Afferent Nerve Ending Density in the Human Laryngeal Mucosa: Potential Implications on Endoscopic Evaluation of Laryngeal Sensitivity

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1 Dysphagia (2015) 30: DOI /s ORIGINAL ARTICLE Afferent Nerve Ending Density in the Human Laryngeal Mucosa: Potential Implications on Endoscopic Evaluation of Laryngeal Sensitivity Giovanni Ruoppolo Ilenia Schettino Antonella Biasiotta Rocco Roma Antonio Greco Pietro Soldo Dario Marcotullio Alessandro Patella Emanuela Onesti Marco Ceccanti Francesca Albino Carla Giordano Andrea Truini Marco De Vincentiis Maurizio Inghilleri Received: 25 February 2014 / Accepted: 4 December 2014 / Published online: 19 December 2014 Ó Springer Science+Business Media New York 2014 Abstract Laryngeal sensitivity is crucial for maintaining safe swallowing, thus avoiding silent aspiration. The sensitivity test, carried out by fiberoptic endoscopic examination of swallowing, plays an important role in the assessment of dysphagic patients. The ventricular folds appear to be more sensitive than the epiglottis during the sensitivity test. Therefore, this study aimed to investigate the mechanical sensitivity of the supraglottic larynx. In seven healthy adults undergoing microlaryngoscopy to remove vocal cord polyps, we excised mucosal samples from the epiglottis and ventricular folds. We measured afferent nerve fiber density by immunoelectron microscopy. All of the subjects underwent an endoscopic sensitivity test based on lightly touching the laryngeal surface of the epiglottis and ventricular folds. The discomfort level was self-rated by the subjects on the visual analog scale. Samples were fixed and stored in cryoprotectant solution at 4 C. Sections were stained with the protein gene product 9.5, a pan-neuronal selective marker. Nerve fiber density was calculated as the number of fibers per millimeter length of section. The mean nerve fiber density was higher in ventricular samples than in epiglottis samples (2.96 ± 2.05 vs 0.83 ± 0.51; two-sided p = 0.018). The mean visual analog scale scores were significantly higher for touching the ventricular folds than for touching the epiglottis (8.28 ± 1.11 vs G. Ruoppolo I. Schettino A. Greco P. Soldo D. Marcotullio A. Patella F. Albino M. De Vincentiis Otorhinolaryngology Section, Department of Sensorial Organs, Sapienza University, Viale del Policlinico, 155, Rome, Italy I. Schettino ileniaschettino@libero.it A. Greco antonio.greco@uniroma1.it P. Soldo pietro.soldo@uniroma1.it D. Marcotullio dario.marcotullio@uniroma1.it A. Patella patella321@gmail.com F. Albino fra.albino@gmail.com M. De Vincentiis marco.devincentiis@uniroma1.it G. Ruoppolo (&) Via Nomentana, 401, Rome, Italy giovanni.ruoppolo@uniroma1.it A. Biasiotta E. Onesti M. Ceccanti A. Truini M. Inghilleri Department of Neurology and Psychiatry, Sapienza University, Viale Università 30, Rome, Italy E. Onesti emanuela.onesti@uniroma1.it M. Ceccanti marco.ceccanti@yahoo.it A. Truini andrea.truini@uniroma1.it M. Inghilleri maurizio.inghilleri@uniroma1.it R. Roma Pediatric Otolaryngology, Children Hospital Bambino Gesù, Piazza Sant Onofrio, 4, Rome, Italy romarocco@gmail.com C. Giordano Department of Radiological Sciences, Oncology and Anatomical Pathology, Sapienza University, Viale del Policlinico, 155, Rome, Italy carla.giordano@uniroma1.it

2 140 G. Ruoppolo et al.: Nerve Endings in the Human Laryngeal Mucosa 4.14 ± 1.21; two-sided p = 0.017). The higher sensitivity of the ventricular region should be considered for further refining clinical endoscopic evaluation of laryngeal sensitivity. Keywords Fiberoptic endoscopy Dysphagia Cough PGP protein Introduction In the last few decades, a crucial role of safe deglutition in maintaining respiratory health has become increasingly evident. Although development of pneumonia is considered to be multi-factorial [1], significant relationships have been found between the rate of pneumonia and laryngeal sensory deficits leading to silent aspiration [2]. On clinical assessment, some efforts have been made to increase the ability to detect silent aspiration using the cough reflex test [3, 4]. However, use of the cough reflex test in a well-conducted, randomized, controlled trial has failed to achieve the end goal of reducing pneumonia in post-stroke dysphagia [5]. Modified barium swallow and fiberoptic endoscopic examination of swallowing (FEES) allow reliable evaluation of silent aspiration. Despite the lower effectiveness of FEES in quantifying aspiration compared with modified barium swallow, endoscopic assessment allows direct evaluation of the laryngeal adductor reflex (LAR) [6]. The LAR, which is a brief closure of the true vocal folds, is a sensorimotor response that plays an important role in defending the lower airways during swallowing and protecting the supraglottic larynx from food or fluid aspiration during inspiration or pharyngeal spillage [7]. Nerve endings mediating the LAR arise from the internal branch of the superior laryngeal nerve (ibsln). Sectioning this nerve abolishes the LAR [8]. A surgical study that investigated the ibsln in human cadaver specimens showed that the ibsln lays inferiorly and deeply to the greater cornu of the hyoid. After crossing the thyrohyoid membrane, the ibsln divides into superior, middle, and inferior branches in humans. The superior branch provides sensorial innervations to the piriform sinus, while the middle ramus innervates the mucosa in the aryepiglottic fold and vestibule. The inferior ramus distributes a few branches to the ventricular and infraglottic laryngeal mucosa [9]. Stimulation of the mucosa innervated by the ibsln elicits the LAR [10, 11]. However, the role of the sensory receptors that are involved is unclear. The morphological, topographical, and physiological characteristics of the sensory structures of the laryngeal mucosa have not been definitively categorized. Descriptions of the different receptors vary because of the study of many different types of species [12]. Histological studies have shown the presence of free nerve endings, laminar endings, and taste buds in the laryngeal mucosa [13 15]. Sensory receptors can be subdivided into mechanoreceptors and chemoreceptors [16], but which receptor causes a specific reflex is unclear. However, several studies have shown that the laryngeal reflexes are initiated when mechanical or chemical stimuli contact receptors in the laryngeal mucosa, and these trigger afferent neural activity [12]. The use of sensory evaluation to investigate the LAR in current clinical assessment of dysphagic patients was first proposed by Langmore et al. [17]. In FEES, they included a sensitivity test based on lightly touching the pharyngeal walls, the laryngeal surface of the epiglottis, the aryepiglottic folds, the arytenoids or the vocal folds, or both, using the tip of a probe. As an alternative procedure for achieving a more precise sensory evaluation, Aviv et al. [18, 19] later introduced fiberoptic endoscopic evaluation of swallowing with sensory testing (FEESST), a technique based on delivering air-pulse stimuli to the mucosa innervated by the superior laryngeal nerve to measure the sensory threshold in the hypopharynx. The chosen area was the mucosa in the pyriform sinus and aryepiglottic folds. In recent years, despite the increasing importance of laryngeal sensitivity in patients with neurogenic oropharyngeal dysphagia, only a few studies have addressed this topic and most have evaluated the reliability of air-pulse stimulation [20]. Our interest in deepening the knowledge on laryngeal sensitivity was based on the clinical observation that the ventricular folds, which were not mentioned in the original Langmore protocol [17], appeared to be more sensitive than the epiglottis during the sensitivity test. Therefore, our study aimed to assess whether afferent nerve fiber density in the mucosa of the laryngeal surface of the epiglottis differs from that of the ventricular folds in humans. We excised laryngeal mucosa tissue from the epiglottis and vestibule in healthy adults undergoing microlaryngoscopy to remove benign vocal cord tumors. We measured afferent nerve fiber density by immunoelectron microscopy. An experimental confirmation of a greater sensitivity of the mucosa of the ventricular folds, if strengthened by the results of further studies, might indicate that this area during FEES should be examined when touching the laryngeal surface of the epiglottis fails to trigger the LAR. In addition, data on laryngeal afferent fiber density could also provide basic reference data for future studies on laryngeal sensitivity in neurodegenerative patients with dysphagia. Materials and Methods In seven healthy adults (mean age ± 16.7 years) undergoing direct microlaryngoscopy to remove benign vocal cord tumors, 14 biopsy specimens were taken from healthy laryngeal mucosa, seven from the laryngeal surface

3 G. Ruoppolo et al.: Nerve Endings in the Human Laryngeal Mucosa 141 Fig. 1 Immunohistochemical detection (940 magnification) of intramucosal nervous fiber density in epiglottis (a) and in ventricular fold (b) in a patient with no history or clinical evidence of neurologic diseases. Blue arrows show single nerve fibers arising from submucosa. Blue scale bar 60 lm of the epiglottis, and seven from the ventricular folds. The samples were analyzed by immunoelectron microscopy. We used a selective pan-neuronal marker, the pan-axonal anti-protein-gene product 9.5 (PGP 9.5), to highlight sensory innervation [21 23]. Inclusion criteria included no smoking for at least 10 years, healthy supraglottic laryngeal mucosa documented by videoendoscopy (flexible fiberoptic rhino-laryngoscope Storz 11101), reflux finding score = 0, and no history or clinical evidence of neurological diseases. Laryngeal sensitivity was tested on the day before surgery using FEES, including a sensitivity test, based on lightly touching the laryngeal surface of the epiglottis and the ventricular folds. After elicitation of the LAR reflex at both levels was checked, the subjects were asked to quantify the discomfort level at the two different sites of stimulation, by making a mark on the visual analog scale (VAS). A 10-cm horizontal line, equally divided into 10 levels, was used. On this scale, the starting point (0) represented no discomfort, while the end point (10) indicated intolerable discomfort. All of the polyps were pedunculated and located on the free edge of the vocal fold. In one patient, the polyps were bilateral while monolateral in other patients (on the right side in four patients and on the left side in the remaining two). Laryngeal biopsy specimens were excised during microlaryngoscopy after removal of polyps. After the ventricular fold sample was excised, the laryngoscope was slightly retracted and the epiglottic sample was removed. Excision was always performed on the right side for technical simplicity. Each mucosal specimen measured approximately mm in size. All of the patients gave their written informed consent to participate in the study. The study was performed according to the ethical standards laid down in the Declaration of Helsinki. Laryngeal samples were fixed using Zamboni s solution (paraformaldehyde/picric acid) for 24 h and then stored in cryoprotectant solution at 4 C. Tissue blocks were cut into 50-lm-thick sections, perpendicular to the mucosal surface. This thickness enabled us to use the free-floating technique, where three random nonconsecutive sections were selected from each sample and all immune steps proceeded in 96-well tissue culture plates. The sections were immunostained with PGP 9.5 (diluted 1:1,000; rabbit) on a shaker table at room temperature overnight. After rinsing, sections in TBS solution were stained with a secondary antibody (diluted 1:100; anti-rabbit) for 1 h. Sections were then incubated in blue chromogen for 2 min. After mounting, counterstaining, and coverslipping, we counted nerve fibers at 409 magnification with a bright light microscope (Fig. 1). After measuring the linear mucosal surface (in microns) by a dedicated software (Meta Imaging Series Software, Sunnyvale, CA, USA), nerve fiber density was calculated based on the section surface, using the following formula: Number of fibers=linear surface in l 1:000 ¼ number of fibers per mm length of section: The medium density value in each sample was finally calculated by averaging the density values for the three sections. All values are expressed as mean ± SD and median. Statistical Analysis Differences in VAS scores between touching the epiglottis and ventricular folds and between ventricular versus epiglottis nerve density values were tested with the Wilcoxon signed-rank test for two paired samples. The correlation

4 142 G. Ruoppolo et al.: Nerve Endings in the Human Laryngeal Mucosa Table 1 Patients self-rated VAS scores for discomfort at two different sites of stimulation between age and nerve fiber density was tested with the Spearman s Rank correlation coefficient. p values \0.05 were considered to indicate statistical significance. Data were analyzed with a PC version of the Statistical Package for Social Sciences 16.0 (SPSS, Chicago, IL, USA). Results Mean VAS scores were significantly higher for touching the ventricular folds than for the epiglottis (8.2 ± 1.1 vs 4.1 ± 1.2, p = 0.017). Individual VAS values are shown in Table 1. The mean nerve fiber density (number of fibers/mm length of section) was significantly higher in samples from the ventricular folds than in those from the epiglottis (mean 2.96 ± 2.05 vs 0.83 ± 0.51; median 3.4 [range ] vs 0.88 [range )]; two-sided p = 0.018). The mean ratio between ventricular and epiglottal fiber density was The Spearman s Rank correlation coefficient showed a significant relationship between age and nerve fiber density in ventricular and epiglottal samples (r s =-0.93, p = and r s = , p = 0.036, respectively), indicating a lower nerve fiber density in older patients (Table 2). Discussion Age (years) VAS score (ventricular folds) A B C D E F G VAS score (epiglottis) The starting point (0) represents no discomfort and the end point (10) indicates intolerable discomfort The role of laryngeal sensitivity in preventing aspiration became clearer after introduction of the FEESST, which is used to determine the sensory threshold of the laryngopharynx. Several studies that investigated the correlation between motor function deficits and laryngopharyngeal sensory deficits by means of the sensory test showed that subjects with a severe LAR deficit are at risk for laryngeal aspiration [24, 25]. FEESST is the most accurate tool to Table 2 Patients age and fiber density (number of fibers/millimeter length of section) in the ventricular folds and in the laryngeal surface of the epiglottis, and the ratio between the two areas Age (years) Fiber density (ventricular folds) Fiber density (epiglottis) A B C D E F G VF/E ratio test laryngopharyngeal sensitivity, allowing quantitative assessment. A disadvantage of FEESST is that it requires complex equipment and is unsuitable for widespread use. In fact, the original fiberoptic procedure of the swallowing examination, described by Langmore in 1988, is still widely used because of its portability, accuracy to assess aspiration, and ease of use [20]. Our study aimed to examine the sensitivity of the supraglottic larynx. All of the seven neurologically healthy subjects who were studied had a significantly higher mean laryngeal nerve fiber density in the ventricular folds than in the laryngeal surface of the epiglottis. Laryngeal nerve fiber density significantly differed in all the subjects, despite wide inter-individual variability. In agreement with the difference in nerve fiber density, the mean VAS score was significantly higher for touching the ventricular folds than for the epiglottis. Taking into account the limitations of the subjective method that we used to measure the patients judgment of laryngeal sensitivity, it was useful to confirm the normal sensory function of the laryngeal supraglottic mucosa and the higher sensitivity of the ventricular mucosa in our healthy subjects. The use of a selective pan-neuronal marker (PGP 9.5) did not allow us to identify the different types of receptors. Therefore, we were unable to detect more specific differences between the sensory receptors at the two sites under study. For the same reason, we were not able to establish what types of mechanoreceptors were stimulated by the touch of the probe. Therefore, the present study was not able to clarify the morphological and topographical characteristics of these sensory structures. However, our study aimed to quantitatively measure sensory innervation, rather than conduct a more complex and expensive morphological or physiological study. Because our study was carried out in humans, small mucosal specimens were excised to avoid bleeding or any other injury, further limiting the reliability of a more detailed study.

5 G. Ruoppolo et al.: Nerve Endings in the Human Laryngeal Mucosa 143 Only patients who had benign lesions on the free edge of the vocal folds were included in this study to avoid possible interference between the lesion and the mucosal lining of the supraglottic larynx. This was a preliminary study because of the few subjects available to undergo a laryngeal biopsy for research. However, the finding that there was a difference in laryngeal nerve fiber density between the epiglottis and ventricular folds indicates that future studies should be performed on laryngeal sensitivity in humans. If our data will be confirmed in future studies, the possibility to probe the ventricular region when touching the laryngeal surface of the epiglottis will fail to trigger the LAR will have to be taken into account. Another interesting finding in our study on healthy subjects was the overall reduction in nerve fiber density in three older subjects. However, the ratio between density in the ventricular mucosa and that in the epiglottis remained unchanged. Our sample is too small to generalize to other populations. However, our data are consistent with the agerelated changes in sensory nerve endings in the larynx of rats [15], as well as evidence that epidermal nerve fiber density decreases in a length-dependent manner with advancing age [26, 27]. Because our findings are preliminary, further studies collecting additional samples in healthy subjects are required to confirm sensory loss in the older larynx. Our data could also provide normative values for investigating possible laryngeal sensory fiber depletion in studies on patients with dysphagia related to neurodegenerative disease. Conclusions Assessment of laryngeal sensitivity plays an important role in evaluating neurogenic oropharyngeal dysphagia. 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