The Regenerative Effects of c-Met Agonistic Antibodies in Vocal Fold Atrophy.

BACKGROUND: Atrophy of the vocal folds and the accompanying glottic insufficiency affect the quality of life. Although growth factors have been used to treat muscle atrophy, their effectiveness is limited by their short half-life. METHODS: In total, 15 rabbits and 24 rats were used for the study. The right recurrent laryngeal nerves of all animals were transected. One month following nerve transection, PBS (PBS group), rHGF (HGF group), or a c-Met agonistic antibody (c-Met group) was injected into the paralyzed vocal folds. The larynges of the rabbits were harvested from each group for histologic examination and subjected to PCR analysis. RESULTS: Cross-sectional areas (CSAs) of thyroarytenoid muscles were evaluated. The c-Met group had increased CSAs compared to the PBS and HGF groups, but there were no significant differences compared to normal controls. The expression levels of myogenesis-related genes were evaluated three weeks after the injection. The expression levels of myosin heavy chain IIa were significantly increased in the PBS group, while the expression levels of MyoD were increased in the c-Met group. CONCLUSIONS: The c-Met agonistic antibody showed promise for promoting muscle regeneration in a vocal fold palsy model.

Incidence and Significance of Hypermetabolic PET-CT Findings in Unilateral TVF Motion Impairment.

OBJECTIVE: To determine the incidence and significance of asymmetric hypermetabolic laryngeal findings on positron emission tomography-computed tomography (PET-CT) in patients with unilateral true vocal fold (TVF) motion abnormalities. STUDY DESIGN: Retrospective cohort. SETTING: Single-center tertiary care institution. SUBJECTS AND METHODS: The medical records of patients with unilateral TVF motion abnormalities were reviewed. The incidence of normal and asymmetric hypermetabolic laryngeal findings was calculated in patients who underwent PET-CT and laryngeal examination, operative laryngoscopy with biopsy, or injection medialization laryngoplasty. RESULTS: A total of 135 patients with unilateral TVF motion abnormalities underwent PET-CT. After exclusion of patients who completed new or surveillance imaging for a laryngeal neoplasm (n = 27), asymmetric hypermetabolic findings in the larynx were noted in 21 (19%) cases: 13 (12%) on the contralateral side of the impaired TVF, 8 (7%) on the ipsilateral side. Two (25%) patients with ipsilateral hypermetabolism had concerning subsequent fiberoptic laryngeal examinations prompting operative biopsy. There was no evidence of inflammatory or neoplastic disease in all patients with contralateral hypermetabolic findings. Fifteen patients completed PET-CT scans after injection medialization procedures; 6 (40%) displayed avidity ipsilateral to the side of the injection. The median time from injection to scan was 27 days, as opposed to 193 days in the unremarkable scans (P = .011). CONCLUSION: Contralateral hypermetabolism in patients with unilateral TVF motion abnormalities may represent a false-positive finding. Ipsilateral hypermetabolic uptake without recent fold instrumentation warrants prompt diagnostic evaluation.

Transition of myosin heavy chain isoforms in human laryngeal abductors following denervation.

The objective of this study was to investigate the myofiber subtype transition of human posterior cricoarytenoid (PCA) muscle after the injury to recurrent laryngeal nerve (RLN). PCA muscle specimens were obtained from 38 bilateral vocal fold paralysis patients underwent arytenoidectomy. According to the duration of RLN injury, all the cases were divided into five denervation groups: 6-12 months, 1-2, 2-3, 3-6, and >6 years. The normal PCA muscles from total laryngectomy patients were chosen as controls. Immunofluorescence was adopted to detect the expression level of myosin heavy chain (MHC)-I and MHC-II in PCA muscle. Quantitative real-time PCR was also used to assess the transcriptional level of MHC subtypes (MHC-I, MHC-IIa, MHC-IIb, MHC-IIx, embryonic-MHC, and peri-natal-MHC). Immunofluorescence showed that MHC-I-positive myofibers in denervation groups were much lower than control group, respectively, while MHC-II-positive myofibers were significantly higher than control group (P < 0.05). With the extension of denervation, the number of MHC-I-positive myofibers gradually decreased, while MHC-II gradually increased and peaked in 1- to 2-year group. Transcriptional level of MHC-I, MHC-IIa, and MHC-IIb in denervation groups significantly down-regulated compared with the control (P < 0.05), respectively. However, MHC-IIx, embryonic-MHC, and peri-natal-MHC significantly up-regulated in all denervation groups, and the highest level was in 1- to 2-year denervation group. Data from the present study demonstrated that the maximum transition of MHC subtypes in human PCA muscles occurred in 1-2 years after denervation, suggesting that laryngeal reinnervation before the occurrence of irreversible transition of MHC subtypes could maintain the structural integrity of laryngeal PCA muscles.

Histological growth pattern of and alpha-actinin-4 expression in thyroid cancer.

AIM: To assess the clinicopathological significance of the histological growth pattern (HGP) and α-actinin-4 (ACTN4) expression in thyroid cancer. PATIENTS AND METHODS: We classified 83 thyroid cancer cases into infiltrative margin (IM) and pushing margin (PM) groups according to peripheral tumor margin contour and immunohistochemically determined ACTN4 expression. Correlations between clinical stage and clinicopathological characteristics were analyzed. RESULTS: IM and high ACTN4 expression were observed in 39% and 49% of cancer cases, respectively. Higher clinical stage was significantly correlated with older age, higher T and N factor, preoperative recurrent laryngeal nerve paralysis (pre-RLNP), IM, and poor prognosis. Patients with stage IV disease had significantly poorer prognosis than those with stages I-III. On multivariate analysis, older age, pre-RLNP, and IM correlated with higher clinical stages. IM was significantly correlated with high ACTN4 expression. CONCLUSION: IM, pre-RLNP, and ACTN4 expression could be novel indicators of tumor aggression and prognostic factors of thyroid cancer.

Functional modulation of satellite cells in long-term denervated human laryngeal muscle.

OBJECTIVES/HYPOTHESIS: To evaluate the effects of long-term denervation on satellite cells (SCs) as myogenic stem cells in human posterior cricoarytenoid (PCA) muscle. STUDY DESIGN: Histological investigation of SCs and quantitative assessment of myoD and myogenin, which are two key myogenic regulatory factors. METHODS: According to the course of denervation, denervated PCA muscles of 58 patients who suffered from traumatic unilateral vocal cord paralysis were divided into four groups: group A (6-12 months, 15 cases), group B (13-24 months, 17 cases), group C (25-36 months, 14 cases), and group D (more than 36 months, 12 cases). Normal PCA muscles (12 cases) were used as a control group. Immunofluorescence labeling was used to visualize the SCs. Transcription and protein expression levels of myoD and myogenin were assessed using real-time quantitative polymerase chain reaction and Western blots, respectively. RESULTS: MyoD- and myogenin-positive cells and embryonic myosin heavy chain-positive myofibers were detected in group A and most of the samples of group B. Transcription levels of myoD and myogenin were highly upregulated in groups A and B, whereas groups C and D showed no significant difference as compared to control. Protein expression levels of myoD and myogenin peaked in group A, which was significantly different than group B. In contrast, no expression was observed in the other groups. CONCLUSIONS: Activated SCs contribute to regenerative myogenesis in denervated laryngeal muscles through compensatory mechanisms. Laryngeal muscles exhibit persistent regenerative potential from the viewpoint of muscle SCs after less than 2 years of denervation.

Unilateral vocal fold paralysis in premature infants after ligation of patent ductus arteriosus: vascular clip versus suture ligature.

OBJECTIVES: We investigated risk factors associated with unilateral iatrogenic vocal fold paralysis (IVFP) in the context of ligation of patent ductus arteriosus (PDA) and compared the rates of paralysis between vascular clip and suture ligation procedures. METHODS: We performed a prospective examination of infants with isolated PDA treated surgically during 1995 to 2005. Statistical significance was determined with a 2-tailed t-test. RESULTS: Of 68 PDA ligations, 13 cases of left-sided IVFP were diagnosed, for an overall incidence of 19%. All cases of IVFP occurred in 55 infants who weighed less than 1 kg at birth. Suture ligature was used in 60% of all PDA ligation patients, and vascular clips in 40%. The incidence of IVFP in patients with vascular clips (19%) was similar to the incidence in those with suture ligature (20%). Hoarseness or stridor was present in 69% of patients with IVFP, compared to 17% of normal controls (p <0.001). The rate of aspiration was not increased in the IVFP group; however, 15% of the patients with IVFP had episodes of decreased oxygen saturation, versus 7% of infants with normal vocal fold mobility. CONCLUSIONS: A hoarse infant with a birth weight of less than 1 kg who has undergone PDA ligation should be examined for unilateral IVFP. Vascular clips and suture ligature are associated with similar rates of IVFP.

Neuroprotection using gene therapy to induce vascular endothelial growth factor-A expression.

Engineered zinc-finger protein (ZFP) transcription factors induce the expression of endogenous genes and can be remotely delivered using adenoviral vectors. One such factor, Ad-32Ep65-Flag (Ad-p65), targets and induces expression of vascular endothelial growth factor (VEGF; also called VEGF-A) splice variants in their normal biological stoichiometry. We show that Ad-p65 transfection of primary motor neurons results in VEGF variant expression and a significant increase in axon outgrowth in these cells. Given the neuroprotective effects of VEGF and its ability to increase neurite outgrowth, we examined the efficacy of Ad-p65 to enhance motor neuron regeneration in vivo using rats that have undergone recurrent laryngeal nerve (RLN)-crush injury. Injection of Ad-p65 after RLN crush accelerated the return of vocal fold mobility and the percentage of nerve-endplate contacts in the thyroarytenoid muscle. Overall, adenoviral delivery of an engineered ZFP transcription factor inducing VEGF-A splice variant expression enhances nerve regeneration. ZFP transcription factor gene therapy to increase expression of the full complement of VEGF-A splice variants is a promising avenue for the treatment of nerve injury and neurodegeneration.

Novel mutations in the GDAP1 gene in patients affected with early-onset axonal Charcot-Marie-Tooth type 4A.

We report a detailed study of eight patients from four Italian families presenting with autosomal recessive axonal Charcot-Marie-Tooth disease (AR-CMT2), characterized by early-onset and progressive severe weakness of all limbs. Vocal cord paresis was present in two cases. Sural nerve biopsy performed in three patients showed a severe neuropathy characterized by a predominant axonal involvement. Five novel mutations (p.Gln99stop, p.Gln122Lys, p.Arg125stop, p.Val219Asp, p.Asn297Lys) and one previously reported mutation (p.Leu239Phe) were identified in GDAP1 gene. GDAP1 mutations should be considered both in recessive and sporadic cases of early-onset axonal CMT.

Dysfunction of the recurrent laryngeal nerve and the potential of gene therapy.

Injury to the recurrent laryngeal nerve causes vocal fold paresis or paralysis resulting in poor voice quality, and possibly swallowing dysfunction and/or airway compromise. Injury can occur as part of a neurodegenerative disease process or can be due to direct nerve trauma or tumor invasion. Management depends upon symptoms, the cause and severity of injury, and the prognosis for recovery of nerve function. Surgical treatment techniques can improve symptoms, but do not restore physiologic motion. Gene therapy may be a useful adjunct to enhance nerve regeneration in the setting of neurodegenerative disease or trauma. Remote injection of viral vectors into the recurrent laryngeal nerve is the least invasive way to deliver neurotrophic factors to the nerve's cell bodies within the nucleus ambiguus, and in turn to promote nerve regeneration and enhance both nuclear and nerve survival. The purpose of this review is to discuss the potential role for gene therapy in treatment of the unsolved problem of vocal fold paralysis.

Adenoviral GDNF gene transfer enhances neurofunctional recovery after recurrent laryngeal nerve injury.

To assess the possibility of gene therapy for recurrent laryngeal nerve (RLN) injury, we examined functional and histological recovery after glial cell line-derived neurotrophic factor (GDNF) gene transfer in a rat RLN crush model. Adenoviral vector encoding beta-galactosidase gene (AxCALacZ) or human GDNF gene (AxCAhGDNF) was injected into the crush site of the RLN. Neurons in the nucleus ambiguus on the crushed side were labeled with X-gal or GDNF immnohistochemistry after AxCALacZ or AxCAhGDNF injection. Reverse transcription-polymerase chain reaction analysis revealed expression of human GDNF mRNA transcripts in brainstem tissue containing the nucleus ambiguus on the crushed side after AxCAhGDNF injection. Animals injected with AxCAhGDNF displayed significantly improved motor nerve conduction velocity of the RLN and recovery rate of vocal fold movement at 2 and 4 weeks after treatment as compared to controls. AxCAhGDNF-injected animals showed a significantly larger axonal diameter and improved remyelination in crushed RLN as compared to controls. Adenoviral GDNF gene transfer may thus promote laryngeal function recovery after RLN injury. Inoculation of adenoviral vector containing the GDNF gene at the site of damage soon after nerve injury may assist patients with laryngeal paralysis caused by nerve injury during head and neck surgery.

Vocal cord abnormalities in Williams syndrome: a further manifestation of elastin deficiency.

Williams syndrome (WS) is due to a deletion in the WS critical region at 7q11.23 which includes the elastin gene (ELN). One of the most characteristic features of this disorder is a harsh, brassy, or hoarse voice but the etiology of the vocal characteristics are unknown. We report two patients with WS who had bilateral vocal cord abnormalities, bringing to four the number of children with WS in whom such defects have been documented. We suggest that vocal cord abnormalities may be a far more common feature of WS than has been previously suspected, and that mild vocal cord dysfunction caused by abnormal vocal cord elastin may be the cause of the hoarse voice in this condition.

Laryngeal reinnervation with the hypoglossal nerve. I. Physiology, histochemistry, electromyography, and retrograde labeling in a canine model.

This study was performed to determine whether the hypoglossal nerve (cranial nerve XI [XII]) would serve as a useful donor for laryngeal reinnervation by anastomosis to the recurrent laryngeal nerve (RLN). Twenty hemilarynges in 10 dogs were studied prospectively after XII-RLN anastomosis (group A; n = 5), split XII-RLN anastomosis (group B; n = 3), XII-RLN anastomosis with a 2-cm interposition graft (group C; n = 2), no treatment (group D; n = 5), RLN section (group E; n = 2), or ansa cervicalis-RLN anastomosis (group F; n = 3). Spontaneous activity was observed monthly by infraglottic examination through permanent tracheostomies and was recorded by electromyography. Laryngeal adductory pressure and induced phonation were obtained by stimulating the RLN while passing a pressure transducer balloon or humidified air through the glottis. At sacrifice, the laryngeal muscles were stained for adenosine triphosphatase to determine the ratio of type I to type II fibers. Retrograde labeling of the brain stem was performed with horseradish peroxidase. Infraglottic examination at 6 months showed a full range of adductory motion in groups A and B during the swallow reflex, comparable with that in group D. Groups C and F showed good bulk and tone, but little spontaneous motion. Group E remained paralyzed. Stimulation of the transferred nerves caused more activity in groups A and B than in the other groups; groups C and F partially adducted at high levels. The laryngeal adductory pressure responses of groups A and B were similar to those of group D. The XII-reinnervated larynges were capable of producing normal induced phonation. Retrograde labeling of the RLN showed that the reinnervating axons originated only in the hypoglossal nucleus. Electromyography of the reinnervated adductor muscles confirmed spontaneous activity in the dogs (awake). Histochemical analysis confirmed slow-to-fast transformation of both the posterior and lateral cricoarytenoid muscles, indicating that significant reinnervation occurred. We conclude that the hypoglossal nerve functions well as a donor for adductory reinnervation of the larynx.

Expression of fibroblast growth factor-2 in the nucleus ambiguus following recurrent laryngeal nerve injury in the rat.

OBJECTIVES: To examine fibroblast growth factor-2 (FGF-2) immunoreactivity in the nucleus ambiguus (NA) after three different recurrent laryngeal nerve (RLN) injuries. STUDY DESIGN: Immunohistochemical analysis of FGF-2. METHODS: Thirty adult rats underwent left-sided RLN crush (group A). The left RLN was transected in groups B (n = 30) and C (n = 30); in group C, both nerve stumps were covered with silicone caps. FGF-2 in the NA was assessed as the ratio of the positive areas on the left (operated [O]) and right (unoperated [U]) sides. The ratio (O/U) was measured 1, 3, 7, 14, and 28 days after the procedure. Three rats underwent left-sided RLN exposure and were killed 7 days later (control). RESULTS: Left-sided RLN paralysis occurred until day 28 in group A. In the control group, O/U was approximately 1. In group A, O/U was significantly elevated on day 7; in group B, on days 3, 7, and 14; and in group C, on day 3. O/U in group B was significantly greater than that in group A on days 14 and 28. Maximal FGF-2 immunoreactivity was significantly lower in group C than in groups A and B. CONCLUSIONS: We demonstrated elevated production of FGF-2 in the NA after RLN injury. This endogenous FGF-2 might contribute to preventing lesion-induced neuronal death. Blockage of axonal regeneration might suppress FGF-2 production in the NA. Further understanding of the roles of FGF-2 after RLN injury may contribute to the prevention of neuronal death and facilitation of axonal regeneration.

Myosin heavy chain composition in rat laryngeal muscles after denervation.

OBJECTIVES: The effects of denervation on myosin heavy chain (MHC) expression in specific laryngeal muscles are characterized using gel electrophoresis. Observed temporal changes in MHC composition will then be used as a biologic marker in studies designed to develop strategies for laryngeal reinnervation and gene therapy. STUDY DESIGN: Animal study using an adult rat model for laryngeal paralysis. METHODS: In anesthetized rats the left recurrent and superior laryngeal nerve were divided. Animals were survived for 7, 14, 28, 90, and 180 days. Animals were euthanized and the thyroarytenoid (TA), vocalis (VOC), posterior cricoarytenoid (PCA), lateral cricoarytenoid (LCA), and cricothyroid (CT) muscle excised. Each muscle was processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometric measurements were obtained to determine composition of MHC fiber types. RESULTS: The changes in relative MHC composition are described for each specific laryngeal muscle. In general, a decrease in type IIB and an increase in IIA and IIX are seen after denervation. Expression of IIL in the denervated condition is variable and the relative change in type I is minimal. CONCLUSION: This study supports previous work using rat soleus muscle in which IIA/IIX expression is favored in conditions with decreased neuromuscular activity, and conversely, IIB expression is activity dependent. Expression of type I appears to be independent of neural activity. Further study will be undertaken to quantify expression of MHC components and to study factors modulating expression.

Cardiorespiratory and metabolic responses to exercise in horses with various abnormalities of the upper respiratory tract.

A standardised incremental exercise test was performed by 9 racehorses with idiopathic laryngeal hemiplegia (ILH), 1 horse with maxillary sinus cysts, 1 horse with epiglottic entrapment, 1 horse with a lesion on the vocal folds, and 1 horse with pharyngitis. Two of the horses with ILH were retested after laryngoplasty and ventriculectomy. The findings were compared with those from 20 normal racehorses. Heart rate, plasma lactate concentration, arterial blood gases, stride frequency, oxygen uptake (VO2) and carbon dioxide production were assessed during treadmill exercise on a +10% slope. The group of horses with ILH had significantly (P < 0.01) lower peak VO2 values (136 +/- 5 ml/kg/min) than did the normal group (154 +/- 3 ml/klg/min). These values represent mean +/- sem. Horses with ILH also had significantly higher (P < 0.05) arterial carbon dioxide tensions (PaCO2) at 10 m/s and lower speeds at a heart rate of 200 bpm (V200) than the normal group. The horse with maxillary sinus cysts had higher PaCO2 tension at 10 m/s than normal, and abnormal values for several cardiorespiratory and metabolic indices. Horses with vocal fold lesions, aryepiglottic entrapment and pharyngitis had arterial blood gas and cardiorespiratory indices that were similar to those of normal horses. One horse which underwent corrective surgery for ILH showed improvements in arterial blood gases and cardiorespiratory indices during exercise, while the other horse had values which were the same as, or worse than, values before surgery. We conclude that the measurement of arterial blood gases and cardiorespiratory indices during treadmill exercise is useful in determining the effect on exercise capacity of various upper airway abnormalities in racehorses.

Costal diaphragmatic O2 and lactate extraction in laryngeal hemiplegic ponies during exercise.

Diaphragmatic O2 and lactate extraction were examined in seven healthy ponies during maximal exercise (ME) carried out without, as well as with, inspiratory resistive breathing. Arterial and diaphragmatic venous blood were sampled simultaneously at rest and at 30-s intervals during the 4 min of ME. Experiments were carried out before and after left laryngeal hemiplegia (LH) was produced. During ME, normal ponies exhibited hypocapnia, hemoconcentration, and a decrease in arterial PO2 (PaO2) with insignificant change in O2 saturation. In LH ponies, PaO2 and O2 saturation decreased well below that in normal ponies, but because of higher hemoglobin concentration, arterial O2 content exceeded that in normal ponies. Because of their high PaCO2 during ME, acidosis was more pronounced in LH animals despite similar lactate values. Diaphragmatic venous PO2 and O2 saturation decreased with ME to 15.5 +/- 0.9 Torr and 18 +/- 0.5%, respectively, at 120 s of exercise in normal ponies. In LH ponies, corresponding values were significantly less: 12.4 +/- 1.3 Torr and 15.5 +/- 0.7% at 120 s and 9.8 +/- 1.4 Torr and 14.3 +/- 0.6% at 240 s of ME. Mean phrenic O2 extraction plateaued at 81 and 83% in normal and LH animals, respectively. Significant differences in lactate concentration between arterial and phrenic-venous blood were not observed during ME. It is concluded that PO2 and O2 saturation in the phrenic-venous blood of normal ponies do not reach their lowest possible values even during ME. Also, the healthy equine diaphragm, even with the added stress of inspiratory resistive breathing, did not engage in net lactate production.