Investigation of nanostructural changes following acute injury using atomic force microscopy in rabbit vocal folds

There continues to be a paucity of data regarding the nanostructural changes of vocal fold (VF) collagen after injury. The aim of this study is to investigate the nanostructural and morphological changes in the rabbit VF lamina propria following acute injury using atomic force microscopy (AFM). Unilateral VF injury was performed on 9 New Zealand breeder rabbits. Sacrifice and laryngeal harvest were performed at three time points: 1 day, 3 days, and 7 days after injury. Histology and immunohistochemistry data were collected to confirm extracellular matrix (ECM) changes in rabbit VF. The progressive changes in thickness and D-spacing of VF collagen fibrils were investigated over a 7-day postinjury period using AFM. At post-injury day 1, a fibrin clot and inflammatory cell infiltration were observed at the injured VF. The inflammatory score at postinjury day 1 was highest in injured VF tissue, with a significant decrease at postinjury day 7. The immunoreactivity of inflammatory proteins (COX-2, TNF-α) was observed in VF up to day 7 after injury. AFM investigation showed clustered and disorganized collagen fibrils at the nanoscale resolution at post-injury day 7. Collagen fibrils in injured VF at postinjury day 7 were significantly thicker than control and postinjury days 1 and 3 (P < 0.001). D-spacing of collagen at postinjury day 7 was not studied due to loss of distinct edges resulting from immature collagen deposition. AFM investigation of VF could add valuable information to understanding micromechanical changes in VF scar tissue.