Bilateral Stylopharyngeus Transection Alters Respiratory Airflow in Conscious Rats

Abstract

Background/Objectives: Upper airway patency is a key pathophysiological factor in obstructive sleep apnea (OSA). Research has primarily focused on the role of the genioglossus muscle in maintaining airway patency in OSA. However, hypoglossal nerve stimulation (HNS) therapy, which activates the genioglossus muscle, has been associated with poor outcomes in patients with lateral oropharyngeal collapse. The stylopharyngeus muscle is an upper airway dilator muscle that supports the lateral pharyngeal wall. Its role in maintaining upper airway patency and its effect on normal respiratory airflow is unclear. We hypothesize that bilateral transection of the stylopharyngeus muscles disrupts normal breathing. Currently, no animal model depicting lateral pharyngeal collapse has been reported. This study aims to introduce a novel rodent model with bilateral transection of the stylopharyngeus muscles to examine its effect on respiratory airflow and tracing. Methods: Adult male Sprague Dawley rats were divided into two groups: (1) bilateral stylopharyngeus muscle transection (n = 4) and (2) sham surgery (n = 2). Under anesthesia, the stylopharyngeus muscle was transected bilaterally in the transection group, while only exposure of the muscle was performed in the sham group. Respiratory airflow was measured using whole-body plethysmography before and after surgery, and airflow tracings were analyzed. Results: Significant alterations in respiratory airflow and tracings, particularly a flattening in inspiratory flow and sharp expiratory peaks, were observed on the first post-operative day in the transection group. The flattening of the inspiratory flow persisted over 3 days. No significant changes were noted in the sham group. Conclusions: Bilateral stylopharyngeus muscle transection alters normal airflow in a conscious rodent model, supporting the hypothesis that stylopharyngeus muscle plays a vital role in shaping respiratory airflow. The flattening of the inspiratory airflow is an indication of flow limitations through the upper airway patency due to the loss of stylopharyngeus function.

1. Introduction

The pathophysiology of recurrent obstructive sleep apnea (OSA) includes airway collapsibility [1], muscle responsiveness [2], arousal threshold [3], and loop gain [4,5]. When focusing on collapsibility, the majority of studies have largely focused on the laxity of the genioglossus muscle, which is innervated by the hypoglossal nerve [6,7]. This has led to the development of hypoglossal nerve stimulation (HNS) as a surgical treatment for OSA patients [8]. However, the collapsibility of the airway in OSA patients occurs in a variety of ways, including circumferential, anterior–posterior palatal, genioglossus as well as lateral oropharyngeal collapse as identified under drug-induced sleep endoscopy (DISE) [9]. Patients with complete concentric palatal collapse are contraindicated for HNS due to the increased risk of failure on HNS [10]. Oropharyngeal lateral wall collapse is also associated with poor outcomes on HNS [11].

The stylopharyngeus muscle may be a key muscle in supporting the lateral walls of the airway. It originates from the styloid process and is inserted into the thyroid cartilage and tonsillar capsule. This pharyngeal muscle is innervated by the glossopharyngeal nerve and functions as an upper airway dilator and assists in both swallowing and breathing. Using cine computed tomography (CT), Schwab reported that in healthy patients, changes in upper airway dimensions during breathing are greater in the lateral than in the anteroposterior direction. This predominantly laterally oriented tension on the pharyngeal walls is not seen in OSA patients where the major axis of the apneic airway is oriented in the anterior-posterior dimension [12,13]. Furthermore, in a sleep study of OSA patients, Guilleminault et al. reported that obstructive events began with the loss of stylopharyngeus muscle activity despite the persistence of genioglossal muscle activity during hypopnea [14]. Lastly, Tessier et al. reported that bilateral glossopharyngeal nerve blockade in the horse and, thus, stylopharyngeus muscle dysfunction resulted in more negative peak inspiratory airway pressures in exercise, attributed to nasopharyngeal collapse [15]. These studies suggest the stylopharyngeus muscle may play a key role in OSA pathophysiology.

The surgical approach to treating lateral wall collapse in OSA has evolved in parallel with the recognition that upper airway obstruction may occur at multiple sites. The lateral pharyngeal wall consists of several muscular structures, including the palatoglossus muscle, the palatopharyngeal muscle, the superior pharyngeal constrictor, and the palatine tonsils [16]. In 2003, lateral pharyngoplasty was introduced as a surgical approach targeting the lateral pharyngeal wall for OSA treatment [17]. The superior pharyngeal constrictor is the main structure that is targeted by lateral pharyngoplasty. However, surgical manipulation of the superior pharyngeal constrictor has been implicated as a potential cause of post-operative dysphagia. Following the introduction of lateral pharyngoplasty, less invasive and more function-preserving techniques- such as an expansion sphincter pharyngoplasty [18] and barbed reposition pharyngoplasty [19] have been proposed to improve both efficacy and safety. To minimize complications, particularly dysphagia during lateral pharyngoplasty, surgical modifications have also focused on the preservation of the stylopharyngeus muscle, which courses through the superior pharyngeal constrictor [20]. While the stylopharyngeus muscle has traditionally received attention for its role in swallowing, we found no studies focusing on airflow.

Despite these studies on the role of stylopharyngeus on OSA, its impact on upper airway patency, as well as respiratory airflow and traces, are unknown. Here, we establish a rat model to examine stylopharyngeus dysfunction on respiratory airflow over several days. We will introduce a novel surgical procedure for isolating and transecting the stylopharyngeus muscle and report on the consequences of bilateral stylopharyngeus muscle transection on respiratory flow in conscious rodents.

2. Materials and Methods

Ethical Approval

The surgery and experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Case Western Reserve University and adhered to the guidelines published by the NIH National Research Council (US). Sprague Dawley rats weighing 300–400 g were used in this study. All rats were housed in standard conditions, with a 12 h light/dark cycle and with ad-libitum access to food and water. All respiratory recordings were performed during the light cycle (from 7:00 a.m. to 12:00 p.m.).

Rodent Models

Adult male Sprague–Dawley rats (Envigo Laboratories) weighing 335–400 g were used for surgical development and unrestrained whole-body plethysmography (WBP) recordings. The rats were housed in standard rodent cages in the Animal Resource Center at CWRU, which is an Association for Assessment and Accreditation of Laboratory Animal Care International-approved facility.

Experimental Groups

Rodents were divided into 2 groups: (1) Bilateral transections and (2) Sham. Four animals received bilateral stylopharyngeus muscle transections. Two animals were surgical shams where all anesthesia and surgery were performed to expose the stylopharyngeus muscle, but no transections were made, and the animal was allowed to recover.

Anesthesia Induction

Rodents were inserted into an anesthesia induction chamber with sevoflurane (Covetrus North America, Dublin, OH, USA). Once the appropriate anesthetic plane was reached, the animal was removed and transferred to the surgical area and connected to the sevoflurane anesthesia system via a nose cone at a flow rate of 1.5 L/min in a supine position. Oxygen saturation and heart rate were monitored by PulseOx attached to the paw, and the temperature was monitored by a rectal thermometer (Kent Scientific, Torrington, CT, USA). Once the animal was in the appropriate anesthetic plane and vitals were stabilized, the neck region was shaved, hair was removed from the surgical area, and betadine (Emerson Healthcare, Concord, MA, USA) was applied.

Surgical Procedure

Under sterile conditions and an appropriate anesthetic plane, a scalpel was used to make a rostral to caudal 2.5 cm midline incision between the regions of the mandible and the neck (Figure 1A). Retraction of the epidermal layer revealed fascia and fat (Figure 1B).

Careful dissection with sterile blunt swabs and lateral retraction of the surrounding fat (Figure 1C) revealed two salivary glands (Figure 1D).

Lateral retraction of the salivary glands revealed pharyngeal and digastric muscles (Figure 1E). The anterior belly of the digastric muscle was carefully separated from the pharyngeal muscles and retracted laterally with hooks, revealing the hyoid bone (Figure 1F).

Medial retraction of the pharyngeal muscles revealed the carotid bifurcation (Figure 1G). After locating and gently dissecting the bifurcation of the carotid artery with blunt swabs, the hypoglossal nerve was visually identified as a white, shiny structure with vertical striations (as it is a motor nerve) and located rostral to the internal and external carotid arteries (Figure 1G,H).

Tracing the internal and external carotid arteries and hypoglossal nerve rostrally (Figure 1I) led to the identification of the glossopharyngeal nerve, a thinner, shiny white structure without striations (Figure 1J). When the hyoid bone was retracted rostrally, the tendon of the stylopharyngeus muscle became visible. Careful dissection medial to the tendon revealed the belly of the stylopharyngeus muscle (Figure 1J).

The stylopharyngeus muscle lay rostrally to the glossopharyngeal nerve (Figure 1K), and upon exposure, muscle contractions were observed in coordination with respiratory efforts. Prior terminal surgeries were conducted to confirm the muscle as the stylopharyngeus muscle. Since the stylopharyngeus muscle is innervated by the glossopharyngeal nerve, the transection of the glossopharyngeal nerve abolishes the contraction, ensuring that the correct muscle has been identified (see Video S1). In the conscious transection group, the stylopharyngeus muscle was transected between the belly and tendon (Figure 1L). These procedures were repeated and performed on the other side for the bilateral transection.

In the sham group, both stylopharyngeus muscles were exposed without being transected. Proper identification of the stylopharyngeus muscle was facilitated by gently pulling the trachea medially and gently manipulating the hyoid bone laterally.

After transections/sham surgery, all retractions were removed, muscles were moved back to their original space, and the incision was closed with staples (Figure 1M (bilateral transection) and Figure 1N (sham)).

Recovery

Following the surgery, all animals received subcutaneous injections of medications to manage pain and prevent infection. Carpofen (Patterson Veterinary, Clinton Township, MI, USA) was administered at a dose of 5 mg/kg. Buprenorphine (Patterson Veterinary) was given at a dose of 0.1 mg/kg to further alleviate pain. Additionally, Enrofloxacin (Patterson Veterinary) was administered at a dose of 15–20 mg/kg. After the surgery, rats were placed in recovery cages on a heating pad. They were observed until animals were able to right themselves and maintain sternal recumbency. Subsequently, all rats were observed daily and received analgesics as needed. Rats were housed in cages under controlled temperatures with a 12-h light/dark cycle. All animals were provided free access to a standard laboratory diet and allowed water ad libitum.

Recording of Respiratory Flow (Waveform)

Rodent respiratory flow and waveform were obtained using whole-body flow –through a plethysmography (WBP) chamber (400 mL) (vivoFlow, SCIREQ Scientific Respiratory Equipment Inc., Montréal, QC, Canada). Data was collected using IOX 2.10.4.8 (EMKA Technologies, Paris, France) and analyzed with Spike2 7.01 (CED Software Cambridge Electronic Design Limited, Technical Centre, Cambridge England). Rodents were conscious and unrestrained during the recording. Rodents were recorded for 3 h between 7 a.m. and 12 p.m. (light cycle period). Recordings of the rodents were taken for 3 consecutive days prior to surgery to allow the animals to acclimate and establish baseline airflow traces. After surgery, rodents were allowed to recover overnight, and WBP recordings were taken for the next 3 days.

Data analysis

Airflow recording from the whole-body plethysmography was acquired through IOX 2.10.4.8 (EMKA Technologies, Paris, France) and exported into Spike2 7.01 to assess continuous airflow traces recorded over the 3 h. Epochs of stable breathing of a minimum of 1 min were chosen for cycle trigger averaging (CTA). CTA was triggered off the inspiratory onset to compare the airflow waveform of 10 stable breaths at baseline and day 1, 2, and 3 post-surgery. The inspiratory onset was defined as the point at which the waveform intersects with a horizontal cursor placed at the midpoint of the y-axis, which is airflow, between the end of expiration and the inspiratory peak. CTA offset was set to zero.

Autopsy

Immediately after WBP recording on the 3rd day post transections, we anatomically assessed the transections of both stylopharyngeus. Rodents were anesthetized with urethane (1.5 g/kg), and once the anesthetic plane was reached, the midline incision was reopened, and we proceeded to assess the status of the stylopharyngeus transections. All animals were euthanized after the examination.

3. Results

Respiratory airflow traces are altered after bilateral stylopharyngeus transections

Whole-body plethysmography recordings from a representative bilateral transection rodent and sham are shown in Figure 2 and Figure 3, respectively. The traces over 60 s show that the stable breathing rate did not significantly change (Figure 2A). When expanded, significant changes in airflow traces in post-operative days 1–3 compared to baseline traces can be observed (Figure 2B). This was more evident when CTAs over 10 breaths were performed (Figure 2C). In baseline, inspiratory traces increased to a peak and then dropped during expiration. After transection, inspiratory traces no longer peaked and were flattened and a sharp expiratory peak was observed. This sharp expiratory peak was diminished on day 2 but returned on day 3. However, the flattened inspiratory traces continued through day 3, but the most significant flattening was on post-transection day 1.

In the sham animals, these changes in respiratory airflow traces were not observed (Figure 3). As in Figure 2, the airflow traces on the left represent 60 s breathing (A), and the traces in the middle represent 15 s breathing (B). The waveform averaged over 10 breaths using the cycle trigger average is shown on the right (C). The waveforms from the top to the bottom represent baseline data, post-operative days 1, 2, and 3. Though the expiratory flow (Ce) is a little bit sharper on the post-operative day 1 compared to baseline (Ci), There were less significant airflow waveform changes between baseline and post-operative day 1 than in the bilateral transections group. Post-operative days 2 and 3 don’t have similar changes as day 1.

Autopsy Results:

We were not able to identify muscles near the previous location of the stylopharyngeus muscle and only observed the occipital bone in the bilateral transection group (Figure 4A). In the shams, we were able to identify the stylopharyngeus muscle and observe its contraction (Figure 4B).

4. Discussion

The results of this study show a surgical method of bilateral transection of the rodent stylopharyngeus muscle as an animal model to examine pharyngeal support in upper airway patency. This is the first investigation to use a rodent model to explore the effects of bilateral stylopharyngeus muscle transection on airflow traces, and the results demonstrate that stylopharyngeus muscle dysfunction can significantly alter respiratory dynamics.

Our findings showed respiratory airflow traces are significantly changed with the removal of the lateral support of the pharyngeal wall. The flattening of inspiratory traces is consistent with inspiratory flow limitation traces in humans, as reviewed in [21]. Modulation of inspiratory is a main function of the stylopharyngeus muscle, which is innervated by the glossopharyngeal nerve. We have shown in a decerebrate in situ rodent preparation that the glossopharyngeal nerve activity has both pre-inspiratory and inspiratory activity [22]. Thus, it is possible in our intact conscious rodent model that without the stylopharyngeus muscle to assist in pre-inspiratory and inspiratory function, the inspiratory waveform is altered.

In previous human studies, Guilleminault et al. reported that in OSA patients, the stylopharyngeus muscle exhibited a sudden electromyogram (EMG) silence during obstructive apneas [14]. In these OSA patients, during hypopneas, where the genioglossus muscle remains active, stylopharyngeus EMG activity is silent. In fact, the apnea does not start unless the stylopharyngeus muscle is inactive. This finding indicates that upper airway patency during obstructive events is not solely dependent on the genioglossus muscle, and the stylopharyngeus may play a bigger role than previously known. Our findings extend this concept by demonstrating that stylopharyngeus muscle transection in rodent models results in inspiratory airflow changes that resemble upper airway obstruction, further supporting the idea that both muscles have an active role in maintaining patency as in other species.

The distinct inspiratory plateau observed after stylopharyngeus transection is reminiscent of flow limitation, which has been well-investigated in patients with OSA [21]. Pho et al. reported similar findings of inspiratory flow limitation using a mouse model [23]. They developed an algorithm to detect inspiratory airflow limitation, defining it as characterized by the presence of a plateau at the maximum inspiratory flow during early inspiration. In their study, obstructive hypopneas were associated with a plateau in the inspiratory phase, which was their definition of flow limitation. Our study extends this concept by demonstrating that transection of the stylopharyngeus muscle results in such an inspiratory flow pattern.

Anatomically, the stylopharyngeus muscle supports the tonsillar capsule and the thyroid cartilage, providing lateral tension to the pharyngeal walls in humans. This lateral support, in combination with the anterior tension exerted by the genioglossus muscle, is essential for maintaining airway patency during both wakefulness and sleep [13]. Several surgical techniques, such as lateral pharyngoplasty, expansion sphincter pharyngoplasty, and barbed reposition pharyngoplasty, have been developed to address lateral pharyngeal wall collapse in patients with OSA. While outcomes such as a reduction in the apnea-hypopnea index and improvements in subjective sleepiness have been reported, the contribution of individual muscles, such as the stylopharyngeus, to airflow patterns has not been fully explored. Mesti et al. emphasized the importance of preserving the stylopharyngeus muscle during lateral pharyngoplasty to reduce the risk of post-operative dysphagia [20]. However, their focus was on swallowing function, and the impact of stylopharyngeus preservation on airflow or airway mechanics was not addressed. The loss of stylopharyngeus function, as seen in our current rodent study, likely results in a diminished ability to keep the airway open, leading to a change in airflow traces during inspiration. Schwab et al. highlighted that the airway shape in patients with OSA tends to be vertically elongated compared to the laterally wide airway seen in healthy individuals [12]. In addition, the traces were similar to what is predicted in the Starling Flow Resistor Model in a compromised upper airway [8]. The stylopharyngeus muscle, by contributing to lateral upper airway expansion, likely plays a significant role in maintaining the normal airway shape and preventing airway collapse.

Therapeutic approaches targeting the stylopharyngeus muscle or its innervating glossopharyngeal nerve could theoretically enhance airway stability. Kuna’s findings demonstrated that glossopharyngeal nerve stimulation results in lateral widening of the pharynx, further supporting the significant role of this muscle and nerve in airway mechanics [24]. While current surgical treatments for obstructive sleep apnea OSA do not directly address the stylopharyngeus muscle, its anatomical significance as a key landmark is widely recognized for ensuring surgical precision and minimizing complications [25]. Ultrasound-guided techniques may offer a promising pathway for precise interventions targeting the glossopharyngeal nerve [26]. The stylopharyngeus muscle and glossopharyngeal nerve could potentially serve as alternative or complementary targets to existing treatments, such as hypoglossal nerve stimulation or ansa cervicalis stimulation [8,27]. However, these innovative strategies remain speculative and theoretical at this stage. Further research is essential to assess their feasibility, effectiveness, and clinical applicability in the treatment of OSA.

Interestingly, the biggest changes in airflow were observed on post-operative day 1, then followed by a reduction in these changes by day 3. We confirmed that the stylopharyngeus muscle was not reattached and not contracting by autopsy in the bilateral transection group. This reduction could be best explained by a neural compensation, changing activation in upper airway muscles that can control palatal patency. The compensatory responses to stylopharyngeus dysfunction could be mediated by the negative pressure generated by flow limitation being sensed in the brainstem. This brainstem mechanism responds by coordination of action to maintain a crucial outcome function rather than a specific muscle function, akin to locomotor adaptations.

This study has several limitations. First, we have a small population, which will be expanded in future studies. Two, the absence of pharyngeal critical pressure (Pcrit) and esophageal pressure measurements limits our ability to definitively conclude that the observed airflow changes are due to true upper airway obstruction. Future studies will incorporate these measurements to confirm flow limitation and airway collapse. Additionally, while airflow recordings were conducted during periods of apparent rest, we cannot confirm whether the animals were asleep. The inclusion of electroencephalography (EEG) in future studies would help determine whether the airflow changes are sleep-related, which would further validate this rodent model for studying sleep-related breathing disorders like OSA.

5. Conclusions

In summary, our study reports that stylopharyngeus muscle transection in a healthy conscious rat model leads to significant changes in respiratory airflow, including the appearance of inspiratory plateaus and sharp expiratory flow. This is the first known demonstration of stylopharyngeus transection as a surgical technique in rodents, and while there are limitations in terms of mechanisms for acute and adaptive response (inspiratory, expiratory, critical pressure for collapsibility), it presents a working model to investigate airway instability, in particular flow limitation, potentially relevant to human snoring and obstructive sleep apnea.