Fig 1.
Rat model of recurrent laryngeal nerve regeneration.
Sprague-Dawley rats (n = 37) divided into a control group (n = 4) and a bridged group (n = 33). This bridged group was further divided into a 1-mm gap group, a 3-mm gap group, and a 5-mm gap group (n = 11 each). In the bridged groups, nerve stumps were drawn 0.5 mm at a time from both ends into silicon tubes of different lengths and filled with type I collagen gel. In the control group, the nerve stumps were ligated using a thread.
Fig 2.
Scores for vocal fold movements.
Vocal fold movements are assessed on a five-point scale based on the criteria described by Tessema et al. The position of the arytenoid cartilage during expiration and inspiration is marked with a white-dotted line and a yellow-dotted line, respectively. 0 = no vocal fold movement; 1 = slight vocal fold movements; 2 = < 50% abduction of vocal folds; 3 = > 50% abduction, unsmooth vocal fold movements; 4 = vocal fold movements and positions indistinguishable from normal.
Table 1.
Scores for vocal fold movements in each group.
Fig 3.
Macroscopic appearance of regenerated tissue in the tubes.
At 15 weeks, we euthanized the rats, removed the silicon tubes, and macroscopically examined the regenerated tissues. The regenerated tissue connected the nerve stumps in all the examined rats. The center of the regenerated tissue was constricted. The tissue was thickest in the 1-mm group, followed by the 3-mm and 5-mm groups.
Fig 4.
Appearance of regenerated tissue in tubes under an optical microscope.
(A) At 15 weeks, the regenerated tissue inside the tubes was removed, and samples obtained from the centers of the tissues were prepared (toluidine blue staining). The tissue is surrounded by fibroblasts, and regenerated nerve fibers are observed in the center of the tissue. Scale bar: 50 μm. (B) We measured and compared the diameters of the regenerated tissues in the tubes. The diameter of the regenerated tissue is significantly more in rats in the 1-mm group than that in those in the 3-mm or 5-mm groups. ** p < 0.01, **** p < 0.001 (one-way ANOVA, Tukey’s multiple comparisons test).
Fig 5.
Appearance of regenerated tissue in tubes under an electron microscope.
We prepared sections of regenerated tissues in tubes 15 weeks after treatment and observed the sections under an electron microscope. We observed the presence of myelinated and unmyelinated nerves. Scale bar: 500 nm.
Fig 6.
Microscopic evaluation of thyroarytenoid muscle.
(Upper row) Low magnification: The thyroarytenoid muscle, in which the muscle fiber cross-sectional area is measured, is surrounded by a dotted line. Thyroarytenoid muscle atrophy is more severe on the treated side than that on the untreated side. Thyroarytenoid muscle on the treated side atorophied compared on the untreated side. Scale bar: 300 μm. (Middle row): High magnification: Magnified images of the boxes in the upper row. Atrophy of single muscle fibers can be observed. Scale bar: 100 μm (Lower row): The binarized thyroarytenoid muscle, and T/U ratios.
Fig 7.
Typical images of thyroarytenoid muscle tissue on the treated side in all groups.
(A) The degree of atrophy of the thyroarytenoid muscle in all groups was assessed using hematoxylin and eosin staining. All examined rats demonstrated overall atrophy of the thyroarytenoid muscle on the treated side. (B) The T/U ratios in the 1-mm, 3-mm, and 5-mm groups were 76.4 ± 5.1%, 69.6 ± 6.1%, and 63.6 ± 6.0%, respectively. Thus, atrophy was the least severe in the 1-mm group, followed by the 3-mm and 5-mm groups, with a significant difference between the 1-mm and 5-mm groups. ** p < 0.01, (one-way analysis of variance, Tukey’s multiple comparisons test).
Fig 8.
Typical electromyograms of rats in the 1-mm, 3-mm, and 5-mm groups. (Upper row): Treated side; (Lower row): Untreated side. In the 5-mm group, no potentials were evoked on the treated side. In the 1-mm and 3-mm groups, although potentials were evoked on the treated side, they were smaller than the action potentials on the untreated side, and the period of latency was longer than that on the untreated side.
Fig 9.
Typical case demonstrating the temporary restoration of vocal fold movements (1-mm group, #3).
(A) Time series of vocal fold movements (B) Optical microscope images of regenerated tissue (scale bar: 50 μm) (C) Electron microscope images (scale bar: 500 nm) (D) Evoked electromyogram. (A) Restoration of vocal fold movements observed at 5 weeks and 10 weeks, but disappeared at 15 weeks. (B) Optical microscope images and (C) Electron microscope images show nerve regeneration. (D) Observed evoked potentials.