Tsunehisa Ohno

Histologic characterization of human scarred vocal folds.

Vocal fold scarring remains a significant problem. Although several animal models have been developed to improve our understanding of the histopathology, the histologic features of scarred human vocal folds have rarely been reported. The present case studies aimed to define the histologic changes of scarred human vocal folds caused by cordectomy or cordotomy. Ten patients with the scarred vocal folds were involved in this study. Nine patients with early glottic cancer underwent endoscopic cordectomy, and one patient underwent superficial cordotomy for idiopathic scar. The postcordectomy or cordotomy scar was biopsied or resected 3-13 months after the original procedure. After confirming absence of any tumor in cancer patients, the remaining specimens were used in the present study. Histologic examination investigated deposition of extracellular matrix (ECM) including collagen, elastin, hyaluronic acid (HA), fibronectin, and decorin in the lamina propria of the scarred vocal folds. There was a wide range of variation in the deposition of ECM in scarred vocal folds. Excessive and disorganized collagen deposition was observed in most cases that had undergone deep resection of the lamina propria, whereas deposition of collagen was mild and well organized after superficial resection. Decorin was retained in all cases after superficial cordectomy or cordotomy, but varied after deep resection. Deposition of elastin, HA, and fibronectin varied regardless of depth of injury. Histology of scarred vocal folds may vary with degree of injury and individual healing mechanism.

Drug delivery system of hepatocyte growth factor for the treatment of vocal fold scarring in a canine model.

Objectives: Vocal fold scarring remains a therapeutic challenge. Previous studies have indicated that hepatocyte growth factor (HGF), a strong antifibrotic element, has therapeutic potential for restoring scarred vocal folds. To enhance the effect of HGF in vivo, we developed a novel drug delivery system (DDS) in which HGF is embedded in gelatin hydrogel and continuously released over a period of 2 weeks. In the present study we investigated the therapeutic efficacy of the HGF DDS on vocal fold scarring by using a canine model. Methods: The vocal folds of 8 beagles were unilaterally scarred by stripping the entire layer of the lamina propria. The contralateral vocal folds were kept intact as normal controls. One month after the procedure, hydrogels (0.5 mL) containing 1 μg of HGF were injected into the scarred vocal folds of 4 dogs (HGF-treated group), whereas hydrogels containing saline solution were injected in the other 4 dogs (sham group). Histologic and vibratory examinations were completed for each group 6 months after the initial surgery. Results: The excised larynx experiments showed significantly better vibration in terms of mucosal wave amplitude and glottal closure in the HGF-treated group compared to the sham group. Histologic evaluation of the vocal folds indicated remarkable reduction in collagen deposition and tissue contraction, with favorable restoration of hyaluronic acid and elastin in the HGF-treated group. Conclusions: The present findings suggest that the novel HGF DDS may provide favorable effects in restoring the vibratory properties of scarred vocal folds.

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.

Age-Associated Changes in the Expression and Deposition of Vocal Fold Collagen and Hyaluronan

Objectives: We investigated age-associated changes in the expression and deposition of collagen and hyaluronan (hyaluronic acid; HA) in aged vocal folds. Methods: Thirty Sprague-Dawley rats were involved in this study. For gene expression analyses, 15 animals were divided into 3 age groups of young (2 month), adult (9 month), and elderly (18 month) rats. Real-time polymerase chain reaction testing was used to quantify the messenger RNA expression of procollagen types I and III, matrix metalloproteinases 2 and 9, and HA synthases 1, 2, and 3. The remaining 15 animals were divided into 3 similar age groups and underwent histologic analyses designed to investigate age-associated changes in the deposition of collagen and HA. Results: The results revealed down-regulated expression of procollagen types I and III, matrix metalloproteinases 2 and 9, and HA synthases 1, 2, and 3 in adult and elderly vocal folds, compared to young vocal folds. Histologically, staining of collagen was dense in the vocal folds of adult and elderly rats, and HA was less dense in the vocal folds of adult and elderly rats than in young rats. Conclusions: A slowdown in the expression of procollagens and matrix metalloproteinases was associated with dense collagen in aged vocal folds, as observed in elderly humans. A similar decrease in the expression of genes that code for HA synthase was consistent with a low density of extracellular matrix HA in the vocal folds of elderly rats.

Gene expression of transforming growth factor-β1 and hepatocyte growth factor during wound healing of injured rat vocal fold†‡

To investigate the expression of genes coding transforming growth factor (TGF)‐β1, hepatocyte growth factor (HGF), and c‐Met, its membrane‐spanning tyrosine kinase receptor, during the inflammatory, proliferative, and remodeling phases of wound healing in the injured rat vocal fold.

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.

Effects of raised-intensity phonation on inflammatory mediator gene expression in normal rabbit vocal fold.

OBJECTIVE: To investigate the hypothesis that a transient episode of raised-intensity phonation causes a significant increase in vocal fold inflammatory messenger RNA (mRNA) expression in vivo. STUDY DESIGN: Prospective animal study. SETTING: Laboratory. SUBJECTS AND METHODS: Ten New Zealand White breeder rabbits received 30 minutes of experimentally induced modal or raised-intensity phonation, followed by a 30-minute recovery period. A separate group of five rabbits served as sham controls. Real-time polymerase chain reaction was performed to investigate the mRNA expression of interleukin 1β (IL-1β), transforming growth factor β (TGFβ1), and cyclooxygenase-2 (COX-2). Separate one-way analysis of variance (ANOVA) tests were used to investigate differences in gene expression across groups, with an appropriate alpha correction of 0.016 to control for type I error. Significant main effects were further examined using Fishers least significant difference. RESULTS: ANOVA revealed that there were differences for IL-1β, TGFβ1, and COX-2 between sham control, modal phonation, and raised-intensity phonation (P < 0.0001). Pairwise comparisons revealed that the expression of IL-1β, COX-2, and TGFβ1 increased significantly during raised-intensity phonation, compared to modal phonation and sham control (P < 0.0001). CONCLUSION: Results provided support for the hypothesis that a transient episode of raised-intensity phonation causes a significant increase in vocal fold inflammatory mRNA expression. Future studies will investigate the signal transduction pathways and mechanisms regulating the vocal fold inflammatory response. The long-term goal of these studies is to advance understanding of the molecular and cellular events underlying phonation-related tissue alterations.

Regenerative effects of basic fibroblast growth factor on extracellular matrix production in aged rat vocal folds.

Objectives We investigated acute changes in extracellular matrix (ECM) gene expression and histologic changes in the deposition of collagen and hyaluronan (hyaluronic acid; HA) after basic fibroblast growth factor (bFGF) treatment of the aged rat vocal fold. Methods For the polymerase chain reaction (PCR) experiments, we divided ten 18-month-old Sprague-Dawley rats into two groups that received serial injections of sham (saline solution) or bFGF (2 ng/uL) and euthanized them 2 weeks after the initial injection to investigate acute changes in ECM gene expression. We treated a separate group of 5 animals unilaterally and sacrificed them 4 weeks after the initial injection to investigate histologic changes in the deposition of collagen and HA. Results Real-time PCR revealed significantly up-regulated HA synthase (HAS)-2, HAS-3, matrix metalloproteinase (MMP)-2, and procollagen type I gene expression in the bFGF treatment group as compared to the sham treatment group. Histologic staining revealed significantly increased deposition of HA in the bFGF-treated vocal fold as compared to the sham-treated vocal fold. No differences in ECM collagen levels were observed between treatment sides. Conclusions Basic fibroblast growth factor induced the up-regulation of HAS-2, HAS-3, MMP-2, and procollagen type I. Histologically, aged vocal folds treated with bFGF revealed increased deposition of HA as compared to sham-treated vocal folds.

Regeneration of Aged Rat Vocal Folds using Hepatocyte Growth Factor Therapy

We investigated acute changes in extracellular matrix gene expression and histologic changes in the deposition of collagen and hyaluronan (HA) from hepatocyte growth factor (HGF) treatment of the aged rat vocal fold. We hypothesized that: 1) HGF induces matrix metalloproteinase gene expression, which might contribute to the downregulation of collagen; and 2) HGF induces hyaluronan synthase (HAS) gene expression, which might play a role in the upregulation of extracellular matrix HA.