three distinct cell strata with unique marker profiles are present within the stratified squamous epithelium of the true vocal fold. We used these definitions to establish that cell proliferation is restricted to certain cell types and layers within the epithelium. These distinct cell types are reproducible across five normal adult larynges. CONCLUSION: We have established that three layers of cells are present within the normal adult stratified squamous epithelium of the true vocal fold. Furthermore, replicating cell populations are largely restricted to the parabasal strata within the epithelium. This delineation of distinct cell populations will facilitate future studies of vocal fold regeneration and cancer. LEVEL OF EVIDENCE: N/A.

Impact of Irradiation on laryngeal hydration and lubrication in rat larynx.

OBJECTIVES: The larynx is susceptible to irradiation, which causes significant vocal fold (VF) edema and dehydration shortly after radiotherapy for head and neck cancers. However little is known about radiation-induced damage to VF liquid homeostasis. To evaluate the effects of irradiation on VF hydration and lubrication, we investigated changes in water transporters (aquaporins [AQPs]) and mucin production in vivo and ex vivo, as well as morphometric changes in the laryngeal mucosa and glands of irradiated rat larynges. STUDY DESIGN: Animal study. MATERIALS AND METHODS: Local irradiation at 18 Gy was delivered to rat larynges. Histologic changes in laryngeal mucosa and glands were observed by light microscopy, and the distributions of AQPs and mucin were investigated by immunofluorescence staining 3 months after irradiation. Early effects on gene regulation of AQPs and mucin were evaluated by quantitative real-time polymerase chain reaction of the extirpated VFs and subglottic laryngeal mucosa at 12, 24, and 72 hours after irradiation. RESULTS: Laryngeal glands exhibited severe atrophic changes and showed decreased density throughout the irradiated larynx. The expression of AQP1, 4, 5, and mucin in VFs, as well as AQP5 and mucin in submucosal laryngeal glands, decreased significantly 3 months after irradiation. An ex vivo study revealed that the gene expression of AQP5 in VF tissues was significantly downregulated at 12 hours postirradiation. CONCLUSION: Laryngeal irradiation induces damage in laryngeal mucosal barriers and alters laryngeal liquid homeostasis, which may be one reason for vocal dysfunction following irradiation. LEVEL OF EVIDENCE: N/A.

Utility of cell viability assays for use with ex vivo vocal fold epithelial tissue.

OBJECTIVES/HYPOTHESIS: Ex vivo models are routinely used to investigate the barrier function of the vocal fold epithelium. However, there are limited reports on assays that can be used to investigate the effect of clinically relevant challenges on vocal fold epithelial tissue viability. Our objective was to determine the utility of two assays routinely used in cell culture-a cellular metabolic activity assay and a cell membrane integrity assay-to investigate the viability of ex vivo porcine vocal fold epithelium. STUDY DESIGN: Prospective, ex vivo animal study. METHODS: Porcine vocal folds were exposed to acrolein, hydrochloric acid, or hydrogen peroxide challenge. An untreated, sham challenge was included as a control. Assays including metabolic activity, cell membrane integrity, and histology were used to determine whether challenges reduced epithelial viability as compared to sham. RESULTS: Cell membrane integrity and metabolic activity assays detected reductions in viability following hydrochloric acid and hydrogen peroxide challenges but not acrolein challenge as compared to sham. No challenge produced significant changes in epithelial appearance as evidenced by light microscopy. CONCLUSIONS: Metabolic activity and cell membrane integrity assays are valuable tools that can be used to evaluate the viability of ex vivo vocal fold epithelial tissue following clinically relevant challenges. As viability is reduced, the ability of epithelial tissue to maintain its barrier function is compromised. Accurate assessment of viability may provide us clues into understanding mechanisms underlying vocal fold epithelial injury and disease. LEVEL OF EVIDENCE: NA Laryngoscope, 125:E180-E185, 2015.

Mechanical regulation of human vocal fold stellate cells.

OBJECTIVE: It is generally accepted that tensile and compressive strains have direct effects on cell morphology and structure, including changes in cytoskeletal structure and organization. Cytoskeletons play the role of mechanoreceptor of the cells. Vocal fold stellate cells (VFSCs) in the human maculae flavae (MFe) are inferred to be involved in the metabolism of extracellular matrices essential for the viscoelasticity of the vocal fold mucosa. Our previous studies have supported the hypothesis that the tension caused by phonation (vocal fold vibration) regulates the behavior of the VFSCs. The microstructure of the intermediate filaments and the expression of their proteins were investigated in VFSCs in the MFe, which had remained unphonated since birth. METHODS: Three adult vocal fold mucosae that had remained unphonated since birth were investigated by immunohistochemistry and electron microscopy. RESULTS: The intermediate filaments of the VFSCs were fewer in number. The expression of their characteristic proteins (vimentin, desmin, and glial fibrillary acidic protein) was also reduced. CONCLUSION: Vocal fold vibration seems to affect VFSC morphology and structure, such as cytoskeletal structure and organization. This supports the hypothesis that vocal fold vibration regulates VFSC behavior in the human MFe. In addition to chemical factors, mechanical factors also appear to modulate VFSC behavior.