Three-dimensional live imaging of Atoh1 reveals the dynamics of hair cell induction and organization in the developing cochlea.

During cochlear development, hair cells (HCs) and supporting cells differentiate in the prosensory domain to form the organ of Corti, but how one row of inner HCs (IHCs) and three rows of outer HCs (OHCs) are organized is not well understood. Here, we investigated the process of HC induction by monitoring Atoh1 expression in cochlear explants of Atoh1-EGFP knock-in mouse embryos and showed that only the cells that express Atoh1 over a certain threshold are selected for HC fate determination. HC induction initially occurs at the medial edge of the prosensory domain to form IHCs and subsequently at the lateral edge to form OHCs, while Hedgehog signaling maintains a space between IHCs and OHCs, leading to formation of the tunnel of Corti. These results reveal dynamic Atoh1 expression in HC fate control and suggest that multi-directional signals regulate OHC induction, thereby organizing the prototype of the organ of Corti.

Idgenes are required for morphogenesis and cellular patterning in the developing mammalian cochlea.

Inhibitor of differentiation and DNA-binding (Id) proteins, Id1 to Id4, function in the regulation of cellular proliferation and differentiation. Id proteins have been shown to interact with bHLH proteins and other proteins involved in regulating cellular proliferation and differentiation, suggesting a widespread regulatory function. Id1-3 are known to be expressed in the prosensory domain of developing cochlea. However, the roles of Id genes in cochlear development are not fully elucidated. The deficiency of any of the Id1-3 genes individually has little effect on the cochlear development, and therefore the functional redundancy among these genes have been presumed to explain the absence of phenotype. Here, we show that conditional knockout of Id1/2/3 genes (Id TKO) causes major defects in morphogenesis and cellular patterning in the development of mammalian cochlea. Id TKO cochlea was 82% shorter than control, and both decreased proliferation and increased cell death caused the hypomorph. Sox2-positive prosensory domain was formed in Id TKO cochlea, but the formation of the medial-lateral (central-peripheral) axis was disturbed; the boundary between the medial and lateral compartments in the prosensory domain was partially doubled; the number of inner hair cells per unit length increased, and the number of outer hair cells decreased. Furthermore, the lateral non-sensory compartment expressing Bmp4 and Lmo3 was missing. Thus, the patterning of the lateral epithelium was more affected than the medial epithelium. These results suggested that Id genes are crucial for morphogenesis of the cochlea duct and patterning of the lateral epithelium in the developing cochlea. Further analyses by quantitative RT-PCR and immunostaining using cochlear explants with a Bmp pathway inhibitor revealed that the Bmp-Id pathway originates from the lateral non-sensory compartment and promotes outer hair cell differentiation.

Integrated regulation of Toll-like receptor responses by Notch and interferon-gamma pathways.

Toll-like receptor (TLR) responses are regulated to avoid toxicity and achieve coordinated responses appropriate for the cell environment. We found that Notch and TLR pathways cooperated to activate canonical Notch target genes, including transcriptional repressors Hes1 and Hey1, and to increase production of canonical TLR-induced cytokines TNF, IL-6, and IL-12. Cooperation by these pathways to increase target gene expression was mediated by the Notch-pathway component and transcription factor RBP-J, which also contributed to lethality after endotoxin injection. TLR- and Notch-induced Hes1 and Hey1 attenuated IL-6 and IL-12 production. This Hes1- and Hey1-mediated feedback inhibitory loop was abrogated by interferon-gamma (IFN-gamma), which blocked TLR-induced activation of canonical Notch target genes by inhibiting Notch2 signaling and downstream transcription. These findings identify new immune functions for RBP-J, Hes, and Hey proteins and provide insights into mechanisms by which Notch, TLR, and IFN-gamma signals are integrated to modulate specific effector functions in macrophages.

Hedgehog signaling regulates prosensory cell properties during the basal-to-apical wave of hair cell differentiation in the mammalian cochlea.

Mechanosensory hair cells and supporting cells develop from common precursors located in the prosensory domain of the developing cochlear epithelium. Prosensory cell differentiation into hair cells or supporting cells proceeds from the basal to the apical region of the cochleae, but the mechanism and significance of this basal-to-apical wave of differentiation remain to be elucidated. Here, we investigated the role of Hedgehog (Hh) signaling in cochlear development by examining the effects of up- and downregulation of Hh signaling in vivo. The Hh effector smoothened (Smo) was genetically activated or inactivated specifically in the developing cochlear epithelium after prosensory domain formation. Cochleae expressing a constitutively active allele of Smo showed only one row of inner hair cells with no outer hair cells (OHCs); abnormal undifferentiated prosensory-like cells were present in the lateral compartment instead of OHCs and their adjacent supporting cells. This suggests that Hh signaling inhibits prosensory cell differentiation into hair cells or supporting cells and maintains their properties as prosensory cells. Conversely, in cochlea with the Smo conditional knockout (Smo CKO), hair cell differentiation was preferentially accelerated in the apical region. Smo CKO mice survived after birth, and exhibited hair cell disarrangement in the apical region, a decrease in hair cell number, and hearing impairment. These results indicate that Hh signaling delays hair cell and supporting cell differentiation in the apical region, which forms the basal-to-apical wave of development, and is required for the proper differentiation, arrangement and survival of hair cells and for hearing ability.

Cooperative functions of Hes/Hey genes in auditory hair cell and supporting cell development.

Notch-mediated lateral inhibition has been reported to regulate auditory hair cell and supporting cell development from common precursors. While the Notch effector genes Hes1, Hes5 and Hey1 are expressed in the developing cochlea, inactivation of either of them causes only mild abnormality, suggesting their functional redundancy. To explore the roles of Hes/Hey genes in cochlear development, we examined compound heterozygous or homozygous mutant mice that lacked Hes1, Hes5 and Hey1 alleles. We found that a reduction in Hes/Hey gene dosage led to graded increase of hair cell formation. However, if at least one allele of Hes1, Hes5 or Hey1 was intact, excessive hair cells were accompanied by overproduction of supporting cells, suggesting that the hair cell increase does not occur at the expense of supporting cells, and that each Hes/Hey gene functions to induce supporting cells. By contrast, when all alleles of Hes1, Hes5 and Hey1 were inactivated, the number of hair cells increased more drastically, whereas that of supporting cells was unchanged compared with control, suggesting that supporting cell formation was balanced by their overproduction and fate conversion into hair cells. The increase of the cell numbers seemed to occur after the prosensory domain formation in the mutants because the proliferation state and the size of the prosensory domain were not affected. Thus, Hes1, Hes5 and Hey1 cooperatively inhibit hair cell formation, and one allele of Hes1, Hes5 or Hey1 is sufficient for supporting cell production probably by lateral inhibition in the sensory epithelium. Strikingly, Hes/Hey mutations lead to disorganized cell alignment and polarity and to hearing loss despite hair cell overproduction. These results suggest that Hes/Hey gene dosage is essential not only for generation of appropriate numbers of hair cells and supporting cells by controlling cell proliferation and lateral inhibition but also for the hearing ability by regulating the cell alignment and polarity.

Retrograde ERK activation waves drive base-to-apex multicellular flow in murine cochlear duct morphogenesis.

A notable example of spiral architecture in organs is the mammalian cochlear duct, where the morphology is critical for hearing function. Genetic studies have revealed necessary signaling molecules, but it remains unclear how cellular dynamics generate elongating, bending, and coiling of the cochlear duct. Here, we show that extracellular signal-regulated kinase (ERK) activation waves control collective cell migration during the murine cochlear duct development using deep tissue live-cell imaging, Förster resonance energy transfer (FRET)-based quantitation, and mathematical modeling. Long-term FRET imaging reveals that helical ERK activation propagates from the apex duct tip concomitant with the reverse multicellular flow on the lateral side of the developing cochlear duct, resulting in advection-based duct elongation. Moreover, model simulations, together with experiments, explain that the oscillatory wave trains of ERK activity and the cell flow are generated by mechanochemical feedback. Our findings propose a regulatory mechanism to coordinate the multicellular behaviors underlying the duct elongation during development.

Stalling interkinetic nuclear migration in curved pseudostratified epithelium of developing cochlea.

The bending of epithelial tubes is a fundamental process in organ morphogenesis, driven by various multicellular behaviours. The cochlea in the mammalian inner ear is a representative example of spiral tissue architecture where the continuous bending of the duct is a fundamental component of its morphogenetic process. Although the cochlear duct morphogenesis has been studied by genetic approaches extensively, it is still unclear how the cochlear duct morphology is physically formed. Here, we report that nuclear behaviour changes are associated with the curvature of the pseudostratified epithelium during murine cochlear development. Two-photon live-cell imaging reveals that the nuclei shuttle between the luminal and basal edges of the cell is in phase with cell-cycle progression, known as interkinetic nuclear migration, in the flat region of the pseudostratified epithelium. However, the nuclei become stationary on the luminal side following mitosis in the curved region. Mathematical modelling together with perturbation experiments shows that this nuclear stalling facilitates luminal-basal differential growth within the epithelium, suggesting that the nuclear stalling would contribute to the bending of the pseudostratified epithelium during the cochlear duct development. The findings suggest a possible scenario of differential growth which sculpts the tissue shape, driven by collective nuclear dynamics.

A rat excised larynx model of vocal fold scar.

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 (P(th)), glottal resistance (R(g)), glottal efficiency (E(g)), 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 P(th), reduced E(g), and intralarynx vibratory amplitude and area asymmetry. The bFGF group was characterized by P(th) below control-group levels, E(g) comparable with control, and vocal fold vibratory amplitude and area symmetry comparable with control. The sham group was characterized by P(th) comparable with control, E(g) superior to control, and vocal fold vibratory amplitude and area symmetry comparable with control. CONCLUSIONS: The excised larynx model 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.

Roles of vitamin A and macula flava in maintaining vocal folds.

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 a 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.

Vitamin A deficiency causes metaplasia in vocal fold epithelium: a rat study.

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.

Characterization of Hair Cell-Like Cells Converted From Supporting Cells After Notch Inhibition in Cultures of the Organ of Corti From Neonatal Gerbils.

The senses of hearing and balance depend upon hair cells, the sensory receptors of the inner ear. Hair cells transduce mechanical stimuli into electrical activity. Loss of hair cells as a result of aging or exposure to noise and ototoxic drugs is the major cause of noncongenital hearing and balance deficits. In the ear of non-mammals, lost hair cells can spontaneously be replaced by production of new hair cells from conversion of supporting cells. Although supporting cells in adult mammals have lost that capability, neonatal supporting cells are able to convert to hair cells after inhibition of Notch signaling. We questioned whether Notch inhibition is sufficient to convert supporting cells to functional hair cells using electrophysiology and electron microscopy. We showed that pharmacological inhibition of the canonical Notch pathway in the cultured organ of Corti prepared from neonatal gerbils induced stereocilia formation in supporting cells (defined as hair cell-like cells or HCLCs) and supernumerary stereocilia in hair cells. The newly emerged stereocilia bundles of HCLCs were functional, i.e., able to respond to mechanical stimulation with mechanotransduction (MET) current. Transmission electron microscopy (TEM) showed that HCLCs converted from pillar cells maintained the pillar cell shape and that subsurface cisternae, normally observed underneath the cytoskeleton in outer hair cells (OHCs), was not present in Deiters' cells-derived HCLCs. Voltage-clamp recordings showed that whole-cell currents from Deiters' cells-derived HCLCs retained the same kinetics and magnitude seen in normal Deiters' cells and that nonlinear capacitance (NLC), an electrical hallmark of OHC electromotility, was not detected from any HCLCs measured. Taken together, these results suggest that while Notch inhibition is sufficient for promoting stereocilia bundle formation, it is insufficient to convert neonatal supporting cells to mature hair cells. The fact that Notch inhibition led to stereocilia formation in supporting cells and supernumerary stereocilia in existing hair cells appears to suggest that Notch signaling may regulate stereocilia formation and stability during development.

Immuno-scanning electron microscopy of collagen types I and III in human vocal fold lamina propria.

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.

Prenatal vitamin A deficiency causes laryngeal malformation in rats.

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.

Collagen subtypes in human vocal folds.

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.

Histological study of acute vocal fold injury in a rat model.

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.

Postnatal development of rat vocal folds.

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.

Cell production in injured vocal folds: a rat study.

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, alpha-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.

Immediate inflammatory response and scar formation in wounded vocal folds.

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 1beta, interferon gamma, tumor necrosis factor alpha, nuclear factor kappa beta, transforming growth factor beta, 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 1beta, nuclear factor kappa beta, tumor necrosis factor alpha (4 and 8 hours), and transforming growth factor beta (72 hours). Interferon gamma 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.

Histological Effect of Basic Fibroblast Growth Factor on Chronic Vocal Fold Scarring in a Rat Model.

OBJECTIVES: Vocal fold scarring is one of the most challenging laryngeal disorders to treat and there are currently no consistently effective treatments available. Our previous studies have shown the therapeutic potential of basic fibroblast growth factor (bFGF) for vocal fold scarring. However, the histological effects of bFGF on scarred vocal fold have not been elucidated. The aim of this study was to examine the histological effects of bFGF on chronic vocal fold scarring. METHODS: Sprague-Dawley rats were divided into phosphate buffered saline (sham) and bFGF groups. Unilateral vocal fold stripping was performed and the drug was injected into the scarred vocal fold for each group 2 months postoperatively. Injections were performed weekly for 4 weeks. Two months after the last injection, larynges were harvested and histologically analyzed. RESULTS: A significant increase of hyaluronic acid was observed in the vocal fold of the bFGF group compared with that of the sham group. However, there was no remarkable change in collagen expression nor in vocal fold contraction. CONCLUSION: Significant increase of hyaluronic acid by local bFGF injection was thought to contribute to the therapeutic effects on chronic vocal fold scarring.

Histologic characterization of rat vocal fold scarring.

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.