How To: Does Intralesional Steroid Injection Effectively Mitigate Vocal Fold Scarring in a Rabbit Model?

INTRODUCTION

The increasing prevalence of professional voice users has led to a corresponding increase in voice-related issues, notably vocal cord polyps. Laryngomicrosurgery (LMS) is a common treatment for these conditions, often employing fiber optic lasers such as potassium-titanyl-phosphate (KTP) and pulse dye laser (PDL) in outpatient departments (OPDs). The use of lasers in these procedures can damage the vocal folds, however, leading to scarring.

In vocal fold scars, abnormal collagen levels increase, whereas those of elastin and hyaluronic acid decrease, leading to compromised vocal fold stiffness and viscosity due to the disorganization of the structure of the lamina propria. Various treatments are available for vocal fold scarring, including injections of autologous fat, collagen, or corticosteroids; scar excision; topical mitomycin-C application; and vascular stripping, but their effectiveness is limited. Therefore, the prevention of scarring becomes a crucial focus.

Steroid injections, known for their potent anti-inflammatory effects, are commonly used after laryngeal surgery, but conclusive evidence of their effectiveness in preventing scarring is lacking. Therefore, the primary objective of our study was to determine the effect of steroid injections in preventing vocal fold scarring. The secondary objective was to evaluate the differences in the preventive effects on vocal fold scarring based on the type of steroid used. Accordingly, the independent variables were the presence of steroid injection and the type of steroid injected, whereas the dependent variables were changes in the transcriptional, histological, and functional levels of vocal fold scars in a rabbit model.

MATERIALS AND METHODS

Ethics Statement: All animal experiments received prior approval from the Institutional Animal Care and Use Committee of Soonchunhyang University Bucheon Hospital. Care for all rabbits was in accordance with the Guidelines on the Care and Use of Laboratory Animals of Soonchunhyang University College of Medicine (IRB No. SCHBCA2022–09).

Animal Model of Vocal Fold Scar and Steroid Injection

The study involved 42 male New Zealand white rabbits, aged six months and weighing 2.5-3.0 kg. Each rabbit underwent bilateral vocal fold scar surgery using a 532-nm diode laser. Then, 28 were randomized into groups to receive an additional corticosteroid (dexamethasone or triamcinolone) injection into the superficial layer of the lamina propria, with 14 animals per group. The remaining 14 rabbits, which underwent the same surgery without additional treatment, served as negative controls. Ten rabbits from each group were subjected to high-speed vibration testing and subsequent histological analysis. Four rabbits from each group were used for real-time polymerase chain reaction (PCR) to assess the expression levels of extracellular matrix (ECM) genes (Fig. 1).

Surgical Procedures

All 42 rabbits were locally anesthetized prior to the experiment. They received intramuscular injections of 1 mL/kg Rompun (xylazine hydrochloride; Bayer Korea, Seoul, Korea) and ketamine (ketamine hydrochloride; Yuhan Ketamine 50 Injection; Yuhan Co., Korea). Following anesthesia, a long nasal speculum was inserted through the oral cavity, connected to a telescope (30°, 2.7 mm × 30 cm; Karl Storz Co., Tuttlingen, Germany), providing an unobstructed view of the vocal folds.

The lamina propria layer of both vocal folds was subjected to injury using a 532-nm diode laser (Quanta D8 532 diode lasers; Quanta System, Solbiate Olona, Italy; 600 µm diameter). The laser was set to 8 W pulses, with a pulse width of 50 ms and 2 pulses/s. The laser was applied from a fiber tip positioned 1-2 mm from the vocal fold, inducing a superficial injury. Each side of the vocal folds received a total of 20 joules of energy.

The treatment groups, Dexa and Triam, subsequently received 0.1-mL corticosteroid injections (either dexamethasone 5 mg/1 mL or triamcinolone 40 mg/1 mL) into the superficial layer of the lamina propria. The control group did not receive any additional treatment. Following surgery, all rabbits were moved to a recovery table and monitored for one to two hours, with 2 L/h of oxygen administered via nasal prong. All rabbits were euthanized four weeks following the surgery.

High-Speed Vocal Fold Vibration Test

Supraglottal structures, including the false vocal folds, epiglottis, and aryepiglottic folds, were removed from 10 harvested larynges. To ensure proper arytenoid approximation, the vocal folds were medialized using suture adduction of the arytenoids with 5–0 nylon sutures. A small, custom-fitted pipette tip was used for each trachea to prevent air leakage, thereby directing air through a tube to the glottis to seal the trachea. Oxygen from a compressed gas tank was delivered via a flow controller (Flow Gentle+; Koike Medical, Tokyo, Japan). The larynges were irrigated with phosphate-buffered saline to maintain moisture during the tests. After confirming the flow rate at which consistent and audible phonation with a stable mucosal wave was obtained for the vocal folds in all groups, all experiments were conducted at this same flow rate.

The maximum amplitude difference (MAD) was measured, reflecting the difference in mucosal wave amplitude. High-speed video endoscopy was performed using a Mega Speed X9 PRO Camera (Mega Speed Corporation, Mississauga, Canada) at a frame rate of 5,000 frames per second, allowing for detailed analysis of the vocal fold vibrations. The high-speed recordings were processed using TEMA 4.2 software (Image Systems Motion Analyses, Linköping, Sweden) to derive and analyze the vibrational amplitudes. Using a steel ruler positioned in front of the larynx and a high-precision calibrated grid displayed on the screen, the highest and lowest amplitudes of each vocal fold vibration cycle were measured and compared across groups. Measurements were taken from an imaginary line connecting the anterior commissure and the midlines of both arytenoids to the mid-membranous vocal fold region of the highest amplitude. All images were resized to equalize the anterior-to-posterior lengths of the vocal folds. The averages of the highest and lowest amplitudes from both vocal folds over approximately 100 cycles were compared among the three groups.

Histological Study

Ten larynges from each group after the vibration study were embedded in paraffin and sectioned coronally at a thickness of 6 µm. Slides including the middle regions of the membranous vocal folds underwent histological analysis. Staining was performed using hematoxylin and eosin and Masson’s trichrome to quantify the pixels of vocal fold and collagen density (CD) in each group.

The vocal folds were examined at a magnification of 20× using a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan) equipped with a Jenoptik color camera (ProgRes C10 Plus, Jena, Germany). ImageJ software (version 1.43u; National Institutes of Health, Bethesda, Md., USA) was used to calculate the total number of stained vocal fold regions. A single-blinded pathologist determined the CD ratio, dividing the number of CD pixels by the total number of pixels in the entire vocal fold, to minimize measurement bias.

The concept of cross-sectional area (CSA) was introduced as an indirect method to assess the volumes of the vocal folds. The mean CSA was determined by counting pixels corresponding to the true vocal fold in the slides, offering a partial representation of changes in vocal fold volume.

Real-Time PCR of ECM Genes

Eight vocal fold samples from four larynges in each group were analyzed using real-time PCR to assess gene expression in the ECM. After dissection, specimens were immediately placed in RNAlater Stabilization Solution and kept on ice until transfer. After 24-hour storage at 4°C, specimens were moved to −20°C until further processing. Homogenization was performed with the Bead Ruptor Elite (Omni International), ensuring a temperature below 4°C using liquid nitrogen. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, Calif., USA), and reverse transcription was performed using ReverTra Ace qPCR RT master mix with gDNA Remover (Toyobo, Osaka, Japan). For the reaction, 1-µg total RNA was utilized in a 10 µL reaction volume, and the generated cDNA was diluted with diethyl pyrocarbonate in 1:5 ratio (cDNA: DEPC). qPCR was done with SYBR Green real-time PCR master mix (Toyobo) on a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, Calif.) (Table I). The qPCR machine was set up with three steps: denaturation at 95°C, primer annealing at 60°C, and extension at 72°C. The reaction was run for 40 cycles and followed by melt curve analysis to ensure single-target amplification. Briefly, all the products generated during the amplification reaction were melted at 95°C, then annealed at 55°C, and subjected to a gradual increase in temperature to 95°C. Gene expression levels were normalized against the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The ∆CT values were calculated using the formula: CT target – CT GAPDH. Relative fold changes were determined using the 2−∆∆CT method.

Statistical Analysis

Statistical analyses were conducted using SPSS (version 18.0; IBM, Armonk, N.Y., USA), with the significance threshold set at p < 0.05. Means and standard deviations were calculated for each group. The Kruskal–Wallis test was used to compare scores across treatment groups and controls. Where statistical significance was observed, post hoc analysis with the Bonferroni multiple comparison test was employed to identify significant differences between pairs of values, with p-values adjusted to account for multiple comparisons. For the CSA analysis, relative normalization was applied to the treatment group data using the mean and standard deviation of the control group data to account for heterogeneity in laryngeal anatomy among rabbits.

RESULTS

High-Speed Vocal Fold Vibration Test

A significant difference in the mean maximum amplitude was observed between groups (p < 0.05). Post hoc analysis indicated that the mean maximum amplitude was significantly higher in both the Dexa group (1040 ± 128 µm) and the Triam group (1085 ± 129 µm) than in the control group (684 ± 181 µm) (p < 0.05) (Fig. 2). No significant differences were found between the treatment groups.

Histological Data

Significant differences were noted in both the mean CD ratio and CSA among the groups (p < 0.05). In post hoc analysis, the mean CD ratio was significantly higher in controls (43.5 ± 7.2%) than in the other groups (p < 0.05). No significant differences were noted between the treatment groups. The normalized mean CSA score of the Triam group was significantly lower than that of the controls (p < 0.05). No significant intergroup differences were observed between other groups.

Real-Time PCR of ECM Genes

Real-time PCR analysis showed significant increases in HAS2 gene expression in the treatment group compared with controls (p < 0.05). Conversely, the expression levels of the gene encoding collagen type IV were notably lower in the treatment group (p < 0.05).

CONCLUSION

Corticosteroid injection notably increased the levels of mRNAs encoding HAS2 and MMP-9. From a histological perspective, the CD ratio was lower in the treatment groups, and there was a significant improvement in MAD. This indicates that corticosteroid injection substantially enhanced vocal fold vibration. We conclude that corticosteroid injection acts as a preventive measure against vocal fold scarring by promoting wound remodeling and reducing fibrogenesis. Additionally, the observed atrophy in triamcinolone-injected vocal folds underscores a potential adverse effect of glucocorticoids on vocal fold tissue, warranting further investigation.