Lesley C. French

Effect of Hepatocyte Growth Factor on Gene Expression of Extracellular Matrix during Wound Healing of the Injured Rat Vocal Fold

Objectives: We performed a prospective, sham-controlled animal study to investigate the effects of hepatocyte growth factor (HGF) manipulation of the extracellular matrix on vocal fold gene expression during acute injury. Methods: Bilateral vocal fold wounds were created in 40 rats. The rats were randomly assigned to 1 of 2 groups (sham treatment or HGF treatment) and received treatment of the injured area at the time of wounding and on alternate post-treatment days. The injured vocal fold specimens were harvested on post-treatment days 1, 3, 7, and 14. We used real-time reverse transcription polymerase chain reaction to quantify messenger RNA expression of transforming growth factor (TGF)–β1, procollagen types I and III, hyaluronan synthase (HAS)–1, HAS–2, and HAS-3. Results: A multivariate analysis of variance revealed a significant interaction between treatment group and post-treatment day for TGF-β1, procollagen type I, procollagen type III, and HAS-2. Post hoc testing revealed significantly lower expression of procollagen type III and significantly higher expression of HAS-2 on post-treatment day 14 in the HGF treatment group than in the sham treatment group. Conclusions: Results provide evidence of HGF treatment effects on procollagen type III and HAS-2 gene expression pathways.

Experimentally induced phonation increases matrix metalloproteinase-1 gene expression in normal rabbit vocal fold.

Objectives An in vivo rabbit model was used to study the effect of 3 hours of experimentally induced phonation on messenger RNA expression of the normal vocal fold. Study Design Prospective; animal model. Subjects and Methods Ten rabbits received experimental phonation for 3 hours, followed by 1 hour of recovery. A separate group of 5 rabbits served as no-phonation controls. We measured messenger RNA expression of matrix metalloproteinase-1, MMP-9, and interleukin-1β using real-time reverse-transcribed polymerase chain reaction. Gene expression ratios from phonation and control animals were assessed with the Mann-Whitney U test. Results Phonation (77 ± 3 dB; 429 ± 141 Hz) resulted in increased matrix metalloproteinase-1 gene expression from rabbits receiving experimental phonation compared with controls, and a nonsignificant increase in matrix metalloproteinase-9 and interleukin-1β gene expression. Conclusion Matrix metalloproteinases play a role in maintaining tissue homeostasis. Investigation of cellular responses to experimental phonation may provide insight into how matrix metalloproteinases and other extracellular matrices contribute to maintenance of the vocal fold and development of pathology.

Extracellular Matrix Gene Expression after Vocal Fold Injury in a Rabbit Model

Objectives: We determined the expression of matrix metalloproteinase (MMP)–1, MMP-9, collagen types I and III, and fibronectin from rabbit vocal folds after injury. Methods: Thirty rabbits were involved in this study. Five animals were assigned to each time period. Noninjured vocal fold specimens were collected as a control. Gene expression was analyzed at 2, 4, 8, 24, 48, and 72 hours after injury by use of real-time polymerase chain reaction. Results: Compared to 2 hours after injury, MMP-1 expression was increased at 4, 8, 24, 48, and 72 hours. Compared to 4 hours, MMP-1 expression was increased at 8, 24, 48, and 72 hours. Compared to the control specimens, MMP-9 expression was increased at 4, 8, 24, 48, and 72 hours. Compared to 2 hours, MMP-9 expression was increased at 4, 8, 24, 48, and 72 hours. Compared to 2 and 4 hours, collagen type I expression was increased at 72 hours. Collagen type III expression was increased at 72 hours compared to 2, 4, and 8 hours. Compared to 2 hours, fibronectin expression was increased at 24, 48, and 72 hours. Conclusions: Results revealed time-dependent changes in expression of MMP-1, MMP-9, collagen types I and III, and fibronectin from rabbit vocal folds after injury. Future experiments are planned to investigate the effects of phonation on expression of these genes after injury.

Model of Evoked Rabbit Phonation

Objectives: We describe a method for eliciting phonation in an in vivo rabbit preparation using low-frequency, bipolar pulsed stimulation of the cricothyroid muscles with airflow delivered to the glottis. Methods: Ten New Zealand White breeder rabbits weighing 3 to 5 kg were used in this study. The cricothyroid muscles were isolated bilaterally, and separate pairs of anode-cathode hooked-wire electrodes were inserted into each muscle. A Grass S-88 stimulator and 2 constant-current PSIU6 isolation units were used to deliver bipolar square wave pulses to each cricothyroid muscle, with airflow delivered to the glottis through a cuffed endotracheal tube. Results: Phonation was evoked with a 50-Hz, 4-mA stimulus train of 1-ms pulses delivered to each cricothyroid muscle. The pulse trains were on for 2 seconds and were repeated every 5 seconds over a period of 180 minutes. Airflow was delivered at 143 cm3/s, producing phonation measuring 71 to 85 dB sound pressure level. Conclusions: Evoked phonation is feasible in rabbits by use of bipolar stimulation of the cricothyroid muscles with airflow delivered to the glottis. The in vivo rabbit preparation described may provide a useful small animal option for studies of evoked phonation. From the level and consistency of the adduction observed, we hypothesize that current spreading to the underlying adductor muscles and nerves resulted in neural pathway involvement beyond discrete activation of the cricothyroid muscle, providing sufficient approximation of the vocal folds for phonation.

Tracheotomy in the First Year of Life: Outcomes in Term Infants, the Vanderbilt Experience

OBJECTIVE: In an era emphasizing critical care of preterm infants, we characterize the indications and outcomes of tracheotomies performed in the first year of life in term infants compared to preterm infants. METHODS: Retrospective study of 127 tracheotomies performed in the first year of life at a tertiary-care childrens hospital between 1988-2004. RESULTS: Mean gestational ages of the term and preterm groups were 38.97 and 29.71 weeks, respectively (P < 0.001). Indications for tracheotomy were upper airway abnormalities in 53% for the term group. The number of subsequent airway procedures required was 1.39 in the term group, achieving decannulation in 36.3%, with a 20.5% mortality rate. CONCLUSION: Compared to preterm infants, the term decannulation rate was favorable, as chronic lung disease was uncommon. However, non-tracheotomy-related mortalities remained high. SIGNIFICANCE: Tracheotomies are often performed for relief of upper airway obstruction, and congenital and acquired comorbidities not related to tracheotomy are associated with adverse outcomes in term infants. EBM rating: C-4 ©2006 American Academy of Otolaryngology–Head and Neck Surgery Foundation, Inc. All rights reserved.

Tracheotomy in the preschool population: indications and outcomes.

OBJECTIVE: Although more tracheotomy procedures are performed within the first year of life than in any other age group, preschool-aged children requiring tracheotomy remain understudied. We characterize the indications and outcomes for patients between the ages of 3 and 6 years undergoing tracheotomy. METHODS: Out of 480 pediatric tracheotomy procedures performed at a tertiary-care hospital between 1988 and 2004, 15 patients underwent primary tracheotomy between 3 and 6 years of age. RESULTS: Most (60%) procedures were performed for pulmonary toilet. Upper-airway obstruction represented the second most common indication (40%), and trauma necessitated tracheotomy procedures more often than had been predicted (40%). The decannulation rate was 40%; 2 patients died. CONCLUSION: Trauma contributed to both upper-airway obstruction as well as requirements for pulmonary toilet. These procedures performed secondary to trauma will likely continue to increase. SIGNIFICANCE: Tracheotomy procedures in the preschool population remain uncommon; however, nearly half of those studied were performed as a direct result of otherwise preventable trauma.

Localization of Phonation Integration Center in Rabbits

Problem The midbrain periaqueductal gray (PAG) serves as a phonation integration center, coordinating respiratory, laryngeal, and orofacial activity during vocalization. The objective of this pilot study was to explore electrical excitation of the midbrain PAG for eliciting vocalization using an in-vivo rabbit preparation and determine the feasibility of using image guidance for improved localization of target midbrain structures. Methods Pilot experiments were conducted using four New Zealand White (NZW) breeder rabbits. In the first experiment, a stereotaxic frame was used to guide stimulation of the midbrain PAG without the use of image guidance. Decisions regarding track placements were directed by a stereotaxic atlas of the NZW rabbit brain. Histologic stains of axial brain sections were then obtained to determine electrode track positioning. In a subsequent experiment, magnetic resonance imaging (MRI) and computed tomography (CT) scans were obtained and used to localize midbrain structures for advanced planning capabilities. Results Without the use of image guidance, phonation was feasible, measuring 80–100 Hz using electrical excitation of the midbrain PAG (0.5 to 1.5 mA, 200 Hz, 2 second train duration). However, precise localization of the midbrain PAG proved technically challenging. Using CT and MRI data, scans could be loaded onto an image guided surgical system and combined using an image fusion tool for advanced planning. Conclusion The authors demonstrate the feasibility of electrical excitation of the midbrain PAG to produce vocalization in rabbits using a stereotaxic brain atlas and offer an alternative approach using image guidance for advanced planning and more precise localization of target midbrain structures. Significance The biomechanical forces of phonation and dynamic nature of vocal fold remodeling have created a need for chronic animal preparations to investigate these reparative processes in-vivo. Image guided techniques may improve the feasibility of brain stimulation techniques for eliciting and studying time dependent changes in vocal fold remodeling. Support NIH-NIDCD R03 DC008400.

R432 – Gene Expression of the Remodeling Rat Vocal Fold Wound:

Problem The authors investigated the expression of key extracellular matrix genes after vocal fold wounding in a rat model to better understand the reparative mechanisms of tissue repair during the remodeling phase of vocal fold injury. Methods Bilateral vocal fold wounds were created in 30 rats. Injured vocal fold specimens were harvested 1, 3, 7, 14, 28, and 56 days after wounding. 5 unwounded rats were used to establish baseline for polymerase chain reaction (PCR). The authors used real-time PCR to quantify messenger RNA expression of procollagen type I, III, interleukin-1 beta (IL-1 beta), decorin, and hyaluronan synthase (HAS) −1, −2, and −3. Analysis of variance was used to detect main effects for gene expression. Post-hoc tests were used to make comparisons between time points. Results Procollagen type I expression was decreased from baseline on post-injury day 1, 28, and 56. Procollagen type III was decreased on post-injury day 1 and 56, and increased from baseline on post-injury day 14. IL-1 beta expression was increased from baseline on post-injury day 1, 3, and 7. Decorin expression was decreased from baseline on post-injury day 1, 3, 7, and 56. HAS-1 expression was decreased from baseline at all post-injury time points. HAS-2 expression was increased from baseline on post-injury day 3, and decreased from baseline on post-injury day 14, 28, and 56. HAS-3 expression was decreased from baseline on post-injury day 1, 28, and 56. Conclusion Findings provide temporal changes in the expression of key extracellular matrix genes during a remodeling phase of vocal fold injury in a rat wound model. Significance Vocal fold wound models provide a means for investigating tissue reparative processes and molecular mechanisms controlling synthesis and degradation of the vocal fold extracellular matrix. Support Vanderbilt University Medical Center.

Nerve-Induced Rabbit Phonation: Temporal Phonation Stability

Problem In-vivo phonation using animals with layered structure similarities and phonation characteristics akin to humans are needed for the study of vocal fold reparative processes. The authors’ previous studies have demonstrated the feasibility of eliciting vocalization in a flow-controlled in-vivo rabbit preparation. In the current study, the authors provide information relative to temporal stability of nerve elicited rabbit phonation. Methods Eight New Zealand White breeder rabbits received pulsatile current to the laryngeal nerves with airflow delivered at a constant rate. Measurements of vocal intensity and fundamental frequency were recorded every 5, 30, and 90 minutes after the onset of phonation. Pearson correlations were used to investigate relationships among vocal intensity and fundamental frequency. Results Phonation was elicited with fundamental frequency and intensity ranging from 300 to 1000 Hz and 68 to 80 dB SPL, respectively at 5 minutes, 55 to 840 Hz and 70 to 79 dB SPL at 30 minutes, and 51 to 438 Hz and 68 to 76 dB SPL measured at 90 minutes. Correlation coefficients revealed predictable relationships among the dependent variables over time. Conclusion Continuous, audible phonation within the range of human phonation was produced over a 90 minute duration using an in-vivo rabbit preparation. Expected relationships between vocal intensity and fundamental frequency were observed over time. Significance Considering the vocal fold microarchitectural properties and phonation characteristics of the rabbit, the in-vivo preparation described may provide a useful small animal option for investigations of phonation dependent tissue remodeling, maintenance of the vocal fold lamina propria, and development of pathology. Support NIH-NIDCD R03 DC008400.