Cell death is a regulated mechanism of eliminating cells to maintain tissue homeostasis. This study described 2 methodological procedures for evaluating cell death in the epithelium of immobilized, approximated and vibrated vocal folds from 12 New Zealand white breeder rabbits. The gold standard technique of transmission electron microscopy evaluated high-quality ultrastructural criteria of cell death and a common immunohistochemical marker, the terminal deoxynucleotidyl transferase dUTP nick end labeling method, to confirm cell death signaling. Results revealed that ultrastructural characteristics of apoptotic cell death, specifically condensed chromatin and apoptotic bodies, were observed after vocal fold vibration and approximation. Although episodes of necrosis were rare, few enlarged cell nuclei were present after vibration and approximation. The vocal fold expresses an immunohistochemical marker for apoptosis along the apical surface of the epithelium. This study provides a solid foundation for future investigations regarding the role of cell death in vocal fold health and disease.

1.
ApopTag® fluorescein in situ apoptosis detection kit (2012).
2.
Ashkenazi, A., V.M. Dixit (1998) Death receptors: signaling and modulation. Science 281: 1305-1308.
3.
Bartlett, R.S., J.D. Gaston, T.Y. Yen, S. Ye, C. Kendziorski, S.L. Thibeault (2015) Biomechanical screening of cell therapies for vocal fold scar. Tissue Eng Part A 21: 2437-2447.
4.
Cory, S., J.M. Adams (2002) The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2: 647-656.
5.
Denecker, G., D. Vercammen, W. Declercq, P. Vandenabeele (2001) Apoptotic and necrotic cell death induced by death domain receptors. Cell Mol Life Sci 58: 356-370.
6.
Dikkers, F.G., C.E. Hulstaert, J.A. Oosterbaan, F.J. Cervera-Paz (1993) Ultrastructural changes of the basement membrane zone in benign lesions of the vocal folds. Acta Otolaryngol 113: 98-101.
7.
Elmore, S. (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35: 495-516.
8.
Gaston, J., B. Quinchia Rios, R. Bartlett, C. Berchtold, S.L. Thibeault (2012) The response of vocal fold fibroblasts and mesenchymal stromal cells to vibration. PloS One 7: e30965.
9.
Ge, P.J., L.C. French, T. Ohno, D.L. Zealear, B. Rousseau (2009) Model of evoked rabbit phonation. Ann Otol Rhinol Laryngol 118: 51-55.
10.
Gray, S.D. (2000) Cellular physiology of the vocal folds. Otolaryngol Clin North Am 33: 679-698.
11.
Gray, S., I. Titze (1988) Histologic investigation of hyperphonated canine vocal cords. Ann Otol Rhinol Laryngol 97: 381-388.
12.
Hellquist, H.B. (1997) Apoptosis in epithelial hyperplastic laryngeal lesions. Acta Otolaryngol Suppl 527: 25-29.
13.
Hirano, M. (1974) Morphological structure of the vocal cord as a vibrator and its variations. Folia Phoniatr 26: 89-94.
14.
Hirvikoski, P., E. Kumpulainen, J. Virtaniemi, R. Pirinen, L. Salmi, P. Halonen, R. Johansson, V.M. Kosma (1999) Enhanced apoptosis correlates with poor survival in patients with laryngeal cancer but not with cell proliferation, Bcl-2 or p53 expression. Eur J Cancer 35: 231-237.
15.
Hooper, C.E. (1956) Cell turnover in epithelial populations. J Histochem Cytochem 4: 531-540.
16.
Kojima, T., C.V. Valenzuela, C.K. Novaleski, M. Van Deusen, J.R. Mitchell, C.G. Garrett, M.P. Sivasankar, B. Rousseau (2014a) Effects of phonation time and magnitude dose on vocal fold epithelial genes, barrier integrity, and function. Laryngoscope 124: 2770-2778.
17.
Kojima, T., M. Van Deusen, W.G. Jerome, C.G. Garrett, M.P. Sivasankar, C.K. Novaleski, B. Rousseau (2014b) Quantification of acute vocal fold epithelial surface damage with increasing time and magnitude doses of vibration exposure. PLoS One 9: e91615.
18.
Kotby, M.N., A.M. Nassar, E.I. Seif, E.H. Helal, M.M. Saleh (1988) Ultrastructural features of vocal fold nodules and polyps. Acta Otolaryngol 105: 477-482.
19.
Lalaoui, N., L.M. Lindqvist, J.J. Sandow, P.G. Ekert (2015) The molecular relationships between apoptosis, autophagy and necroptosis. Semin Cell Dev Biol 39: 63-69.
20.
Lungova, V., J.M. Verheyden, J. Herriges, X. Sun, S.L. Thibeault (2015) Ontogeny of the mouse vocal fold epithelium. Dev Biol 399: 263-282.
21.
Madara, J.L. (1990) Maintenance of the macromolecular barrier at cell extrusion sites in intestinal epithelium: physiological rearrangement of tight junctions. J Membr Biol 116: 177-184.
22.
Majno, G., I. Joris (1995) Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 146: 3-15.
23.
Marcelino, F.C., D.T. Oliveira (2005) Histopathological changes of vocal folds induced by chronic pollutant exposure: an experimental study. J Voice 19: 529-533.
24.
Marchiando, A.M., L. Shen, W.V. Graham, K.L. Edelblum, C.A. Duckworth, Y. Guan, M.H. Montrose, J.R. Turner, A.J. Watson (2011) The epithelial barrier is maintained by in vivo tight junction expansion during pathologic intestinal epithelial shedding. Gastroenterology 140: 1208-1218.e1201-1202.
25.
Martinez, M.M., R.D. Reif, D. Pappas (2010) Detection of apoptosis: a review of conventional and novel techniques. Anal Methods 2: 996-1004.
26.
Martins, R.H., J. Defaveri, M.A. Custodio Domingues, E.S.R. de Albuquerque, A. Fabro (2010) Vocal fold nodules: morphological and immunohistochemical investigations. J Voice 24: 531-539.
27.
Martins, R.H., J. Defaveri, M.A. Domingues, R. de Albuquerque e Silva (2011) Vocal polyps: clinical, morphological, and immunohistochemical aspects. J Voice 25: 98-106.
28.
Pellettieri, J., A. Sanchez Alvarado (2007) Cell turnover and adult tissue homeostasis: from humans to planarians. Ann Rev Genet 41: 83-105.
29.
Rousseau, B., A. Suehiro, N. Echemendia, M. Sivasankar (2011) Raised intensity phonation compromises vocal fold epithelial barrier integrity. Laryngoscope 121: 346-351.
30.
Van Den Berg, J. (1958) Myoelastic-aerodynamic theory of voice production. J Speech Hearing Res 1: 227-244.
31.
Watson, A.J., C.A. Duckworth, Y. Guan, M.H. Montrose (2009) Mechanisms of epithelial cell shedding in the mammalian intestine and maintenance of barrier function. Ann NY Acad Sci 1165: 135-142.
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