Establishing principles of macromolecular crowding for in vitro fibrosis research of the vocal fold lamina propria.

OBJECTIVES/HYPOTHESIS: Vocal fold fibrosis represents a major disease burden. Screening of antifibrotic compounds could be facilitated by an in vitro fibrogenesis system. Limitations of existing models might be overcome by implication of the excluded volume effect. STUDY DESIGN: In-vitro study. METHODS: Vocal fold fibroblasts obtained from rats' lamina propria were cultured in four different settings: in standard medium, under "crowded" conditions by adding inert macromolecules, under external administration of transforming growth factor (TGF)ß-1, and under a combination of both. After 5 days, supernatant and cell layer were collected and analyzed by enzyme-linked immunosorbent assay. Immunofluorescence was additionally performed. RESULTS: Collagen-alpha1(I) deposition increased significantly under crowded conditions and after administration of TGFß-1. Amounts of collagen in the cell layer were significantly higher under crowding conditions with TGFß-1 compared to administration of TGFß-1 alone. CONCLUSION: Crowding enhanced collagen deposition, resulting in more favorable conditions for studying fibrogenesis. This can be the first step toward developing a robust in vitro model for testing antifibrotic compounds. LEVEL OF EVIDENCE: NA.

Vocal fold fibroblast response to growth factor treatment is age dependent: results from an in vitro study.

INTRODUCTION: Vocal fold (VF) fibroblasts (VFFs) are the central target in developing new strategies for treatment of VF injury and scarring. Nevertheless, only little is known about the basic biological characteristics of these cells. The aim of this study was to explore the impact of age of VFFs on the response to external growth factor stimulation. STUDY DESIGN: In vitro cell study using a rat model. METHODS: VFFs were extracted from young and aged rat VF 3 months after establishing unilateral injury. Resulting scar fibroblasts (SFs) and normal fibroblasts (NFs) were subsequently cultured separately with or without the addition of hepatocyte growth factor (HGF). After 24 and 72 hours, the production of hyaluronic acid (HA) was examined in the supernatant culture media using enzyme-linked immunosorbent assay. RESULTS: Only cultured SF and NF from young animals could be stimulated significantly in the production of HA by HGF. Within these, average percentage increase was higher in NF compared with SF. CONCLUSION: The response of VFFs in cell culture to growth factors stimulation is highly depending on the age of the animals. This is another step in a nearer characterization of scar VFF and could furthermore be an important point when estimating the success of an intervention. Age-depending effects must be considered as an important factor in developing possible therapeutic agents for VF scarring.

Age effects on extracellular matrix production of vocal fold scar fibroblasts in rats.

Vocal fold (VF) fibroblasts are the central subject of interest in fibrogenesis and wound healing after VF injury. Scar fibroblasts (SF) exhibit an aberrant production of several extracellular matrix (ECM) components which lead either to VF fibrosis or scarless wound healing. This study aimed to investigate the role of age at the time of injury on ECM production of SF. This is designed as an animal study. VF injury was established unilaterally in eight male Sprague-Dawley rats [3 months of age (n = 4), 11 months of age (n = 4)], while the other side was left intact. Three months after injury the larynges were excised and fibroblasts were extracted from VF [normal fibroblasts (NF)­scar fibroblasts (SF)] and cultured in vitro. After first passage, VF fibroblasts were plated in 24-well plates and levels of hyaluronic acid (HA) and collagen type I were determined enzymatically from supernatant after 24 and 72 h. Cultured SF from younger animals produced significantly higher levels of HA compared to NF fibroblasts from the same animals. HA concentrations of the older animals did not differ significantly between the NF and SF cultures, but the range in SF cultures was large. In contrast to previous studies, we found that even 3 months after VF injury cultured SF from young animals expressed higher levels of HA in comparison to SF from older animals. No difference in collagen levels were observed between the younger and older animals. Age of animals is an essential factor during VF healing and has to be considered for study design.

Subjective and objective vocal parameters in women with polycystic ovary syndrome.

INTRODUCTION: Despite the relatively high prevalence of the polycystic ovary syndrome (PCOS) affecting up to 15% of all women of reproductive age, only little is known about vocal changes related to this endocrinologic disorder that is characterized by biochemical or clinical hyperandrogenism, impaired cycles, and/or polycystic ovaries. The aim of our study was to describe objective and subjective vocal changes in women with a diagnosed PCOS compared with a control group. MATERIALS AND METHODS: Our study group comprised 34 women-24 cases with confirmed PCOS and 10 controls in whom PCOS was excluded. A complete endocrinologic laboratory status was obtained by all participants; study procedures included a videolaryngostroboscopy, voice recording, and completion of the German version of the Voice Handicap Index. RESULTS: A trend toward lower mean fundamental frequency was detectable, but this failed statistical significance. No differences were found in the objective and subjective voice parameters. CONCLUSION: Elevated serum levels of androgens, as found in women with PCOS, were shown not to have an impact on the subjective and objective voice parameters.

Vitamin D status is associated with disease-free survival and overall survival time in patients with squamous cell carcinoma of the upper aerodigestive tract.

UNLABELLED: Considerable clinical and experimental data suggest that vitamin D plays a role in pathogenesis and progression of cancer; nevertheless clinical data for head and neck squamous cell carcinoma (HNSCC) are not available. The aim of our study was to associate tumor and clinical characteristics with status of vitamin D [25-hydroxyvitamin D (25(OH)D)]. 88 patients with newly diagnosed squamous cell carcinoma of the head and neck were included in the study. A blood draw was taken at the time of diagnosis. The observation period lasted from 2006 until 2010. RESULTS: When compared to a cohort from an epidemiological study, serum levels of 25(OH)D were significantly reduced in patients with HNSCC. We furthermore found disease-free survival, as well as overall survival times to be significantly associated with 25(OH)D levels. In conclusion, results from our study suggest an influence of vitamin D status on cancer incidence, as well as on mortality of HNSCC.

Subjective breathing impairment in unilateral vocal fold paralysis.

INTRODUCTION: Dysphonia is considered a major symptom of unilateral vocal fold paralysis (UVFP). Besides this, many patients complain of further symptoms such as dysphagia and dyspnea, which might not be expected to such an extent. The aim of this survey was to elucidate these symptoms in a cohort of patients with UVFP. SUBJECTS AND METHODS: Sixty-three patients (22 men, 41 women) suffering from UVFP were interviewed. Therefore we developed a questionnaire dealing with each of the three symptom categories: voice production, swallowing and breathing. RESULTS: All of the surveyed patients reported voice impairment, almost 60% complained of swallowing problems after the onset of paralysis. Seventy-five percent reported a subjectively impaired breathing sensation, not just phonatory dyspnea but during everyday physical activity as well. DISCUSSION: Our study revealed a certain discrepancy between objectively assessed laryngoscopic findings and subjective symptoms. A majority of patients suffered from an impairment in each of the three laryngeal functions (dysphonia, dysphagia and dyspnea). The latter two differ from the classic approach to this condition but must be considered as well in clinical diagnostics and therapy.

First report of intralaminar tumorlike lipomatous lesions of the thyroid cartilage.

INTRODUCTION: We present two cases of tumorlike lipomatous lesions in the thyroid cartilage. Both presented as circumscribed tumors by ultrasonography and were identified accidentally. METHODS: Two tumorlike lesions were identified in two different patients. Additionally, computed tomography and magnetic resonance imaging scans of the laryngeal skeleton were performed. Surgery was performed in one patient, and the lesion was excised. Histopathology revealed fatty tissue with several foci of hematopoietic cells. CONCLUSION: This is the first report of intralaminar tumorlike lipomatous lesions in the thyroid cartilage. These should be included in the differential diagnoses of primary cartilaginous lesions of the laryngeal skeleton.

Sex hormones and the elderly male voice.

The objective was to describe influences of sex hormones on the male voice in an elderly cohort. Sixty-three elderly males were recruited to undergo assessment of voice parameters, stroboscopy, voice-related questionnaires, a blood draw, and an ultrasound examination of the laryngeal skeleton. The group was divided into men with normal hormonal status and men with lowered levels of sex hormones, called hypogonades. Depending on the level of androgens, voice parameters did not differ. In subjects with decreased levels of estrogens, a significant increase in mean fundamental frequency, as well as changes of highest and lowest frequency plus a shift of the frequency range could be detected. We could detect significant changes of voice parameters depending on status of estrogens in elderly males. Androgens appear to have no impact on the elderly male voice. To our knowledge, this is the first prospective study that correlates sex hormones with voice parameters in elderly men.

Treatment of functional ventricular fold phonation by temporary suture lateralization.

Ventricular fold phonation (VFP) is a phenomenon characterized by phonation using the false vocal folds. Besides a substitution voice due to loss of the true vocal folds-so called compensatory type-the noncompensatory types have a psychogenic, functional, or habitual background. Therapeutic options for these cases so far comprise voice therapy, pharmacological therapy (injection of anesthetics or botulinum toxin), and, in case of failure, surgical resection of the ventricular folds. Indication for aggressive surgical interventions is usually difficult, as there is always a risk of producing an irreversible state with an even worse situation. We present two cases of functional (psychogenic) VFP treatment refractory to conservative treatment. Lateralization of the ventricular folds by an endo-extralaryngeal temporary suture, similar to that used in bilateral vocal fold paralysis, was performed. Immediately after the procedure, a constant phonation at glottic level could be achieved in both cases. Sutures were removed 4 days after surgery, and phonation remained at the glottic level. Vocal fold phonation could be stabilized in the long run, and both patients recovered completely under additional voice therapy.

Curved rigid laryngoscope: missing link between direct suspension laryngoscopy and indirect techniques?

Microlaryngoscopy is the standard procedure for endolaryngeal surgery. The advantages are a steady operating field, bimanual handling and stereoscopic view in high-resolution magnification. The major drawback is that the oropharyngeal structures have to be brought into an unnatural position by the straight rigid laryngoscope with considerable forces occurring. These forces can lead to tissue injuries or even make a microlaryngoscopic operation impossible. To overcome these disadvantages, a few case studies using curved rigid laryngoscopes are published. However, there is still a lack of information to what extent curved rigid laryngoscopes could actually improve the endolaryngeal exposure with less forces occurring. It was the aim of this study to gain basic data on the forces that are needed for endolaryngeal exposure with a prototype of a curved rigid laryngoscope and to compare the occurring forces with straight laryngoscopes. In 30 consecutive patients scheduled for routine microlaryngoscopic procedures the curved laryngoscope was inserted and occurring forces were measured by a tension spring balance in four different head positions. A standard straight laryngoscope was inserted afterwards and measurements were taken again. Our results showed that the occurring forces could be reduced significantly in each head position when compared with a standard straight laryngoscope. Similarly, the anterior commissure could be exposed in a significantly higher percentage with the curved laryngoscope. In conclusion, we could show that even with a moderate-curved rigid laryngoscope a significant reduction of the forces to the oro-pharyngeal tissues can be obtained and that endolaryngeal exposure is possible in virtually all patients. Bimanual precise operations should be possible in the common way like in standard microlaryngoscopy with the only difference of not using a microscope, but operating via a monitor. We do not think that traditional microlaryngoscopy with straight instruments can or should be replaced by curved laryngoscopes, but these techniques could bridge the gap to indirect techniques in particular in specialised institutions dealing frequently with difficult patients and situations.

Laryngo-tracheal profile: a new method for assessing laryngo-tracheal stenoses.

We present the clinical application of a new method for objective assessment of both grade and length of laryngo-tracheal stenoses (LTS) on a CT-based skeletonization algorithm, called laryngo-tracheal profile (LTP). Extraction of the laryngo-tracheal tract (LTT)-medial axis was performed after S-CT scanning. Orthogonal to the medial axis, the LTT cross-profile was computed, and the length and degree of LTS were presented as line charts. Clinical application of this newly developed method is demonstrated on three patients who had to undergo preoperative assessment before surgical treatment of tracheal stenoses. LTP provides an objective method of assessment of both the length and degree of tracheal stenoses in precise correlation to defined anatomical landmarks. This method provides important additive information for preoperative evaluation as well as for monitoring of therapeutical success. Current methods used so far are able to evaluate the severity of LTS, but do not provide exact quantitative assessment of complex LTS. Especially in CT-scans of strong curved passages, where an overestimation of the cross-sectional area results by an oblique cut of the tubular structure, LTP may overcome this problem by a simple post processing skeletonization algorithm.

Herniation of the mylohyoid muscle.

During ultrasound examinations in patients with dysphagia, deficiencies in the mylohyoid muscle with herniation of the sublingual gland were found, which may be mistaken as soft tissue tumors. Between the years 2001 and 2003, 205 half-heads used in dissection courses were examined to determine the location and contents of these gaps. In 25 of these cadaveric specimens, the hiatus (of variable size) appeared as small fissures between the divided fibers of the mylohyoid. In 18 cases (72%), the sublingual gland slipped through these deficiencies and occurred in the front part of the submandibular triangle. From the inferior surface of the muscle, the submental artery also coursed through the separated muscle fibers. The herniations of 7 specimens (28%) were found without any contents, neither with gland perforation nor with a submental artery. The following study points out the spatial relation of the salivary glands to the floor of the mouth and the clinical significance, and some factors referred to the development of herniations are discussed.

Short-term therapeutic trial of proton pump inhibitors in suspected extraesophageal reflux.

Pharyngoesophageal gastric acid reflux is thought to initiate chronic posterior laryngitis. The gold standard for measuring gastric reflux is dual-channel 24-hour pH monitoring. This is a time-consuming, inconvenient, expensive method that is not available in all areas. New therapeutic regimes that make use of proton pump inhibitors (PPIs) have proven to be therapeutically efficient for control of acid reflux. Twenty-four consecutive patients with chronic voice disorders and signs of posterior laryngitis were selected for therapy. Twenty-four hour pH monitoring was performed independently before the therapy. The trial therapy consisted of all patients receiving pantoprazole, 40 mg once daily for 6 weeks. Immediately following the therapy a statistically significant (p < 0.05) improvement was observed in all patients. This improvement was analyzed retrospectively by comparison with the results of 24-hour pH monitoring. In 71% of the patients the 24-hour pH-monitoring gave a positive result showing a high number of patients with extraesophageal reflux in our study group. Patients with positive results of pH-monitoring responded in a statistically significant manner (p < 0.05) to the pantoprazole therapy, whereas those patients without detected reflux did not. A 3-month follow-up of the patients with a positive result of the pH-monitoring confirmed the improvement. No patients reported adverse effects. A 6-week treatment with pantoprazole can be clinically justified. It helps to save time and reduce costs, allows for selection of reflux-negative patients for alternative therapy, and may prevent inadequate treatment of patients with false-negative pH monitoring. Twenty-four hour pH monitoring is still recommended for patients unresponsive to this trial therapy.

Spiral-CT-based assessment of tracheal stenoses using 3-D-skeletonization.

PURPOSE: Demonstration of a technique for three-dimensional (3-D) assessment of tracheal-stenoses, regarding site, length and degree, based on spiral computed tomography (S-CT). PATIENTS AND METHODS: S-CT scanning and automated segmentation of the laryngo-tracheal tract (LTT) was followed by the extraction of the LTT medial axis using a skeletonization algorithm. Orthogonal to the medial axis the LTT 3-D cross-sectional profile was computed and presented as line charts, where degree and length was obtained. Values for both parameters were compared between 36 patients and 18 normal controls separately. Accuracy and precision was derived from 17 phantom studies. RESULTS: Average degree and length of tracheal stenoses was found to be 60.5% and 4.32 cm in patients compared with minor caliber changes of 8.8% and 2.31 cm in normal controls (p << 0.0001). For the phantoms an excellent correlation between the true and computed 3-D cross-sectional profile was found (p << 0.005) and an accuracy for length and degree measurements of 2.14 mm and 2.53% respectively could be determined. The corresponding figures for the precision were found to be 0.92 mm and 2.56%. CONCLUSION: LTT 3-D cross-sectional profiles permit objective, accurate and precise assessment of LTT caliber changes. Minor LTT caliber changes can be observed even in normals and, in case of an otherwise normal S-CT study, can be regarded as artifacts.