楯谷 一郎

Objectives/Hypothesis: Therapeutic challenges exist in the management of vocal fold scarring. We have previously demonstrated the therapeutic potential of hepatocyte growth factor (HGF) in the management of acute phase vocal fold scarring using a novel hydrogel-based HGF drug delivery system (DDS). However, the effect of HGF on matured vocal fold scarring remains unclear. The current study aims to investigate the effect of HGF-DDS on chronic vocal fold scarring using a canine model. Study Design: Animal model. Methods: Vocal folds from eight beagles were unilaterally scarred by stripping the entire layer of the lamina propria; contralateral vocal folds were kept intact as normal controls. Six months after the procedures, hydrogels (0.5 mL) containing 1 μf HGF were injected into the scarred vocal folds of four dogs (HGF-treated group). Hydrogels containing saline solution were injected into the other four dogs (sham group). Histological and vibratory examinations on excised larynges were completed for each group 9 months after the initial surgery. Results: Experiments conducted on excised larynges demonstrated significantly better vibrations in the HGF-treated group in terms of mucosal wave amplitude. Although phonation threshold pressure was significantly lower in the HGF-treated group compared with the sham group, no significant differences were observed in the normalized glottal gap between HGF-treated and sham groups. Histological examinations of the HGF-treated vocal folds showed reduced collagen deposition and less tissue contraction with favorable restoration of hyaluronic acid. Conclusions: Results suggest that administration of HGF may have therapeutic potential in the treatment of chronic vocal fold scarring. © 2009 The American Laryngological, Rhinological and Otological Society, Inc.

Objectives: Treatment of vocal fold scarring remains a therapeutic challenge. Our group previously reported the efficacy of treating injured vocal folds by implantation of bone marrow-derived stromal cells containing mesenchymal stem cells. Appropriate scaffolding is necessary for the stem cell implant to achieve optimal results. Terudermis is an atelocollagen sponge derived from calf dermis. It has large pores that permit cellular entry and is degraded in vivo. These characteristics suggest that this material may be a good candidate for use as scaffolding for implantation of cells. The present in vitro study investigated the feasibility of using Terudermis as such a scaffold. Methods: Bone marrow-derived stromal cells were obtained from GFP (green fluorescent protein) mouse femurs. The cells were seeded into Terudermis and incubated for 5 days. Their survival, proliferation, and expression of extracellular matrix were examined. Results: Bone marrow-derived stromal cells adhered to Terudermis and underwent significant proliferation. Immunohistochemical examination demonstrated that adherent cells were positive for expression of vimentin, desmin, fibronectin, and fsp1 and negative for beta III tubulin. These findings indicate that these cells were mesodermal cells and attached to the atelocollagen fibers biologically. Conclusions: The data suggest that Terudermis may have potential as stem cell implantation scaffolding for the treatment of scarred vocal folds. © 2009 Annals Publishing Company. All rights reserved.

Purpose: To develop and evaluate a rat excised larynx model for the measurement of acoustic, aerodynamic, and vocal fold vibratory changes resulting from vocal fold scar. Method: Twenty-four 4-month-old male Sprague-Dawley rats were assigned to 1 of 4 experimental groups: chronic vocal fold scar, chronic vocal fold scar treated with 100-ng basic fibroblast growth factor (bFGF), chronic vocal fold scar treated with saline (sham treatment), and unscarred untreated control. Following tissue harvest, histological and immunohistochemical data were collected to confirm extracellular matrix alteration in the chronic scar group; acoustic, aerodynamic, and high-speed digital imaging data were collected using an excised larynx setup in all groups. Phonation threshold pressure (Pth), glottal resistance (R g), glottal efficiency (Eg), vibratory amplitude, and vibratory area were used as dependent variables. Results: Chronically scarred vocal folds were characterized by elevated collagen Types I and III and reduced hyaluronic acid abundance. Phonation was achieved, and data were collected from all control and bFGF-treated larynges; however, phonation was not achieved with 3 of 6 chronically scarred and 1 of 6 saline-treated larynges. Compared with control, the chronic scar group was characterized by elevated Pth, reduced Eg, and intralarynx vibratory amplitude and area asymmetry. The bFGF group was characterized by Pth below control-group levels, Eg comparable with control, and vocal fold vibratory amplitude and area symmetry comparable with control. The sham group was characterized by Pth comparable with control, Eg superior to control, and vocal fold vibratory amplitude and area symmetry comparable with control. Conclusions: The excised larynxmodel reported here demonstrated robust deterioration across phonatory indices under the scar condition and sensitivity to treatment-induced change under the bFGF condition. The improvement observed under the sham condition may reflect unanticipated therapeutic benefit or artifact. This model holds promise as a tool for the functional characterization of biomechanical tissue changes resulting from vocal fold scar and the evaluation of experimental therapies. © American Speech-Language-Hearing Association.

Summary: As the number of interventions for vocal fold scar grows and with the advancement of mathematical modeling, greater accuracy and precision in the measurement of vocal fold pliability will become essential. Although indirect pliability measures have been used successfully, direct measurement of tissue pliability is essential. Indirect measurement with parallel plate technology has limitations; it requires the tissue to be removed from the surrounding framework, allows no site specificity, and offers no future for in vivo use in animals or humans. We tested the linear skin rheometer (LSR) in the evaluation of vocal fold pliability. We measured site-specific rheology of vocal folds thereby creating "pliability maps" in human, dog, and rat cadaveric larynges under conditions of altered stiffness; the canine vocal folds possessed sulci, the rat vocal fold was stiff secondary to controlled biopsy, and the human vocal fold was injected with trichloroacetic acid. Histology was performed to confirm the site and type of canine sulci. We found that the LSR reliably detected stiffness in the vocal folds of all species and created "pliability maps" consistent with previous data and clinical observations. The LSR should prove useful in the evaluation of vocal fold pliability for ex vivo and ultimately for in vivo applications. © 2009 The Voice Foundation.

Objectives: We have previously demonstrated the therapeutic potential of hepatocyte growth factor (HGF) in the treatment of vocal fold scarring, although how exogenous HGF affects gene expression of endogenous HGF or extracellular matrix components in the vocal fold fibroblasts remains unclear. In this in vitro study, we aimed to clarify this aspect in order to better understand the effects of HGF on the vocal folds. Methods: Fibroblasts were obtained from the lamina propria of the vocal folds of 5 Sprague-Dawley rats and were cultured with HGF at concentrations of 100, 10, 1, and 0 ng/mL. The cells were collected on days 1, 3, and 7, and the expression of endogenous HGF, its receptor c-Met, transforming growth factor-β1 (TGF-β1), procollagen types I and III, and hyaluronic acid synthase (HAS)-1 and HAS-2 messenger RNAs (mRNAs) was examined by real-time reverse transcription-polymerase chain reaction. Results: The expression of endogenous HGF and HAS-1 mRNAs increased significantly when exogenous HGF was administered at a concentration of 1 ng/mL. On day 1, the expression of TGF-β1 and HAS-2 mRNAs increased significantly in response to 1 ng/mL HGF. Conclusions: Exogenous HGF triggered the up-regulation of endogenous HGF, TGF-β1, HAS-1, and HAS-2 mRNAs in vocal fold fibroblasts. © 2009 Annals Publishing Company. All rights reserved.

Botulinum neurotoxin (BoNT) injection into the thyroarytenoid (TA) muscle is a commonly performed medical intervention for adductor spasmodic dysphonia. The mechanism of action of BoNT at the neuromuscular junction is well understood, however, aside from reports focused on myosin heavy chain isoform abundance, there is a paucity of data addressing the effects of therapeutic BoNT injection on the TA muscle proteome. In this study, 12 adult Sprague Dawley rats underwent unilateral TA muscle BoNT serotype A injection followed by tissue harvest at 72 h, 7 days, 14 days, and 56 days postinjection. Three additional rats were reserved as controls. Proteomic analysis was performed using 2-D SDS-PAGE followed by MALDI-MS. Vocal fold movement was significantly reduced by 72 h, with complete return of function by 56 days. Twenty-five protein spots demonstrated significant protein abundance changes following BoNT injection, and were associated with alterations in energy metabolism, muscle contractile function, cellular stress response, transcription, translation, and cell proliferation. A number of protein abundance changes persisted beyond the return of gross physiologic TA function. These findings represent the first report of BoNT-induced changes in any skeletal muscle proteome, and reinforce the utility of applying proteomic tools to the study of system-wide biological processes in normal and perturbed TA muscle function. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.

Objectives: The roles of vitamin A in the vocal fold epithelium are not well documented, although vitamin A has been used as a conservative treatment for laryngeal leukoplakia. The purpose of this study was to analyze the roles of vitamin A in vocal fold epithelial differentiation. Methods: Vitamin A-deficient (VAD) rats were generated, and the abnormality of their vocal fold epithelium was examined by hematoxylin and eosin staining and immunohistochemical analysis for keratin 10 and transglutaminase (TGase) 1. Results: The VAD experimental rats exhibited orthokeratosis of the vocal fold epithelium. Keratin 10 and TGase 1 were up-regulated in the epithelium of the VAD rats. Conclusions: It is suggested that vitamin A suppresses TGase 1 expression in normal vocal folds to inhibit keratinization, and that the TGase 1 up-regulation caused by vitamin A deficiency may be related to the formation of metaplasia in the laryngeal epithelium. © 2008 Annals Publishing Company. All rights reserved.

Objectives: Inflammatory factors are key mediators of wound healing processes following injury, and their modulation may improve healing outcomes. The objective of this study was to characterize in vivo inflammatory factor and extracellular matrix (ECM) messenger RNA (mRNA) expression levels 1 hour after vocal fold injury. Methods: Five Sprague-Dawley rats were subjected to bilateral vocal fold injury, 5 rats were reserved as uninjured controls, and 1 rat was subjected to unilateral vocal fold injury and reserved for histology. Tissue was harvested 1 hour after injury. Real-time reverse transcription-polymerase chain reaction was performed to examine the mRNA expression profiles of inflammatory factors nuclear factor kappa beta (NF-κβ), interferon gamma (IFN-γ), cyclooxygenase 2 (COX-2), transforming growth factor beta isoform 1 (TGF-β1), tumor necrosis factor alpha (TNF-α), and interleukin 1 beta (IL-1β), as well as ECM genes hyaluronic acid synthase (HAS) 1, HAS-2, procollagen 1, procollagen 3, and elastin, in the injured samples compared with the uninjured controls. Results: Injury resulted in subepithelial bleeding throughout the vocal fold. The COX-2, TNF-α, IL-1β, and HAS-1 mRNA expression levels were significantly up-regulated 1 hour after injury compared with the uninjured controls. Conclusions: Inflammatory factor and ECM gene expression changes occur in vocal fold wound sites as early as 1 hour after injury. These results should inform future efforts to attenuate vocal fold scarring via the modulation of inflammatory factors. © 2008 Annals Publishing Company. All rights reserved.

Objectives: Vitamin A plays important roles in development, growth, and regeneration. Vitamin A-storing stellate cells have been identified in several organs. The functional roles of vitamin A in the vocal folds are still unknown, although vitamin A-storing vocal fold stellate cells have been observed in the macula flava of human and rat vocal folds. The purpose of this study was to investigate the roles of vitamin A in vocal folds. Methods: Vitamin A-deficient rats were generated, and the vocal folds were examined histologically. Messenger RNA was extracted from the vocal folds and analyzed by real-time polymerase chain reaction. Results: Immunohistochemical analysis of normal vocal folds revealed expression of retinoic acid receptor α in vocal fold stellate cells. The cells in the macula flava of vitamin A-deficient rats showed a larger nucleus/cytoplasm ratio than did those of vitamin A-sufficient rats, but messenger RNA expression of major extracellular matrix components in the macula flava of vitamin A-deficient rats did not present a remarkable change except for procollagen type I. Expression of hyaluronic acid, collagen types I and III, and elastin did not show a significant change in vitamin A-deficient rat vocal folds. Conclusions: These results indicate that vitamin A is not essential to maintaining the extracellular matrix of normal adult vocal folds, although vocal fold stellate cells participate in vitamin A storage. © 2008 Annals Publishing Company. All rights reserved.

Objectives: Our previous research demonstrated that vitamin A might be related to vocal fold development. The purpose of this study was to determine whether vitamin A deficiency affects prenatal laryngeal development in rats. Methods: Two considerations were necessary in designing a study using a rat model: for embryonic survival, vitamin A is necessary through day 10 of gestation, and laryngeal formation occurs primarily after day 11. Thus, we created a rat model that developed vitamin A deficiency after embryonic day 11. Ten pregnant rats (5 vitamin A-deficient rats and 5 control rats) were studied. Embryos were collected at embryonic day 18.5 and analyzed histologically. Results: Eighteen percent of the vitamin A-deficient embryos were alive and demonstrated laryngotracheal cartilage malformation, incomplete separation of the glottis, and/or laryngoesophageal clefts. Conclusions: These results document the important role played by vitamin A in laryngeal development. © 2007 Annals Publishing Company. All rights reserved.

Purpose: The present study was a methodological study designed to reveal the dynamic mechanisms of phonation instability pressure (PIP) using bifurcation analysis. Phonation pressure range (PPR) was also proposed for assessing the pressure range of normal vocal fold vibrations. Method: The authors first introduced the concept of bifurcation on the basis of a symmetric vocal fold model and then applied the bifurcation analysis to data from excised larynges. By recording acoustic signals from 10 excised larynges, the authors measured phonation threshold pressure (PTP), PIP, and PPR at the bifurcation pressure points from the spectrograms as subglottal pressure was progressively increased. Furthermore, to investigate the effects of vocal fold elongation on PTP, PIP, and PPR, the authors manipulated the elongation of the vocal folds at 0%, 5%, 10%, and 15% of the resting vocal fold length. Results: The authors found that PTP, PIP, and PPR were effectively determined using the bifurcation analysis. When vocal fold elongation was increased from 0% to 15%, PTP was significantly increased (p < .001), PIP was not significantly changed (p = .54), and PPR was significantly decreased (p = .003). Conclusion: PIP and PPR represent important parameters to assess phonation instability. Bifurcation analysis represents a valuable procedure for revealing the mechanisms behind PTP, PIP, and PPR and for investigating the effects of vocal fold biomechanical parameters on these 3 pressure parameters. © American Speech-Language-Hearing Association.

Objectives: This study was undertaken to identify the types of collagen fibrils in the extracellular matrix of the human vocal fold lamina propria. Methods: Human vocal folds were obtained from 3 autopsy cases less than 65 years of age. The vocal fold specimens were labeled by primary antibodies of anti-type I and anti-type III collagens, and then by secondary antibody conjugated with 15 nm colloidal gold. The specimens were observed with a scanning electron microscope. Secondary electron imaging and backscatter electron imaging of high-resolution field emission scanning electron microscopy were used to detect gold particles indicating immunolabeling. Results: Type III collagen-labeling gold particles were abundant on the fibrils constructing collagenous fibers, whereas type I collagen-labeling gold particles were sparsely present on fibrils in collagenous fibers. A few reticular fibers were labeled by both collagen type I and collagen type III. Conclusions: The results suggest that collagen type I coexists with collagen type III in fibrils of both collagenous fibers and reticular fibers. © 2007 Annals Publishing Company. All rights reserved.

Objectives: Vocal fold scarring is the major cause of voice disorders after voice surgery or laryngeal trauma. The role of inflammatory factors in vocal fold wound healing and fibrosis has not been adequately investigated. Scarless wound healing has been associated with decreased inflammatory responses. To understand scar formation and develop reliable treatments, it is necessary to control extracellular matrix production and inflammation. Thus, we examined the inflammation profile and extracellular matrix production in wounded vocal folds in the acute phase of wound healing. Methods: Vocal fold stripping was performed on 30 Sprague-Dawley rats. Vocal fold tissue was collected at 5 time points (4, 8, 16, 24, and 72 hours). We examined the in vivo messenger RNA expression profile of inflammatory factors interleukin 1β, interferon γ, tumor necrosis factor a, nuclear factor κβ, transforming growth factor β, and cyclooxygenase 2, as well as hyaluronic acid synthases 1 and 2, procollagen subtypes I and III, and elastin synthase in scarred vocal folds after injury, compared to normal vocal folds, using real-time reverse transcription-polymerase chain reaction. Results: The inflammatory factors showed a time-dependent sequence of expression peaks, starting with interleukin 1β, nuclear factor κβ, tumor necrosis factor α (4 and 8 hours), and transforming growth factor β (72 hours). Interferon γ decreased at 24 hours. Correspondingly, hyaluronic acid synthase 1 expression peaked first (4 and 8 hours), whereas hyaluronic acid synthase 2 expression peaked at 16 hours and again at 72 hours. Procollagen I expression peaked at 72 hours, whereas procollagen III decreased from 8 to 16 hours but peaked at 72 hours. Cyclooxygenase 2 expression was elevated, whereas elastin expression remained constant. Conclusions: The results show a clear profile of vocal fold inflammation with corresponding changes in extracellular matrix production. © 2006 Annals Publishing Company. All rights reserved.

Summary: Phytochemical constituents of medicinal plants demonstrate inhibition of tissue and bacterial hyaluronidase. Echinacoside is a caffeoyl conjugate of Echinacea with known anti-hyaluronidase properties. The purpose of this study was to investigate the wound healing effects of Echinacea on vocal fold wound healing and functional voice outcomes. Pig animal model. Methods: Vocal fold injury was induced in 18 pigs by unilateral vocal fold stripping. The uninjured vocal fold served as control. Three groups of six pigs randomly received a topical application of 300, 600, or 1200 mg of standardized Echinacea on the injured side. Animals were euthanized after 3, 10, and 15 days of wound healing. Phonation threshold pressure and vocal economy measurements were obtained from excised larynges. Treatment outcomes were examined by comparing the animals receiving treatment with a set of 19 untreated and 5 historical controls. Treatment effects on wound healing were evaluated by histologic staining for hyaluronan and collagen. Treated larynges revealed improved vocal economy and phonation threshold pressure compared with untreated larynges. Histologically, treated vocal folds revealed stable hyaluronan content and no significant accumulation of collagen compared with control. Findings provide a favorable outcome of anti-hyaluronidase treatment on acute vocal fold wound healing and functional measures of voice. © 2006 The Voice Foundation.

Objectives: The collagen subtypes in human vocal folds are of particular interest, because each collagen subtype has different features that make it uniquely suited for performing specific tissue tasks and each collagen subtype can affect the tissue properties of the vocal fold lamina propria, Methods: Human vocal folds from 5 autopsy cases (less than 65 years old) were examined by immunohistochemistry for collagen types I, III, IV, and V and elastin. Results: Collagen type III was distributed throughout the whole lamina propria. Type I was found just beneath the basal membrane, in the deep layer of the lamina propria and in the anterior and posterior maculae flavae. Types IV and V were present in the epithelial and endothelial basal membrane. Three-dimensional images from thick specimens reconstructed with confocal microscopy showed 2 distinct patterns: type III fibers were wavy, collagenous fibers, as previously observed in the vocal folds, and type I fibers were thinner than type III fibers. These results suggest that type III fibers help maintain the lamina propria structure and that type I fibers provide the tensile strength required around the basal membrane and vocal ligament to maintain the vocal fold shape while withstanding vibratory forces. © 2006 Annals Publishing Company. All rights reserved.

Objectives: We used an acute vocal fold injury in a rat model to characterize vocal fold wound healing by studying the expression pattern of the extracellular matrix components in the vocal fold lamina propria. Methods: Vocal fold stripping was performed unilaterally in 27 Sprague-Dawley rats. The vocal folds were harvested at 5 time points (1, 3, 5, 7, and 14 days) and histologically analyzed by Alcian blue stain, trichrome stain, and immunofluorescence with antibodies to collagen type I, collagen type III, and fibronectin. Results: Re-epithelialization occurred by day 3 and was complete by day 14. Granulation tissue was formed by day 3. Hyaluronic acid and collagen type I appeared in injured vocal folds by day 3, peaked at day 5, and thereafter decreased. Collagen type III and fibronectin appeared by day 1 and continued to be intense at all time points after day 3. Conclusions: These results suggest that the expression of these extracellular matrix components peaks in the period around days 3 to 5, and that the characteristics of wound healing in the vocal fold are similar to those in the skin in the early phases, but differ during the subsequent remodeling phase. © 2006 Annals Publishing Company. All rights reserved.

Objectives: In this study we aimed to determine the feasibility of using a rat model for the study of postnatal vocal fold (VF) development. Methods: Eighteen male rats that were 3 days old, 3 weeks old, or 8 months old were analyzed histologically with Alcian blue stain used for detecting hyaluronic acid, elastin-van Gieson stain for elastin, Oil Red O and gold chloride stains for vitamin A-containing lipid droplets, and immunohistochemistry for vimentin (general fibroblast marker) and collagen types I and III. Results: The macula flava (MF) was observed as a mass of cells that expressed vimentin intensively in the cytoplasm. The MF showed denser hyaluronic acid and collagen type I than did the midmembranous portion of the VF lamina propria. Clear developmental changes were evident in the MF and other regions. The vimentin-positive cells of the 3-day-old MF were mainly oval-shaped and had less cytoplasm, whereas those of the 8-month-old MF were spindle- and stellate-shaped and had more cytoplasm, similar to that reported in humans. Vitamin A-containing lipid droplets were limited to the 3-week-old and 8-month-old MFs and were not present in the 3-day-old VF. Conclusions: These results suggest that a rat model is useful in studying VF development and that vitamin A is related to the maturity of the VF. © 2006 Annals Publishing Company. All rights reserved.

Objectives: Fibroblasts are reported to play an important role in producing the extracellular matrix of the vocal fold. However, no reports have focused on how and where these cells are generated in the vocal fold after injury. To reveal the characteristics of vocal fold cell production, we investigated cell proliferation in the acute phase of wound healing. Methods: Using a telescope for guidance, we made an incision in the middle region of the vocal fold tissue in 24 rats and performed immunohistochemical staining for vimentin, α-smooth muscle actin, and 5-bromo-2-deoxyuridine. Results: After injury, epithelialization occurred with a peak at day 1, and fibroblasts proliferated in the lamina propria with a peak at day 3, whereas those in the macula flava did not show any increased proliferation. Conclusions: It is suggested that the fibroblasts in the macula flava have functions different from those of fibroblasts in the lamina propria and that the macula flava does not serve as a cell source for the vocal fold in response to injury. © 2006 Annals Publishing Company. All rights reserved.

To evaluate the effectiveness of hyperfractionation for T2 glottic cancer from a viewpoint of laryngeal preservation, we analyzed 21 patients (twice-a-day group) who were treated with hyperfractionation between 1992 and 1998 and compared the results with those of 27 patients (once-a-day group) treated with conventional once-a-day radiation between 1987 and 1992. In the twice-a-day group, radiation was performed with two fractions of 1.2 Gy/day up to a total dose of 72-74.4 Gy. In the once-a-day group, radiation was performed with a fraction of 2 Gy/day up to a total dose of 66 Gy. If radiation was ineffective at 40 Gy, it was stopped, and surgical treatment was carried out. Kaplan-Meier estimates were used for the analysis of the survival rate and laryngeal preservation rate, and the results were compared. In the once-a-day group, the 5-year survival rate was 92.3%. The 5-year laryngeal preservation rate was 51.8%, and it was 60.3% in 20 patients who had undergone full-dose radiation (once-a-day full-dose group). In the twice-a-day group, no major complication, such as laryngeal necrosis, was seen in any case, and the 5-year survival rate was 95.3%. The 5-year laryngeal preservation rate was 95.3%, and it was significantly better than that of both the once-a-day group and the once-a-day full-dose group. Hyperfractionation is considered to be useful for preserving the larynx for the treatment of T2 glottic cancer. © Springer-Verlag 2005.

Aged vocal folds have been reported to have dense collagen deposition and decreased hyaluronic acid (HA) in the lamina propria. These characteristics are thought to contribute to vocal problems that occur with age (presbyphonia). To restore better viscoelasticity to aged vocal folds, an intervention that might increase HA and decrease collagen production from aged vocal fold fibroblasts would appear to be a potentially useful approach. Our previous in vitro study has revealed that basic fibroblast growth factor (bFGF) consistently stimulates HA production and decreases collagen production from aged rat vocal fold fibroblasts. The present in vivo study examined the effects of intracordal injection of bFGF into aged rats' vocal folds in terms of restoration of HA and collagen distribution in the lamina propria. We injected bFGF transorally into the lamina propria of (unilateral) vocal folds. The injection was repeated 4 times weekly, and rats were painlessly sacrificed 1 week, 1 month, and 2 months after the final injection. Histologic examination revealed that bFGF significantly increased the HA content of the lamina propria up to 2 months, but showed no effect on collagen, even after 2 months. Because it might take longer for excessive collagen to be degraded, further studies are necessary to clarify the long-term effect on collagen. A drug delivery system for bFGF also needs to be developed to maximize its effect in the future. The present study suggested at least a positive effect of bFGF in restoring the HA content in the aged vocal fold lamina propria.

This study aimed to clarify the characteristics of rat vocal fold scarring by examining the alteration of key components in the extracellular matrix: hyaluronic acid, collagen, and fibronectin. Under monitoring with a 1.9-mm-diameter telescope, unilateral vocal fold stripping was performed, and larynges were harvested at 2, 4, 8, and 12 weeks after operation. The vocal folds were histologically analyzed with Alcian blue stain, trichrome stain, and immunofluorescence of collagen type I, collagen type III, and fibronectin. The scarred vocal folds showed less hyaluronic acid and more collagen types I and III than did the controls at all time points. Type III was stable for 12 weeks, while type I declined until 8 weeks and thereafter remained unchanged. Fibronectin increased for 4 weeks and then decreased; it was close to the control level at 8 and 12 weeks. These results suggest that the tissue remodeling process in scarred vocal folds slows down around 2 months after wounding.

To examine the functional effects of hyaluronan and collagen alterations in acute vocal fold scar, we injured 15 pig larynges by vocal fold mucosa stripping. At 3, 10, and 15 days after operation, we performed excised larynx experiments to measure phonation threshold pressure (PTP) and vocal economy (an acoustic output-cost ratio; OCR), and then performed hyaluronan and collagen assays. Five uninjured larynges were used as excised controls. Hyaluronan was reduced in the scarred vocal folds through 15 days of wound healing. Collagen was increased at day 15. The PTP was increased and OCR was decreased in scarred larynges, indicating decreased vocal efficiency and ease of phonation. Thus, PTP and OCR were sensitive to the biomolecular changes in acute vocal fold scar. Hyaluronan was more susceptible than collagen to acute tissue ultrastructural alterations. These findings may provide a rationale for increasing hyaluronan in acute vocal fold scar to improve postoperative vocal outcomes.

Since 1990, we have performed steroid injection into the vocal fold by fiberoptic laryngeal surgery (FLS) under local anesthesia. In this study, the usefulness of this method was evaluated in 28 patients with vocal nodules. Under monitoring using a fiberoptic laryngoscope, a curved injection needle was inserted via the oral cavity and steroid was injected. Endoscopic findings showed that the vocal nodule had disappeared in 17 patients of the 27 patients and decreased in 10 after injection. The maximum phonation time was 10.9 s before operation and 13.9 s after operation, showing a significant increase (P<0.05), and the mean flow rate also showed a significant improvement (P<0.05). The patients' self-rating concerning hoarseness demonstrated great improvement after injection. This technique can be performed under local anesthesia in combination with voice therapy on an outpatient basis, and it is considered to be useful for treating vocal nodules. © Springer-Verlag 2003.

20歳男.右耳下部に発生した10cmを超える巨大な石灰化上皮腫例であった.腫瘤は広頸筋の直下の層にまで達していたが,耳下腺よりは浅層に存在していた.術中迅速診断で石灰化上皮腫と診断された.術後は顔面神経麻痺を認めず,経過は良好であり,術後4年経過現在再発は認めていない

This study aimed to examine the possibility of restoration of spiral ganglion neurons, which transmit sound stimulation to the brain, by transplantation of fetal neural stem cells (NSCs) into the modiolus of cochleae. Fetal mouse NSCs expressing green fluorescence were injected into the modiolus of cisplatin-treated cochleae of mice. The temporal bones were collected 14 days after transplantation, and provided histological examination. The cell fate of transplants was determined by immunohistochemistry for a neural or glial cell-marker. Histological analysis 2 weeks after transplantation revealed robust survival of transplant-derived cells in the modiolus of the cochlea. NSCs injected in the basal portion of cochleae migrated as far as the apical end of the modiolus. Grafted NSCs expressing a neural cell marker were identified, but the majority of grafted NSCs differentiated into glial cells. These findings suggest the possible use of NSCs in cell therapy for restoration of spiral ganglion neurons. However, further treatments are required to increase the number of NSC-derived neurons in the modiolus to realize functional recovery.

This study investigated surgical procedures for cell transplantation into the mouse inner ear. Female C57BL/6 mice were used as recipient animals. Fetal mouse neural stem cells expressing green fluorescence were used as donor cells. Two methods, an injection of transplants from the lateral semicircular canal (LSCC) and from the cochlear lateral wall (CLW), were examined. Two weeks after transplantation, the distribution of transplant-derived cells in the cochlea was examined. Effects on auditory function were assessed by measurement of auditory brain stem responses (ABRs). Cochleae receiving cell transplantation from the LSCC exhibited robust survival of transplant-derived cells mainly in the scala vestibuli and scala tympani. Transplantation from the LSCC caused elevation of ABR thresholds by less than 10 dB SPL. However, transplantation from the CLW resulted in considerable hearing loss, even though transplant-derived cells settled in the scala media. These findings demonstrate that an approach from the LSCC can be utilized for cell transplantation into the perilymph without causing apparent auditory disorder, while an approach from the CLW delivers cells to the endolymph but appears to cause auditory dysfunction.

This study investigated surgical procedures for cell transplantation into the mouse inner ear. Female C57BL/6 mice were used as recipient animals. Fetal mouse neural stein cells expressing green fluorescence were used as donor cells. Two methods, an injection of transplants from the lateral semicircular canal (LSCC) and from the cochlear lateral wall (CLW), were examined. Two weeks after transplantation, the distribution of transplant-derived cells in the cochlea was examined. Effects on auditory function were assessed by measurement of auditory brain stein responses (ABRs). Cochleae receiving cell transplantation from the LSCC exhibited robust survival of transplant-derived cells mainly in the scala vestibuli and scala tympani. Transplantation from the LSCC caused elevation of ABR thresholds by less than 10 dB SPL. However, transplantation from the CLW resulted in considerable hearing loss, even though transplant-derived cells settled in the scala media. These findings demonstrate that an approach from the LSCC can be utilized for cell transplantation into the perilymph without causing apparent auditory disorder, while an approach from the CLW delivers cells to the endolymph but appears to cause auditory dysfunction.

This study aimed to examine the possibility of restoration of spiral ganglion neurons, which transmit sound stimulation to the brain, by transplantation of fetal neural stem cells (NSCs) into the modiolus of cochleae. Fetal mouse NSCs expressing green fluorescence were injected into the modiolus of cisplatin-treated cochleae of mice. The temporal bones were collected 14 days after transplantation, and provided histological examination. The cell fate of transplants was determined by immunohistochemistry for a neural or glial cell-marker. Histological analysis 2 weeks after transplantation revealed robust survival of transplant-derived cells in the modiolus of the cochlea. NSCs injected in the basal portion of cochleae migrated as far as the apical end of the modiolus. Grafted NSCs expressing a neural cell marker were identified, but the majority of grafted NSCs differentiated into glial cells. These findings suggest the possible use of NSCs in cell therapy for restoration of spiral ganglion neurons. However, further treatments are required to increase the number of NSC-derived neurons in the modiolus to realize functional recovery.

Calcifying epithelioma (pilomatrixoma) is a relatively uncommon benign tumor. It usually presents as a slow-growing subcutaneous or intradermal nodule measuring less than 3 cm. In this report, we present a 20-year-old male with giant pilomatrixoma in the subauricular region. We performed en bloc resection of the tumor, which measured 12 x 9 cm. Histopathologically, this tumor did not show any malignant findings. There has not been any evidence of recurrence since the treatment.

Loss of sensory hair cells in the inner ear is a major cause of permanent hearing loss, since regeneration of hair cells rarely occurs in mammals. The aim of this study was to examine the potential of neural stem cell transplantation to restore inner ear hair cells in mice. Fetal neural stem cells were transplanted into the mouse inner ear after drug-induced injury. Histological analysis demonstrates that the majority of grafted cells differentiated into glial or neural cells in the inner ear. Strikingly, however, we show that grafted cells integrate in vestibular sensory epithelia and express specific markers for hair cells. This finding suggests that transplanted neural stem cells have the potential to differentiate and restore inner ear hair cells. © 2003 Lippincott Williams & Wilkins.

To elucidate cortical correlates of vestibulo-ocular reflex (VOR) modulation, we observed cortical activation during fixation suppression and habituation of caloric vestibular nystagmus in 12 normal subjects, using PET. Significant positive correlation between regional cerebral blood flow (rCBF) and slow phase eye velocity of caloric nystagmus was observed in the middle and posterior insula, inferior parietal lobule, temporal pole, right fusiform gyrus, lingual gyrus, and cerebellar vermis and hemisphere. The rCBF increase in the insular region and the inferior parietal lobule was lateralized depending on the direction of the nystagmus. Caloric nystagmus was suppressed as a result of visual fixation, during which time the area around the right frontal eye field, temporal pole, inferior temporal gyrus, a broad area in the visual cortex, including fusiform and lingual gyrus, cerebellar uvula/nodulus and flocculus, exhibited positive correlation with fixation suppression of caloric nystagmus, while vestibular cortices exhibited negative correlation. The caloric nystagmus habituated with repetition of stimulation. With habituation, we observed activation in the right anterior cingulate gyrus, left superior parietal lobule and right cuneus, and deactivation in the anterior insula, cingulate gyrus, inferior parietal lobule and occipito-temporal visual cortex. The region that showed significant co-activation with fixation suppression and habituation of caloric nystagmus was the right cuneus, and significant co-deactivation was observed in the anterior insula, cingulate gyrus, inferior parietal lobule and middle temporal visual cortex. The present results support previous observations that the parieto-insular cortex and inferior parietal lobule are involved in processing of vestibular information, and, in addition, suggest that activation may depend on the direction of nystagmus. Deactivation of vestibular cortices during visual fixation supports the concept of inhibitory visual - vestibular interaction in the cortex. Significant activation of the cingulate, superior parietal and visual cortices, and cerebellar vermis accompanying reduction of caloric response with repeated stimuli suggests possible involvement of these regions in vestibular habituation. Common activation of the cuneus in visual cortex and deactivation of vestibular and visuo-spatial association cortices by both visual suppression and habituation of VOR suggests that these two mechanisms are not completely independent but may share some cortical and subcortical regions.

We examined cortical activation by speech in patients with moderate inner ear hearing loss using PET to investigate the response of the language network to insufficient speech input. We made two word lists, well-perceived words and poorly-perceived words, and measured rCBF during monaural presentation of these words. Well-perceived words activated bilateral temporal lobes, bilateral inferior frontal gyri (IFG) and left angular gyrus (AG) regardless of the ear stimulated, Poorly-perceived words activated contralateral temporal lobe and bilateral IFG, while little or no activation was observed in the ipsilateral temporal lobe and left AG. Insufficient activation of the temporal lobe ipsilateral to the ear stimulated might correlated with less accurate word comprehension in patients with inner ear hearing loss. © 2003 Lippincott Williams & Wilkins.

Descending necrotizing mediastinitis occurs secondary to deep neck infection, and the primary focus of infection is mostly located in the tonsil, pharynx and carious tooth. DNM following acute epiglottitis is quite rare, with only one case reported. We treated an 84-year old female with an acute epiglottitis followed by DNM. She was successfully treated by drainages with cervical surgery combined with thoracotomy and cervical surgery.

With significant development of mouse genomics and the availability of transgenic and knockout mice, the mouse will be the preferred animal model for inner ear research. However, few studies have used mice as experimental animals for examination of hair cell degeneration, because of their relative resistance to ototoxic agents and difficulties in surgical treatment. This study presents a model for induction of apoptotic cell death in sensory epithelia of the mouse inner ear using injection of neomycin into the posterior semicircular canal. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay revealed that local application of neomycin produced sufficient induction of apoptotic cell death in both auditory and vestibular epithelia over a definite time course. Supplementation of the general caspase-inhibitor significantly reduced induction of TUNEL-positive cells, indicating caspase-dependency of apoptotic cell death observed in the present model. In addition, the approach to the posterior semicircular canal was an easy technique, and sham-operation induced no significant induction of TUNEL-positive cells. This model, hence, enables the use of various genetic tools in studies for mechanisms of hair cell apoptosis. © 2002 Elsevier Science B.V. All rights reserved.

In the auditory system, efforts to reduce degeneration of spiral ganglion neurons have the immediate objective of improving clinical benefits of cochlear implants, which are small devices designed to stimulate spiral ganglion neurons electronically. Recent studies have indicated several neurotrophins can enhance survival of spiral ganglion neurons. However, the strategy for application of neurotrophins in inner ear is still a matter of debate. In this study, we examined the potential of cell therapy as a strategy for application of neurotrophins in the inner ear. Neural stem cells obtained from green fluorescent protein-transgenic mice were used as donor cells. Medium containing neural stem cells was injected into mouse inner ear. Histological analysis 4 weeks later revealed that transplant-derived cells survived in inner ear and that most transplant-derived cells in the cochlea had differentiated into glial cells. Moreover, expression of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor was observed in transplant-derived cells. These findings indicate that transplantation of neural stem cells can be a useful strategy for application of neurotrophins in inner ear. © 2003 Lippincott Williams & Wilkins.

Recent advances in stem cells research and its application to medicine are reviewed. Stem cells are composed of embryonic stem cells (ES cells) and somatic stem cells, in which neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and other forms have already been identified. Stem cells have a possibility of regenerating damaged organs, which were difficult to transplant, and may be able to serve as a source of donor organ to be used for transplantation. In nervous systems, ES cells, neural stem cells and mesenchymal stem cells are investigated to regenerate damaged central and peripheral neurons. ES cells and neural stem cells are reported to form synapses with host neurons when they are transplanted into the brain or spinal cords. Mesenchymal stem cells are reported to differentiate into neurons in vivo and are expected as a source of cells used for stem cell therapy in the future. The possibility of stem cell transplantation for the treatment of inner ear hearing loss is also discussed.

Objective - Since 1990, we have performed steroid injections into the vocal fold under topical anesthesia using fiberoptic laryngeal surgery (FLS) in an outpatient clinic. The aim of this study was to retrospectively assess the usefulness of this treatment method in 44 patients with mild Reinke's edema. Material and Methods - Using fiberoptic monitoring of the larynx, a curved injection needle was inserted via the oral cavity and triamcinolone acetonide was injected into Reinke's space of the bilateral vocal fold. Results - Remission or improvement was observed in almost all patients in terms of both patients' self-rating of hoarseness and endoscopic vocal fold findings. The maximum phonation time was a mean of 9.0 s before operation and 11.4 s after operation, and this increase was significant (p < 0.01). Voice pitch also improved, from 168 to 181 Hz, in female patients, and this increase was also significant (p < 0.05). Conclusion - Steroid injection is considered to be useful for treating mild Reinke's edema.

We developed a technique of fiberoptic laryngeal surgery for the treatment of vocal process granulomas. In this system, the granuloma can be removed relatively easily and repeatedly under topical anesthesia on an outpatient basis. We treated 27 patients for a total of 4 intubation granulomas and 23 contact granulomas. Ten of the 23 contact granulomas recurred after the initial surgery, but the intubation granulomas did not recur. Most of the recurrent lesions were resolved by fewer than 3 procedures, and all patients were finally cured. Although conservative therapies such as voice therapy and proton pump inhibitors have recently prevailed, surgical removal remains useful in treating vocal process granulomas. Fiberoptic laryngeal surgery facilitates repeated surgical procedures.

8例の良性縦隔甲状腺腫に対し頸部からのアプローチで腫瘍摘出術を行った.腫瘍の存在部位は右葉が4例,左葉3例,左葉下極に接した異所性甲状腺腫が1例であった.腫瘍の最大径は52〜120mmで,CT上腫瘍の下端が大動脈弓のレベルに達しているもの6例,達していないもの2例であった.全例頸部からのアプローチのみで腫瘍を摘出でき,術後に出血,反回神経麻痺等の合併症を生じた症例もなかった

The etiology of spasmodic dysphonia (SD) is still unknown. In the present study, cortical function of a 59-year-old male patient with adductor type SD was examined during phonation with positron emission tomography (PET). Magnetic resonance imaging showed no organic abnormality in the brain. However, PET showed remarkable activities during phonation in the left motor cortex, Broca's area, the cerebellum, and the auditory cortices, whereas the supplementary motor area (SMA) was not activated. The SMA is known to function for motor planning and programming and is usually activated in normal phonation. Several previous reports have shown that the damage of the SMA caused a severe disturbance of voluntary vocalization. In the present case, it was suggested that the functional deficit of the SMA might be related to SD. © 2001 by W.B. Saunders Company.