The present study was conducted on 25 male Sprague-Dawley rats (weight, 320–350 g) purchased from the Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The rats were maintained in the animal laboratory of the Shanghai Jiao Tong University affiliated Shanghai First People's Hospital (Shanghai, China) and all surgical procedures were performed in the animal operating room. The experiment was approved in accordance with the Institutional Animal Care and Use guidelines of Shanghai Jiao Tong University by the Animal Experimental Committee of First People's Hospital affiliated with the Shanghai Jiao Tong University of Medicine. The animals were treated humanely. All rats were maintained under standardized laboratory conditions in groups of 5 rats, with food and water available ad libitum. The humidity was 60% and the air temperature was 25°C, with a 12 h light/dark cycle.

Following intraperitoneal anesthesia with sodium pentobarbital (40 mg/kg body weight; Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China), telescopic video laryngoscopy (BF-1T160; Olympus Corporation, Tokyo, Japan) was performed to confirm normal motion of the vocal fold with breathing. Following removal of the hair on the neck, a vertical midline cervical incision was made with a sharp blade (Tongkang Medical Instrument Co., Ltd., Shanghai, China). To expose the tracheoesophageal groove, the salivary glands and infrahyoid muscles were reflected laterally using iris hooks (Tongkang Medical Instrument Co., Ltd.). Subsequently, the right RLN was carefully isolated from the tracheoesophageal groove. The animals were divided into two groups, with 5 rats/group. For the denervation group, the right RLN was resected at the level of the seventh tracheal ring, with the removal of a 5-mm segment. The muscle layers and skin were sutured separately. For the control group, the incision was sutured following identification of the right RLN. Telescopic video laryngoscopy was repeated to confirm paralysis of the right vocal fold. On occasions where the right vocal fold exhibited movement, the rat was excluded from further investigation. An intraperitoneal injection of benzylpenicillin sodium (10⁵ U/kg body weight; North China Pharmaceutical Co., Ltd., Shijiazhuang, China) was administered to all rats to prevent infection. All the rats were monitored following surgery, fed a standard diet, and allowed to heal for 3, 6, 10 or 16 weeks (n=5 for each group and time period).

At 3, 6, 10 and 16 weeks, five rats with transection injuries were anesthetized again, as described above. Previous studies have demonstrated that the effects of anesthesia on vocal fold mobility are minimal (52,53). Video laryngoscopy was performed to assess the vocal fold motion and glottis morphology during spontaneous breathing. The movements observed via transoral endoscopy were divided into five different categories ranging from grade 0 to 4 using the following criteria: grade 0, no vocal fold movement; grade 1, slight vocal fold movement; grade 2, <50% abduction of vocal fold; grade 3, >50% abduction of vocal fold; and grade 4, normal vocal fold movement. The final scores were obtained by two investigators who were blinded to the animal grouping and time since denervation. In case of disagreement, a third investigator's score was obtained. Normal movement of the vocal fold was measured using five rats and a double-blind assay was performed to prevent subjective bias.

Images were captured for measurement of the arytenoid cartilages (Fig. 1). The angles between the arytenoid cartilage structures were calculated (54) using images of maximal adduction and abduction (Fig. 2).

Animals were maintained in a quiet environment for one day prior to euthanasia. Vocalization lasting 20 sec was recorded by stimulating the right hind limb using forceps every 4 h following surgery. The procedures were performed in an identical environment by the same investigator using the same equipment, crush pressure (5 N), and distance between the recorder and mouth (10 cm). A capture tool (Cool Edit Pro v2.1; Adobe Systems, Inc., San Jose, CA, USA) was used to record the sound fragments, followed by acoustic space analysis using Photoshop CC software, version 14.0 (Adobe Systems, Inc.). Calculations were performed as follows: Spectral area (%) = area of postoperative rats/area of control rats. Amplitude (%) = amplitude of postoperative rats/amplitude of control rats.

Following euthanasia using sodium pentobarbital (120 mg/kg body weight), the larynx was excised. Under a dissecting microscope (SZ51-ILST-SET; Olympus Corporation), the thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles were removed for immunostaining of BDNF and GDNF. The TA and PCA muscles were selected for the current study as the TA muscles are hypothesized to be the most affected laryngeal muscles following resection of the RLN, and the PCA muscle is the sole abductor muscle (55). At each time-point, six sections (7 µm thick) from each rat were prepared for staining. Sections were pretreated with 0.3% H₂O₂ and preincubated in 5% bovine serum albumin (Wuhan Boster Biological Technology, Co., Ltd., Hubei, China). The sections were then incubated with a rabbit monoclonal antibody against BDNF (dilution, 1:75; ab108319; Abcam, Cambridge, MA, USA) or a rabbit polyclonal antibody against GDNF (dilution, 1:100; sc-328; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), overnight at 4°C, followed by incubation for 15 min at 37°C with the horseradish peroxidase-conjugated secondary antibody (GK500711; Genetic Technology Co., Ltd., Shanghai, China) according to the manufacturer's instructions. Following incubation with 3,3′-diaminobenzidine, the sections were dehydrated, dried and coverslipped.

Two observers blinded to the grouping examined each section separately using a morphometric workstation running Image Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). Following calibration of the optical density, the images were recorded at a final magnification of ×100. The area, mean density and relative optical density of immunolabeling were obtained for each specimen. The microscope illumination was calibrated to ensure that the relative optical density values were within the linear response range.

The right RLN was harvested. The left nerve served as a control and was harvested at the same level. The RLN was isolated and fixed in 4% (vol/vol) formaldehyde for 48 h, embedded in paraffin and 7-µm sections were cut from each segment, and examined by modified Bielschowsky staining (56). The slices were placed in silver nitrate solution for impregnation for 30 min and restored using formaldehyde. Silver ammonia was added, which was followed by another restoration. Finally, the sections were toned with gold chloride and fixed by sodium thiosulfate (Shanghai Yuanye Biotechnology Co., Ltd.). As a result, the axons appeared black. For each time-point, six sections from each rat were prepared for imaging. The diameter and number of axons were calculated using Photoshop CC software, version 14.0 (Adobe Systems, Inc.), and the longest and shortest axons in addition to the mean axon diameter were analyzed.

Each outcome value was summarized as the mean ± standard deviation. A one-way analysis of variance with Tukey's multiple comparison tests was used for statistical analysis of the measures of vocal fold movement and vocalization using SPSS version 20.0 (IBM SPSS, Armonk, NY, USA). The immunostaining and histological results were analyzed using the independent-samples t-test. P<0.05 was considered to indicate a statistically significant difference.

All animals survived until the predetermined endpoint. All incisions healed well without infection. Nerve ends were surrounded by scar adhesions, with no significant bulges.

All denervated animals demonstrated immediate right vocal fold paralysis. No obvious vocal cord movement was recorded at any of the time intervals following transection of the RLN. A significant difference was indicated for angles between the arytenoid cartilage structures for the denervation and control groups (P<0.01). The angles of maximal abduction were 21.97±5.30, 22.34±6.79, 22.56±4.94, 25.94±6.92 and 40.60±9.36, at 3, 6, 10 and 16 weeks and in the control group, respectively (Fig. 3A), and the maximal adduction angles were 13.14±5.54°, 11.70±4.52°, 10.29±3.41°, 9.12±3.99° and 4.90±0.22°, at 3, 6, 10 and 16 weeks and in the control group, respectively (Fig. 3B). The vocal fold movements on the side of nerve injury did not return to normal during the study.

The voice spectra of the denervation and control groups are presented in Fig. 4. The vocalization sounded sharp and high-pitched in the control rats, whereas it was hoarse and deep in the denervation groups. The spectral analysis of the control group demonstrated a high amplitude with a wide, continuous waveform (Fig. 4A). Following RLN injury, the spectrum became narrow with a low amplitude. However, the amplitude and width of the continuous waveform increased gradually with time (Fig. 4B–E).

The spectral area (calculated as a percentage) in the denervation group increased gradually over time, with values at 3, 6, 10 and 16 weeks of 21.07±14.41, 25.04±7.50, 33.22±13.89 and 40.55±15.36%, respectively (Fig. 5A; P<0.01 vs. the control group). The postoperative amplitudes at 3, 6, 10, and 16 weeks were 60.43±41.49, 73.62±18.72, 87.02±23.40 and 95.74±6.38%, respectively (Fig. 5B). Over time, the amplitude percentages increased (P<0.05), although there was no significant difference between the control group and the 16-week denervation group (P=0.0540).

Figs. 6 and 7A present data for the expression of BDNF in the TA and PCA muscles. In the TA muscles, BDNF expression levels decreased slowly over time. However, the expression of BDNF significantly decreased at 3 weeks following injury. Although the expression increased at 6 weeks, it did not reach the baseline level.

Expression of GDNF (Figs. 7B and 8) in the TA was similar to that in the PCA muscle from 3 to 10 weeks; however, at rest, there was a marginal increase in GDNF expression levels in the PCA muscle.

Compressed or degenerating axons were observed in certain cases. Fig. 9 demonstrates axons embedded in a fibrous structure. At 16 weeks, the RLN had fewer axons (~81.00±7.07 axons) than the control nerves (Fig. 10). The axon diameters of the RLN sections were recorded. The mean diameter of the axons was 4.38±1.1.75 µm in normal rats; the right RLN exhibited a reduction for 6 weeks, followed by a slight increase. RLN regrowth was not correlated with vocal fold motion.