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Objectives: Vocal fold scar remains a therapeutic challenge. Vocal fold fibroblasts (VFFs) secrete extracellular matrix (ECM), and transforming growth factor-beta 1 (TGF-β1)-mediated fibroblast to myofibroblast differentiation is central to the development of fibrosis. The transient receptor potential (TRP) channel superfamily is a group of nonselective cation channels, and activation of TRP ankyrin 1 (TRPA1) channel has been shown to have antifibrotic effects through TGF-β1/Smad signaling in various organs. This study aimed to elucidate expression of TRPA1 and the impact of TRPA1 activation on TGF-β1/Smad signaling in VFFs., Methods: Vocal folds were dissected from 10-week-old, male Sprague-Dawley rats and primary VFFs were established. TRPA1 was examined in VFFs and lamina propria via immunostaining. VFFs were treated with allyl isothiocyanate (AITC, TRP channel agonist, 10 -5  M) ± TGF-β1 (10 ng/ml) ± A-967079 (selective TRPA1 channel antagonist, 5.0 × 10 -7  M) for 4 or 24 h. Trpa1, Smad3, Smad7, Col1a1, Acta2, and Has1 mRNA expression were quantified via qPCR., Results: TRPA1 was expressed in cultured VFFs and the lamina propria. TGF-β1 administration significantly increased Trpa1 compared to control. AITC alone did not alter Smad3, Smad7, Acta2, or ECM related genes. However, the combination of AITC and TGF-β1 significantly increased Smad3 and decreased Smad7 and Acta2 compared to TGF-β1 alone; A-967079 significantly reduced this response., Conclusions: VFFs expressed TRPA1, and the activation of TRPA1 regulated TGF-β1/Smad signaling in VFFs. These findings provide preliminary insights into potential anti-fibrotic mechanisms of TRPA1 activation through TGF-β1/Smad signaling in VFFs., Level of Evidence: NA Laryngoscope, 134:4593-4598, 2024., (© 2024 The American Laryngological, Rhinological and Otological Society, Inc.)

Background: The relationship between the tumor-immune microenvironment and systemic inflammatory markers, including neutrophil-to-lymphocyte ratio (NLR), is unclear., Methods: We examined the characteristics of systemic inflammatory markers and tumor immune microenvironments in relation to treatment outcomes in 29 consecutive patients with recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) who received pembrolizumab, using 14-marker multiplex immunohistochemistry and image cytometry., Results: NLR ≥4.5 (high NLR) at pretreatment status significantly correlated with short overall survival (OS) and progression-free survival-2 (PFS2) and malnutrition status. High NLR in peripheral blood was significantly correlated with low lymphoid cell and high tumor-associated macrophage counts in tissues, especially myeloid-to-lymphoid cell ratios, suggesting an association between circulating and intratumoral immune complexity profiles., Conclusions: This study suggests a link between NLR in circulating blood, systemic nutritional status, and immune composition within the tumor., (© 2024 Wiley Periodicals LLC.)

Intratumoral immune profiles are related to prognosis and therapeutic efficacy, and could result in personalized treatments based on biomarkers. To develop a multiplex, quantitative, and rapid tissue evaluation method based on the clinically established standard immunohistochemistry (IHC), a 6-marker rapid multiplex IHC was developed based on our previously reported 14-marker multiplex IHC by reducing the number of labels and accelerating the staining procedure. First, fewer labels were required to identify the same immunological features linked to prognosis in 14-marker multiplex IHC analyses. The six selected markers showed a significant correlation with the 14 markers in the immune classification. Next, a rapid staining protocol was developed by optimizing the reaction temperature, chromogen, and washing time, allowing the completion of 6-marker analysis in 5 h and 49 min, as opposed to the several days required for conventional multiplex IHC. Validation of benign tonsil and head and neck cancer tissues revealed a significant correlation between rapid and conventional 6-makrer multiplex IHC in terms of staining intensities, densities of T cells, macrophages, lymphoid/myeloid immune cell ratios, and spatial profiles of intratumoral immune infiltrates. This method may enable quantitative assessment of the tumor-immune microenvironment on a clinically feasible time scale, which promotes the development of tissue biomarker-guided therapeutic strategies., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Takahiro Tsujikawa reports financial support was provided by Japanese 10.13039/100009950Ministry of Education, Culture, Sports, Science, and Technology. Takahiro Tsujikawa reports financial support was provided by 10.13039/100009619Japan Agency for Medical Research and Development. Takahiro Tsujikawa reports financial support was provided by Public Promoting Association Asano Foundation for Studies on Medicine. Takahiro Tsujikawa reports financial support was provided by Research Promotion Award from the Japanese Society of Otorhinolaryngology-Head and Neck Surgery, Inc. Takahiro Tsujikawa reports a relationship with 10.13039/100004334Merck Biopharma that includes: consulting or advisory and speaking and lecture fees. Takahiro Tsujikawa reports a relationship with 10.13039/100009947Merck Sharp & Dohme Corp that includes: speaking and lecture fees. Takahiro Tsujikawa reports a relationship with 10.13039/501100003769Eisai Co Ltd that includes: speaking and lecture fees. Takahiro Tsujikawa reports a relationship with 10.13039/501100013170Ono Pharmaceutical Co Ltd that includes: speaking and lecture fees. Takahiro Tsujikawa reports a relationship with 10.13039/100008021Bristol Myers Squibb Co that includes: speaking and lecture fees. Takahiro Tsujikawa reports a relationship with 10.13039/100030815Otsuka Pharmaceutical Factory Inc that includes: speaking and lecture fees. Saya Shibata reports a relationship with Screen Holdings Co Ltd that includes: employment. Hiroshi Ogi reports a relationship with Screen Holdings Co Ltd that includes: employment. Eiichi Konishi reports a relationship with Roche Diagnostics that includes: consulting or advisory. Eiichi Konishi reports a relationship with 10.13039/100010795Chugai Pharmaceutical Co Ltd that includes: speaking and lecture fees. Kyoko Itoh reports a relationship with Screen Holdings Co Ltd that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Objectives: Age-related voice changes are characterized as breathy, weak and strained, and a deterioration in vocal function in the elderly has been putatively linked to a reduced intensity of speech. They contribute to undesirable voice changes known as presbyphonia. These changes are caused by histological alterations in the lamina propria of the vocal fold mucosa and atrophy of the thyroarytenoid muscle, as well as by decreased respiratory support. There are several clinical studies on presbylarynx dysphonia showing the effectiveness of voice therapy. However, physiological changes of the presbylarynx following voice therapy have not been verified. The purpose of this prospective study was to demonstrate the clinical effectiveness of voice therapy for rehabilitating presbylarynx dysphonia, using vocal function assessments and thyroarytenoid muscular activity detection on laryngeal electromyography (LEMG)., Methods: 10 patients who were diagnosed with aged vocal fold atrophy from ages 60 to 87 years (mean age: 72 years) underwent approximately 12 weeks of voice therapy, mainly using forward-focused voice and vocal resistance training. Stroboscopic examination, aerodynamic assessment, acoustic analysis, Voice Handicap Index (VHI)-10, and LEMG were performed pre- and post-voice therapy. Vocal fold vibratory amplitude (VFVA) was measured by image analysis from the stroboscopic examinations. Turns analysis during steady phonation on LEMG was also assessed., Results: Maximum phonation time, subglottic pressure, jitter, shimmer, VFVA, and VHI-10 significantly improved after voice therapy. The number of turns per second on LEMG also significantly increased., Conclusion: Our data suggest that voice therapy may improve vocal function and thyroarytenoid muscle activity in patients with aged vocal fold atrophy., (Copyright © 2021 The Voice Foundation. Published by Elsevier Inc. All rights reserved.)

Objectives: The Japanese herbal medicine kyoseihatekigan (KHG) has been used to alleviate the symptoms of croaky voice and globus hystericus, and each of its components has anti-inflammatory and antioxidant effects. However, the mechanisms underlying these beneficial actions of KHG on the vocal folds remain largely unknown. We examined the effects of KHG on rat vocal fold wound healing and assessed its anti-inflammatory and antioxidant properties., Study Design: Animal model., Methods: The vocal folds of Sprague-Dawley rats were unilaterally injured under endoscopy. Rats were divided into three groups based on KHG dosing from pre injury day 4 to post injury day 3: 0 mg/kg/day (sham group), 500 mg/kg/day (1% KHG group) and 1000 mg/kg/day (2% KHG group). Histologic changes were examined to assess the degree of inflammation and oxidative stress at day 3, and fibrosis at day 56. In addition, gene expression related to pro-inflammatory cytokines and transforming growth factor-beta1 (TGF-β1) signaling was examined by quantitative real-time polymerase chain reaction (qPCR)., Results: Histologic analysis showed that the 1% and 2% KHG treatments significantly decreased cell infiltration and the 4-hydroxy-2-nonenalx-immunopositive area, and increased hyaluronic acid at day 3. Both KHG treatments significantly decreased fibrosis at day 56. qPCR revealed that mRNA of interleukin-1β and cyclooxygenase-2 were significantly suppressed at day 1 and TGF-β1 mRNA was significantly downregulated at day 5 in both KHG groups., Conclusions: The current findings suggest that KHG has anti-inflammatory and antioxidant effects in the early phase of vocal fold wound healing, which can lead to better wound healing with less scar formation., (Copyright © 2021 The Voice Foundation. Published by Elsevier Inc. All rights reserved.)

Objectives: To examine the sustained effects of oropharyngeal capsaicin stimulation on the regulation of swallowing, we recorded the swallowing-related nerve activities during continuous infusion of capsaicin solution into the oropharynx., Methods: In 33 in situ perfused brainstem preparation of rats, we recorded the activities of the vagus, hypoglossal, and phrenic nerves during fictive swallowing. The interburst intervals (IBIs) of the swallowing-related nerves during sequential pharyngeal swallowing (sPSW) elicited by electrical stimulation of the superior laryngeal nerve (SLN) during concurrent capsaicin stimulation of 10, 1, and 0.1 μM (n = 28) were compared with those during oropharyngeal infusion of saline (control) (n = 5)., Results: The IBIs during SLN-induced sPSW were reduced at 5 min after initiation of continuous infusion of 10 and 1 μM capsaicin solution. The IBIs showed significant decreases to -25.8 ± 6.9%, -25.9 ± 5.3, -18.3 ± 3.7, and -12.0 ± 1.6 at 30 min following 1 μM capsaicin stimulation at SLN stimulus conditions at 5 Hz of 1.2 times threshold, 10 Hz of 40 μA, 5 Hz of 60 μA, and 10 Hz of 60 μA, respectively. Continuous capsaicin stimulation of 0.1 μM solution did not show significant sustained effects., Conclusion: Pharmacological stimulation of capsaicin could provide time-dependent effects on the likelihood of swallowing, particularly subserving sustained facilitation of swallowing reflex with appropriate concentration of capsaicin., Level of Evidence: NA Laryngoscope, 134:305-314, 2024., (© 2023 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives: Local injection of glucocorticoids (GCs) into the vocal folds has been used for treating the vocal fold lesions. While the positive effects on vocal fold nodules, polyps, or scarring have been clinically reported, some concern remains around the potential adverse effects such as vocal fold atrophy, and the mechanisms remain unclear. The present study examined the histology and gene expression of locally injected GC into the vocal folds in rats., Methods: Thirteen-week-old male Sprague-Dawley rats were used in the experiments. Triamcinolone acetonide (TAA) or saline were administered repeatedly to the right vocal folds at a weekly interval, and rats were euthanized one week after the last administration for histological examination. Genetic examination was assessed hyaluronic acid (HA) metabolism at 1 or 3 days after a single TAA injection by quantitative real-time polymerase chain reaction (qRT-PCR)., Results: The group which underwent four TAA injections showed a significant decrease in HA in the lamina propria (LP), thickness of the LP and total cell numbers of the LP compared with the saline group. In contrast, there was no significant difference in the area of collagen accumulation and the thyroarytenoid muscle, although there was a tendency of atrophy of the muscle. After single injection of TAA, qRT-PCR showed a significant decrease in the expression of HA synthases, Has2 and Has3., Conclusions: The current animal study first demonstrates that repeated intracordal injection of GCs may lead to atrophy of vocal folds caused by decrease of deposition of HA in the LP and decrease of gene expression of Has., (Copyright © 2021 The Voice Foundation. Published by Elsevier Inc. All rights reserved.)

Objectives: Effective treatments for vocal fold fibrosis remain elusive. Tamoxifen (TAM) is a selective estrogen receptor modulator and was recently reported to have antifibrotic actions. We hypothesized that TAM inhibits vocal fold fibrosis via altered transforming growth factor beta 1 (TGF-β1) signaling. Both in vitro and in vivo approaches were employed to address this hypothesis., Methods: In vitro, vocal fold fibroblasts were treated with TAM (10 -8 or 10 -9  M) ± TGF-β1 (10 ng/ml) to quantify cell proliferation. The effects of TAM on genes related to fibrosis were quantified via quantitative real-time polymerase chain reaction. In vivo, rat vocal folds were unilaterally injured, and TAM was administered by oral gavage from pre-injury day 5 to post-injury day 7. The rats were randomized into two groups: 0 mg/kg/day (sham) and 50 mg/kg/day (TAM). Histological changes were examined on day 56 to assess tissue architecture., Results: TAM (10 -8  M) did not affect Smad3, Smad7, Acta2, or genes related to extracellular matrix metabolism. TAM (10 -8 or 10 -9  M) + TGF-β1, however, significantly increased Smad7 and Has3 expression and decreased Col1a1 and Acta2 expression compared to TGF-β1 alone. In vivo, TAM significantly increased lamina propria area, hyaluronic acid concentration, and reduced collagen deposition compared to sham treatment., Conclusions: TAM has antifibrotic potential via the regulation of TGF-β1/Smad signaling in vocal fold injury. These findings provide foundational data to develop innovative therapeutic options for vocal fold fibrosis., Level of Evidence: NA Laryngoscope, 133:2248-2254, 2023., (© 2022 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives: Functional dysphonia (FD) varies in terms of vocal behavior and treatment efficacy. So-called hypofunctional dysphonia is characterized by insufficient subglottal pressure which causes a lack of driving power needed to vibrate the vocal folds leading to weak voice or aphonia in severe cases. While voice therapy is the initial treatment, some patients fail to respond to it. Interferential current (IFC) stimulation has been used as part of rehabilitation by physical therapists to reduce the progressive pain. IFC stimulation has also been developed as a laryngeal sensory stimulation device to modify the swallowing function by triggering swallowing reflex. Many researchers have shown recently in animal studies that laryngeal afferent inputs, such as vocal fold vibrations, subglottic pressure, flow rate, and vocal fold location affect vocal motor pattern and voice quality. However, IFC stimulation as a laryngeal afferent has not been verified. Herein, we assessed the effects of IFC stimulation to the neck on difficult functional dysphonia., Methods: Six patients with refractory FD with insufficient subglottic pressure were assessed in this study. All six cases were females and two of them presented with aphonia. All cases were initially treated by voice therapy (VTx) such as flow phonation, water resistance therapy, or tube phonation for 2 months to increase subglottic pressure; however, this resulted in poor improvement in voice. We additionally performed VTx with concurrent application of IFC stimulation to the neck for 3 months, and the effects on voice were evaluated., Results: VTx with IFC stimulation resulted in improved voice in all cases, demonstrating the improvement in maximum phonation time, subglottic pressure, and voice handicap index-10., Conclusions: Results from this clinical study suggest that VTx with IFC stimulation may be useful for adjusting vocal function in patients with FD caused by insufficient subglottic pressure., Competing Interests: CONFLICTS OF INTEREST None., (Copyright © 2022 The Voice Foundation. Published by Elsevier Inc. All rights reserved.)

Objectives: To examine the role of neurons of the pontine respiratory group (PRG) overlapping with the Kölliker-Fuse nucleus in the regulation of swallowing, we compared the activity of swallowing motor activities and interneuron discharge in the dorsal swallowing group in the medulla before and after pharmacological inhibition of the PRG., Methods: In 23 in situ perfused brainstem preparation of rats, we recorded the activities of the vagus (VNA), hypoglossal (HNA), and phrenic nerves (PNA), and swallowing interneurons of the dorsal medulla during fictive swallowing elicited by electrical stimulation of the superior laryngeal nerve or oral water injection. Subsequently, respiratory- and swallow-related motor activities and single unit cell discharge were assessed before and after local microinjection of the GABA-receptor agonist muscimol into the area of PRG ipsilateral to the recording sites of swallowing interneurons., Results: After muscimol injection, the amplitude and duration of swallow-related VNA bursts decreased to 71.3 ± 2.84 and 68.1 ± 2.76 % during electrically induced swallowing and VNA interburst intervals during repetitive swallowing decreased. Similar effects were observed for swallowing-related HNA. The swallowing motor activity was similarly qualitatively altered during physiologically induced swallowing. All 23 neurons were changed in either discharge duration or frequency after PRG inhibition, however, the general discharge patterns in relation to the motor output remained unchanged., Conclusion: Descending synaptic inputs from PRG provide control of the primary laryngeal sensory gate and synaptic activity of the PRG partially determine medullary cell and cranial motor nerve activities that govern the pharyngeal stage of swallowing., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Objectives/hypothesis: Vocal fold (VF) scar and sulcus cause severe vocal problems, but optimal methods have not been established. Total replacement of the mucosa is required particularly for cases in which the whole lamina propria is occupied by severe fibrosis and vibratory function is totally lost. The amniotic membrane (AM) has been proven to have regenerative potential, as it contains stem cells and growth factors. The current study investigated the biocompatibility and effects of AM for regeneration of the VF mucosa., Study Design: In vitro and in vivo studies., Methods: Vocal fold fibroblasts (VFFs) from 13 Sprague-Dawley rats were seeded on AM and subjected to histology and immunohistochemistry, and gene expressions in the VFFs on AM were examined in in vitro study. Twelve New Zealand White rabbits were used in in vivo study. VFs were stripped down and were reconstructed with AM. The regenerative effects were examined 3 months later by histological examination., Results: In vitro study indicated VFFs survived on AM and stained positively for Ki67, vimentin, and fibronectin. Gene expressions of Has1, Has2, and Hgf were significantly increased in the VFFs on AM compared with the other groups. The in vivo study indicated AM-transplanted VFs showed a significantly higher density of hyaluronic acid and lower density of collagen compared with sham VFs., Conclusions: The current preliminary study suggests biocompatibility and possible regenerative effects of AM for VFs., Level of Evidence: NA Laryngoscope, 132:2017-2025, 2022., (© 2021 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives: Medialization procedures, such as type I thyroplasty, arytenoid adduction, and vocal fold injection, are popular treatments for dysphonia due to unilateral vocal fold paralysis (UVFP). However, dysphonia occasionally persists after medialization procedures owing to tension imbalance. This tension imbalance causes diplophonia, asymmetry and aperiodic vibrational flutter in travelling wave motion. Currently, there is no established treatment for tension imbalance. We herein report two cases with residual dysphonia due to tension imbalance following medialization for chronic UVFP, and another case presenting with dysphonia due to tension imbalance following chronic unilateral vocal fold paresis., Methods: Three patients underwent voice therapy using flow phonation to facilitate increased airflow management in speech as well as forward oral resonance by focusing on balanced airflow. Phonatory outcomes were evaluated using stroboscopic findings, aerodynamic and acoustic measures, as well as self-rating., Results: Aerodynamic assessments, acoustic findings and self-ratings improved in all three cases after voice therapy. Stroboscopic findings prior to voice therapy showed asymmetric vibration with glottic gap, which was improved after voice therapy. Fundamental frequency (F0) also increased post-therapy., Conclusions: In a previous canine study, it was shown that enhanced breath support with expiratory airflow resulted in increased F0, suggesting that enhanced breath support could increase vocal fold tension. The increased F0 achieved in the present cases following voice therapy may increase vocal fold tension with breath support. Thus, voice therapy using flow phonation may be effective for supporting vocal fold tension and improving dysphonia due to tension imbalance following UVFP and paresis., (Copyright © 2020 The Voice Foundation. Published by Elsevier Inc. All rights reserved.)

Oropharyngeal swallowing is centrally mediated by a swallowing central pattern generator (Sw-CPG) in the medulla oblongata. The activity of the Sw-CPG depends on the sensory inputs determined by physical and chemical bolus properties. Here we investigate the sensory-motor integration during swallowing arising from different sensory sources. To do so we electrically stimulated the superior laryngeal nerve and we triggered swallowing with oral injections of distilled water or capsaicin solution and extracellularly recorded from swallowing interneurons in arterially perfused brainstem preparations of rats. We recorded the activities of 40 neurons, while monitoring the motor activities of the phrenic, vagal and hypoglossal nerves. Eighteen neurons responded to electrical stimulation of the ipsilateral superior laryngeal nerve, and 6 neurons were excited by oral fluid injection, while 16 non-respiratory neurons did not receive afferent inputs to either electrical or physiological stimuli. The cellular activities displayed by swallowing interneurons during electrical and physiological stimulation of pharyngeal and laryngeal afferent input reveal complex adaptations of the timing of firing patterns and frequencies. The modulation of neuronal activity is likely to contribute to the coordination of efficient bolus transfer during the pharyngeal stage of swallowing., (Copyright © 2021 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.)

Objectives/hypothesis: Vocal fold fibrosis remains a significant clinical challenge. Estrogens, steroid hormones predominantly responsible for secondary sexual characteristics in women, have been shown to alter wound healing and limit fibrosis, but the effects on vocal fold fibrosis are unknown. We sought to elucidate the expression of estrogen receptors and the effects of estrogens on TGF-β1 signaling in rat vocal fold fibroblasts (VFFs)., Study Design: In vitro., Methods: VFFs were isolated from 10-week-old, male Sprague-Dawley rats, and estrogen receptor alpha (ERα) and G protein-coupled receptor 30 (GPR30) were examined via immunostaining and quantitative polymerase chain reaction (qPCR). VFFs were treated with estradiol (E2, 10 -7 , 10 -8 or 10 -9  M) ± transforming growth factor beta 1 (TGF-β1, 10 ng/mL). ICI 182,780 (ICI, 10 -7  M) or G36 (10 -7  M) were employed as antagonists of ERα or GPR30, respectively. qPCR was employed to determine estrogen receptor-mediated effects of E2 on genes related to fibrosis., Results: ERα and GPR30 were expressed in VFFs at both the protein and the mRNA levels. E2 (10 -7  M) did not alter Smad3, Smad7, Acta2 mRNA, or extracellular matrix related genes. However, the combination of E2 (10 -8  M) and TGF-β1 significantly increased Smad7 (P = .03) and decreased Col1a1 (P = .04) compared to TGF-β1 alone; this response was negated by the combination of ICI and G36 (P = .009)., Conclusions: E2 regulated TGF-β1/Smad signaling via estrogen receptors in VFFs. These findings provide insight into potential mechanisms of estrogens on vocal fold injury with the goal of enhanced therapeutics for vocal fold fibrosis., Level of Evidence: NA Laryngoscope, 131:2285-2291, 2021., (© 2020 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives: We aimed to examine the effect of unilateral inhibition of the medullary dorsal swallowing networks on the activities of swallowing-related cranial motor nerves and swallowing interneurons., Methods: In 25 juvenile rats, we recorded bilateral vagal nerve activity (VNA) as well as unilateral phrenic and hypoglossal activity (HNA) during fictive swallowing elicited by electrical stimulation of the superior laryngeal nerve during control and following microinjection of the GABA agonist muscimol into the caudal dorsal medulla oblongata in a perfused brainstem preparation. In 20 animals, swallowing interneurons contralateral to the muscimol injection side were simultaneously recorded extracellularly and their firing rates were analyzed during swallowing., Results: Integrated VNA and HNA to the injection side decreased to 49.0 ± 16.6% and 32.3 ± 17.9%, respectively. However, the VNA on the uninjected side showed little change after muscimol injection. Following local inhibition, 11 out of 20 contralateral swallowing interneurons showed either increased or decreased of their respective firing discharge during evoked-swallowing, while no significant changes in activity were observed in the remaining nine neurons., Conclusion: The neuronal networks underlying the swallowing pattern generation in the dorsal medulla mediate the ipsilateral motor outputs and modulate the contralateral activity of swallowing interneurons, suggesting that the bilateral coordination of the swallowing central pattern generator regulates the spatiotemporal organization of pharyngeal swallowing movements., Level of Evidence: NA Laryngoscope, 131:2187-2198, 2021., (© 2020 American Laryngological, Rhinological and Otological Society Inc, "The Triological Society" and American Laryngological Association (ALA).)

Objectives/hypothesis: Vocal fold atrophy, scar, and sulcus reduce the vibratory function of the vocal fold mucosa, which causes severe refractory dysphonia. We have reported encouraging preliminary results using an intracordal injection of basic fibroblast growth factor (bFGF) and showed improvement in phonatory parameters and voice. The present study summarizes our experience with 100 cases of stiffened vocal folds that were treated with bFGF injections., Study Design: Retrospective chart review with Interstitial Review Board (IRB) approval., Methods: Local injection of bFGF was performed in 100 cases of vocal fold pathology, which included 43 cases of vocal fold atrophy, 41 cases with scar, and 16 cases with sulcus. Ten micrograms of bFGF were injected into the vocal folds under topical anesthesia 4 times in each patient. Therapeutic outcomes were examined with maximum phonation time (MPT), voice handicap index-10 (VHI-10), and GRBAS scale., Results: MPT, VHI-10, and GRBAS scores significantly improved in all pathology groups. An improvement on the VHI-10 greater than five points was observed in 82% of atrophy cases, 78% of scar cases, and 67% of sulcus cases. Improvement on the VHI-10 was significantly better in the atrophy group than the scar or sulcus groups. The mild/moderate cases of scar and sulcus showed better improvement than severe cases., Conclusions: The current large case series indicates positive effects of intracordal injection of bFGF for improvement of voice with no severe adverse events. The effects appeared best for cases of atrophy, while the treatment of severe scar and sulcus requires further improvement., Level of Evidence: 4 Laryngoscope, 131:2059-2064, 2021., (© 2020 American Laryngological, Rhinological and Otological Society Inc, “The Triological Society” and American Laryngological Association (ALA).)

Similar to the hypertrophic scar and keloids, the efficacy of glucorticoids (GC) for vocal fold injury is highly variable. We previously reported dexamethasone enhanced the pro-fibrotic effects of transforming growth factor (TGF)-β as a potential mechanism for inconsistent clinical outcomes. In the current study, we sought to determine the mechanism(s) whereby GCs influence the fibrotic response and mechanisms underlying these effects with an emphasis on TGF-β and nuclear receptor subfamily 4 group A member 1 (NR4A1) signaling. Human VF fibroblasts (HVOX) were treated with three commonly-employed GCs+ /-TGF-β1. Phosphorylation of the glucocorticoid receptor (GR:NR3C1) and activation of NR4A1 was analyzed by western blotting. Genes involved in the fibrotic response, including ACTA2, TGFBR1, and TGFBR2 were analyzed by qPCR. RNA-seq was performed to identify global changes in gene expression induced by dexamethasone. GCs enhanced phosphorylation of GR at Ser211 and TGF-β-induced ACTA2 expression. Dexamethasone upregulated TGFBR1, and TGFBR2 in the presence of TGF-β1 and increased active NR4A1. RNA-seq results confirmed numerous pathways, including TGF-β signaling, affected by dexamethasone. Synergistic pro-fibrotic effects of TGF-β were observed across GCs and appeared to be mediated, at least partially, via upregulation of TGF-β receptors. Dexamethasone exhibited diverse regulation of gene expression including NR4A1 upregulation consistent with the anti-fibrotic potential of GCs.

Objectives: Voice therapy with semioccluded vocal tract exercises (SOVTE) has a long history of use in singers and nonsingers with dysphonia. SOVTE with increased vocal tract impedance leads to increased vocal efficiency and economy. Although there is a growing body of research on the physiological impact of SOVTE, and growing clinical sentiment about its therapeutic benefits, empirical data describing its potential efficacy in singers and nonsingers are lacking. The objective of the current study is to evaluate vocal tract function and voice quality in singers and nonsingers with dysphonia after undergoing SOVTE., Methods: Patients who were diagnosed with functional dysphonia, vocal fold nodules and age-related atrophy were assessed (n = 8 singers, n = 8 nonsingers). Stroboscopic examination, aerodynamic assessment, acoustic analysis, formant frequency, and self-assessments were evaluated before and after performing SOVTE., Results: In the singer group, expiratory lung pressure, jitter, shimmer, and self-assessment significantly improved after SOVTE. In addition, formant frequency (first, second, third, and fourth), and the standard deviation (SD) of the first, second, and third formant frequency significantly improved. In the nonsinger group, expiratory lung pressure, jitter, shimmer, and Voice Handicap Index-10 significantly improved after SOVTE. However, no significant changes were observed in formant frequency., Conclusions: These results suggest that SOVTE may improve voice quality in singers and nonsingers with dysphonia, and SOVTE may be more effective at adjusting the vocal tract function in singers with dysphonia compared to nonsingers., (Copyright © 2019 The Voice Foundation. Published by Elsevier Inc. All rights reserved.)

Objectives: Development of novel vocal fold (VF) therapeutics is limited by a lack of standardized, meaningful outcomes. We hypothesize that automated microindentation-based VF biomechanical property mapping matched to histology permits quantitative assessment., Study Design: Ex vivo., Methods: Twelve anesthetized New Zealand white rabbits underwent endoscopic right VF injury. Larynges were harvested/bisected day 7, 30, or 60 (n = 4/group), with four uninjured controls. Biomechanical measurements (normal force, structural stiffness, and displacement at 1.96 mN) were calculated using automated microindentation mapping (0.3 mm depth, 1.2 mm/s, 2 mm spherical indenter) with a grid overlay (>50 locations weighted toward VF edge, separated into 14 zones). Specimens were marked/fixed/sectioned, and slides matched to measurement points., Results: In the injury zone, normal force/structural stiffness (mean, standard deviation [SD]/mean, SD) increased from uninjured (2.2 mN, 0.64/7.4 mN/mm, 2.14) and day 7 (2.7 mN, 0.75/9.0 mN/mm, 2.49) to day 30 (4.3 mN, 2.11/14.2 mN/mm, 7.05) and decreased at 60 days (2.7 mN, 0.77/9.1 mN/mm, 2.58). VF displacement decreased from control (0.28 mm, 0.05) and day 7 (0.26 mm, 0.05) to day 30 (0.20 mm, 0.05), increasing at day 60 (0.25 mm, 0.06). A one-way ANOVA was significant; Tukey's post hoc test confirmed day-30 samples differed from other groups (P < 0.05), consistent across adjacent zones. Zones far from injury remained similar across groups (P = 0.143 to 0.551). These measurements matched qualitative histologic variations., Conclusion: Quantifiable VF biomechanical properties can be linked to histology. This technological approach is the first to simultaneously correlate functional biomechanics with histology and is ideal for future preclinical studies., Level of Evidence: NA Laryngoscope, 130:454-459, 2020., (© 2019 The American Laryngological, Rhinological and Otological Society, Inc.)

Objective: We hypothesize that Smad3 is a master regulator of fibrosis in the vocal folds (VFs) and RNA-based therapeutics targeting Smad3 hold therapeutic promise. Delivery remains challenging. We previously described a novel synthetic peptoid oligomer, lipitoid L0 , complexed with siRNA to improve stability and cellular uptake. An advantage of these peptoids, however, is tremendous structural and chemical malleability to optimize transfection efficiency. Modifications of L0 were assayed to optimize siRNA-mediated alteration of gene expression., Methods: In vitro, Smad3 knockdown by various lipitoid variants was evaluated via quantitative real-time polymerase chain reaction in human VF fibroblasts. Cytotoxicity was quantified via colorimetric assays. In vivo, a rabbit model of VF injury was employed to evaluate the temporal dynamics of Smad3 knockdown following injection of the L0 -siRNA complex., Results: In vitro, similar reductions in Smad3 expression were established by all lipitoid variants, with one exception. Sequence variants also exhibited similar nontoxic characteristics; no statistically significant differences in cell proliferation were observed. In vivo, Smad3 expression was significantly reduced in injured VFs following injection of L0 -complexed Smad3 siRNA at 1 day postinjection. Qualitative suppression of Smad3 expression persisted to 3 days following injury, but did not achieve statistical significance., Conclusions: In spite of the chemical diversity of these peptoid transfection reagents, the sequence variants generally provided consistently efficient reductions in Smad3 expression. L0 yielded effective, yet temporally limited knockdown of Smad3 in vivo. Peptoids may provide a versatile platform for the discovery of siRNA delivery vehicles optimized for clinical application., Level of Evidence: NA., (© 2019 The Authors. Laryngoscope Investigative Otolaryngology published by Wiley Periodicals, Inc. on behalf of The Triological Society.)

Objectives/hypothesis: Direct glucocorticoid (GC) injection for vocal fold (VF) scarring has evolved as a therapeutic strategy, but the mechanisms underlying the antifibrotic effects remain unclear. GCs act via the glucocorticoid receptor (GR), which is phosphorylated at multiple serine residues in a hormone-dependent manner to affect bioactivity. We hypothesize that GCs regulate SMAD signaling via GR phosphorylation in vocal fold fibroblasts (VFFs)., Study Design: In vitro., Methods: Human VFFs were treated with dexamethasone (DM; 10 -5 -10 -7 M) ± transforming growth factor (TGF)-β1 (10 ng/mL). RU486 (10 -6 M) was employed to isolate the regulatory effects of GR. Total GR, Ser 211 , and Ser 203 phosphorylation was examined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunocytochemistry. Quantitative polymerase chain reaction was employed to determine GR-mediated effects of DM on genes related to fibrosis., Results: Total GR and Ser 211 phosphorylation was observed predominantly in the nucleus 1 hour after DM administration. DM decreased total GR expression, but Ser 203 and Ser 211 phosphorylation increased. RU486 limited the effects of DM. SMAD3 and SMAD7 mRNA expression significantly decreased 4 hours after DM administration (P < 0.05); this response was negated by RU486. COL1A1 remained unchanged, and ACTA2 significantly increased following 24 hours of DM treatment (P < 0.05)., Conclusion: DM regulated TGF-β1 signaling via altered SMAD3 and SMAD7 expression. This response was associated with altered GR phosphorylation. These findings provide insight into the mechanisms of steroidal effects on vocal fold repair; ultimately, we seek to enhance therapeutic strategies for these challenging patients., Level of Evidence: NA Laryngoscope, 129:E187-E193, 2019., (© 2018 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives/hypothesis: Our laboratory recently described NR4A1 as an endogenous inhibitor of TGF-β-induced vocal fold (VF) fibrosis. Our prior report described the temporal expression of NR4A1 during VF healing in vivo and the effects of NR4A1 knockdown on fibroplastic cell activities in vitro. Based on these findings, we hypothesized that cytosporone-B (Csn-B), an NR4A1 agonist, may hold significant therapeutic potential., Study Design: In vitro., Methods: Human VF fibroblasts were exposed to TGF-β1+/-Csn-B. Expression of genes related to fibrosis were quantified. In addition, contraction was assayed as a surrogate for the fibrotic phenotype in our cell line., Results: TGF-B1 stimulated COL1A1 and ACTA2, as expected. Csn-B significantly downregulated TGF-β1-mediated upregulation of these genes (P = .009, P = .03, respectively). Csn-B had no effect on genes related to TGF-β/Smad signaling. Csn-B also decreased the TGF-β1-mediated contractile phenotype in our cells (P = .004)., Conclusions: NR4A1 is an endogenous inhibitor of fibrosis in the vocal folds and Csn-B, as an NR4A1 agonist, may evolve as an ideal, therapeutic candidate for this challenging condition., Level of Evidence: NA Laryngoscope, 128:E425-E428, 2018., (© 2018 The American Laryngological, Rhinological and Otological Society, Inc.)

Sensory-motor control of the pharyngeal swallow requires sensory afferent inputs from the pharynx and larynx evoked by introducing bolus into the pharynx. Patients with reduced sensitivity of the pharynx and larynx are likely to have a swallowing impairment, such as pre-swallow aspiration due to delayed swallow triggering. Interferential current stimulation applied to the neck is thought to improve the swallowing function of dysphagic patients, although the mechanism underlying the facilitatory effect of such stimulation remains unknown. In the present study, we examined the changes in the elicitability of swallowing due to the stimulation and the responses of the swallowing-related neurons in the nucleus tractus solitarius and in the area adjacent to the stimulation in decerebrate and paralyzed guinea pigs. The swallowing delay time was shortened by the stimulation, whereas the facilitatory effect of eliciting swallowing was attenuated by kainic acid injection into the nucleus tractus solitarius. Approximately half of the swallowing-related neurons responded to the stimulation. These data suggest that the interferential current stimulation applied to the neck could enhance the sensory afferent pathway of the pharynx and larynx, subserving excitatory inputs to the neurons of the swallowing pattern generator, thereby facilitating the swallowing reflex.

The medullary vagal motor nuclei, the nucleus ambiguus (NA) and dorsal motor nucleus of the vagus (DMV), innervate the respiratory and gastrointestinal tracts. We conducted immunohistochemical analysis of expression of the androgen receptor (AR) and estrogen receptor α (ERα), in relation to innervation of the trachea and esophagus via vagal motor nuclei in mice. AR and ERα were expressed in the rostral NA and in part of the DMV. Tracing experiments using cholera toxin B subunit demonstrated that neurons of vagal motor nuclei that innervate the trachea and esophagus express AR and ERα. There was no difference in expression of sex steroid hormone receptors between trachea- and esophagus-innervating neurons. These results suggest that sex steroid hormones may act on vagal motor nuclei via their receptors, thereby regulating functions of the trachea and esophagus.

The retrotrapezoid/parafacial respiratory group (RTN/pFRG) located ventral to the facial nucleus plays a key role in regulating breathing, especially enhanced expiratory activity during hypercapnic conditions. To clarify the roles of the RTN/pFRG region in evoking coughing, during which reflexive enhanced expiration is produced, and in swallowing, during which the expiratory activity is consistently halted, we recorded extracellular activity from RTN/pFRG neurons during these fictive behaviors in decerebrate, paralyzed, and artificially ventilated guinea pigs. The activity of the majority of recorded respiratory neurons was changed in synchrony with coughing and swallowing. To further evaluate the contribution of RTN/pFRG neurons to these nonrespiratory behaviors, the motor output patterns during breathing, coughing, and swallowing were compared before and after brain stem transection at the caudal margin of RTN/pFRG region. In addition, the effects of transection at its rostral margin were also investigated to evaluate pontine contribution to these behaviors. During respiration, transection at the rostral margin attenuated the postinspiratory activity of the recurrent laryngeal nerve. Meanwhile, the late expiratory activity of the abdominal nerve was abolished after caudal transection. The caudal transection also decreased the amplitude of the coughing-related abdominal nerve discharge but did not abolish the activity. Swallowing could be elicited even after the caudal end transection. These findings raise the prospect that the RTN/pFRG contributes to expiratory regulation during normal respiration, although this region is not an essential element of the neuronal networks involved in coughing and swallowing., (Copyright © 2015 the American Physiological Society.)

There is accumulating evidence that fibroblasts are target cells for steroids such as sex hormones and corticoids. The characteristics of fibroblasts vary among tissues and organs. Our aim in this study is to examine differences in responses to steroid hormones among fibroblasts from different cervicothoracic regions. We compared the actions of steroid hormones on cultured fibroblasts from the vocal folds, which are considered to be the primary target of steroid hormones, and the trachea and esophagus in adult male rats. Expression of steroid hormone receptors (androgen receptor, estrogen receptor α, and glucocorticoid receptor) was identified by immunofluorescence histochemistry. Androgen receptor was much more frequently expressed in fibroblasts from the vocal fold than in those from the trachea and esophagus. Cell proliferation analysis showed that administration of testosterone, estradiol, or corticosterone suppressed growth of all 3 types of fibroblasts. However, mRNA expression for extracellular matrix-associated genes, including procollagen I and III and elastin, and hyaluronic acid synthase I was elevated only by addition of testosterone to fibroblasts from the vocal fold. These results indicate that each steroid hormone exerts region-specific effects on cervicothoracic fibroblasts with different properties through binding to specific receptors.

Because there is no absolute indicator of the nutritional status and prognosis in patients with severe aspiration problems, it is quite difficult to arrive at a true long-time prognosis. By performing surgery for intractable aspiration on such patients, both the prognosis and QOL of the patients could be expected to improve. In our department, we have experienced patients dying within 6 months after surgery. In these cases, the patient's preoperative nutritional status was not good. Therefore, we consider that, when we adopt this procedure, there should be some indicators we should use which could have an effect on the prognosis of such nutritionally-challenged patients. In patients who underwent surgery for intractable aspiration; we examined the relationship between their survival and the prognostic nutritional index (PNI) which is an indicator of the risk of complications such as post-operative events in the surgical field. We investigated the relationship between the prognosis and the postoperative indicators of each of the following: WBC, CRP, serum albumin level, and PNI. Out of a total of 31 cases, the average O-PNI of eight cases in which death occurred was 29.45, and the average of six cases in which death occurred within 6 months after surgery was 28.26. The average O-PNI of the survivors was 36.01. A significant association was noted between the early postoperative deaths and some of the four indicators namely that serum albumin level and O-PNI. Based on the ROC curve, the O-PNI offered higher precision than the albumin level. The cut-off value of the O-PNI value for early postoperative mortality rate was 32. The early postoperative mortality rate was 44.4% in patients with less than 32 O-PNI in the preoperative examination, but if it were O-PNI 32 or more, the early postoperative mortality rate was 9.1%, significantly lower. Therefore, O-PNI could be useful as one of the prognostic evaluation factors in the case of preoperative surgery for intractable aspiration. Based on the O-PNI score, it was possible to evaluate the survival benefit associated with this operative procedure. We showed a treatment algorithm based on the preoperative O-PNI value. We believe there is a necessity to develop preoperative effective nutritional therapy as a future issue.

To examine the relationship between the neuronal networks underlying respiration and non-respiratory behaviors such as vocalization and airway defensive reflexes, we compared the activity of respiratory neurons in the ventrolateral medulla during breathing with that during non-respiratory behaviors including vocalization, swallowing, and coughing in guinea pigs. During fictive vocalization the activity of augmenting expiratory neurons ceased, whereas the other types of expiratory neurons did not show a consistent tendency of increasing or decreasing activity. All inspiratory neurons discharged in synchrony with the phrenic nerve activity. Most of the phase-spanning neurons were activated throughout the vocal phase. During fictive swallowing, many expiratory and inspiratory neurons were silent, whereas many phase-spanning neurons were activated. During fictive coughing, many expiratory neurons were activated during the expiratory phase of coughing. Most inspiratory neurons discharged in parallel with the phrenic nerve activity during coughing. Many phase-spanning neurons were activated during the expiratory phase of coughing. These findings indicate that the medullary respiratory neurons help shape respiratory muscle nerve activity not only during breathing but also during these non-respiratory behaviors, and thus suggest that at least some of the respiratory neurons are shared among the neuronal circuits underlying the generation of breathing and non-respiratory behaviors., (Copyright © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)