Characterization of submucosal lesions using optical coherence tomography in the rabbit subglottis

OBJECTIVE: To evaluate the efficacy of optical coherence tomography in differentiating between several simulated subglottic lesions, using an ex vivo rabbit laryngotracheal model. DESIGN: Laryngotracheal complexes were harvested from euthanized rabbits and divided into the following 4 groups: (1) control, (2) submucosal collagen injection (simulating scar formation), (3) dehydration and rehydration (simulating edema), and (4) repeated intubation trauma. The subglottic region was imaged using optical coherence tomography. Images were later correlated with conventional histologic findings. RESULTS: The epithelium, basement membrane, lamina propria, perichondrium, and cartilage (cricoid and tracheal) were clearly imaged. In group 2, an increase in the thickness of the lamina propria was observed, in addition to a characteristic optical pattern of the injected collagen. Dehydration (in group 3) produced a visible reduction in the thickness of the lamina propria, while rehydration of the same specimen with distilled water revealed a significant increase in submucosal swelling. Repeated intubation (in group 4) resulted in tissue edema that was seen as wavy heterogeneous thickening of the lamina propria. Edema produced by repeated intubation or distilled water immersion was easily differentiated from native and collagen-injected tissues. CONCLUSION: Optical coherence tomography successfully identifies the microstructure layers of the subglottis and can differentiate between edema and increased collagen deposition in the rabbit model.     

Subglottic wound healing in a new rabbit model of acquired subglottic stenosis

OBJECTIVES: It is difficult to develop models of subglottic stenosis in small animals that ensure reliable stenosis. We therefore sought to establish a new animal model of subglottic stenosis in rabbits and observe subglottic wound healing. METHODS: Using a diode laser under endoscopic visualization after anterior tracheal incision, we induced full-thickness injury to the subglottis in 60 rabbits: 20 on the anterior 120 degrees, 20 on the posterior 120 degrees, and 20 circumferentially. The animals were painlessly sacrificed 2 or 4 weeks later, and their gross and histologic findings were compared with those of each other and with those of 16 age-matched nonwounded controls. RESULTS: Of the 60 animals, 22 died, primarily of acute airway obstruction and mostly in the circumferential group. All of the injured rabbits showed subglottic stenosis compared with controls, ranging from 12% to 56% reduction in cross-sectional area in the 120 degrees injured groups and from 32% to 82% reduction in the 360 degrees injured group. The significant narrowing of the subglottic lumina resulted in substantial early mortality in the circumferential group. Histologic examination showed mucosal ulceration, inflammation, and formation of granulation tissues during the acute phase after wounding, and collapse of the injured cartilage and submucosal thickening and fibrosis at later times. CONCLUSIONS: Subglottic healing and stenosis depend on the extent of cartilaginous injury. This model may be useful for developing methods to treat subglottic stenosis.     

Microdebrider resection of bilateral subglottic cysts in a pre-term infant: a novel approach

Acquired subglottic cysts are a rare cause of stridor in infants. The two major risk factors for development of these cysts are prematurity and history of intubation. Microlaryngeal decompression and carbon dioxide laser resection of these cysts have been the most common treatment methods with recurrence rates as high as 43% [J. Lim, W. Hellier, J. Harcourt, S. Leighton, D. Albert, Subglottic cysts: the Great Ormond Street experience, Int. J. Pediatr. Otorhinolaryngol. 67 (2003) 461-465]. Carbon dioxide laser therapy also carries the risk of airway fire, injury to adjacent structures, and possible delayed scarring. We present a case of bilateral subglottic cysts in a premature infant with progressive stridor, treated using a microdebrider, and review the literature regarding the treatment of these lesions.     

Hylan B gel restores structure and function to laser-ablated canine vocal folds

OBJECTIVES: We evaluated cross-linked hyaluronic acid (hylan B gel) as a scaffold for tissue regeneration and mucosal wave restoration in carbon dioxide laser-ablated canine vocal folds. METHODS: Five beagles underwent stroboscopy before ablation of the left vocal fold with a carbon dioxide laser. Four weeks later, stroboscopy was repeated before and after submucosal injection of hylan B gel into the left vocal fold of 4 animals and of saline solution in 1 animal. Stroboscopy was repeated 12 weeks later, and histologic analysis was performed. RESULTS: Four weeks after laser ablation, all animals had soft tissue defects and absence of mucosal waves. Hylan B injection restored mucosal waves, and saline injection did not. Twelve weeks after injection, hylan B-injected larynges had tissue regeneration and mucosal waves, and the saline-injected larynx had neither. Histology showed regenerated lamina propria with residual foci of hylan B in the hylan B-injected larynges and dense submucosal scar in the saline-injected animal. CONCLUSIONS: Submucosal hylan B gel injection in laser-ablated canine vocal folds restored tissue volume and mucosal waves and facilitated functional tissue regeneration over 12 weeks. Hylan B gel may have utility as a soft tissue scaffold for rehabilitation of phonatory function in vocal folds with lamina propria defects.     

人羊膜上皮细胞移植用于兔损伤声带组织修复的初步研究

目的 研究人羊膜上皮细胞(human amniotic epithelial cells,hAEC)移植入兔声带损伤组织内的生长分布特点,探索hAEC促进声带损伤后修复再生的潜能.方法 分离和培养hAEC,慢病毒增强型绿色荧光蛋白(1entivirus enhanced green fluorescent protein,Lenti-EGFP)基因转染作标记.建立深及声韧带的兔声带损伤模型,设hAEC移植组(13侧声带)、损伤对照组(13侧声带)及正常对照组(4侧声带).荧光显微镜下连续观察hAEC在声带内的存活、分布情况,应用HE染色和免疫组化染色分析胶原、纤维连接蛋白等主要细胞外基质在损伤后3个月时的含量及分布.结果 hAEC原代培养6 d后呈铺路石样生长,植入声带损伤组织后可在固有层内持续存活2个月,细胞呈纵向排列,有趋向性.声带损伤2个月时免疫荧光显示hAEC移植组兔肌细胞标志结蛋白荧光阳性,提示hAEC向肌细胞分化;同时Ⅲ型胶原荧光阳性,提示hAEC植入后具有分泌Ⅲ型胶原功能.光镜观察见hAEC移植后3个月兔胶原纤维密度和排序较损伤对照组改善,但未及正常;免疫组化染色示hAEC移植组纤维连接蛋白含量和分布介于损伤对照组和正常对照组之间.结论 hAEC可在异种动物声带损伤组织内持续存活、生长,并有向声带组织分化和分泌部分细胞外基质的潜能,可能促进声带损伤后的修复再生.     

人羊膜上皮细胞移植用于兔损伤声带组织修复的初步研究

目的 研究人羊膜上皮细胞(human amniotic epithelial cells,hAEC)移植入兔声带损伤组织内的生长分布特点,探索hAEC促进声带损伤后修复再生的潜能.方法 分离和培养hAEC,慢病毒增强型绿色荧光蛋白(1entivirus enhanced green fluorescent protein,Lenti-EGFP)基因转染作标记.建立深及声韧带的兔声带损伤模型,设hAEC移植组(13侧声带)、损伤对照组(13侧声带)及正常对照组(4侧声带).荧光显微镜下连续观察hAEC在声带内的存活、分布情况,应用HE染色和免疫组化染色分析胶原、纤维连接蛋白等主要细胞外基质在损伤后3个月时的含量及分布.结果 hAEC原代培养6 d后呈铺路石样生长,植入声带损伤组织后可在固有层内持续存活2个月,细胞呈纵向排列,有趋向性.声带损伤2个月时免疫荧光显示hAEC移植组兔肌细胞标志结蛋白荧光阳性,提示hAEC向肌细胞分化;同时Ⅲ型胶原荧光阳性,提示hAEC植入后具有分泌Ⅲ型胶原功能.光镜观察见hAEC移植后3个月兔胶原纤维密度和排序较损伤对照组改善,但未及正常;免疫组化染色示hAEC移植组纤维连接蛋白含量和分布介于损伤对照组和正常对照组之间.结论 hAEC可在异种动物声带损伤组织内持续存活、生长,并有向声带组织分化和分泌部分细胞外基质的潜能,可能促进声带损伤后的修复再生.

Abstract：

Objective To investigate the survival, growth and distribution of human amniotic epithelial cells (hAEC) after injected into injured rabbit vocal folds, in addition, to assess the ability of hAEC to affect the components of lamina propria extracellular matrix (ECM) and prevent vocal fold scarring.Methods hAEC were isolated from human amnion and marked by Lenti-EGFP. Fifteen New Zealand rabbits were used for this experiment. EGFP-hAEC was injected into the left injured vocal folds in thirteen rabbits, and the contralateral thirteen vocal folds experienced an injured procedure only (" injured untreated control"), and four vocal folds were left as untreated controls. The survival, distribution, differentiation potential and secretion function of hAEC were examined by immunofluorescence method. HE staining and immunohistochemical staining were performed for the evaluation of collagen and fibronectin respectively.Results hAEC showed a cobblestone-like growth. After implanted into the injured vocal folds, hAEC could survive in vocal fold lamina propria for 2 months. The immunofluorescence analysis showed the evidence of hAEC differentiation into muscle cells as well as secretion the ECM protein. Three months postoperatively, the density of collagen was higher in the injured untreated control folds than that in the injured vocal folds injected with hAEC and the untreated controls. Besides, the content of fibronectin in the injured untreated control group was significantly increased. Conclusions hAEC survived in the vocal folds lamina propria,and had the potentiality to differentiate into vocal folds tissue and secret some ECM components. The histological improvement caused by the injected cells demonstrate that hAEC had the ability to promote the repairment and regeneration of injured vocal folds.     

Subglottic cysts in the premature infant

Supraglottic cysts in the newborn are a well-recognized entity, but subglottic cysts have been rarely reported. Over the past 6 years we have observed subglottic cysts in nine patients with relatively long intubations from the neonatal intensive care unit of two university hospitals. Most frequently the patients were extubated and did well for weeks or months, but then they had progressive biphasic stridor. On endoscopy the patients had a subglottic stenosis that was irregular but with a smooth mucosal lining. Usually the cysts were apparent, but in two patients the mucosa was thickened and the patients were treated as a subglottic stenosis with tracheostomy. These subglottic cysts were recognized at the time of laryngotracheoplasty. In six patients the cysts were managed either by marsupialization with cup forceps, endoscopic diathermy, or carbon dioxide laser without recurrence. It is our belief that this condition is most likely due to scarring and obstruction of mucus glands of the subglottic area from prolonged intubation. This entity should be recognized and looked for in the neonate who has an acquired subglottic stenosis and should first be treated conservatively with endoscopic marsupialization.     

A comparison of the carbon dioxide and neodymium: YAG lasers in a canine model of acquired subglottic stenosis

In a previously defined canine model of subglottic stenosis, two different wavelength lasers--the carbon dioxide (CO2) and the neodymium-yttrium aluminum garnet (Nd:YAG)--were used as the principal surgical therapy, and the results were compared. Two lathyrogenic agents, colchicine and penicillamine, were also used in a subset of each treatment group in order to determine their effect as an adjunct to laser therapy in the treatment of subglottic stenosis. The CO2 laser treatment group showed an overall improvement; the Nd:YAG laser treatment group showed a worsening of subglottic stenosis. The dogs treated with the lathyrogenic agents had no added benefit from the use of these agents.     

Viscoelastic measurements after vocal fold scarring in rabbits--short-term results after hyaluronan injection

CONCLUSIONS: The scarring model resulted in significant damage and elevated viscoelasticity of the lamina propria. Hyaluronan preparations may alter viscoelasticity in scarred rabbit vocal folds. OBJECTIVES: Vocal fold scarring results in stiffness of the lamina propria and severe voice problems. The aims of this study were to examine the degree of scarring achieved in the experiment and to measure the viscoelastic properties after injection of hyaluronan in rabbit vocal folds. MATERIALS AND METHODS: Twenty-two vocal folds from 15 New Zealand rabbits were scarred, 8 vocal folds were controls. After 8 weeks 12 of the scarred vocal folds received injections with 2 types of cross-linked hyaluronan products and 10 scarred folds were injected with saline. After 11 more weeks the animals were sacrificed. After dissection, 15 vocal folds were frozen for viscoelastic measurements, whereas 14 vocal folds were prepared and stained. Measurements were made of the lamina propria thickness. Viscoelasticity was measured on intact vocal folds with a linear skin rheometer (LSR) adapted to laryngeal measurements. RESULTS: Measurements on the digitized slides showed a thickened lamina propria in the scarred samples as compared with the normal vocal folds (p<0.05). The viscoelastic analysis showed a tendency to stiffening of the scarred vocal folds as compared with the normal controls (p=0.05). There was large variation in stiffness between the two injected hyaluronan products.     

Prevention of vocal fold scarring by topical injection of hepatocyte growth factor in a rabbit model

OBJECTIVES/HYPOTHESIS: Vocal fold scarring disrupts the layer structure of the vocal fold lamina propria that is essential for optimal mucosal vibration. Prevention of vocal fold scarring remains challenging. Hepatocyte growth factor (HGF) has strong antifibrotic activity. The authors' previous studies have found that HGF stimulates hyaluronic acid production and suppresses collagen production from vocal fold fibroblasts, suggesting that HGF has therapeutic potential in prevention of vocal fold scarring. The present study aimed to demonstrate the effects of HGF on vocal fold scarring in an in vivo rabbit model. STUDY DESIGN: Animal experiment. METHODS: The vocal fold mucosa was stripped unilaterally in 20 rabbits, then HGF or saline (sham-treated group) was immediately injected into the injured site. At 6 months after the procedure, histological, rheological, and physiological examinations of vibratory behavior were completed. RESULTS: Histological examination revealed excessive collagen deposition and disorganized elastin in the sham-treated group, whereas the HGF-treated group presented with better wound healing exhibiting less collagen deposition. Contraction of the injured vocal folds observed in the sham-treated group did not occur in the HGF-treated group. Rheological data indicated that the HGF-treated vocal folds were less stiff and viscous compared with the sham-treated group. Mucosal vibration of HGF-treated vocal folds appeared much better than the sham-treated group in terms of phonation threshold pressure, vocal efficiency, mucosal wave amplitude, and glottal closure. CONCLUSION: Hepatocyte growth factor proved to be useful in preventing vocal fold scarring and maintaining viscoelastic shear properties of the vocal fold.     

Treatment of vocal fold scarring: rheological and histological measures of homologous collagen matrix

The current treatment options for dysphonia secondary to vocal fold scarring are limited. Few studies address changes in the lamina propria, which is critical to vocal fold biomechanical properties and voice production. Using rheological and histological measures of homologous collagen matrix (HCM)-injected vocal folds, we assessed HCM's potential for providing bulk and restoring biomechanical performance. Twenty rabbits underwent bilateral vocal fold scarring. After 10 weeks of scar maturation, the rabbits had unilateral injections of HCM or saline solution. Ten weeks after the injections, histological studies revealed well-defined collagen globules distributed throughout the lamina propria and underlying muscular tissue. Significantly more procollagen was observed in the HCM-treated group. No significant differences in elastic shear modulus or dynamic viscosity were found between the treatment groups. This study demonstrates that HCM is an inert, relatively stable injectate that may serve well for medialization but does not appear to improve the dynamic properties of the lamina propria.     

Can Mitomycin C Really Prevent Airway Stenosis?

BACKGROUND/OBJECTIVES: Mitomycin C (MMC) is used in the prevention of upper airway stenosis. However, the efficacy of MMC is still debatable, and the potential complications of MMC use have been scarcely reported. We evaluated the efficacy and early complications of MMC in a wound model of the subglottis. STUDY DESIGN: A randomized animal study. METHODS: Sixty rabbits underwent a full-thickness injury to the posterior subglottis by diode laser. The animals were randomly assigned to three groups according to the different topical treatments of MMC 0.4 or 10 mg/mL or saline alone for 5 minutes. Animals were killed at 4 weeks, and gross and histologic findings were compared among different groups and 10 age-matched, non-wound, normal controls. RESULTS: Thirty-two of the 60 (53%) animals died mainly of acute airway obstruction by necrotic debris, sloughs on, or cartilage collapse of the unhealed posterior subglottis during early weeks after wounding, higher in the MMC-treated animals (67%) than in the wound controls (25%) (P = .007). This resulted from the significant delay of wound healing in the MMC-treated groups compared with the wound controls (P = .012). The degree of subglottic stenosis was comparable among different groups (P > .8), although collagen deposition was significantly lower in the MMC-treated groups compared with the untreated controls (P = .036). CONCLUSIONS: There was a significant risk of acute airway obstruction from delayed wound healing in rabbits that received subglottic laser wounding and MMC treatment. This risk should be considered in the use of MMC in clinical practice.     

Proximal large-bore jet ventilation for laryngeal laser surgery

The carbon dioxide surgical laser has made many open upper airway operations obsolete. However, new laser-related hazards have been encountered, the most serious of which is fire in the airway. Venturi jet ventilation removes that risk and provides an unobstructed surgical field. Indications and techniques for the use of jet ventilation with carbon dioxide laser surgery remain controversial, particularly whether jet ventilation should be used in papillomatosis or in the severely obstructed airway, and whether proximal and distal catheter techniques are equally safe and effective. We report the successful use of proximal large-bore Venturi jet ventilation in 327 consecutive patients undergoing microlaryngeal laser surgery. The technique is ideal for pediatric patients with subglottic stenosis or recurrent respiratory papillomas and has not caused distal seeding in the latter group. We recommend the use of low-pressure ventilation, even in relatively obstructed airways. The experimental evidence reported here corroborates the clinical viewpoint expressed; the technique is safe, simple, and uncomplicated if used with care.     

Hepatocyte growth factor and its receptor c-met in rat and rabbit vocal folds

Vocal fold fibrotic scar is characterized by fibrosis of the lamina propria and epithelium, and is difficult to treat. Hepatocyte growth factor (HGF) has antifibrotic activity and has received attention as a possible therapeutic alternative to treat fibrosis. In this study, in order to clarify whether HGF can be involved in vocal fold scarring, we examined the existence of HGF and its receptor, c-Met, in rat vocal folds, and then the activity of HGF in rabbit injured vocal folds, using immunohistochemistry and enzyme-linked immunosorbent assay. We found HGF and c-Met on epithelial cells and gland cells of the rat vocal folds. On the injured vocal folds of rabbits, little HGF was observed immediately after injury, but prominent activity occurred simultaneously with reepithelialization of the vocal fold mucosa on days 10 to 15. The activity of HGF was observed on fibroblasts in the lamina propria, as well as the epithelium. It is suggested that HGF in the vocal folds is produced by the fibroblasts and delivered to the epithelium. The implication of these findings is that HGF is involved in wound healing of the vocal fold, and may provide an alternative approach in preventing and treating vocal fold scarring.     

Fibroblast transplantation in the airway: implications for subglottic stenosis

OBJECTIVE: Because subglottic stenosis (SGS) represents one of the most challenging pathologies confronting the pediatric otolaryngologist, our laboratory is investigating the role fibroblasts play in mucosal scar formation in the course of SGS development. Our objective is to establish cell transplantation into the subglottic mucosal wound bed as a viable tool for examining the cellular processes that underlie the development of SGS. DESIGN: A series of 2 animal experiments, with animals assigned to a control, vehicle-only, or cell-treated group. SETTING: John G. Rangos Sr Research Center, Children's Hospital of Pittsburgh, Pittsburgh, Pa. SUBJECTS: Twenty-six New Zealand white rabbits. This animal model has been well established in the study of SGS formation. INTERVENTIONS: Fluorescently labeled exogenous fibroblasts were transplanted into the injured subglottis of the rabbits. RESULTS: Exogenous fibroblasts derived from fetal and adult dermis and subglottic mucosa were successfully transplanted into the injured subglottic mucosa of adult rabbits. Transplanted fibroblasts survived into the latter stages of wound healing (at 14 and 21 days) and appeared to be associated with a mild inflammatory cell influx and active remodeling of the mucosal wound bed. CONCLUSION: Cell transplantation is a viable tool for the study of fibroblast activity in the mucosal wound bed.     

Imaging the mucosa of the human vocal fold with optical coherence tomography

OBJECTIVES: Discerning the layered microstructure of the vocal folds is critical for effective phonomicrosurgery. Optical coherence tomography (OCT), a noncontact, noninvasive technology that provides cross-sectional images by means of backscattered light, offers the potential for delineating these layers in vivo. METHODS: The glottal mucosa of 3 human cadaver larynges was imaged with conventional OCT and polarization-sensitive OCT (PS-OCT). Images were obtained through the epithelium and lamina propria. RESULTS: Although the superficial layer of the lamina propria appeared quite homogeneous, the outer surface of the superficial lamina propria was correlated with an increase in backscatter with OCT. The superficial lamina propria and vocal ligament were correlated with a marked increase in tissue birefringence with PS-OCT. CONCLUSIONS: This preliminary study demonstrates the capacity of OCT and PS-OCT for visualizing the layered microstructure of the vocal fold mucosa. We believe that these imaging techniques will have applications in the exploration of solutions to vocal fold scarring and in imaging vocal fold disorders in the clinic and operating room.     

Congenital subglottic hemangiomas: a treatment update

Congenital subglottic hemangiomas are rare lesions associated with a significant degree of morbidity and mortality. Although various methods of treatment have been proposed, no one single modality has yet been shown to be uniformly effective in treating this life-threatening condition. Amongst the myriad of suggested treatments, the carbon dioxide laser, as originally proposed by Healy, et al., appears to have the highest degree of success with the lowest incidence of complications. The purpose of this paper is to review a large experience at a single institution with the use of the carbon dioxide laser for treatment of congenital subglottic hemangiomas.     

Avoiding intubation in the injured subglottis: the role of heliox therapy

Intubation in the child presenting with severe viral tracheobronchitis or prior subglottic injury can be detrimental to the child and the subglottis. Intubation may lead to further mucosal ischemia, scar, subglottic stenosis, or failed extubation requiring a tracheotomy. Heliox is a combination of helium and oxygen that produces less-dense gas exchange. Its use leads to a decrease in turbulent airflow, which may obviate the need for intubation. Here we report our experience using heliox as initial therapy in 14 consecutive children presenting with severe airway distress and the need for intubation. (Five had viral tracheobronchitis, 5 had inflammatory exacerbation of subglottic stenosis, and 4 had acute iatrogenic subglottic injury.) In 10 of the 14 children, intubation, which can lead to mucosal injury and scarring, was avoided by the use of heliox therapy. Of the 4 children in whom heliox therapy failed, 3 had a prior history of subglottic stenosis. Heliox is a relatively safe and reliable alternative to intubation of children with severe subglottic edema or injury. Heliox should be considered before intubation for selected children with subglottic inflammation and severe airway distress.     

Efficacy of bronchoscopic carbon dioxide laser surgery for benign strictures of the trachea

There have been conflicting reports in our literature concerning the efficacy of bronchoscopic carbon dioxide laser surgery for the treatment of benign strictures of the trachea. We have examined our experience in the management of this disease over a 2 1/2-year period; in all cases, our initial management was performed utilizing the rigid, ventilating bronchoscope with the universal endoscopic coupler and carbon dioxide laser. Eight of 14 patients were successfully managed in this study; retrospective analysis of our results revealed that the presence of one or more of the following four factors was extremely important in predicting an unfavorable prognosis of patients with tracheal stenosis managed endoscopically with the carbon dioxide laser: 1. loss of cartilaginous support; 2. stenosis length greater than 1 cm; 3. circumferential scarring; and 1. carinal involvement.     

Laryngotracheal injury following cricothyroidotomy

Airway complications following elective cricothyroidotomy were reviewed in 48 adult cardiothoracic surgery patients. A 52% incidence of airway complications was found and manifested by failed or delayed decannulation, extensive subglottic granulation tissue, stenosis, vocal cord paralysis, and aspiration pneumonia. The most common cause for decannulation difficulty was subglottic stenosis (50%). Several risk factors were specifically identified, including a period of cricothyroid cannulation exceeding 30 days, the presence of diabetes, and advanced age. These findings suggest that airway sequelae following cricothyroidotomy in cardiothoracic surgery patients is higher than previously reported. Indications and risk factors for cricothyroidotomy are discussed.