Clinicopathological evaluation of intralaryngeal ultrasonography]

We used a miniature ultrasonic probe to study its diagnostic application in the detection of laryngeal lesions. Using the immersion method and an extracted human larynx, we evaluated ultrasonic images, identified the layered structure of vocal folds, and examined clinical cases following the study of diagnostic application. By the immersion method, ultrasonic images revealed that the membranous vocal fold was separated into three layers. We identified the histological structures of these three layers by comparing the ultrasonic images with the corresponding histologies. The first layer (hyperechoic) was the epithelial and the superficial layer of the lamina propria; the second layer (hypoechoic) was the vocal ligament; and the third layer (hyperechoic) was the deep layer of the lamina propria. On the basis of this study, we then evaluated ultrasonographic images of clinical cases. Benign lesions such as vocal nodules or vocal polyps were noted on the layered structure of the mucosa, especially, polyps as hyperechoic images. Furthermore, glottic cancer lesions demonstrated hypoechoic images and sometimes revealed destruction of the layered structures. These results suggest that intralaryngeal ultrasonography can be useful as a clinical application for the detection of tumor involvement in the vocal folds.     

Clinical assessment of intralaryngeal ultrasonography.

OBJECTIVES: To assess the clinical use of intralaryngeal ultrasonography. STUDY DESIGN: A retrospective study was conducted of 16 patients with laryngeal lesions treated by laryngomicrosurgery. METHODS: An intraluminal ultrasonic tomography apparatus was connected to a radial scanning 30-MHz miniaturized probe. Under general anesthesia, ultrasonic images were obtained using the filling method. RESULTS: In cases of benign disease, such as vocal cord nodules or polyps, the layers of the mucosa could be identified. Characteristic internal echoes were noted in cases of hemorrhagic polyps, vocal cord cysts, and vocal cord cancer. In case of hemorrhagic polyps, hyperechoic regions were noted within the lesions. In cases of vocal cord cysts, internal echoes were absent, and posterior echoes were mildly enhanced. In cases of vocal cord cancer, infiltration beyond the mucosa could be visualized. CONCLUSIONS: Laryngeal lesions can be diagnosed by intralaryngeal ultrasonography using the filling method. Although it does not replace the combination of conventional endoscopy and a critical evaluation of the clinical symptoms of the individual disease, it can profitably complement them. Intralaryngeal ultrasonography can help in determining the extent of tumor involvement during microscopic laser surgery performed under general anesthesia. Confirmation of the results of this pilot study with a larger series of patients is desirable.     

Distribution of collagen in the lamina propria of the human vocal fold

OBJECTIVES: To describe the arrangement of collagen fibers in the lamina propria of the human vocal fold. STUDY DESIGN: Analysis of the lamina propria of the vocal fold obtained from human cadavers. METHODS: The Picrosirius-polarization method was used to visualize collagen fibers. RESULTS: Analysis of 20 human vocal folds by the Picrosirius-polarization method permitted the visualization of two fiber populations arranged in three layers in the lamina propria: two layers of thick, strongly birefringent collagen fibers (collagen type I), one immediately below the epithelium and another more dense layer in the deep region superficially to the vocal muscle, penetrating between muscle fibers. The third layer consisted of fine, weakly birefringent fibers (collagen type III) located between the two layers of thick fibers. In addition, the collagen fibers in the lamina propria showed an intertwined network arrangement in the form of a "wicker basket." CONCLUSIONS: This basket-like configuration better explains how the vocal fold is able to stretch even though it contains nonstretchable fibers and to modulate the frequency of the voice under the action of the intrinsic musculature of the larynx. Segmental areas of disarray of the basket-like structure of the collagen layers were systematically observed in older patients. Thus, it is possible that vocal alterations occurring in the elderly might be the result of a loss of histoarchitectural arrangement of the collagen system and its relationship with the lamina propria and underlying musculature.     

Microendoscopy of Reinke's space

OBJECTIVES: Contemporary surgical treatment of the superficial layer of the lamina propria or Reinke's space is most commonly performed through an incision in the overlying vocal fold epithelium. This approach may disrupt normal tissue, induce scarring, and allow extrusion of implanted materials. Previously reported external approaches to Reinke's space required either a laryngofissure or a "minithyrotomy" for access. These surgical approaches were performed without direct imaging of Reinke's space. Instruments placed below the vocal fold epithelium via this external approach were visualized through the translucent vocal fold epithelium. We designed this study to identify the feasibility of limited-access surgery of the lamina propria using microendoscopes placed into Reinke's space through an external approach. METHODS: A cadaveric human larynx was dissected, and microendoscopes were directly advanced into Reinke's space through a subepithelial puncture of the cricothyroid membrane, as well as lateral fenestration through the thyroid cartilage. RESULTS: Photodocumentation of the undersurface of vocal fold epithelium, the opposing surface of the vocal ligament, and the intervening Reinke's space was successfully accomplished. CONCLUSIONS: Advances in both microendoscopes and accompanying instrumentation permit access to the superficial layer of the lamina propria without disrupting the overlying epithelium. This approach to microendoscopy of Reinke's space may allow for more effective surgical treatment of cysts, chronic edema, vascular abnormalities, atrophy, scarring, and sulcus vocalis.     

Imaging of the calf vocal fold with high-frequency ultrasound

Objectives/Hypothesis: High‐frequency ultrasound imaging offers the potential for assisting in the diagnosis and treatment of vocal fold pathology if it allows aspects of vocal fold microstructure to be visualized noninvasively. The objective of this study was to assess the ability of high‐frequency ultrasound to image vocal fold anatomy and injected biomaterials. Study Design: The vocal folds of two excised calf larynges were imaged ex vivo and compared with corresponding histological sections. Methods: High‐frequency ultrasound imaging was performed under saline submersion using 40 and 50 MHz transducers, and corresponding cryostat cross‐sections were stained with H&E, Trichome, and Verhoeff's Van Gieson stains. Results: The epithelial surface, lamina propria, and underlying muscle were easily identified with the high‐frequency ultrasound as verified with histological sections representing each imaged region. The arytenoid cartilage vocal process can also be clearly distinguished from the surrounding tissue, as can the full extent of injected biomaterials within the superficial lamina propria. Useful ultrasound resolution was obtained to depths of at least 10 mm within the tissue with the 40 MHz transducer. Conclusions: This preliminary study demonstrates the capability of high‐frequency ultrasound to image the layered anatomy of the calf vocal fold and to discern materials injected into the superficial lamina propria, indicating that this technology holds a strong potential for use in phonosurgery.     

Histological structure of vocal fold lamina propria in foetal larynx

Previous investigations of vocal fold lamina propria in adult human larynx have revealed a highly specific organization and distribution of the connective fibres. The authors tried to elicit the developmental characteristics of that process using a material of foetal larynxes, ranging in foetal age from 13 to 23 weeks. The specimens were embedded in paraffin and then serially sectioned in both horizontal and vertical plane. The specific technique of differentiating elastic fibres from collagen fibres was utilized. The distribution and organization of connective tissue in foetal vocal fold lamina propria differed from that in adults and no straight elastic fibres were demonstrated as the first sign of later differentiation.     

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.     

正常儿童喉部的超声解剖学研究

目的探讨正常儿童喉部的超声影像学表现。方法对照研究3个儿童离体喉标本相应断面的声像图与组织大切片,确认甲状软骨、杓状软骨、环状软骨、会厌软骨、会厌前间隙、声门旁间隙、室带、声带、喉室、杓肌的超声表现;然后对33名正常儿童进行喉部超声检查,确认各结构声像图表现。结果正常儿童甲状软骨为盾形等回声结构;杓状软骨为低回声结构,横断面为镰刀形,纵切面为类三角形;横断面环状软骨为"n"形低回声结构,纵切面显示环状软骨板为类长方形等回声结构;会厌软骨为细带状等回声结构;会厌前间隙和声门旁间隙是高回声结构;声带为长三角形等回声结构;室带为高回声结构。杓肌表现为双侧杓状软骨后方的等回声。结论超声对儿童喉部甲状软骨、杓状软骨、环状软骨、会厌软骨、会厌前间隙、声门旁间隙、室带、声带和杓肌显示良好。     

Ageing of the vibratory tissue of human vocal folds

Sixty-four human larynges ranging in age between 70 and 104 years were investigated histologically. The results were incorporated into our previous data for younger age groups. Discussion was focused on the mucosa around the vocal fold edge. The following tendencies were observed with ageing: (1) the membranous vocal fold shortens in males; (2) the mucosa thickens in females; (3) the cover of the vocal fold thickens in females; (4) edema develops in the superficial layer of the lamina propria in both sexes; (5) the intermediate layer of the lamina propria thins and its contour becomes deteriorated in males; (6) elastic fibers in the intermediate layer become less dense and atrophy in males; (7) the deep layer of the lamina propria thickens in males; (8) collagenous fibers in the deep layer become denser and fibrotic in males. The degree of these geriatric changes vary from individual to individual.     

Collagen fiber and versican distribution within the lamina propria of fetal vocal folds

OBJECTIVES: To analyze the presence and distribution of collagen fibers and versican in human vocal fold lamina propria of fetal larynges. STUDY DESIGN: Cross sectional analysis of cadaveric vocal folds of human fetuses. METHODS: Seven fetal larynges obtained from 28- to 36-week-old fetuses were analyzed with the Picrosirius-polarization method, immunohistochemistry, and image analysis. RESULTS: Collagen fibers within the lamina propria exhibited a monolaminar distribution pattern and spatial arrangement in "wicker basket." Versican distribution was larger in the superficial and intermediate layers when compared to the deep layer. CONCLUSION: Our findings suggest that collagen and versican distribution and arrangement within the lamina propria in the developing fetus are important for vocalization at birth.     

Comparative Histology and Vibration of the Vocal Folds: Implications for Experimental Studies in Microlaryngeal Surgery

Objectives/Hypothesis To determine the most suitable animal model for experimental studies on vocal fold surgery and function by a histological comparison of the microflap surgical plane and laryngeal videostroboscopy (LVS) in different species of animals. A second goal was to determine how the layered vocal fold structure in humans and three different animal species affects surgical dissection within the lamina propria. Study Design Prospective laboratory. Methods Three larynges each from dogs, monkeys, and pigs were compared with three ex vivo human larynges. Microflap surgery was performed on one vocal fold from each larynx. Both the operated and nonoperated vocal folds were examined histologically using stains specific for elastin, mature collagen, and ground substance. Based on the histological results, LVS was performed on two dogs and two pigs after first performing a tracheotomy for ventilation and airflow through the glottis. Arytenoid adduction sutures were placed to facilitate vocal fold adduction. Results The distributions of the collagen and elastin fibers were found to differ among the species with concentrations varying within species. Unlike the human vocal fold, which has a higher elastin concentration in the deeper layers of the lamina propria, both the pig and the dog had a thin band of elastin concentrated just deep to the basement membrane zone in the superficial layer. Just deep to this thin band, the collagen and the elastin were less concentrated. The monkey vocal fold had a very thin mucosal layer with less elastin throughout the mucosa. The microflap dissections in each of the dog, pig, and human vocal folds were similar, being located within that portion of the superficial lamina propria where the elastin and mature collagen are less concentrated. The microflap plane in the monkey vocal fold was more deeply located near the vocalis fibers. Despite the differences in elastin concentration, the microflap plane in both the dog and the pig was found to be similar to that in humans. The dog anatomy was much more suitable for microsuspension laryngoscopy and stroboscopic examination. The dog vocal folds vibrated in a similar fashion to human vocal folds with mucosal waves and vertical phase differences, features not seen in the pig vocal folds. Conclusions Based on both the histological and stroboscopic results, the dog was believed to be a more suitable animal model for studies on vocal fold surgery, acknowledging that no animal's laryngeal anatomy is identical to that of the human. The dog LVS model presented allows for longitudinal laryngeal studies requiring repeated examinations at multiple time periods with histological correlation applied at sacrifice.     

Does Hyaluronic Acid Distribution in the Larynx Relate to the Newborn's Capacity for Crying?

Objectives/Hypothesis: The newborn is heavily dependent on voice communication and therefore has relatively higher vocal demands and expenditures than the adult, the loudness output per mass performance exceeds that of the adult, and the newborn larynx exhibits significant histological and biochemical differences. The neonatal larynx is capable of sustaining relatively greater pitch and loudness than the adult over longer periods of time. This ability may be related to a more compact arrangement of collagen within the lamina propria, less interstitial space, and a uniform distribution of hyaluronic acid (HA). As HA is the primary determinant of vocal fold viscosity and water content, the distribution of HA in the superficial portion of the neonatal vocal fold is hypothesized to be related to newborn crying endurance. Our objective was to examine the histological structure and the quantity and arrangement of HA within the lamina propria of the pediatric larynx and review the relevant physiology of hyaluronic acid and its impact on voice production. Study Design: Histological and digital subtraction analysis. Methods: Intact, neonatal larynges were sourced from fresh cadaveric specimens. Trichrome stain was used to assess the collagen content and location in the tissues. HA was stained using a colloidal iron staining technique with and without incubation with bovine testicular hyaluronidase. Average optical density was calculated in tissue before and after treatment with hyaluronidase, and the stain intensity ratio was calculated. Results: A total of 14 larynges were suitable for examination, eight males and six females. Histological examination revealed a uniform appearance of the vocalfold without evidence of a distinct vocal ligament or layered structure. Colloidal iron staining revealed an even distribution of HA throughout the vocal fold with no significant difference between quadrants. Images of the colloidal iron-stained tissue had a mean pixel intensity of 82 of 255. Slides of vocal fold tissue treated with hyaluronidase revealed a pixel intensity of 106 of 255 for a 22% mean difference in stain intensity (P < .01). Conclusions: The identification of the layered structure of the adult lamina propria has raised a number of questions as to the development and purpose of the human larynx. Based on histological observations from the current study, possible explanations for the physiological differences include differences in the distribution and tissue concentration of HA and consequently dynamic viscosity, oncotic affinity for water, and less intercellular space in the superficial lamina propria.     

In vivo optical coherence tomography of the human larynx: normative and benign pathology in 82 patients

OBJECTIVES: Optical coherence tomography (OCT) is an emerging imaging modality that combines low-coherence light with interferometry to produce cross-sectional images of tissue with resolution about 10 mum. Patients undergoing surgical head and neck endoscopy were examined using a fiberoptic OCT imaging probe to study and characterize microstructural anatomy and features of the larynx and benign laryngeal pathology in vivo. STUDY DESIGN: Prospective clinical trial. MATERIALS AND METHODS: OCT imaging of the larynx was performed in 82 of 115 patients who underwent surgical endoscopy for various head and neck pathologies. The OCT device employs a 1.3 microm broadband light source (FWHM, 80 nm). The frame rate is 1 Hz. Imaging was performed using a handheld probe placed in near contact with the target site. The maximum axial and lateral dimensions for the region of interest imaged were 2.5 mm x 6 mm, with resolutions of 10 microm. Simultaneously, conventional endoscopic images were obtained to provide anatomic correlation with OCT images and histology. Optical micrometry was performed to measure the epithelium thickness. RESULTS: Systematic OCT imaging of laryngeal structures and subsites provided information on the thickness of the epithelium, integrity of the basement membrane, and structure of the lamina propria. Microstructural features identified included glands, ducts, blood vessels, fluid collection/edema, and the transitions between pseudostratified columnar and stratified squamous epithelium. The mean epithelial thickness of laryngeal subsites was calculated: true vocal cord (129 microm), false vocal cords (124 microm), aryepiglottic fold (177 microm), subglottis (98 microm), and epiglottis (185 microm). True vocal cord pathology imaged included Reinke's edema, papillomatosis, polyps, mucous cysts, and granulation tissue. Subglottic imaging identified boundaries between epithelium, lamina propria, and cartilage. The OCT images compared favorably with conventional histopathology. CONCLUSION: OCT has the unique ability to image laryngeal tissue microstructure and can detail microanatomic changes in benign, premalignant, and malignant laryngeal pathologies. OCT holds the potential to guide surgical biopsies, direct therapy, and monitor disease, particularly when office-based systems are developed. This is a promising imaging modality to study the larynx.     

An experimental study of the circulation of the vocal fold on phonation

The ischemic change of the vocal fold on phonation was studied with an oxygen electrode using the canine vocal fold. Oxygen pressures of a limited area of the vocal fold were measured in the lamina propria and vocal muscle at the middle of the membranous portion. The results are summarized as follows: oxygen pressure in each area decreased on phonation as compared with respiratory movement, an ischemic change of the vocal fold occurred on phonation, and the response in the lamina propria was less than that of the vocal muscle.     

Collagen subtypes in human vocal folds

OBJECTIVES: The collagen subtypes in human vocal folds are of particular interest, because each collagen subtype has different features that make it uniquely suited for performing specific tissue tasks and each collagen subtype can affect the tissue properties of the vocal fold lamina propria. METHODS: Human vocal folds from 5 autopsy cases (less than 65 years old) were examined by immunohistochemistry for collagen types I, III, IV, and V and elastin. RESULTS: Collagen type III was distributed throughout the whole lamina propria. Type I was found just beneath the basal membrane, in the deep layer of the lamina propria and in the anterior and posterior maculae flavae. Types IV and V were present in the epithelial and endothelial basal membrane. Three-dimensional images from thick specimens reconstructed with confocal microscopy showed 2 distinct patterns: type III fibers were wavy, collagenous fibers, as previously observed in the vocal folds, and type I fibers were thinner than type III fibers. These results suggest that type III fibers help maintain the lamina propria structure and that type I fibers provide the tensile strength required around the basal membrane and vocal ligament to maintain the vocal fold shape while withstanding vibratory forces.     

3D structure of the macula flava in the human vocal fold

OBJECTIVE: To investigate the 3D structure of the macula flava in the human vocal fold. MATERIAL AND METHODS: The 3D structure of the macula flava in the human vocal fold was investigated by means of scanning electron microscopy using a chemical digestion method (modified sodium hydroxide maceration method) and the results compared with those obtained using light microscopy. RESULTS: The macula flava of the human vocal fold comprised a dense mass of cells and extracellular matrices. Many cells were stellate in shape and possessed cytoplasmic processes. Extracellular matrices were composed of collagenous, reticular and elastic fibers, and ground substances (hyaluronic acid). Many fibers ran in various directions. The stellate cells were surrounded by these extracellular matrices. Amorphous materials were attached to the surface of the stellate cells. Extracellular matrices in the macula flava extended to those in the lamina propria of the vocal fold mucosa. CONCLUSION: Anterior and posterior maculae flavae in the human vocal fold mucosa are postulated to be involved in the metabolism of extracellular matrices, which are essential to the viscoelasticity of the lamina propria (the vibrating tissue) of the human adult vocal fold mucosa.     

Injection laryngoplasty according to the pathologic condition

Injection laryngoplasty is done based on the pathologic condition of the larynx. Autologous fat was harvested and endolaryngeal microsurgery conducted for injection laryngoplasty under general anesthesia. For glottic incompetence caused by bilateral atrophy of vocal fold mucosa lamina propria, autologous fat was injected into vocal fold mucosa and into the muscle just below mucosa. For glottic incompetence with a unilateral midcord gap caused by unilateral atrophy of the vocalis muscle, fat was injected into the thyroarytenoid muscle at the membranous portion of the vocal fold. In a patient with a unilateral midcord gap and a large posterior gap, autologous fat was injected into the thyroarytenoid muscle lateral to the oblong fovea of the arytenoid cartilage to arytenoid adduction. For glottic incompetence with a unilateral midcord gap and a large posterior gap, consequently afflicted with voice disorder and aspiration, fat was injected into the vocal fold, false vocal fold, aryepiglottic fold of the larynx, and the medial wall of the piriform sinus of the hypopharynx. Lipoinjection into the vocal fold, false vocal fold, and aryepiglottic fold enabled laryngeal closure. Lipoinjection into the piriform sinus lowered its capacity and residual food was reduced and pharyngeal clearance on the affected side was improved. The injected portion and the amount of injected material should be modified at injection laryngoplasty based on the pathologic condition of the larynx.     

The anisotropic nature of the human vocal fold: an ex vivo study

The purpose of this study was to measure the relationship between the shear elastic properties of vocal fold with respect to the direction of applied stress. There is extensive published material that quantifies the shear viscoelastic properties of the vocal fold, but as much of these data were obtained using rotating parallel plate rheometers, which are unable to resolve out difference of the shear elastic behaviour with respect to direction, there is very little data that indicates anisotropic behaviour. To overcome this gap in knowledge, the team devised an apparatus that is capable of applying a shear stress in a known direction. A series of measurements were taken at the mid-membranous position, in the transverse and longitudinal directions. Point-specific measurements were performed using fourteen human cadaver excised larynges, which were hemi-sectioned to expose the vocal fold. An extremely low sinusoidal shear force of 1 g was applied tangentially to the membrane surface in both the longitudinal and transverse direction, and the resultant shear strain was measured. With the probe applied to the intact vocal fold, the average ratio of the elasticity in the transverse with respect to the longitudinal direction was 0.55. Further investigation using histological staining of collagens in the lamina propria indicates that there is a visible difference in the general alignment of collagen fibres when comparing the coronal and the sagittal sections. Our conclusion is that there is a quantifiable difference between the shear elastic response of the lamina propria in the longitudinal and transverse directions, and that this could be explained by the difference in alignment of collagen fibres within the lamina propria.     

自体脂肪间充质干细胞移植促进声带修复再生的实验研究

目的 探讨自体脂肪间充质干细胞移植入损伤声带后的生长分布及对声带固有层及其主要细胞外基质变化的影响特点.方法 对34只实验用兔(68侧声带)的53侧声带进行全麻支撑喉镜下声带锐性损伤.分离培养、鉴定及定向诱导20只实验用兔脂肪间充质干细胞.将自体脂肪间充质干细胞植入20侧损伤声带后,示踪观察脂肪间充质干细胞在声带固有层内的生长分布;同时以单纯支架(胶原或透明质酸凝胶)植入18侧受损声带(胶原10侧,透明质酸8侧)及单纯损伤15侧声带作为对照组.术后15 d～12个月时采用HE染色、Masson染色、Alcian Blue染色及免疫组化染色观察声带形态学变化及声带固有层胶原、透明质酸、纤维连接蛋白的含量及分布变化.结果 脂肪间充质干细胞呈梭形贴壁生长,具有多向分化潜能,植入损伤兔声带后持续分布于声带固有层.损伤声带在脂肪间充质于细胞植入6个月时形态接近正常,12个月时部分组织学结构类似正常;声带细胞外基质中的胶原于植入3个月内含量有增高趋势,无序分布于固有层,后逐渐减少至正常水平附近,12个月时部分分布略显不规则;透明质酸于植入40 d含量亦有增高趋势,分布于固有层各层,6个月含量减至正常水平附近,局限于固有层浅中层;纤维连接蛋白于植入后始终在固有层内散在分布,含量在40 d内有增高趋势,后逐渐减少,12个月时含量接近正常.单纯声带损伤3个月后局部开始出现瘢痕挛缩,以胶原纤维为主的大量纤维组织增生,12个月时仍紊乱分布于声带固有层各层.单纯支架植入组变化介于二者之间.结论 脂肪间充质干细胞植入兔损伤声带后具有促进声带细胞外基质分泌、合理分布及部分有序化排列的功能,具有促进声带修复再生的作用.     

Development of the human true vocal fold: depth of cell layers and quantifying cell types within the lamina propria

OBJECTIVES: We sought to further describe the development of the 3-layered human vocal fold in children and to quantify macrophage and myofibroblast concentrations in each layer. METHODS: We used an optical analysis software package to examine 8 longitudinally sectioned human vocal folds that had been fixed in formalin (ages 2 days to 14 years). RESULTS: The 2-day-old vocal fold contained only a monolayer of cells. This became a bilayer by 5 months, and a trilayer began to become evident by 7 years. The percent of total depth represented by the superficial layer of the lamina propria (SLP) gradually decreased with age. The SLP made up 22% of the total lamina propria by age 7 years; this percentage approximates that in the adult vocal fold. Macrophages and myofibroblasts were predominately found in the SLP, and began to be apparent by 11 months of age. CONCLUSIONS: These results help describe the development of human voice and may have implications as to when phonosurgical therapy can be considered for children.