Uncovering the neural control of laryngeal activity and subglottic pressure in anaesthetized rats: insights from mesencephalic regions.

To assess the possible interactions between the dorsolateral periaqueductal gray matter (dlPAG) and the different domains of the nucleus ambiguus (nA), we have examined the pattern of double-staining c-Fos/FoxP2 protein immunoreactivity (c-Fos-ir/FoxP2-ir) and tyrosine hydroxylase (TH) throughout the rostrocaudal extent of nA in spontaneously breathing anaesthetised male Sprague-Dawley rats during dlPAG electrical stimulation. Activation of the dlPAG elicited a selective increase in c-Fos-ir with an ipsilateral predominance in the somatas of the loose (p < 0.05) and compact formation (p < 0.01) within the nA and confirmed the expression of FoxP2 bilaterally in all the domains within the nA. A second group of experiments was made to examine the importance of the dlPAG in modulating the laryngeal response evoked after electrical or chemical (glutamate) dlPAG stimulations. Both electrical and chemical stimulations evoked a significant decrease in laryngeal resistance (subglottal pressure) (p < 0.001) accompanied with an increase in respiratory rate together with a pressor and tachycardic response. The results of our study contribute to new data on the role of the mesencephalic neuronal circuits in the control mechanisms of subglottic pressure and laryngeal activity.

Negative prognostic behaviour of PD-L1 expression in tongue and larynx squamous cell carcinoma and its significant predictive power in combination with PD-1 expression on TILs.

BACKGROUND: Biomarkers that can predict outcome will improve the efficacy of treatment for HNSCC patients. In this regard, we retrospectively evaluated the prognostic effect of PD1, PD-L1, and CD45RO in tongue and larynx squamous cell carcinomas. METHODS: FFPE tissue blocks of 63 larynx and 40 tongue squamous cell carcinoma samples were selected, cut into 3 µm sections, and immunohistochemically stained for PD1, PD-L1, and CD45RO. The slides were evaluated by an expert pathologist, and results were analysed using Chi-square, univariate, and multivariable Cox regression methods. RESULTS: TC-PD-L1 expression (P = 0.001) and its expression intensity (P = 0.002) were significantly correlated with a higher percentage of PD-1 + tumor infiltrating lymphocytes. In univariate survival analysis, TC-PD-L1 and its expression intensity had a significant impact on both DFS (HR: 0.203; P = 0.003 and HR: 0.320; P = 0.005) and OS (HR: 0.147; P = 0.002 and HR: 0.322; P = 0.005). Based on the multivariate analysis, PD1 (DFS: HR: 3.202; P = 0.011, OS: HR: 2.671; P = 0.027) and TC-PD-L1 (DFS: HR: 0.174; P = 0.006, OS: HR: 0.189; P = 0.009) were found to be independent prognostic markers. In the second part, scoring systems were defined based on the expression status of PD1 and PD-L1. Patients with higher scores were expected to have longer DFS and OS. In multivariate analysis, the PD1/TC-PD-L1 (DFS: P = 0.001, OS: P = 0.003) scoring systems showed superior prognostic effects. Interestingly, at the highest levels of this score, none of the patients experienced recurrence or cancer-caused death. CONCLUSION: Collectively, this study suggests negative prognostic behaviour for TC-PD-L1 protein and introduces the PD-1/TC-PD-L1 scoring system as a strong prognostic marker in OS and DFS prediction of tongue and larynx HNSCC patients.

The Follicular Dendritic Cells and HPV 18 Interrelation in Head and Neck Squamous Cell Carcinomas of the Larynx.

Background and Objectives: Even if they are cells of controversial origin (mesenchymal, perivascular, or fibroblastic), follicular dendritic cells (FDC) are present in all organs. The aim of this study was to establish the FDC expression pattern and its interrelation with HPV 18 expression in laryngeal squamous cell carcinoma (LSCC). Materials and Methods: Fifty-six cases of LSCC were evaluated by simple and double immunostaining. The following score was used: 0 (negative or few positive cells), 1 (10-30% of positive cells), 2 (30-50% of cells), and 3 (over 50% of cells). Results: The expression of CD 21-positive cells with dendritic morphology (CDM) was noticed in the intratumoral area of conventional (well and poorly differentiated types and HPV 18 positive cases with a value of 2 for the score) and papillary types (HPV-18 negative cases with a score of 1). The highest value of 2 for the score of CDM in HPV-18 positive cases was found in the peritumoral area of well- and poorly-differentiated conventional LSCCs. A significant correlation was found between scores of CDM from the intratumoral area and those of the peritumoral area (p = 0.001), between CDM and non-dendritic morphology cells (NDM) of the intratumoral area (p = 0.001), and between HPV-18 status and peritumoral NDM cells (p = 0.044). Conclusions: The FDC and NDM cell score values of intratumoral and peritumoral areas may represent important parameters of LSCCs. This may contribute to a better stratification of laryngeal carcinoma cases and the individualized selection of clinical treatment protocols.

Differential Markers of Subpopulations of Epithelial Cells of the Larynx in Squamous Cell Carcinoma.

In squamous cell carcinoma of the larynx, the population of epithelial cells in the tumor tissue is initially heterogeneous and, in addition to tumor cells invading the organ mucosa, includes normal epithelial cells of protein-mucous glands and cells of the stratified epithelium covering the mucous membrane. A search for differential markers to separate these subpopulations was carried out. The surface marker CD44 and cytokeratins 5 and 17 that are often used to verify carcinoma cells, are common markers for all epithelial cells of the larynx. In highly differentiated carcinoma, subpopulations of normal and tumor epithelial cells can be separated by the level of expression of cytokeratins 10 and 18 and nuclear markers Ki-67 and p63. However, in moderately differentiated carcinoma, tumor cells and normal cells of the basal layer of the stratified epithelium covering the mucous membrane of the larynx have similar phenotypes, which should be taken into account when conducting experimental studies.

Ki-67, p-53, E-Cadherin, and ß-Catenin expression of advanced glotto-subglottic and supraglottic larynx carcinomas.

Background: Identifying tumor markers that can be used to determine the biological behavior of tumors and predicting their prognosis may be helpful in choosing treatment strategies. Besides the differences in the embryological and histological anatomy of the larynx in this regard, the possibility of molecular causes that can explain the different clinical behaviors has always been a question for the scientific world. Aim: In this study, we aimed to investigate whether there were any immunohistochemically molecular differences among laryngeal carcinoma cases originating from two different anatomical regions of the larynx. Patients and Methods: The study group consisted of 43 patients. The rate of supraglottic cancers was 41.8%, while the rest had glotto-subglottic tumors. Ki67, ß-catenin, E-cadherin, and p53 were examined in pathology preparations obtained by laryngectomy surgeries. The data obtained were analyzed by comparing factors that may affect the prognosis of the disease and between tumors originating from the two different anatomical regions. Results: We did not see any statistically significant difference between groups for stage and grade of tumor, tumor recurrence rate, or lymphovascular or perineural invasion rated in terms of the investigated markers. In addition, there was no statistically significant difference between the two distinct groups in survival analysis. Conclusions: With these results, our study differs from some studies in the literature, and we think that this difference could be because the cases in our study consisted of advanced stage tumors and the groups investigated had similar survival rates.

Long-time remission of laryngeal Rosai-Dorfman disease with thalidomide: a report of three cases.

OBJECTIVE AND IMPORTANCE: Rosai-Dorfman disease (RDD) is a benign and rare non-Langerhans cell histiocytic proliferative disorder. Laryngeal involvement is an unusual site of extranodal involvement of RDD. Laryngeal RDD can cause life-threatening airway obstruction that requires effective control of the disease. In this study, we report three cases of laryngeal RDD with excellent and durable responses to thalidomide. CLINICAL PRESENTATION: Patient 1 was a 39-year-old male who presented with a two-year history of nasal obstruction. Patient 2 was a 26-year-old woman who presented complaining of a hoarse voice for one year. Patient 3 was a 24-year-old man who presented with complaints of a hoarse voice and progressing dyspnea for five months. Electronic laryngoscopy revealed submucous nodular lesions in the nasal cavity, nasopharynx, and larynx of the three patients. Biopsy of the lesions showed large histiocytes with abundant pale cytoplasm which were S-100 and CD68 positive consistent with RDD. INTERVENTION: Before thalidomide treatment, patient 1 received chemotherapy and six times surgical excision due to the recurrence of laryngeal lesions. Patient 2 failed steroid treatment. Patient 3 underwent an emergency tracheostomy due to airway obstruction. All three patients then received thalidomide 100 mg/d treatment and achieved satisfactory and durable responses with the longest follow-up of 45 months. CONCLUSION: Thalidomide may induce long-term remission in laryngeal RDD.

Inactivation of Lats1 and Lats2 highlights the role of hippo pathway effector YAP in larynx and vocal fold epithelium morphogenesis.

Proliferation and differentiation of vocal fold epithelial cells during embryonic development is poorly understood. We examined the role of Hippo signaling, a vital pathway known for regulating organ size, in murine laryngeal development. Conditional inactivation of the Hippo kinase genes Lats1 and Lats2, specifically in vocal fold epithelial cells, resulted in severe morphogenetic defects. Deletion of Lats1 and Lats2 caused abnormalities in epithelial differentiation, epithelial lamina separation, cellular adhesion, basement membrane organization with secondary failed cartilage, and laryngeal muscle development. Further, Lats1 and Lats2 inactivation led to failure in differentiation of p63+ basal progenitors. Our results reveal novel roles of Hippo-Lats-YAP signaling in proper regulation of VF epithelial fate and larynx morphogenesis.

Irisin Association with Ki-67, MCM3 and MT-I/II in Squamous Cell Carcinomas of the Larynx.

BACKGROUND: Current studies indicate irisin role in carcinogenesis. The aim of the study was to investigate the expression of irisin in LSCCs and to determine its association with clinicopathological factors, as well as recognized markers of proliferation, i.e., Ki-67 and MCM3,5,7 and MT-I/II proteins. MATERIAL AND METHODS: The research material consisted of 140 cases of LSCCs, 57 cases of laryngeal papillomas (BLs) and 14 controls (benign hypertrophic changes). Tissue microarrays were used to perform IHC. Western blot and immunofluorescence were performed in laryngeal cancer cell lines and normal keratinocytes. RESULTS: Irisin expression levels were significantly increased in LSCC compared to BLs (p < 0.0001) and controls (p = 0.001). We noted a positive moderate and weak correlation between irisin and Ki-67, MCM3 and MT-I/II. We observed an elevated level of irisin expression with increasing tumor size (T1-2 vs. T3-4; p = 0.0348). The levels of irisin were higher in N0 than in N1 and N2-3 (p = 0.0031 and p = 0.0457, respectively). Our in vitro study revealed a higher level of irisin in Larynx Epidermoid Carcinoma 2 (HEp-2) cells compared to the control Normal Human Keratinocyte (HaCat) cell line. CONCLUSIONS: Increased irisin expression levels in LSCC and its correlation with clinicopathological and proliferation factors may indicate the potential role of irisin as a biomarker in the diagnostic process of LSCC.

Proteomic and Genomic Methylation Signatures of Idiopathic Subglottic Stenosis.

OBJECTIVE: Idiopathic subglottic stenosis (iSGS) is a chronic inflammatory condition that causes dyspnea and affects middle-aged women of White race and non-Latino or Hispanic ethnicity. To better characterize its phenotype and pathogenesis, we assessed the proteomic and genomic methylation signatures of subglottic tissue collected from iSGS patients compared to controls. STUDY DESIGN: Molecular analysis of clinical biospecimens. METHODS: We collected subglottic tissue biopsies from 12 patients during direct laryngoscopy, immediately prior to surgical treatment of iSGS; as well as from 4 age-, sex-, and race/ethnicity-matched control patients undergoing other direct laryngoscopic procedures. We isolated protein and genomic DNA, acquired proteomic data using label-free quantitative mass spectrometry techniques, and acquired genome-wide methylation data using bisulfite conversion and a microarray platform. We compared molecular profiles across the iSGS and control groups, and with respect to clinical course in the iSGS group. Eight of the 12 iSGS patients underwent subsequent blood collection and plasma isolation for further assessment. RESULTS: Proteomic analysis revealed 42 differentially abundant proteins in the iSGS biopsies compared to controls, inferring enrichment of biological pathways associated with early wound healing, innate immunity, matrix remodeling, and metabolism. Proteome-based hierarchical clustering organized patients into two iSGS and one control subgroups. Methylation analysis revealed five hypermethylated genes in the iSGS biopsies compared to controls, including the biotin recycling enzyme biotinidase (BTD). Follow-up analysis showed elevated plasma BTD activity in iSGS patients compared to both controls and published normative data. CONCLUSION: iSGS exhibits distinct proteomic and genomic methylation signatures. These signatures expand current understanding of the iSGS phenotype, support the possibility of disease subgroups, and should inform the direction of future experimental studies. LEVEL OF EVIDENCE: Not applicable Laryngoscope, 131:E540-E546, 2021.

Coaction of TGF-ß1 and CDMP1 in BMSCs-induced laryngeal cartilage repair in rabbits.

Bone marrow mesenchymal stem cells (BMSCs) are well-known for tissue regeneration and bone repair. This study intended to evaluate the potential efficiency BMSCs in poly(lactide-co-glycolide) (PLGA) scaffolds for the treatment of laryngeal cartilage defects. BMSCs were isolated and identified, and added with 10 ng/mL transforming growth factor-beta1 (TGF-ß1) or/and 300 ng/mL CDMP1 to coculture with PLGA scaffolds. The chondrogenic differentiation, migration, and apoptosis of BMSCs were detected under the action of TGF-ß1 or/and CDMP1. After successful modeling of laryngeal cartilage defects, PLGA scaffolds were transplanted into the rabbits correspondingly. After 8 weeks, laryngeal cartilage defects were assessed. Levels of collagen II, aggrecan, Sox9, Smad2, Smad3, ERK, and JNK were detected. The TGF-ß1 or/and CDMP1-induced BMSCs expressed collagen II, aggrecan, and Sox9, with enhanced cell migration and inhibited apoptosis. In addition, laryngeal cartilage defect in rabbits with TGF-ß1 or/and CDMP1 was alleviated, and levels of specific cartilage matrix markers were decreased. The combined effects of TGF-ß1 and CDMP1 were more significant. The TGF-ß1/Smad and ERK/JNK pathways were activated after TGF-ß1 or/and CDMP1 were added to BMSCs or rabbits. In summary, BMSCs and PLGA scaffolds repair laryngeal cartilage defects in rabbits by activating the TGF-ß1/Smad and ERK/JNK pathways under the coaction of TGF-ß1 and CDMP1.

Association of mRNA Expression Levels of LRP1 and Actin-Binding Proteins with the Development of Laryngeal and Laryngopharyngeal Squamous Cell Carcinoma.

We analyzed the association of the level of mRNA expression of the main endocytosis receptor LRP1 and actin-binding proteins (ezrin, profilin-1, cofilin-1, and adenylyl cyclase-associated protein 1) with the development and metastasis of laryngeal and laryngopharyngeal squamous cell carcinoma. The mRNA expression was evaluated in paired tissue samples using quantitative reverse transcription real-time PCR (RT-qPCR) and SYBR Green reagents. The study included 38 patients with stage T1-4N0-1M0 laryngeal and laryngopharyngeal squamous cell carcinoma and 10 patients with chronic hyperplastic laryngitis or grade II-III epithelial dysplasia. The expression of LRP1 in patients with laryngeal and laryngopharyngeal squamous cell carcinoma depended on the stage of the tumor process. Against the background of low expression of LRP1 mRNA, the relationship between cofilin 1 and profilin 1 expression became stronger (r=0.08; p=0.05) and a correlation between cofilin 1 and esrin expression (r=0.7; p=0.05) appeared. Studies on a larger patient cohort are required to make a definite conclusion on the role of LRP1 in the development of laryngeal and laryngopharyngeal squamous cell carcinoma.

Relationship between the Levels of mRNA Expression for Protein Phosphatase 1B and Proteins Involved in Cytoskeleton Remodeling in Squamous Cell Carcinoma of the Larynx and Hypopharynx.

We analyzed the expression of genes encoding proteins involved in cytoskeleton remodeling (RND3, SNAI1, vimentin, cofilin, adenylate cyclase-associated protein 1, ezrin, and profilin) depending on the level of expression of protein phosphatase 1B (PPM1B) mRNA on the example of squamous cell carcinoma of the larynx and hypopharynx. Against the background of a high level of PPM1B expression, a significantly high level of profilin expression was noted. Metastasis correlated with the level of snai1 expression, while relapse after combination treatment was negatively associated with the level of vimentin expression. The obtained new data can reflect molecular peculiarities of the tumor growth in squamous cell carcinoma of the larynx and hypopharynx.

Mechanisms of larynx and vocal fold development and pathogenesis.

The larynx and vocal folds sit at the crossroad between digestive and respiratory tracts and fulfill multiple functions related to breathing, protection and phonation. They develop at the head and trunk interface through a sequence of morphogenetic events that require precise temporo-spatial coordination. We are beginning to understand some of the molecular and cellular mechanisms that underlie critical processes such as specification of the laryngeal field, epithelial lamina formation and recanalization as well as the development and differentiation of mesenchymal cell populations. Nevertheless, many gaps remain in our knowledge, the filling of which is essential for understanding congenital laryngeal disorders and the evaluation and treatment approaches in human patients. This review highlights recent advances in our understanding of the laryngeal embryogenesis. Proposed genes and signaling pathways that are critical for the laryngeal development have a potential to be harnessed in the field of regenerative medicine.

Vitamin B12b Enhances the Cytotoxicity of Diethyldithiocarbamate in a Synergistic Manner, Inducing the Paraptosis-Like Death of Human Larynx Carcinoma Cells.

We have shown that hydroxycobalamin (vitamin В12b) increases the toxicity of diethyldithiocarbamate (DDC) to tumor cells by catalyzing the formation of disulfiram (DSF) oxi-derivatives. The purpose of this study was to elucidate the mechanism of tumor cell death induced by the combination DDC + В12b. It was found that cell death induced by DDC + B12b differed from apoptosis, autophagy, and necrosis. During the initiation of cell death, numerous vacuoles formed from ER cisterns in the cytoplasm, and cell death was partially suppressed by the inhibitors of protein synthesis and folding, the IP3 receptor inhibitor as well as by thiols. At this time, a short-term rise in the expression of ER-stress markers BiP and PERK with a steady increase in the expression of CHOP were detected. After the vacuolization of the cytoplasm, functional disorders of mitochondria and an increase in the generation of superoxide anion in them occurred. Taken together, the results obtained indicate that DDC and B12b used in combination exert a synergistic toxic effect on tumor cells by causing severe ER stress, extensive ER vacuolization, and inhibition of apoptosis, which ultimately leads to the induction of paraptosis-like cell death.

Increased Expression of Estrogen Receptor Beta in Idiopathic Progressive Subglottic Stenosis.

BACKGROUND/OBJECTIVES: Idiopathic progressive subglottic stenosis (IPSS) predominantly affects females in perimenopause. It has, therefore, been hypothesized that estrogen is involved in its pathogenesis. There are two main types of estrogen receptors: ER-α and ER-ß. Abnormal variants of ER-ß have previously been shown to be associated with poor wound healing. Estrogen receptors have recently been identified in subglottic tissue samples, with elevated levels of ER-α and progesterone receptors, and no expression of ER-ß, in stenotic specimens reported in one study. The objective of this study was to confirm the presence of estrogen receptors in the subglottis and investigate levels of expression and types of estrogen receptors in normal and stenotic subglottic tissue. METHODS: Subglottic tissue was obtained from three female and one male cadaver without laryngotracheal pathology to serve as controls. Subglottic tissue specimens from five female patients with IPSS were also analysed. Immunofluorescence stains for ER-α and ER-ß were performed on specimens. Staining patterns were compared qualitatively and semi-qualitatively between control and IPSS specimens. RESULTS: Immunofluorescence stains demonstrated the presence of both ER-α and ER-ß in subglottic tissue. IPSS specimens demonstrated significantly greater staining intensity of ER-α in the epithelium and ER-ß in glands and ducts compared to controls. CONCLUSIONS: This study confirms the presence of estrogen receptors in the subglottis. Increased expression of ER-α in the epithelium and ER-ß in glands and ducts in IPSS compared to controls may help to explain the predisposition to stenosis in these individuals. LEVEL OF EVIDENCE: 3b Laryngoscope, 130:2186-2191, 2020.

Comparative analysis of a panel of biomarkers related to protein phosphatase 2A between laryngeal squamous cell carcinoma tissues and adjacent normal tissues.

Laryngeal squamous cell carcinoma (LSCC) is the most common type of head and neck squamous cell carcinoma (HNSCC) worldwide. Protein phosphatase 2A (PP2A) dysfunction has been widely reported in a broad range of malignancies due to its distinctive role in miscellaneous cellular processes. However, it is poorly understood whether aberrant alterations of PP2A are involved in the network of oncogenic events in LSCC. Here, we detected a panel of PP2A-associated proteins using western blot in both laryngeal squamous cell carcinoma tissues and paired adjacent normal tissues from patients (Data S1). We found that phospho-PP2A/C (Y307), α4, cancerous inhibitor of protein phosphatase 2A (CIP2A), Akt, ezrin, phospho-ezrin (T567), 14-3-3, and focal adhesion kinase (FAK) showed increased expression levels in carcinoma tissues relative to normal tissues, while phospho-Akt (T308) showed decreased levels. Our study, thus, provides a rationale for targeting PP2A to develop novel therapies and proposes a combination of interrelated biomarkers for the diagnostic evaluation and prognosis prediction in LSCC.

[Carcinogenicity study of CD133(+)CD44(+) laryngeal cancer stem cells and identification of related microRNAs].

Objective: To compare the carcinogenic abilities of CD133(+)CD44(+) laryngeal cancer stem cells and general laryngeal cancer stem cells and to identify the mechanism underlying the action of miRNAs. Methods: Solid tumor-derived laryngeal carcinoma stem cells and Hep-2-derived laryngeal carcinoma stem cells were cultured, and CD133(+)CD44(+) laryngeal cancer stem cells were sorted by flow cytometry. Boden chamber invasion assay, cell migration assay and tumor formation assay were then performed to compare the invasion, migration and tumorigenic abilities of CD133(+)CD44(+) laryngeal cancer stem cells and general laryngeal cancer stem cells. And then, miRNAs isolated from two laryngeal cancer stem cells were detected and analysed with miRNA chip. Results: (1)In Boyden chamber invasion assay, the cell invasion rate of CD133(+)CD44(+) laryngeal cancer stem cells was obviously higher (80.2%±2.3% vs. 63.9%±3.2%, t=5.011, P=0.027); (2)CD133(+)CD44(+) laryngeal cancer stem cells also had higher mobility in cell migration assay (82.9%±1.1% vs. 70.9%±0.6%, t=4.514, P=0.031); (3)In tumor formation assay, the tumor formation rate of CD133(+)CD44(+) laryngeal cancer stem cells was also higher (80% vs. 50%). What's more, we identified 15 miRNAs that were significantly upregulated in CD133(+)CD44(+) laryngeal cancer stem cells and 3 miRNAs that were significantly downregulated in CD133(+)CD44(+) laryngeal cancer stem cells, compared with normal laryngeal cancer stem cells. Conclusions: CD133(+)CD44(+) laryngeal cancer stem cells have stronger invasion, migration and tumorigenic abilities compared with normal laryngeal cancer stem cells, and the difference of miRNAs' expression is one of the possible causes.

Direct Visualization of Laryngeal Mucociliary Clearance in Adults.

OBJECTIVES: Mucociliary clearance is a protective mechanism of the respiratory tract that facilitates the removal of foreign particles and microorganisms. There is a paucity of data on the mucociliary clearance in the adult larynx. Our study aims to visualize and describe the mucociliary clearance of the adult larynx in healthy subjects. METHODOLOGY: Subjects were identified from a volunteer database. Exclusion criteria included laryngeal disease, previous laryngeal surgery, recent upper respiratory infection, and current smoking. A high-definition videolaryngoscope was used to visualize the larynx. The larynx was topicalised with local anesthetic. Methylene blue was placed on both false vocal cords and at the petiole of the epiglottis. Dye clearance was recorded and analyzed. RESULTS: In total, 10 participants participated, 7 men and 3 women, with a mean age of 42 ± 15.7 years (range: 25-71). The average reflux symptom index score was 1.4. Clearance of the dye from the false vocal cords followed a uniform lateral flow, up onto the aryepiglottic folds. The dye from the petiole had minimal vertical movement. Swallowing cleared dye from the aryepiglottic folds. The average time for dye clearance to the aryepiglottic fold was 2.21 ± 1.14 minutes. CONCLUSIONS: This is the first study visualizing the mucociliary clearance of the larynx. Ciliary directionality was consistent in the participants studied, with dye moving superolateral from the false cords to the aryepiglottic fold. Swallowing was an effective mechanism for clearance from the endolarynx, when the dye had reached the aryepiglottic fold. Future research can study potential alterations in laryngeal mucociliary clearance in chronic disease states.

Injectable basic fibroblast growth factor-loaded alginate/hyaluronic acid hydrogel for rejuvenation of geriatric larynx.

Increase in the geriatric population has led to an increase in the number of elderly patients with laryngeal atrophy and dysfunction. Symptoms of voice change, dysphagia, and aspiration pneumonia negatively influence patient's health status, quality of life, and life span. Injection laryngoplasty used to treat laryngeal dysfunctions does not recover intrinsic functions of the larynx. Thus, we fabricated an injectable basic fibroblast growth factor (bFGF)-loaded alginate (ALG)/hyaluronic acid (HA) hydrogel for inducing rejuvenation of geriatric laryngeal muscles. Optimal in situ-forming bFGF-loaded ALG/HA hydrogel for injection laryngoplasty was prepared and the release profile of bFGF was analyzed. For in vivo analysis, the bFGF-loaded ALG/HA hydrogel was injected into the laryngeal muscles of 18-month-old Sprague-Dawley rats. The rejuvenation efficacy of bFGF-loaded ALG/HA hydrogel in geriatric laryngeal muscle tissues 4- and 12-weeks post-injection was evaluated by quantitative polymerase chain reaction (qPCR), histology, immune-fluorescence staining and functionality analysis. The bFGF-loaded ALG/HA hydrogel induced an increase in the expression of myogenic regulatory factor-related genes, hypertrophy of muscle fiber, proliferation of muscle satellite cells, and angiogenesis and decreased interstitial fibrosis. Administration of the bFGF-loaded ALG/HA hydrogel caused successful glottal gap closure. Thus, the bFGF-loaded ALG/HA hydrogel could be a promising candidate for laryngoplasty aimed at rejuvenating geriatric larynx. STATEMENT OF SIGNIFICANCE: In this manuscript, optimal in situ-forming bFGF-loaded ALG/HA hydrogel for injection laryngoplasty was prepared and the release profile of bFGF was analyzed. Herein, we introduced the materials and methods of injection laryngoplasty for geriatric rat experiment. In addition, we studied effects of bFGF-loaded ALG/HA hydrogel on the therapeutic rejuvenation of geriatric rat larynx. The bFGF-loaded ALG/HA hydrogel induced an increase in the expression of myogenic regulatory factor-related genes, hypertrophy of muscle fiber, proliferation of muscle satellite cells, and angiogenesis and decreased interstitial fibrosis. Furthermore, our functional analysis through the high-speed camera setup demonstrated that the administration of the bFGF-loaded ALG/HA hydrogel induced successful glottal gap closure. Thus, the bFGF-loaded ALG/HA hydrogel could be a promising candidate for injection laryngoplasty with therapeutic effects.

The Relevance of Assessing the Cell Proliferation Factor Ki-67 in Squamous Cell Carcinoma of the Larynx.

The aim of study was to investigate the expression of the proliferation factor Ki-67 and its relationship with histological grade, cancer stage, and treatment outcome in squamous cell carcinoma of the larynx. Samples from 78 patients with laryngeal cancer were analysed retrospectively. Paraffin sections of tumors were immunohistochemically stained for Ki-67 expression. The patients were divided in two groups according to the proliferative factor values (a low Ki-67 index group - Ki-67≤34 and high Ki-67 index group-Ki-67 >34). Statistical analysis of the data shows significant correlation among histological tumor grade and the value of the Ki-67 proliferative index. There was no correlation between tumoral Ki-67 expression and diagnosis, stage of the disease, or treatment outcome. In conclusion, Ki-67 expression in laryngeal cancer is not the most reliable marker for making precise diagnosis and predicting the clinical course.