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Real-time Light-guided Vocal Fold Injection via Cricothyroid Membrane in Unilateral Vocal Fold Paralysis: A Human Pilot Study

Purpose: Vocal fold injection (VFI) via the cricothyroid (CT) membrane is used in the treatment of various VF diseases. The technical challenges of this technique are mainly related to the invisibility of the needle. Recently, real-time light-guided VFI (RL-VFI) was developed for simultaneous injection under light guidance in the CT approach. Herein, we present the first clinical trial of RL-VFI investigating the feasibility and safety of the new technique in unilateral vocal fold paralysis (VFP). Methods: This was a prospective pilot study that enrolled 40 patients, who were treated with RL-VFI for unilateral VFP between September 2020 and August 2021. Adverse events were monitored during the procedure and for 4 weeks postoperatively. Voice Handicap Index-10, GRBAS scale, aerodynamic study, and acoustic analysis were evaluated to compare the voice improvement after 4 weeks with the baseline values. Results: The needle tip was intuitively identified by the red light, and the mean procedure time was 95.6 ± 40.6 seconds for the initial injection, while it required 79.2 ± 70.5 seconds for the additional injection. The injection was proceeded under light guidance without additional manipulation after the needle reached the intended point. No acute and delayed adverse events were reported. Among the 40 patients, 36 completed voice analysis after 4 weeks. Subjective and objective voice parameters, including voice handicap index, GRBAS scale, maximum phonation time, mean expiratory airflow, fundamental frequency, jitter, shimmer, and noise-to-harmonics ratio improved significantly after RL-VFI ( P < 0.05) while expiratory volume was retained. Conclusion: RL-VFI is feasible and safe for treating patients with unilateral VFP. This technique is anticipated to improve the precision and safety of the CT approach in the treatment of unilateral VFP. This study provides a rationale for further structured clinical studies.


INTRODUCTION
The vocal fold injection (VFI) technique has been utilized as an effective treatment option for unilateral vocal fold paralysis (VFP) since its introduction over a century ago [1,2]. This minimally invasive technique has been re-emphasized due to recent advancements in endoscopic technology and injection materials [3,4].
Currently, VFI has been popularly used in the management for unilateral VFP, and its indications is broadening to a variety of vocal fold (VF) pathologies [1,5].
Various approaches have been reported for VFI, including transcutaneous (cricothyroid [CT] membrane, trans-thyroid cartilage [TT], and thyrohyoid [TH] membrane approaches), transoral, and transnasal approaches [6,7]. Each approach has its advantages and limitations, and the choice of an approach usually depends on the surgeon's preference and the patient's anatomical condition [8,9]. Among these approaches, some laryngologist prefers the CT membrane approach because the submucosal pathway may reduce the risk of bleeding, laryngospasm, and injectate spillage. Moreover, it has accelerated the spread of office based VFI [3,8,9].
The CT approach has been used in the treatment of various VF diseases. In this technique, precise injection at a designated location is crucial for effective augmentation and avoiding mis-injection [10]. However, the needle tip is not visible because it moves inside the VF during the CT approach. Thus, most laryngologists discern the location of the needle tip indirectly using physical maneuvers such as CT membrane palpation and the distortion of the VF configuration [11,12]. Due to the invisibility of the needle tip, the precise localization of the needle is very difficult in the CT approach and requires a high level of experience with a steep learning curve [8,13]. Furthermore, technical proficiency of the CT approach necessitates an understanding of the anatomical orientation linking exterior landmarks with internal laryngeal anatomy [14].
Some researchers suggested that the invisibility of the needle tip is the primary reason for the technical limitation in CT approach [5,8]. To overcome this limitation, Cha et al. conceptualized a new technique that allows simultaneous injection under light guidance and coined "Real-time Light-guided VFI" (RL-VFI) for this technique [8]. In previous studies, the concept models for RL-VFI were developed [8], and the technique was further verified in an ex vivo and in vivo canine model [9,15], demonstrating that the device for RL-VFI might be feasible and safe by providing precise localization and visual-motor feedback.
Recently, the Ministry of Food and Drug Safety in South Korea approved the RL-VFI device for clinical use.  The procedure times of RL-VFI were subsequently measured as two steps, aiming and injection. The aiming time was defined as the time from needle insertion in the skin to needle placement at the target point (just lateral to the vocal process). The injection time was defined as the time from the needle placement at the target point to injection completion. The sum of the two measurements computed the total procedure time.
The amount of injection material was determined based on the clinical findings of VF during the procedure.
After sufficient injection, 10-20% of preinjected volume was excessively injected considering the absorbed or dispersed amount after injection. If the augmentation was insufficient after the initial injection with 1 mL of commercialized volume, an additional injection was performed for proper medialization by replacing the used syringe with a new prefilled one using the same injector. The total injection volume was measured as the actual volume that was inserted into the vocal fold, excluding the volume of the dead space (0.3 mL) formed in the connector of the device. The procedure time for the additional injection was measured using the manner described earlier.

Voice analysis
The voice outcome of RL-VFI was evaluated prior to and 4 weeks following the procedure. Voice evaluation methods included voice handicap index-10 (VHI-10), perceptual evaluation (GRBAS scale: grade, roughness, breathiness, asthenia, and strain), acoustic analysis, and aerodynamic study. Pre-and post-operative voice data were analyzed for acoustic analysis and aerodynamic study using the Computerized Speech Lab and the Phonatory Aerodynamic System (Model 4500 and Model 6600, PENTAX Medical).  Change percentage was obtained from the change value compared to the baseline (pre-operative) value. A paired two-tailed student t-test was used to compare the differences in the voice outcomes pre-and postoperatively.

Continuous
Statistical analyses were conducted using the IBM Statistical Product and Service Solutions, version 20.0 (IBM Corp., Armonk, NY, USA); P-values <0.05 were considered statistically significant.

RESULTS
Out of 74 eligible patients with unilateral VFP, 9 did not meet the inclusion criteria and 25 refused to participate. Finally, 40 patients were enrolled, and they underwent RL-VFI treatment, of which 4 did not attend the scheduled voice evaluation 4 weeks postoperatively. The participants visited the hospital within 3 months and delayed adverse events for 4 weeks were monitored. Thus, 40 patients were included for the safety evaluation and procedure time measurement, and 36 were evaluated for postoperative voice outcomes (Fig. 2).

Patient demographics
This study included 32 men (80%) and 8 women (20%), with a mean age of 61.2 ± 13.0 years. VFP was mostly observed on the left side (n = 35, 87.5%) and mainly caused by thoracic surgery involving aorta (n = 9, 22.5%), lung or esophagus (n = 9, 22.5%). Participants with iatrogenic cause of VFP were mainly referred from the cardiac surgery department of our institution for adjuvant VFI therapy. Mediastinal lymph node metastases (n = 14, 35.0%) and idiopathic (n = 7, 17.5%) were other etiologies for VFP. The mean onset of VFP was 5.9 ± 14.5 months, which mainly occurred in less than 6 months (Table 1).

Feasibility and safety of RL-VFI
The sequential steps of the RL-VFI were demonstrated in Figure 3. In the representative case, the left VF was paralyzed in the paramedian position and had a bowed edge (Fig. 3A). When the needle entered the skin and approached the CT membrane, the needle tip was identified by the red light emitted through the subglottic mucosa (Fig. 3B). The light on the subglottic mucosa provided information regarding the spatial orientation and intuitively helped prevent the needle from penetrating the mucosa. The needle was gently advanced into the paraglottic space and the thyroarytenoid muscle. The light could be clearly identified when it reached the target point just lateral to the vocal process (Fig. 3C). After confirming the placement of the needle at the target point under light guidance, HA was injected into the thyroarytenoid muscle (Fig. 3D). As the injectate spread, the A c c e p t e d A r t i c l e light dispersed more through it due to the higher transmittance of the injectate as compared to the adjacent tissues. The extent of the injectate was clearly indicated by light dispersion (Fig. 3E). The procedure was completed safely, and the injector was withdrawn. Thus, the left VF was effectively medialized with the injectate after the procedure (Fig. 3F).
If the initial injection did not medialize the VF sufficiently, an additional injection was administered for further augmentation, because each prefilled syringe contains 1.0 mL of HA. The additional RL-VFI was performed in the same way by the used syringe with a new prefilled one using the same injector. The needle tip was clearly traceable through the injected material with dispersed light. Further augmentation of the VF could be accomplished without difficulty under light guidance (Fig. 4).
The mean volume of injectate used was 1.0 ± 0.4 mL. Additional injections were needed in 29/40 patients because further augmentation was required after the initial injection. The mean aiming time, injection time, and total procedure time during the initial injection were 22.6 ± 18.4, 73.6 ± 32.8, and 95.6 ± 40.6 seconds, respectively, and those during the additional injection were 17.2 ± 15.0, 62.0 ± 69.3, and 79.2 ± 70.5 seconds, respectively ( Table 2). RL-VFI was performed with no acute complications for all participants. During the follow-up period, there were no procedure-related delayed adverse events or unscheduled hospital visits.
However, expiratory volume retained its level after operation, and acoustic analysis of voice break, subharmonic, and voice irregularity did not show significant change.
A c c e p t e d A r t i c l e

DISCUSSION
The CT approach is a minimally invasive and effective method, wherein the needle is directly inserted into the VF through the skin and CT membrane. Although its operative concept is simple, the procedure can be technically challenging [14]. The insertion point of the needle is first determined by external landmarks, and then the needle placement in the larynx is indirectly identified using the altered configuration of the VF on laryngoscopy [12]. Because the needle is invisible until penetrating the mucosa or approaching the VF submucosa, it may require several attempts even for experienced laryngologists [14]. This invisibility of the needle tip might be the crucial drawback of the CT approach [5,8].
Identifying the needle tip in the VF is the most difficult step of this approach, which is usually presumed based on the distortion of VF configuration [3], creating undesirable clinical situations. Incorrect placement of the needle tip can lead to failure to reduce the VF gap [10], and penetration of the VF mucosa may cause adverse effects such as spillage of the injectate, hematoma, and laryngeal spasm [3]. Inadvertent mis-injection in the extra-glottic space is a rare deleterious complication of pulmonary embolism [16], while mis-injection in the Reinke's space may cause unfavorable outcomes requiring laryngeal microsurgery [17].
Improved precision of VFI may make the procedure reliable and predictable. Various efforts have been made to increase the accuracy of the CT approach [5,11,14]. Jin et. al conducted an anthropometric study for the CT approach using three-dimensionally reconstructed computed tomography [14]. They provided anatomical information regarding the distance and angle of the path of the needle during the approach. Electromyographyguided VFI is proposed as a good option for which the long-term results have already been validated [18], whereas VFI under ultrasonographic guidance is offered to provide a real-time assessment of the position of the needle [19]. Additionally, Hoffman et al. suggested an innovative idea of transillumination of the VF, which provided direct visual feedback in this approach [5]. Although this technique was proven competent in localizing the needle tip, the fiberoptic cable had to be removed before delivering the injectate. However, it seems difficult to apply this removal and reinsertion technique in real practice.
RL-VFI was conceptualized to perform simultaneous injection into the VF with the precise placement of the needle tip under light guidance [8]. The RL-VFI device allows accurate localization of the needle tip and facilitates material delivery without further manipulation of the needle after determining the target point of injection. An ex vivo study using a prototype RL-VFI device successfully demonstrated accurate localization of the needle tip [8,9]. Further research on an in vivo canine model presented successful results with various A c c e p t e d A r t i c l e transcutaneous approaches [15], with histologic analysis of the canine larynx confirming the presence of the injectate in the thyroarytenoid muscle, without any evidence of thermal damage to the surrounding tissues.
Based on the results of preclinical studies [8,9,15], this study was designed as a pilot study, a first trial applied to human for the purpose of investigating the feasibility and safety of RL-VFI.
This feasibility study confirmed a clear visualization and precise localization of the needle tip during RL-VFI in clinical practice. The needle tip was easily identified during the entire procedure from penetration of the CT membrane to approaching the target point in the VF mucosa (Fig. 3). During RL-VFI, the brightness scale and scattering pattern of the light provided information on the needle depth. When the needle was positioned deeper from the surface, the light appeared blurred because it scattered during transmission. The light became more intensified and focused as the needle tip approached the mucosa. This pattern could give intuitive intraoperative guidance for the depth orientation and could help prevent inadvertent mucosal penetration.
Moreover, navigating the needle through the injected material presented a stronger dispersion of light, which helped to estimate the extent of the injectate in real time. Although indicating the accurate orthotopic location in the injection material is limited, the light-guidance still provides information of the needle entering the "space" of injected material through a light dispersion. Additional injection into any point in this space makes the material-filled sphere expand radially. Thus, it could facilitate controlling the dose precisely during injecting HA ( Fig. 3 and 4).
To ensure the applicability of RL-VFI in real practice, the procedure times of RL-VFI were measured as two steps, aiming and injection. The aiming step, involving needle insertion in the skin and needle placement at the target point, is vital in conventional VFI via the CT approach. In the conventional CT approach, the needle tip position is approximated by the movement of the VF free edge when swinging the syringe horizontally [11,12].
This skill can only be acquired through clinical experience, and it follows a steep learning curve [8,13]. This step usually takes a long time to determine the placement of the needle at the intended point because laryngologists usually depend on indirect physical maneuvers such as the distortion of the VF configuration [11,12]. RL-VFI adjunctively supports this process by providing intuitive optical signs for locating the needle tip. The mean aiming time was 22.6 ± 18.4 seconds in this study, which is encouraging for possibly improving the clinical efficacy of the CT approach. The injection step involves injectate delivery into the VF. It is performed simultaneously while assessing the augmentation degree. The participants were asked to phonate during augmentation to determine the appropriate augmentation degree. During phonation, the light assisted in maintaining the needle tip at the intended point and helped to achieve delicate augmentation. The total A c c e p t e d A r t i c l e procedure time was appropriate for the office-based setting, and this quantitative result supports the applicability of the procedure.
VFI for unilateral VFP improves voice outcomes as assessed by VHI-10, GRBAS scale, aerodynamic studies, and acoustic analysis [20][21][22][23]. For the acoustic analysis, we presented a total of 10 values consisting of 2 values from each measurement related to fundamental frequency, frequency and amplitude perturbation, noise and tremor evaluation, and voice break, subharmonic, and voice irregularity, while maximum phonation time, mean expiratory airflow, mean sound pressure level, aerodynamic efficiency, expiratory volume were presented as aerodynamic aspects of dysphonia [24,25]. In this study, most of the voice parameters related to unilateral VFP improved significantly after 4 weeks when compared with the baseline values. The total GRBAS scale has shown significant improvement owing to improved results in grade, roughness, and breathiness scale. The participants were mostly referred after undergoing thoracic surgery (aorta, lung) or were receiving palliative care for metastatic disease, and therefore, the majority of the participants' underlying lung function was deteriorated as evidenced by the expiratory volume ( Table 3). The low postoperative VHI-10 and MPT values appear to be associated to the patient group characteristics as recruited in a tertiary referral institution that regularly supports cancer patients. Nevertheless, significant changes in VHI-10 and functional parameters such as MPT and MEA can be regarded an improvement in voice function representing an effective augmentation.
VFI is known to be a rather safe procedure but can lead to several adverse events such as injectate spillage, needle penetration, hematoma, superficial injection into the subepithelial space, or other severe complications [3,16,17]. In our study, such complications were avoided with the aid of the light source. The light guidance was especially helpful for avoiding penetration of the mucosa or mis-injection. Furthermore, as was histologically presented in the previous in vivo study [15], clinical evidence of thermal damage (tissue shrinkage, burn) was not observed in any of our participants. clinical evidence of thermal damage (tissue shrinkage, burn) was not observed in any of our participants. Moreover, there were no significant bleeding or regurgitation events, related to the larger diameter of the needle (23-gauge) compared to conventional needle diameter (25-gauge). Although it should be further validated with a larger sample, RL-VFI is expected to help improve the safety of VFI by advanced precision based on our results.
The clinical application of RL-VFI could improve the CT approach in various aspects. First, the blind nature of the procedure can be overcome with light guidance, leading to improved safety and efficacy. RL-VFI would ensure guiding the needle to the appropriate location in the larynx and preventing inadvertent penetration of the VF mucosa or mis-injection at unintended locations. This would also improve the accessibility of various A c c e p t e d A r t i c l e anatomical locations. Second, RL-VFI can be applied in VFI training and could shorten the learning curve by providing visual feedback [26]. This will eventually lead to reducing the disparity in clinical results between expert and novice laryngologists because the needle location can be easily identified by intuitive guidance rather than personal experience. Therefore, RL-VFI may improve the reproducibility of VFI via the CT approach, regardless of clinical experience. Moreover, the visual feedback will also be helpful in treating patients with distorted anatomy such as thick neck or scar tissue which are difficult even for highly experienced laryngologists. Finally, when counseling patients, RL-VFI would help them to understand the procedure intuitively and be reassured.
Although our clinical results of RL-VFI may be promising, our study has several limitations. First, the sample size was small. Potential adverse events may not have occurred in this study, and the functional outcomes might have been overestimated. Second, the research followed a single-armed design. Thus, the functional results need further verification. Although improvements in voice outcomes after VFI have been acknowledged in several studies, the between-study heterogeneity of voice outcome is large, and the specific values may vary depending on patient factors, etiology, and injection skills [27]. After this study, we will validate the efficacy of RL-VFI based on the clinical outcome such as the duration of adequate voice and the patient's convenience in a randomized controlled study. Third, only the CT approach for RL-VFI was validated among various transcutaneous approaches in this study. Although other transcutaneous approaches, including TT and TH, have been successfully applied with RL-VFI in a previous preclinical study [15], they were not assessed in this study. Lastly, we have also encountered a technical issue related to the device during the trial.
The injector on the device was designed to connect to an optic fiber through a connector, leaving the dead space (0.3 mL) where it attaches to the syringe. In 9 (22.5%) participants with injection volume between 0.7 mL and 1.0 mL, additional filler and cost were required due to this technical issue of the dead space. This technical issue for dead space needs to be amended in the advanced version of the device. In the future, a structured analysis of long-term voice outcomes and comparison of various approaches with the conventional VFI are warranted to verify the potential advantages of RL-VFI.
In conclusion, RL-VFI is feasible and safe for treating patients with unilateral VFP. This technique is anticipated to improve the precision and safety of the CT approach in the treatment of unilateral VFP. This study provides a rationale for further structured clinical studies.     Procedure time of the initial injection with RL-VFI, sec (mean ± SD) The aiming time 22.6 ± 18.4 The injection time 73.6 ± 32.8 The total procedure time 95.6 ± 40.6 Procedure time of the additional injection with RL-VFI, sec (mean ± SD)*