Reinnervation of motor endplates and increased muscle fiber size after human insulin-like growth factor I gene transfer into the paralyzed larynx.

Current surgical strategies for the treatment of laryngeal paralysis are limited by the muscle atrophy associated with denervation. Moreover, attempts at reinnervation have not effected significant change in surgical outcome. To address this clinical problem, we have developed a rat laryngeal paralysis model to study novel gene transfer strategies. Using this model, the human insulin-like growth factor I (hIGF-I) gene was introduced into paralyzed rat laryngeal muscle to assess the benefit of sustained local hIGF-I production. A muscle-specific nonviral vector containing the alpha-actin promoter and hIGF-I gene was used in formulation with a polyvinyl-based delivery system and injected into paralyzed adult rat laryngeal muscle. Twenty-eight days after a single injection, gene transfer efficiency, muscle fiber size, motor endplate length, and nerve-to-motor endplate contact were evaluated. Gene transfer was detected in 100% of injected animals by PCR. Gene transfer with expression, as measured by RT-PCR for hIGF-I mRNA, occurred in 81.3 % of injected animals. When compared with controls, hIGF-I-transfected animals presented a significant increase in muscle fiber diameter [17.56 (+/-0.97 SD) microm versus 14.70 (+/-1.43 SD) microm; p = 0.0002], a significant decrease in motor endplate length [20.88 (+/-1.42 SD) microm versus 25.41 (+/-3.19 SD) microm; p = 0.0025], and a significant increase in percentage of endplates with nerve contact (20.3% (+/-13.9 SD) versus 4.4% (+/-4.2 SD); p = 0.0079). In the context of laryngeal paralysis, gene therapy represents a tremendous opportunity to augment current surgical treatment modalities by preventing or reversing muscle atrophy, and by enhancing nerve sprouting and reinnervation.

Expression of extraocular-superfast-myosin heavy chain in rat laryngeal muscles.

Superfast myosin heavy chain (MHC), which is found in jaw-closing muscle and extraocular muscle (EOM), may also be found in rat laryngeal muscles. Immunostaining and Western blot using anti-EOM antibody were performed to identify and localize EOM MHC in laryngeal muscles. Specific reactivity of laryngeal IIL MHC was confirmed by Western blot and on immunostaining, all fibers in the lateral part of thyroarytenoid muscle reacted with EOM antibody. A scattered pattern of positive fibers was observed in the medial part of the thyroarytenoid, the posterior cricoarytenoid and the lateral cricoarytenoid muscles. EOM MHC was not detected in the cricothyroid muscle. The expression of EOM MHC in rat laryngeal muscle is consistent with the functional demands of the airway protection reflex.

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.

Myosin heavy chain composition in human laryngeal muscles.

OBJECTIVES: Myosin heavy chain (MHC) composition of human thyroarytenoid (TA), lateral cricoarytenoid (LCA), interarytenoid (IA), vocalis, posterior cricoarytenoid (PCA), and cricothyroid muscles were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western bolt techniques. The presence of superfast MHC was also assessed using antibodies directed against the extraocular MHC. STUDY DESIGN: MHC protein was analyzed using fresh human laryngeal muscles. METHODS: Laryngeal muscles excised from cadavers were processed for SDS-PAGE. The composition of MHC isoforms was determined by densitometry. Western blot was carried out to identify specific bands. RESULTS: MHC types IIA and IIB are the predominant MHC components in human laryngeal muscles. The adductor muscles--TA, LCA, and IA--have a higher percentage of type IIB MHC and a lower percentage of type I when compared with the abductor--PCA. The rank file order for type IIB MHC composition (TA > LCA > or = IA > PCA) is the same in all specimens. A band migrating between type IIA and type I was observed in several specimens. Although similar to type IIL in rats, this atypical band did not react with anti-extraocular MHC antibody on Western blot. CONCLUSION: Characterization of laryngeal muscles determined by the composition of MHC is correlated with function and neural input. Human laryngeal muscle is characterized by a predominance of fast-type MHCs in laryngeal closing muscle and mixed fast-slow type MHCs in respiratory and phonatory muscle groups. Although an atypical myosin band similar to type IIL (superfast) MHC in rat was identified, it did not react with anti-extraocular MHC antibody.

Modulation of myosin heavy chains in rat laryngeal muscle.

OBJECTIVES: To test the hypothesis that myosin heavy chain (MHC) composition is a biological marker indicative of appropriate and functional reinnervation. STUDY DESIGN: Age-matched adult rats were randomized for prospective study under three experimental conditions. METHODS: In adult rats, three experimental conditions were surgically created, including transient recurrent laryngeal nerve (RLN) crush injury, RLN transection and repair, and cricoarytenoid joint fixation with intact RLN. Animals were survived for 30, 90, and 180 days. At each interval, vocal fold mobility was assessed by rigid microlaryngoscopy. Laryngeal electromyography (EMG) was performed before euthanasia. The thyroarytenoid and posterior cricoarytenoid muscles were then excised, each muscle was processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and MHC composition was determined. RESULTS: Thirty days after nerve crush injury, three of six animals regained vocal fold mobility and normal MHC composition. Impaired vocal fold motion in three of six animals was associated with MHC composition characteristic of denervation. At 90 and 180 days, normal vocal fold motion and normal MHC composition were observed in all animals. Following nerve transection and repair, impaired vocal fold motion and MHC composition characteristic of denervation were observed in all animals, despite evidence of reinnervation on EMG. Following joint fixation, alteration in MHC composition consistent with denervation was observed only at 30 days, as was evident in the nerve crush model. CONCLUSION: Temporary injury and vocal fold immobilization result in transient shifts in MHC composition. Nerve transection and repair result in persistent alteration of MHC composition and vocal fold dysfunction. The expression of normal MHC composition is dependent on the condition of appropriate neural contact and functional reinnervation.

Three-dimensional reconstruction of the feline larynx with serial histologic sections.

This paper reviews a new technique to develop high-resolution three-dimensional (3-D) images of the larynx using histological sections. Three-dimensional computer-reconstructed histological sections of the cat are used in this study to evaluate the recurrent laryngeal nerve (RLN) in its true anatomic course, with emphasis on its relationship to surrounding structures (laryngeal framework). A cat model was used because of specimen availability and technical ease of tissue preparation. Computer-reconstructed serial histologic sections add a new dimension to the study of laryngeal anatomy, laryngeal trauma, and growth patterns of laryngeal cancer. The technical aspects of three-dimensional reconstruction and future applications are discussed.

The sublabial transseptal transsphenoidal approach to sellar and parasellar lesions.

Multiple surgical approaches to the sella turcica have been described. The sublabial transseptal transsphenoidal approach provides a safe, cosmetically acceptable route to the sella with excellent exposure. Since 1976, we have employed this technique for the surgical treatment of pituitary neoplasms and parasellar tumors. The University of Washington experience with the sublabial transseptal transsphenoidal approach to the sella in a series of 253 patients is reported with respect to the otolaryngological aspects of the surgical technique, results, and complications of the procedure. In addition, a literature review of major studies that used this procedure is included, and the reported results and complications are compared to those of the present study.

IGF-1 gene transfer into denervated rat laryngeal muscle.

OBJECTIVES: To demonstrate gene transfer in rat laryngeal muscle using a reporter gene, beta-galactosidase, and a muscle-specific expression system containing the human IGF-1 (hIGF-1) gene sequence and to investigate the myotrophic and neurotrophic effects of hIGF-1 gene transfer in denervated rat laryngeal muscle. METHODS: In 8 adult rats, a polyvinyl-based formulation containing beta-galactosidase DNA was injected into denervated thyroarytenoid muscle. Twelve animals were similarly administered a polyvinyl-based formulation containing a muscle-specific expression system and hIGF-1 DNA. Twelve animals were injected with isotonic sodium chloride solution, and all animals survived for 1 month. The production of beta-galactosidase and hIGF-1 was detected using immunohistochemical techniques. The effects of hIGF-1 on motor endplates and nerve sprouting were assessed using cholinesterase or silver staining and immunostaining for growth-associated protein (GAP-43). mals by immunostaining, X-gal histochemical staining, or both. In frozen section specimens, hIGF-1 immunoreactivity was positive in 3 of 8 animals. In sequential sections, GAP-43 was localized to areas of hIGF-1 expression in 2 of the 3 hIGF-1-positive specimens. Increased nerve sprouting and motor endplate contact occurred in 2 of 4 animals treated with hIGF-1. CONCLUSIONS: Gene transfer into laryngeal muscle was demonstrated using a polyvinyl-based formulation containing a muscle-specific gene expression system. Preliminary findings indicate a positive effect on motor endplates, nerve sprouting, and the expression of GAP-43 in animals treated with the hIGF-1 vector. This study establishes a foundation for investigating hIGF-1 gene transfer as a novel treatment of laryngeal paralysis. Further studies are necessary to quantify myotrophic and neurotrophic effects and to establish therapeutic benefit.

Diagnosis and treatment of obstructive sleep apnea of the larynx.

To determine the mechanism for obstructive sleep apnea in two patients with clinical abnormalities of laryngeal function, airflow dynamics during sleep were analyzed. The site of airway obstruction was assessed by examining pressure gradients across specific airway segments. The relation between maximal inspiratory airflow and nasal pressure was analyzed to determine (1) the critical pressure, a measure of the collapsibility of the laryngeal airway, and (2) the effect of nasal continuous positive airway pressure on airflow during sleep. Large inspiratory pressure gradients developed during sleep between the supraglottic and pleural spaces, indicating that collapse had occurred in the larynx. Elevated critical pressures of -6.4 and +1.2 cm H2O, respectively, occurred in the two patients. When the nasal pressure was raised to 10 cm H2O, normal levels of tidal airflow occurred, and obstructive apneas were eliminated. These findings indicate that sleep apnea was caused by laryngeal airflow obstruction that resulted from elevations in the collapsibility of the larynx. The response to nasal continuous positive airway pressure suggested that laryngeal sleep apnea was similar to pharyngeal sleep apnea in pathophysiologic characteristic and response to treatment.

Human insulinlike growth factor 1 gene transfer into paralyzed rat larynx: single vs multiple injection.

OBJECTIVE: To compare the biological effects of single vs multiple treatment of rat denervated laryngeal muscle with human insulinlike growth factor 1 (hIGF1) gene therapy. EXPERIMENTAL METHODS OR DESIGN: A muscle-specific nonviral vector containing the alpha-actin promoter and hIGF1 gene formulated with polyvinyl polymers was injected into denervated adult rat thyroarytenoid muscle. The effects on animals given a single injection (n = 16) vs those given multiple injections (n = 14) vs control groups (n = 18) were evaluated. Twenty-eight days after the first injection, gene expression, muscle fiber size, motor endplate length, and nerve-to-motor endplate contact were evaluated. RESULTS: Gene expression, detected by reverse transcriptase polymerase chain reaction for hIGF1 messenger RNA, occurred in 13 (81%) of 16 animals receiving single injections and 14 (100%) of 14 animals receiving multiple injections. Compared with controls, hIGF1-transfected animals in both single- and multiple-injection groups had a significant increase in the lesser diameter of muscle fiber, a significant decrease in motor endplate length, and a significant increase in the percentage of endplates with nerve contact (P <.05 for all). There was no statistical difference between single- and multiple-injection groups. CONCLUSIONS: Applied to laryngeal paralysis, hIGF1 gene therapy provides an opportunity to augment surgical treatment modalities by the prevention or reversal of muscle atrophy, and enhancement of nerve sprouting and muscle reinnervation. Although the percentage of denervated muscles demonstrating hIGF1 expression was increased following multiple injections, no difference was observed in the biological response compared with that in the single-injection treatment groups. Further investigation will be conducted to assess long-term benefits and physiological responses and to define the limitations of this potentially valuable therapeutic strategy.

Cell adhesion molecules in normal laryngeal development.

Larynges taken from normally developing rats ranging from embryos starting at embryonic day 10 to adults were harvested. Sections of the larynges were prepared with the use of immunohistochemical techniques with primary antibodies directed to the neural cell adhesion molecule; selected sections were exposed to antibodies directed against the embryonic polysialated form of the neural cell adhesion molecule and against L1. Cell adhesion molecules were present throughout embryonic development associated with the recurrent laryngeal nerve. Downregulation of expression was observed after postnatal day 14. We conclude that cell adhesion molecules may play a role in the normal neural development of the larynx in rats.

Pathophysiology and indications for medialization thyroplasty in patients with dysphagia and aspiration.

Medialization thyroplasty is generally considered a phonosurgical procedure for voice augmentation in patients with glottic insufficiency. This article addresses specifically the issue of dysphagia and aspiration in patients with laryngeal paralysis. A retrospective review of patients undergoing medialization thyroplasty is performed. From 1991 to 1995, 84 patients at The Johns Hopkins Medical Institutions underwent medialization thyroplasty for unilateral vocal fold motion impairment. At presentation 48 patients had isolated recurrent laryngeal nerve injury, 26 with combined superior laryngeal nerve/recurrent laryngeal nerve injury and 10 with idiopathic nerve injury. Sixty-one percent of patients had swallowing difficulties. The severity of symptoms is greater in the superior laryngeal nerve/recurrent laryngeal nerve group. Before surgery 13 patients were dependent on feeding tubes. Nine patients improved to the point at which all alimentation was taken by mouth and tube feedings were discontinued after medialization thyroplasty. One patient was subsequently converted to a full oral diet after cricopharyngeal myotomy. Three patients remained dependent on feeding tubes. The pathophysiology of dysphagia including clinical and experimental observations is reviewed. In addition, the nonsurgical and surgical approaches to treatment of patients with laryngeal paralysis are reviewed.

Quest for the aberrant vessel.

In most cases of posterior epistaxis, the terminal branches of the internal maxillary artery (IMA)--principally the sphenopalatine artery--are believed to be the source of hemorrhage. However, we recently treated three patients in which unexpected or aberrant vascular anatomy resulted in persistent posterior epistaxis.

Necrotizing fasciitis of the eyelids.

Necrotizing fasciitis is a destructive soft tissue infection that rarely involves the eyelids. Three cases of necrotizing fasciitis of the eyelids are described. Necrotizing fasciitis was preceded by minor forehead soft tissue trauma in two cases and occurred spontaneously in one. In two patients necrotizing fasciitis was bilateral and involved both the upper and lower eyelids. Review of these cases, in addition to 18 cases previously reported in the English literature, reveals a predominance in females, preceding minor local soft tissue trauma, frequent bilateral involvement, and an association with alcohol abuse and diabetes. In all of the patients, group A beta-hemolytic streptococci were cultured from the wound. Early recognition of the disease process, prompt surgical debridement of the necrotic tissue, aggressive antimicrobial therapy, and delayed skin grafting combine to minimize morbidity.

Craniocervical necrotizing fasciitis.

Craniocervical necrotizing fasciitis (CCNF) is a severe, progressive bacterial infection of the cervical fascia. The most significant manifestations of this disease are extensive fascial necrosis with widespread undermining of the surrounding tissues and extreme systemic toxicity. We are adding three cases to the 29 previously reported cases of CCNF. We will discuss the regional anatomy, focusing on the relationship of the cervical fascial planes to vital structures. Available data suggest that CCNF is a synergistic infection produced in most instances by a combination of facultative anaerobic and obligate anaerobic organisms. Although antimicrobial therapy should provide broad-spectrum activity against mixed flora, treatment also includes aggressive excision and debridement of involved fascia, subcutaneous tissue, and necrotic skin.

Postnatal development of myosin heavy chain isoforms in rat laryngeal muscles.

The developmental transitions of myosin heavy chain (MHC) isoforms of rat posterior cricoarytenoid (PCA), thyroarytenoid (TA), cricothyroid (CT), and lateral cricoarytenoid (LCA) muscles were examined by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot techniques. The muscles were microscopically dissected from animals on postnatal days 0, 3, 7, 10, 14, 21, 28, 35, 45, and 55 and from adult animals. Silver-stained SDS-PAGE gels of each muscle were analyzed densitometrically to measure the composition of MHC isoforms, and Western blot was carried out to identify specific bands. Characterizations of the internal laryngeal muscles determined by the composition of MHCs were correlated with their function in the adult. Temporally, differentiation reflects onset of function. Differentiation of isoforms and transition to adult forms occur first in the TA muscle, followed by the PCA, LCA, and CT muscles. Expression of type IIL was observed only in muscles innervated by the recurrent laryngeal nerve. Postnatally observed developmental differences of myosin phenotypes suggest that regulation of MHC expression is influenced by neural activity or other environmental factors.

Hydroxylapatite laryngeal implants for medialization. Preliminary report.

Laryngeal implantation for medialization has improved our ability to manage the patient with vocal fold motion impairment. We present preliminary data evaluating the use of preformed hydroxylapatite laryngeal implants and instrumentation for rapid determination of implant size and position. A window in the thyroid ala is created by means of a standard fenestra template. One of 5 prosthesis templates is inserted through the window for determination of correct size and position. The corresponding implant is then inserted and secured with a hydroxylapatite shim. Thirty-five patients have been implanted for vocal fold paralysis, and 4 patients were implanted for soft tissue deficits or bowing. Thirty-one of 35 patients have reported subjective improvement (89%). Improvement was demonstrated in 13 of 15 (87%) patients with complete preoperative and postoperative objective voice function measurements. Complications include 1 implant extrusion and 1 case of airway obstruction secondary to edema. Preliminary results indicate that prefabricated hydroxylapatite implants are effective for medialization thyroplasty. Advantages include a readily available implant selection, rapid determination of correct size and position, and improved implant stabilization with a hydroxylapatite shim.

Correlation between stroboscopy and electromyography in laryngeal paralysis.

Twenty patients with vocal fold motion impairment were reviewed to correlate the findings of electromyography (EMG) and stroboscopy. The causes of motion impairment were idiopathic, previous surgery with recurrent laryngeal nerve injury, neck and skull base trauma, and neoplasm. The EMG studies were analyzed to assess the status of innervation of the immobile vocal fold. The presence or absence of the mucosal wave prior to therapeutic intervention was determined with stroboscopic examination. Eight of 10 patients with EMG evidence of reinnervation or partial denervation were found to have mucosal waves, and 3 of 10 patients with EMG evidence of denervation were found to have mucosal waves. Six patients developed mucosal waves after surgical medialization, despite evidence of denervation by EMG criteria. These findings support the premise that tension and subglottic pressure, rather than status of innervation, determine the presence of the mucosal wave.

Laryngeal synkinesis following reinnervation in the rat. Neuroanatomic and physiologic study using retrograde fluorescent tracers and electromyography.

The functional organization of laryngeal motoneurons in the nucleus ambiguous (NA) was evaluated in adult male rats before and after recurrent laryngeal nerve section and reinnervation. Using retrograde double labeling techniques with fluorescent probes, we obtained the number and position of labeled neurons by using the Bioquant 3-D imaging system. Reinnervation was documented by electromyography. In nine control animals vector analysis revealed significant (p less than .05) separation of the posterior cricoarytenoid (PCA) muscle motoneurons and the thyroarytenoid and lateral cricoarytenoid (TA/LCA) muscle motoneurons. The PCA motoneurons were positioned ventromedially in the NA, and TA/LCA motoneurons were found dorsolaterally in the NA. Rostral-caudal separation was not significant. Electromyography revealed phasic electrical activity synchronous with respiration in the PCA, and activity synchronous with deglutition in the TA/LCA. In four animals surviving 15 weeks following recurrent laryngeal nerve section and primary neurorrhaphy, functional organization within the NA was lost and phasic motor unit activity synchronous with respiration was seen in the TA/LCA muscle as well as the PCA. Vector analysis revealed the reinnervating motoneurons for both the PCA and TA/LCA to be positioned dorsolaterally, similar to the control group TA/LCA motoneurons. These findings demonstrate a shift in the topographic organization of laryngeal motoneurons within the NA following reinnervation, with random organization occurring at the neurorrhaphy site.

Comparison of soft tissue response in rabbits following laryngeal implantation with hydroxylapatite, silicone rubber, and Teflon.

This study evaluates the soft tissue response in rabbits following laryngeal implantation for medialization using hydroxylapatite prostheses, carved silicone rubber prostheses, and injectable Teflon. Sixteen rabbits underwent left recurrent laryngeal nerve section for denervation and laryngeal implantation with hydroxylapatite. At 1, 3, 6, and 12 months, 4 animals were painlessly sacrificed and processed for histology. Similarly, animals were implanted with carved silicone rubber prostheses or with Teflon injected through a flap in the thyroid lamina for comparison at 1, 3, and 6 months. In animals implanted with hydroxylapatite, histologic findings include limited acute inflammatory response, thin fibrous encapsulation, and osteogenesis in the region of the fenestra, with lamellar bone bridging the space between the implant and thyroid lamina. With silicone rubber prostheses, there is a limited inflammatory response and fibrous encapsulation of the implant without evidence of osteogenesis. Animals implanted with Teflon demonstrated a classic foreign body reaction with multinucleated giant cells, granuloma formation, and migration of Teflon into surrounding muscle. With respect to soft tissue response, both hydroxylapatite and silicone rubber are less reactive than Teflon. The osteogenesis observed in the presence of hydroxylapatite increases implant stability and minimizes the risk of migration. Conversely, the presence of bone growth may limit the reversibility of medialization procedures performed with hydroxylapatite.