- Email: [email protected]
Developing a Porcine Model for Study of Vocal Fold Scar
Developing a Porcine Model for Study of Vocal Fold Scar Gayle Woodson, Springfield, Illinois Summary: The porcine larynx is very similar in size and structure to that of humans, and wound healing in pigs is very similar to that of humans. However, the pig is not often used in vocal fold scar research because it is difficult to view the vocal folds endoscopically. To further assess the pig as a model for studying vocal scar, we compared the plane of surgical dissection in the mucosa of four porcine vocal folds with that in eight human cadaver larynges. The plane of dissection was quite similar in porcine and human larynges, occurring within the loose layer of the superficial lamina propria. We also compared healing of porcine vocal folds after elevation and replacement of an epithelial flap versus excision of epithelium, leaving an open wound. After 6 weeks, larynges were harvested for histologic examination. There was no significant difference between the mucosa of the normal vocal fold and that of the healed microflap. However, after healing of epithelial excision, there was a depressed scar, with average lamina propria thickness of 302 mm versus 864 mm for the normal fold (P < 0.05). Finally, to document that the mucosal wave can be evaluated in the porcine larynx, we developed a preparation that removes the false vocal folds, to allow ex vivo phonation. Experimentally created scar in the porcine larynx is a favorable model for the study of vocal fold healing and for assessment of treatments for vocal fold scar. Key Words: Larynx–Vocal fold–Scar–Animal model–Pig–Wound healing.
INTRODUCTION Vocal fold scar is one of the most challenging clinical problems currently encountered in laryngology. Many treatment approaches have been used, but no treatment reliably rehabilitates and restores the voice. Clinical study of the pathogenesis and treatment of vocal fold scar is very difficult. Vocal scar is not common, and consequently, in any study, treatment groups are small. Additionally, the defects are varied. Sometimes a scarred vocal fold is grossly normal, but stroboscopy reveals diffuse stiffness, adynamic segments, or asymmetric mucosal wave. In other patients, the scar is a divot that results in a glottal gap on phonation. Scar may also result in a loss of vocal fold volume or a bulky scar that impairs glottic closure. Because of low incidence and heterogeneous pathology, statistical evaluation of treatment outcomes is difficult.1 Animal models offer the opportunity for controlled experiments to create standardized injuries, study wound healing, and quantify the effects of different therapeutic interventions. Vocal fold scar has been studied in rabbits, dogs, and rats. The dog has been a preferred model because of the ease of viewing the larynx in vivo to assess vocal fold vibration.2 However, the structure of the canine vocal fold differs significantly from that of humans. Although no animal studied to date has a vocal ligament such as that seen in humans, the microscopic architecture of the pig is more similar to that of humans than that of dogs, rabbits, or rats. The human vocal fold is uniquely structured for phonation: the epithelium is separated from underlying muscle and ligaAccepted for publication March 16, 2012. From the Department of Surgery, Division of Otolaryngology, Southern Illinois University School of Medicine, Springfield, Illinois. Address correspondence and reprint requests to Gayle Woodson, Division of Otolaryngology, Southern Illinois University School of Medicine, P.O. Box 19662, Springfield, IL 62794-9662. E-mail: [email protected] Journal of Voice, Vol. 26, No. 6, pp. 706-710 0892-1997/$36.00 Ó 2012 The Voice Foundation doi:10.1016/j.jvoice.2012.03.003
ment by specialized lamina propria in which tissue density is stratified to allow the epithelium to vibrate with greater amplitude than deeper structures.3 Collagen density is sparse in the superficial lamina propria and increases with depth into the vocal fold.4 The lamina propria is a continuous layer about 1 mm thick that wraps around the free edge of the vocal fold. Epithelium can be easily stripped from the human vocal fold, and the plane of separation in humans occurs at a consistent level within the lamina propria.5 But in the canine larynx, the pattern of collagen distribution is inverted. Density is greatest in the superficial lamina propria, near the epithelium, and decreases toward the underlying muscle.4 The shape of the canine vocal fold is quite different from that of humans. The layer of lamina propria is much deeper and projects into the lumen as a long shelf, rather than wrapping around the edge of the vocalis muscle.4 And when epithelium is removed from the vocal fold, the level at which tissue shears off is unpredictable.2 Thus, the structure of the canine vocal fold differs significantly from that of humans. The structure of the pig vocal fold is quite similar to that of humans.4 As in humans, the lamina propria is approximately 1 mm thick along the medial edge of the vocal fold. Collagen is loosely organized in the superficial lamina propria and more densely concentrated at deeper levels.2 In addition, there is extensive literature indicating that wound healing in pigs is very similar to that in humans, making the pig an attractive model for the study of therapeutic interventions for wound healing and scar.4 A recent review of 180 published studies testing 25 skin wound therapies concluded that results in the pig have the closest concordance with humans.6 Anatomically and physiologically, porcine skin is quite similar to that of humans. Porcine dermal collagen is biochemically quite similar to human dermal collagen, so much so that porcine collagen is used therapeutically in human wound products.7 Porcine skin and human skin also have very similar concentration and distribution of several antigens including keratins 16 and 10, filaggrin,
Gayle Woodson
Porcine Vocal Scar
collagen IV, fibronectin, and vimentin.8 Physiologically, healing of skin wounds in humans and pigs is similar. Although wounds in most small animals occur primarily through contracture, partial thickness wounds in humans and swine heal largely through re-epithelialization.9 Given the parallels of porcine and human skin, it is not unreasonable to assume that the vocal fold mucosa would also have biological similarity, making the pig a good model for the study of vocal fold healing. As mentioned above, the major objection that has previously been cited against the pig as an animal model for vocal fold scar is that it is very difficult to view the vocal folds with laryngoscopy. The false vocal folds in pigs are very prominent and obscure the true vocal folds. Thus, it is not possible to view the mucosal wave in vivo to document the functional effects of vocal fold scar and the efficacy of treatment. Therefore, we developed a protocol for assessing function by ex vivo phonation in the excised larynx, by debulking the false vocal folds. The present study was intended to demonstrate the feasibility and value of the pig as a model for studying vocal fold scar and the potential for testing the efficacy of surgical and medical treatment of vocal fold scar. The objectives of the experiments were to (1) compare the histology and surgical dissection plane in human and porcine vocal folds, (2) establish a protocol for creating experimental scar in the porcine larynx, and (3) document the feasibility of assessing the mucosal wave in the excised porcine larynx. METHODS Dissection plane in cadaver larynges Four fresh porcine cadaver larynges were bisected in the midline sagittal plane. In one half of each larynx, an epithelial flap was developed with microscissors and excised from the vocal fold edge. The opposite fold was left intact as a control. Epithelium was also excised from one vocal fold in each of seven human cadaver larynges, using microscissor dissection. In four larynges, dissection was as superficial as possible, and the tips of the scissors were oriented toward the surface, with the goal of separating the epithelium from the underlying lamina propria. In three larynges, the tips of the scissors were directed deeply, with the goal of separating the lamina propria from the vocal ligament. As with the porcine larynges, one hemilarynx was left intact as a control. Each hemilarynx was decalcified, fixed in 10% formalin, and embedded in paraffin. Coronal sections of 0.7 mm were prepared and stained alternately with H&E or Verhoeff-van Gieson. In addition, the excised mucosa was similarly fixed and stained. Slides were digitally imaged and then processed with Image J (NIH) to measure the thickness of the lamina propria on the excised epithelium, the larynx after excision of epithelium, and in the intact specimen. Vocal fold healing The study was approved by Animal Care Committee. Subjects were eight minipigs. Under general anesthesia, a vertical midline incision was made to in the anterior neck of each pig to expose the larynx and trachea. A small tracheal cannula was
707 placed for ventilation during the procedure. The larynx was opened by a midline thyrotomy to expose the vocal folds. A longitudinal incision was made laterally on the superior aspect of the left vocal mucosa. Mucosa was elevated from the vibratory edge by blunt dissection in the subepithelial plane. In four animals, this flap was excised. In the other four animals, the medially based flap was replaced and secured with 5-0 chromic sutures. In all animals, the thyroid cartilage was reapproximated, carefully aligning the anterior commissure, and the tracheal cannula was removed. All pigs had a good laryngeal airway at the end of the procedure and tolerated a normal diet after recovery from anesthesia. However, two pigs died a few days after surgery because of stress-related pulmonary edema. The study was completed in six pigs. Thirty days after surgery, the animals were sacrificed. Larynges were excised and bisected in a sagittal plane. Each hemilarynx was grossly inspected and photographed, then decalcified, fixed in 10% formalin, and embedded in paraffin. Coronal sections of 0.7 mm were prepared from the anterior, middle, and posterior third of each vocal fold. Alternate sections were stained with H&E or Verhoeff-van Gieson technique. Slides were examined for collagen and elastin fiber distribution and density, comparing the experimental fold with the control side. Computerized image (NIH Scion) analysis was used to measure mucosal thickness, from the free edge of the vocal fold to the outer surface of the vocalis muscle. Data were analyzed using a paired student t test, comparing values on the experimental and control sides. Feasibility of ex vivo phonation in excised porcine larynges Two fresh porcine larynges were prepared for ex vivo phonation. For each larynx, approximately 4 cm of trachea were preserved in continuity with the larynx. The thyroid cartilage was resected above the glottis, along a line beginning posteriorly above the arytenoid cartilages, and angling anteriorly and caudally to a point just above the anterior commissure. The epiglottis and submucosal tissue of the false vocal folds were excised. The mucosa of the false folds was then sutured laterally to the thyroid cartilage, exposing the superior surface of the vocal folds. Bilateral arytenoid adduction sutures were placed to approximate the vocal processes. The larynx was mounted on a platform, and a polyethylene tube was inserted into the distal trachea and secured with circumferential plastic ligatures. A stroboscopic video camera was mounted above the larynx to view the vocal folds. Phonation was elicited when compressed air was passed through the larynx with variable lateral compression of the thyroid cartilages, and mucosal vibration was recorded. RESULTS Dissection plane In all four porcine larynges, and all human larynges, the plane of dissection was within the lamina propria. Histologic examination of the excised epithelium always included a portion of the superficial lamina propria along its deep surface (Figure 1A).
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Journal of Voice, Vol. 26, No. 6, 2012
Histologic examination of the residual laryngeal specimens after excision of epithelium always revealed a layer of the superficial lamina propria. However, the amount of tissue could not be quantified by measuring the thickness of this layer, as there was distortion at the interface, with distraction of the collagen fibers and patchy separation of lamina propria from underlying soft tissue (Figure 3). Vocal fold healing Gross inspection. After healing of an epithelial flap, the gross appearance of the operated vocal fold was not noticeably different from that of the unoperated side. The surface of the epithelium was smooth and even, and the vibratory edge was sharp. However, after resection of vocal fold epithelium, healing resulted in a depressed scar, with an uneven surface and a rounded edge.
FIGURE 1. Epithelial strip excised from vocal fold mucosa by blunt microscissor dissection. Note attached portion of superficial lamina propria (103). A. Porcine epithelium. B. Human cadaver epithelium.
This was also true in both groups of human larynges (Figure 1B). Figure 2 displays the measurements of the thickness of the excised specimens. Because there was no difference between the two groups of human larynges, that data are combined. The average thickness of the excised strip was 145 m in the human larynx and 194 m in the porcine larynx.
FIGURE 2. Bar graph comparing thickness of excised epithelium from excised porcine and cadaver human larynges.
Histologic analysis. In the vocal folds that had healed after replacement of a microflap, the edge of the vocal fold, as seen in coronal section, was similar to the control vocal folds (Figure 4A and B). However, vocal folds that had healed by secondary intention had a rounded edge, with very thin lamina propria (Figure 4C). Table 1 displays the thickness of the mucosa in the specimens. The average thickness of the mucosa in the control vocal folds was 919.15 m. After replacement of a microflap, mucosal thickness was 879.86 m, compared with an average thickness of 919 m. This difference was not significantly significant. In vocal folds that healed by secondary intention, the average thickness of mucosa was 301.95 m, significantly thinner than the thickness in the control vocal folds (864.33), P < 0.05. The concentration of collagen was somewhat increased in the lamina propria of the scarred vocal folds but not appreciably different in the vocal fold that healed after elevation and replacement of an epithelial flap. Ex vivo phonation in excised larynges The resection of supraglottic cartilage and retraction of the false vocal folds provided a good view of the vocal fold when viewed from above (Figure 5). Phonation could be produced by applying medial compression to both thyroid ala as air was passed through the trachea across the glottis, and the resultant glottic wave was visible during stroboscopic illumination. DISCUSSION In both porcine and human vocal folds, the excised epithelium always contained a portion of the superficial lamina propria. This is consistent with a study by Gray et al,5 who used blunt instruments to dissect the lamina propria in eight human larynges. The plane of dissection was consistently within the lamina propria. This strong attachment of the epithelium to underlying lamina propria in humans has been attributed to anchoring fibers, analogous to the fibers in skin that secure the epidermis to dermis. Anchoring fibers are abnormal in blistering skin disorders, such as epidermolysis bullosa. The vocal fold has no dermis; however, electron microscopy of the basement membrane zone of the vocal fold showed a similar structure to that of the epidermal dermal interface.10 Collagen fibers of the
Gayle Woodson
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Porcine Vocal Scar
FIGURE 3. Coronal sections through larynges after excision of epithelium by blunt dissection (23). Note fragmentation of residual lamina propria and separation from underlying tissues. A. Porcine larynx. B. Human cadaver larynx.
superficial lamina propria pass through loops created by anchoring fibers that originate in the lamina densa of the basement membrane. It is likely that porcine vocal fold mucosa has a similar ultrastructure, given the remarkable similarity of porcine and human skin. Pigskin is anatomically very similar to human skin, with thick and well-developed rete pegs and dermal papillary bodies. There are also similarities in keratinous proteins and lipid composition of the stratum corneum.11–13 Although the superficial lamina propria vocal fold epithelium seems firmly anchored to epithelium, this attachment to tissue is easily disrupted. The results of this study confirmed what has long been recognized clinically: that epithelium can be easily stripped from the vocal fold. It has also been observed that after stripping, the vocal fold tends to heal with scarring, and the vibratory edge becomes stiff. In this study, the histology
of both human and porcine specimens after removal of epithelium showed that the residual lamina propria was disrupted and distorted during this process, with dispersion of fibers and separation from the underlying muscle (Figure 3). In the porcine vocal folds that healed after removing epithelium, the area healed by re-epithelialization, there was very little subepithelial tissue, indicating that most of the residual lamina propria had sloughed or undergone necrosis (Figure 4C). However, when the flap was replaced, healing resulted in a nearly normal layer of lamina propria, indicating that the residual lamina propria was either preserved or had regenerated. This porcine model of scar provides support for the benefits of the epithelial preservation in laryngeal microsurgery. The laryngeal microflap technique was developed based on recognition of the layered structure of the vocal fold mucosa. To remove submucosal pathology, the epithelium is elevated and
FIGURE 4. Coronal sections through porcine hemilarynx. A. Control. B. One month after elevation and replacement of epithelial flap. C. One month after excision of epithelium, with healing by secondary intention.
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Journal of Voice, Vol. 26, No. 6, 2012
TABLE 1. Mean Thickness of Vocal Fold Mucosa Microflap Surgical side (m) Control side (m) Difference (m)
Replaced (P > 0.05)
Excised (P < 0.05)
879.86 919.15 39.29
301.95 864.34 562.39
then replaced at the end of the procedure. This theoretically covers and preserves the lamina propria.14 Although microflap surgery has better results than leaving a wound to close secondarily, Sataloff et al15 have pointed out that the vocal fold is not totally normal following healing of a microflap and presented a case series showing that vocal function is better after the ‘‘micromini-flap’’ technique, which limits epithelial elevation to the smallest possible area. Although Sataloff et al propose that the impaired function after microflap surgery is because of disruption of the basement membrane, the results from the human specimens in this study suggest that the basement membrane is robust and not easily disrupted by surgery. Although it is true that the pig lacks a vocal ligament, similarities in the dissection plane of the lamina propria suggest that the pig is a good model for the study of vocal fold healing and for the study of therapeutic interventions. In particular, the model would be useful for the study of procedures to restore the lamina propria in scarred vocal folds and to rehabilitate the larynx after endoscopic resection of laryngeal cancers.
FIGURE 5. Excised porcine larynx, prepared for ex vivo phonation, viewed from above. Thyroid cartilage has been removed above level of vocal folds. Epiglottis removed. Submucosal fat removed from false vocal folds and mucosa sutured laterally to expose vocal folds.
SUMMARY The pig is a very logical choice for the study of vocal fold scar, as the size of the larynx and histologic structure of the vocal fold mucosa of the pig is more similar to humans than that of other animals studied. Additionally, the process of wound healing in pigs is very similar to that of humans. The current experiments indicate that the dissection plane in the lamina propria is very similar in the porcine and human larynx. This may be because of similarities in the ultrastructure of the basement membrane zone. Experimental scar can be created via a laryngofissure approach by excising epithelium. Elevation and replacement of a microflap has little impact on the structure of the vocal fold. An open vocal fold wound heals by secondary intention, resulting in a depressed scar, with loss of lamina propria. This experimental scar can be used in the future to study therapeutic interventions. Phonatory function can be studied in the excised porcine larynx. We conclude that the pig is an excellent model for the study of vocal fold scar and has excellent potential for assessing the efficacy of treatment.
REFERENCES 1. Hansen JK, Thibeault SL. Current understanding and review of the literature: vocal fold scarring. J Voice. 2006;20:110–120. 2. Garrett CG, Coleman JR, Reinisch L. Comparative histology and vibration of the vocal folds: implications for experimental studies in microlaryngeal surgery. Laryngoscope. 2000;110:814–824. 3. Hirano M. Morphological structure of the vocal cord as a vibrator and its variations. Folia Phoniatr (Basel). 1974;26:89–94. 4. Kurita S, Nagata K, Hirano M. A comparative study of the layer structure of the vocal fold. In: Bless DM, Abbs JH, eds. Vocal Fold Physiology: Contemporary Research and Clinical Issues. San Diego, CA: CollegeHill Press; 1983:3–21. 5. Gray SD, Chan KJ, Turner B. Dissection plane of the human vocal fold lamina propria and elastin fibre concentration. Acta Otolaryngol. 2000;120: 87–91. 6. Sullivan TP, Eaglstein WH, Davis SC, Mertz P. The pig as a model for human wound healing. Wound Repair Regen. 2001;9:66–76. 7. Heinrich W, Lange PM, Stirz T, Iancu C, Heidermann E. Isolation and characterization of the large cyanogen bromide peptides from burns skin graft donor sites. J Burn Care Rehabil. 1996;17:246–251. 8. Wollina U, Berger U, Mahrle G. Immunohistochemistry of porcine skin. Acta histochem. 1991;90:87–91. 9. Hartwell SW. The Mechanism of Healing in Human Wounds. Springfield, IL: Charles Thomas; 1955. 10. Gray SD, Pignatari SN, Harding P. Morphologic ultrastructure of anchoring fibers in normal vocal fold basement membrane zone. J Voice. 1994;8: 48–52. 11. Weinstein GD. Comparison of turnover time and of keratinous protein fractions in swine and human epidermis. In: Bustad LK, McClellan RD, eds. Swine in Biomedical Research. Seattle, WA: Frayn; 1966:287–289. 12. Nicolaides N, Fu HC, Rice GR. The skin surface lipids of man compared with those of eighteen species of animals. J Invest Dermatol. 1968;51: 83–89. 15:886–9. 13. Gray GM, White RJ, Williams RH, Yardley HJ. Lipid composition of the superficial stratum corneum of pig epidermis. Br J Dermatol. 1982;106: 59–63. 14. Zeitels SM, Hillman RE, Bunting GW, Vaughn T. Reinke’s edema: phonatory mechanisms and management strategies. Ann Otol Rhinol Laryngol. 1996;105:533–543. 15. Sataloff RT, Spiegel JR, Heuer RJ, et al. Laryngeal mini-microflap: a new technique and reassessment of the microflap saga. J Voice. 1995;9: 198–204.
INTRODUCTION Vocal fold scar is one of the most challenging clinical problems currently encountered in laryngology. Many treatment approaches have been used, but no treatment reliably rehabilitates and restores the voice. Clinical study of the pathogenesis and treatment of vocal fold scar is very difficult. Vocal scar is not common, and consequently, in any study, treatment groups are small. Additionally, the defects are varied. Sometimes a scarred vocal fold is grossly normal, but stroboscopy reveals diffuse stiffness, adynamic segments, or asymmetric mucosal wave. In other patients, the scar is a divot that results in a glottal gap on phonation. Scar may also result in a loss of vocal fold volume or a bulky scar that impairs glottic closure. Because of low incidence and heterogeneous pathology, statistical evaluation of treatment outcomes is difficult.1 Animal models offer the opportunity for controlled experiments to create standardized injuries, study wound healing, and quantify the effects of different therapeutic interventions. Vocal fold scar has been studied in rabbits, dogs, and rats. The dog has been a preferred model because of the ease of viewing the larynx in vivo to assess vocal fold vibration.2 However, the structure of the canine vocal fold differs significantly from that of humans. Although no animal studied to date has a vocal ligament such as that seen in humans, the microscopic architecture of the pig is more similar to that of humans than that of dogs, rabbits, or rats. The human vocal fold is uniquely structured for phonation: the epithelium is separated from underlying muscle and ligaAccepted for publication March 16, 2012. From the Department of Surgery, Division of Otolaryngology, Southern Illinois University School of Medicine, Springfield, Illinois. Address correspondence and reprint requests to Gayle Woodson, Division of Otolaryngology, Southern Illinois University School of Medicine, P.O. Box 19662, Springfield, IL 62794-9662. E-mail: [email protected] Journal of Voice, Vol. 26, No. 6, pp. 706-710 0892-1997/$36.00 Ó 2012 The Voice Foundation doi:10.1016/j.jvoice.2012.03.003
ment by specialized lamina propria in which tissue density is stratified to allow the epithelium to vibrate with greater amplitude than deeper structures.3 Collagen density is sparse in the superficial lamina propria and increases with depth into the vocal fold.4 The lamina propria is a continuous layer about 1 mm thick that wraps around the free edge of the vocal fold. Epithelium can be easily stripped from the human vocal fold, and the plane of separation in humans occurs at a consistent level within the lamina propria.5 But in the canine larynx, the pattern of collagen distribution is inverted. Density is greatest in the superficial lamina propria, near the epithelium, and decreases toward the underlying muscle.4 The shape of the canine vocal fold is quite different from that of humans. The layer of lamina propria is much deeper and projects into the lumen as a long shelf, rather than wrapping around the edge of the vocalis muscle.4 And when epithelium is removed from the vocal fold, the level at which tissue shears off is unpredictable.2 Thus, the structure of the canine vocal fold differs significantly from that of humans. The structure of the pig vocal fold is quite similar to that of humans.4 As in humans, the lamina propria is approximately 1 mm thick along the medial edge of the vocal fold. Collagen is loosely organized in the superficial lamina propria and more densely concentrated at deeper levels.2 In addition, there is extensive literature indicating that wound healing in pigs is very similar to that in humans, making the pig an attractive model for the study of therapeutic interventions for wound healing and scar.4 A recent review of 180 published studies testing 25 skin wound therapies concluded that results in the pig have the closest concordance with humans.6 Anatomically and physiologically, porcine skin is quite similar to that of humans. Porcine dermal collagen is biochemically quite similar to human dermal collagen, so much so that porcine collagen is used therapeutically in human wound products.7 Porcine skin and human skin also have very similar concentration and distribution of several antigens including keratins 16 and 10, filaggrin,
Gayle Woodson
Porcine Vocal Scar
collagen IV, fibronectin, and vimentin.8 Physiologically, healing of skin wounds in humans and pigs is similar. Although wounds in most small animals occur primarily through contracture, partial thickness wounds in humans and swine heal largely through re-epithelialization.9 Given the parallels of porcine and human skin, it is not unreasonable to assume that the vocal fold mucosa would also have biological similarity, making the pig a good model for the study of vocal fold healing. As mentioned above, the major objection that has previously been cited against the pig as an animal model for vocal fold scar is that it is very difficult to view the vocal folds with laryngoscopy. The false vocal folds in pigs are very prominent and obscure the true vocal folds. Thus, it is not possible to view the mucosal wave in vivo to document the functional effects of vocal fold scar and the efficacy of treatment. Therefore, we developed a protocol for assessing function by ex vivo phonation in the excised larynx, by debulking the false vocal folds. The present study was intended to demonstrate the feasibility and value of the pig as a model for studying vocal fold scar and the potential for testing the efficacy of surgical and medical treatment of vocal fold scar. The objectives of the experiments were to (1) compare the histology and surgical dissection plane in human and porcine vocal folds, (2) establish a protocol for creating experimental scar in the porcine larynx, and (3) document the feasibility of assessing the mucosal wave in the excised porcine larynx. METHODS Dissection plane in cadaver larynges Four fresh porcine cadaver larynges were bisected in the midline sagittal plane. In one half of each larynx, an epithelial flap was developed with microscissors and excised from the vocal fold edge. The opposite fold was left intact as a control. Epithelium was also excised from one vocal fold in each of seven human cadaver larynges, using microscissor dissection. In four larynges, dissection was as superficial as possible, and the tips of the scissors were oriented toward the surface, with the goal of separating the epithelium from the underlying lamina propria. In three larynges, the tips of the scissors were directed deeply, with the goal of separating the lamina propria from the vocal ligament. As with the porcine larynges, one hemilarynx was left intact as a control. Each hemilarynx was decalcified, fixed in 10% formalin, and embedded in paraffin. Coronal sections of 0.7 mm were prepared and stained alternately with H&E or Verhoeff-van Gieson. In addition, the excised mucosa was similarly fixed and stained. Slides were digitally imaged and then processed with Image J (NIH) to measure the thickness of the lamina propria on the excised epithelium, the larynx after excision of epithelium, and in the intact specimen. Vocal fold healing The study was approved by Animal Care Committee. Subjects were eight minipigs. Under general anesthesia, a vertical midline incision was made to in the anterior neck of each pig to expose the larynx and trachea. A small tracheal cannula was
707 placed for ventilation during the procedure. The larynx was opened by a midline thyrotomy to expose the vocal folds. A longitudinal incision was made laterally on the superior aspect of the left vocal mucosa. Mucosa was elevated from the vibratory edge by blunt dissection in the subepithelial plane. In four animals, this flap was excised. In the other four animals, the medially based flap was replaced and secured with 5-0 chromic sutures. In all animals, the thyroid cartilage was reapproximated, carefully aligning the anterior commissure, and the tracheal cannula was removed. All pigs had a good laryngeal airway at the end of the procedure and tolerated a normal diet after recovery from anesthesia. However, two pigs died a few days after surgery because of stress-related pulmonary edema. The study was completed in six pigs. Thirty days after surgery, the animals were sacrificed. Larynges were excised and bisected in a sagittal plane. Each hemilarynx was grossly inspected and photographed, then decalcified, fixed in 10% formalin, and embedded in paraffin. Coronal sections of 0.7 mm were prepared from the anterior, middle, and posterior third of each vocal fold. Alternate sections were stained with H&E or Verhoeff-van Gieson technique. Slides were examined for collagen and elastin fiber distribution and density, comparing the experimental fold with the control side. Computerized image (NIH Scion) analysis was used to measure mucosal thickness, from the free edge of the vocal fold to the outer surface of the vocalis muscle. Data were analyzed using a paired student t test, comparing values on the experimental and control sides. Feasibility of ex vivo phonation in excised porcine larynges Two fresh porcine larynges were prepared for ex vivo phonation. For each larynx, approximately 4 cm of trachea were preserved in continuity with the larynx. The thyroid cartilage was resected above the glottis, along a line beginning posteriorly above the arytenoid cartilages, and angling anteriorly and caudally to a point just above the anterior commissure. The epiglottis and submucosal tissue of the false vocal folds were excised. The mucosa of the false folds was then sutured laterally to the thyroid cartilage, exposing the superior surface of the vocal folds. Bilateral arytenoid adduction sutures were placed to approximate the vocal processes. The larynx was mounted on a platform, and a polyethylene tube was inserted into the distal trachea and secured with circumferential plastic ligatures. A stroboscopic video camera was mounted above the larynx to view the vocal folds. Phonation was elicited when compressed air was passed through the larynx with variable lateral compression of the thyroid cartilages, and mucosal vibration was recorded. RESULTS Dissection plane In all four porcine larynges, and all human larynges, the plane of dissection was within the lamina propria. Histologic examination of the excised epithelium always included a portion of the superficial lamina propria along its deep surface (Figure 1A).
708
Journal of Voice, Vol. 26, No. 6, 2012
Histologic examination of the residual laryngeal specimens after excision of epithelium always revealed a layer of the superficial lamina propria. However, the amount of tissue could not be quantified by measuring the thickness of this layer, as there was distortion at the interface, with distraction of the collagen fibers and patchy separation of lamina propria from underlying soft tissue (Figure 3). Vocal fold healing Gross inspection. After healing of an epithelial flap, the gross appearance of the operated vocal fold was not noticeably different from that of the unoperated side. The surface of the epithelium was smooth and even, and the vibratory edge was sharp. However, after resection of vocal fold epithelium, healing resulted in a depressed scar, with an uneven surface and a rounded edge.
FIGURE 1. Epithelial strip excised from vocal fold mucosa by blunt microscissor dissection. Note attached portion of superficial lamina propria (103). A. Porcine epithelium. B. Human cadaver epithelium.
This was also true in both groups of human larynges (Figure 1B). Figure 2 displays the measurements of the thickness of the excised specimens. Because there was no difference between the two groups of human larynges, that data are combined. The average thickness of the excised strip was 145 m in the human larynx and 194 m in the porcine larynx.
FIGURE 2. Bar graph comparing thickness of excised epithelium from excised porcine and cadaver human larynges.
Histologic analysis. In the vocal folds that had healed after replacement of a microflap, the edge of the vocal fold, as seen in coronal section, was similar to the control vocal folds (Figure 4A and B). However, vocal folds that had healed by secondary intention had a rounded edge, with very thin lamina propria (Figure 4C). Table 1 displays the thickness of the mucosa in the specimens. The average thickness of the mucosa in the control vocal folds was 919.15 m. After replacement of a microflap, mucosal thickness was 879.86 m, compared with an average thickness of 919 m. This difference was not significantly significant. In vocal folds that healed by secondary intention, the average thickness of mucosa was 301.95 m, significantly thinner than the thickness in the control vocal folds (864.33), P < 0.05. The concentration of collagen was somewhat increased in the lamina propria of the scarred vocal folds but not appreciably different in the vocal fold that healed after elevation and replacement of an epithelial flap. Ex vivo phonation in excised larynges The resection of supraglottic cartilage and retraction of the false vocal folds provided a good view of the vocal fold when viewed from above (Figure 5). Phonation could be produced by applying medial compression to both thyroid ala as air was passed through the trachea across the glottis, and the resultant glottic wave was visible during stroboscopic illumination. DISCUSSION In both porcine and human vocal folds, the excised epithelium always contained a portion of the superficial lamina propria. This is consistent with a study by Gray et al,5 who used blunt instruments to dissect the lamina propria in eight human larynges. The plane of dissection was consistently within the lamina propria. This strong attachment of the epithelium to underlying lamina propria in humans has been attributed to anchoring fibers, analogous to the fibers in skin that secure the epidermis to dermis. Anchoring fibers are abnormal in blistering skin disorders, such as epidermolysis bullosa. The vocal fold has no dermis; however, electron microscopy of the basement membrane zone of the vocal fold showed a similar structure to that of the epidermal dermal interface.10 Collagen fibers of the
Gayle Woodson
709
Porcine Vocal Scar
FIGURE 3. Coronal sections through larynges after excision of epithelium by blunt dissection (23). Note fragmentation of residual lamina propria and separation from underlying tissues. A. Porcine larynx. B. Human cadaver larynx.
superficial lamina propria pass through loops created by anchoring fibers that originate in the lamina densa of the basement membrane. It is likely that porcine vocal fold mucosa has a similar ultrastructure, given the remarkable similarity of porcine and human skin. Pigskin is anatomically very similar to human skin, with thick and well-developed rete pegs and dermal papillary bodies. There are also similarities in keratinous proteins and lipid composition of the stratum corneum.11–13 Although the superficial lamina propria vocal fold epithelium seems firmly anchored to epithelium, this attachment to tissue is easily disrupted. The results of this study confirmed what has long been recognized clinically: that epithelium can be easily stripped from the vocal fold. It has also been observed that after stripping, the vocal fold tends to heal with scarring, and the vibratory edge becomes stiff. In this study, the histology
of both human and porcine specimens after removal of epithelium showed that the residual lamina propria was disrupted and distorted during this process, with dispersion of fibers and separation from the underlying muscle (Figure 3). In the porcine vocal folds that healed after removing epithelium, the area healed by re-epithelialization, there was very little subepithelial tissue, indicating that most of the residual lamina propria had sloughed or undergone necrosis (Figure 4C). However, when the flap was replaced, healing resulted in a nearly normal layer of lamina propria, indicating that the residual lamina propria was either preserved or had regenerated. This porcine model of scar provides support for the benefits of the epithelial preservation in laryngeal microsurgery. The laryngeal microflap technique was developed based on recognition of the layered structure of the vocal fold mucosa. To remove submucosal pathology, the epithelium is elevated and
FIGURE 4. Coronal sections through porcine hemilarynx. A. Control. B. One month after elevation and replacement of epithelial flap. C. One month after excision of epithelium, with healing by secondary intention.
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Journal of Voice, Vol. 26, No. 6, 2012
TABLE 1. Mean Thickness of Vocal Fold Mucosa Microflap Surgical side (m) Control side (m) Difference (m)
Replaced (P > 0.05)
Excised (P < 0.05)
879.86 919.15 39.29
301.95 864.34 562.39
then replaced at the end of the procedure. This theoretically covers and preserves the lamina propria.14 Although microflap surgery has better results than leaving a wound to close secondarily, Sataloff et al15 have pointed out that the vocal fold is not totally normal following healing of a microflap and presented a case series showing that vocal function is better after the ‘‘micromini-flap’’ technique, which limits epithelial elevation to the smallest possible area. Although Sataloff et al propose that the impaired function after microflap surgery is because of disruption of the basement membrane, the results from the human specimens in this study suggest that the basement membrane is robust and not easily disrupted by surgery. Although it is true that the pig lacks a vocal ligament, similarities in the dissection plane of the lamina propria suggest that the pig is a good model for the study of vocal fold healing and for the study of therapeutic interventions. In particular, the model would be useful for the study of procedures to restore the lamina propria in scarred vocal folds and to rehabilitate the larynx after endoscopic resection of laryngeal cancers.
FIGURE 5. Excised porcine larynx, prepared for ex vivo phonation, viewed from above. Thyroid cartilage has been removed above level of vocal folds. Epiglottis removed. Submucosal fat removed from false vocal folds and mucosa sutured laterally to expose vocal folds.
SUMMARY The pig is a very logical choice for the study of vocal fold scar, as the size of the larynx and histologic structure of the vocal fold mucosa of the pig is more similar to humans than that of other animals studied. Additionally, the process of wound healing in pigs is very similar to that of humans. The current experiments indicate that the dissection plane in the lamina propria is very similar in the porcine and human larynx. This may be because of similarities in the ultrastructure of the basement membrane zone. Experimental scar can be created via a laryngofissure approach by excising epithelium. Elevation and replacement of a microflap has little impact on the structure of the vocal fold. An open vocal fold wound heals by secondary intention, resulting in a depressed scar, with loss of lamina propria. This experimental scar can be used in the future to study therapeutic interventions. Phonatory function can be studied in the excised porcine larynx. We conclude that the pig is an excellent model for the study of vocal fold scar and has excellent potential for assessing the efficacy of treatment.
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