Radiation fibrosis of the vocal fold: from man to mouse

Abstract

Objectives/hypothesis: To characterize fundamental late tissue effects in the human vocal fold following radiation therapy. To develop a murine model of radiation fibrosis in order to ultimately develop both treatment and prevention paradigms.

Design: Translational study using archived human and fresh murine irradiated vocal fold tissue.

Methods: 1) Irradiated vocal fold tissue from patients undergoing laryngectomy for loss of function from radiation fibrosis was identified from pathology archives. Histomorphometry, immunohistochemistry, and whole-genome microarray, as well as real-time transcriptional analyses, were performed. 2) Focused radiation to the head and neck was delivered to mice in a survival fashion. One month following radiation, vocal fold tissue was analyzed with histomorphometry, immunohistochemistry, and real-time PCR transcriptional analysis for selected markers of fibrosis.

Results: Human irradiated vocal folds demonstrated increased collagen transcription, with increased deposition and disorganization of collagen in both the thyroarytenoid muscle and the superficial lamina propria. Fibronectin were increased in the superficial lamina propria. Laminin decreased in the thyroarytenoid muscle. Whole genome microarray analysis demonstrated increased transcription of markers for fibrosis, oxidative stress, inflammation, glycosaminoglycan production, and apoptosis. Irradiated murine vocal folds demonstrated increases in collagen and fibronectin transcription and deposition in the lamina propria. Transforming growth factor (TGF)-β increased in the lamina propria.

Conclusion: Human irradiated vocal folds demonstrate molecular changes leading to fibrosis that underlie loss of vocal fold pliability occurring in patients following laryngeal irradiation. The irradiated murine tissue demonstrates similar findings, and this mouse model may have utility in creating prevention and treatment strategies for vocal fold radiation fibrosis.

Figure 1

Distorted muscle and collagen architecture in radiated specimens (a) Normal muscle and collagen architecture (b) Mild disorganization (c) Severe disorganization

Figure 2

Increased thyroarytenoid muscle collagen content in radiated specimens vs. controls (n = 12, p=0.011).

Figure 3

Increased superficial lamina propria collagen content in radiated human vocal fold vs. controls (n = 13, p=0.0007).

Figure 4

Decreased thyroarytenoid muscle laminin content in radiated human vocal fold vs. controls (n = 13, p<0.0001).

Figure 5

Increased superficial lamina propria fibronectin content in radiated human vocal fold vs. controls (n = 12, p=0.021).

Figure 6

Collagen 1 mRNA increases in radiated human vocal fold tissue compared to control

Figure 7

Fibronectin mRNA increases in radiated human vocal fold tissue compared to control

Figure 8

Human Vocal Fold Radiation Fibrosis Summary

Figure 9

Pigmentary change localized to cervical region after completion of radiation treatment.

Figure 10

Radiated mice demonstrated higher levels of Collagen deposition: a) Light microscopy (20×) of vocal fold after Trichrome staining with increased collagen density staining blue. b) Integrated optical density, representative of collagen content (n=8, p<0.002)

Figure 10

Radiated mice demonstrated higher levels of Collagen deposition: a) Light microscopy (20×) of vocal fold after Trichrome staining with increased collagen density staining blue. b) Integrated optical density, representative of collagen content (n=8, p<0.002)

Figure 11

Radiated mice larynges exhibit no change in hyaluronic acid (HA) levels compared to control mice: a) Light microscopy (10×) of vocal fold after Alcian blue stain with hyaluronic acid staining blue. b) Integrated optical density, representative of hyaluronic acid content (n=8, p=0.82)

Figure 11

Radiated mice larynges exhibit no change in hyaluronic acid (HA) levels compared to control mice: a) Light microscopy (10×) of vocal fold after Alcian blue stain with hyaluronic acid staining blue. b) Integrated optical density, representative of hyaluronic acid content (n=8, p=0.82)

Figure 12

Radiated murine vocal folds demonstrate increased fibronectin content with immunostaining: a) Light microscopy (10×) of vocal fold after fibronectin immunostain. b) Integrated optical density, representative of hyaluronic acid content (n=4, p<0.02)

Figure 12

Radiated murine vocal folds demonstrate increased fibronectin content with immunostaining: a) Light microscopy (10×) of vocal fold after fibronectin immunostain. b) Integrated optical density, representative of hyaluronic acid content (n=4, p<0.02)

Figure 13

Radiated mice demonstrate significantly higher levels of procollagen and TGF-β mRNA: a) Increase in procollagen mRNA after radiation (n=5, p=0.048) b) Increase in TGF-β mRNA after radiation (n=5, p=0.007)