Respiratory internal kinematics of the tongue base and soft palate in obese minipigs with obstructive sleep apnea

It is largely unknown how the tongue base and soft palate deform to alter the configuration of the oropharyngeal airway during respiration. This study is to address this important gap. After live sleep monitoring of five Yucatan and two Panepinto minipigs to verify obstructive sleep apnea (OSA), eight and four ultrasonic crystals were implanted into the tongue base and soft palate to circumscribe a cubic and square region, respectively. The 3D and 2D deformational changes of the circumscribed regions were measured simultaneously with electromyographic activity of the oropharyngeal muscles during spontaneous respiration under sedated sleep. The results indicated that both obese Yucatan and Panepinto minipigs presented spontaneous OSA, but not in three nonobese Yucatan minipigs. During inspiration, the tongue base showed elongation in both dorsal and ventral regions but thinning and thickening in the anterior and posterior regions, respectively. The widths showed opposite directions, widening in the dorsal but narrowing in the ventral regions. The soft palate expanded in both length and width. Compared to normal controls, obese/OSA ones showed similar directions of deformational changes, but the magnitude of change was two times larger in the tongue base and soft palate, and obese/OSA Panepinto minipigs presented 10 times larger changes in all dimensions of both the tongue base and the soft palate. The distance changes between the dorsal surface of tongue base and soft palate during inspiration increased in normal but decreased in obese OSA minipigs.

palate also plays an important role to regulate the airflow through nose and/or mouth (Stănescu & Rodenstein, 1988).Clinical studies have demonstrated that in obstructive sleep apnea (OSA), respiratory disturbances are more common at the soft palate (Lee & Cho, 2019) but more severe at the tongue base (Kim et al., 2009).Therefore, the internal kinematics of these two structures play a critical role in maintaining or compromising the oropharyngeal airway.However, how these two key structures alter their shapes and spatial relationship to configure the oropharyngeal airway during respiration is largely unknown, especially during sleep.The present study was to address this important gap by using implantable ultrasonic crystals to quantify the respiratory 3D and 2D deformational changes in the tongue base and soft palate respectively during sedated sleep in normal non-OSA and obese OSA minipig models.The hypothesis was that the respiratory deformational changes of the tongue base and soft palate are greater in obese OSA than those in normal non-OSA minipigs.

| Animals and OSA verification
Three normal (two females and one male) and two obese (one male and one female) Yucatan (aged 8-11 months; Premier BioSource), and two obese Panepinto (males, aged 6.5 years, donated by Panepinto & Associate) minipigs were included in the present study.
Panepinto minipig is a crossbreeding between Yucatan and Vietnamese minipig (Panepinto et al., 1978).The ranges of body weights and the body mass index (BMI) were as follows: normal: 46-55 kg with the BMI of 38-39 kg/m 2 ; obese Yucatan: 68-71 kg with the BMI of 50-51 kg/m 2 ; and obese Panepinto: 86-104 kg with the BMI of 49-59 kg/m 2 .The body length was measured from the tip of the snout to the base of the tail, and the BMI was calculated using the formula body weight in kg/body length in meter 2 .All procedures were approved by the Institutional Animal Care and Use Committee of the University of Washington (Protocol# 3393-04).
Before the terminal procedures for the internal kinematics of the tongue base and soft palate, OSA was verified and characterized on all seven minipigs during sedated and spontaneous sleep.The results of the wireless and remote sleep polysomnography and airflow dynamics were published previously (Deng et al., 2020;Liu et al., 2021).In summary, OSA was verified in all four obese minipigs with the apnea-hypopnea index (AHI) ranging from 30-59, and the AHI in three normal minipigs were 0-5.This verification also demonstrated the similarity of respiration parameters during sedated and spontaneous sleep, indicating the sedated sleep can be a surrogate of the spontaneous sleep.

| Terminal procedures
Under sedation with xylazine (4 mg/kg), midazolam (0.5 mg/kg), and butorphanol (0.3 mg/kg), and maintenance with isoflurane 2%-3% in oxygen, the pig was placed supine.A submandibular incision was first made, and subcutaneous tissues were separated to expose the bilateral genioglossus, styloglossus, and thyrohyoideus.A pair of 0.10 mm nickel-chromium wire electrodes (California Fine Wire) with 1 mm bared tip and 2 mm separation between the wires were inserted into each of these three muscles along their fiber direction using a 25G needle.These tongue and hyoid muscles were chosen because they are active during respiration (Fregosi & Ludlow, 2014;Sokoloff, 2004).Then, the two mouth openers were inserted into the bilateral molar region to keep the gape at 35-40 mm, and the tongue was pulled forward to expose the two circumvallate papillae as the border of the tongue base from the tongue body.Eight 2 mm B barbed ultrasound crystals (Sonometrics Co.) were implanted into the tongue base through the submandibular approach.During implantation, a small tunnel was first made by a dull long-beaked straight hemostat, then a barbed crystal was inserted using another longbeaked straight hemostat.The location of each crystal was confirmed by transoral palpation of the tongue base by the fingers of the operator.Therefore, the dorsal surface of the tongue was kept intact without penetration.The implanted crystals in the tongue base were secured by their barbs and leading wires were further sutured to the nearby facial tissues.As shown in Figure 1a, these eight crystals circumscribed a cubic region in the tongue base: crystals #1 and #2 were implanted 2 mm posterior to the two circumvallate papillae and 3 mm underneath the dorsal mucosa; the #3 and #4 crystals were 20 mm posterior to crystals #1 and #2 respectively and located in the dorsal area.Crystals #5, 6, 7, and 8 were placed in the ventral region at a distance of 20 mm from crystals #1, 2, 3, and 4, respectively.In addition, another four crystals were directly implanted into the soft palate to circumscribe a rectangular region with 20 mm separation as well by using a sharp long-beaked hemostat and were secured in place by their barbs as well (Figure 1b).The distances between each pair of the ultrasonic crystals in each dimension were consistently 20 mm across all animals regardless of their body size and BMI and were reconfirmed by the dissection postmortem.The leading wires of the crystals implanted into the tongue base were led out from the submandibular incision and the incision was closed in layers by suturing.The leading wires of the crystals placed in the soft palate exited the mouth via the upper retromolar regions of each side.

| Data recording and analysis
After the instrumentation, the pig was situated in the prone position, and the tongue was placed back to its original position.The electromyographic activity (EMG) electrode leads were connected to the MP150 (BioPac Co.), and the crystal wires were inserted into the Sonometric input box.The deformational changes in the tongue base and soft palate along with EMG activity were recorded for 5-10 min during respiration using a computer running both Acknowledge (Ver.4.0,BioPac) and SonoLab (Sonometrics) programs.Signals from these two recording systems were synchronized by digitalanalog input and output.
Off-line analyses of the crystal signals were performed by selecting 25-30 stable and consecutive respiratory cycles, and the synchronized EMG activity was taken for identifying the inspiratory and expiratory phases of respiration.Since the initial distances between each implanted crystal pair were consistent (20 mm) in all animals (Figure 1a), the change in each crystal pair during each respiratory cycle was calculated and averaged in each animal.In addition to each selected crystal pair in the tongue base and soft palate, the distances between the crystal pairs of the soft palate and the dorsal tongue base were also sampled and calculated to quantify how the spatial relationships between the dorsal surface of the tongue base and the soft palate changed in respiratory phases.SPSS (Ver.19; IBM) for Windows was used for statistical analysis.
Descriptive statistics were calculated for all means, standard deviations, and ranges of deformational changes.The data were further examined to confirm their normal distribution through skewness calculations.One-way analysis of variance was used to examine the differences among the three types of minipigs followed by Tukey post hoc tests for pair-wise comparisons.Spearman correlations were calculated to test the associations between the distance changes in each dimension, AHI and BMI.The significant level was set as p < .05.

| RESULTS
Corresponding to the respiratory rate, the stereotyped deformational changes in the lengths, widths, and thicknesses of the tongue base along with the muscle activity bursts were observed (Figure 2).The respiratory rate was about 16-25 per minute and the ratio of inspiratory/expiratory phases was about 0.65.As marked in Figure 3, both dorsal and ventral lengths of the tongue base increased during the inspiratory phase.
However, the widths of the tongue base increased dorsally and decreased ventrally, indicating widening in the dorsal and narrowing in the ventral tongue base during inspiratory phase.The thicknesses also showed opposite changes in the anterior and posterior tongue base, that is, became thinner in the anterior and thicker in the posterior tongue base.Therefore, the internal kinematic pattern of the tongue base during inspiration presented the following features: elongation, dorsal widening and ventral narrowing, anterior thinning, and posterior thickening.During expiration, all lengths, widths, and thicknesses presented their deformational changes in the opposite directions.
Since respiration is a symmetric movement and no significant differences were found in the deformational changes in the lengths and thickness between the right and left tongue base as shown in Figure 3, the values of both sides were averaged to reflect the overall changes in the dorsal/ventral lengths and anterior/posterior thicknesses.These results show that although the patterns of deformational changes presented similar directions in all minipigs, their deformational ranges were different during respiration.As shown in Figure 4, while the normal Yucatan showed the smallest range from 0.05 to 0.23 mm, obese/OSA Yucatan minipigs exhibited two times larger ranges in elongation (0.14-0.19 mm) with similar ranges to the normal in the anterior thinning and posterior thickening, and smaller widening and narrowing ranges in the dorsal and ventral parts (0.06-0.11 mm), respectively.The two aged obese/OSA Panepinto minipigs presented significantly larger ranges with as much as 6-8 times more (0.71-1.24, p < .05)than the normal group in all lengths, widths, and thicknesses, possibly related to particularly heavy snoring during recording (Figure 5).These deformational changes in the lengths, widths, and thicknesses of the tongue base made the shape circumscribed by the eight implanted ultrasound crystals from an originally cubic to an irregular trapezoid-like shape, featuring a longer and wider top but narrowed bottom, and further tapering sagittally from anteriorly decreased to posteriorly increased thicknesses in the tongue base (Figure 6).
Due to the difficulty of access and the vulnerability of the crystals, data from the soft palate was limited.Similar stereotyped EMG bursts and deformational changes of the soft palate were observed (Figure 7).In the phase of inspiration, the soft palate elongated symmetrically with larger and smaller widening in anterior and posterior regions, respectively in normal Yucatan with the range of 0.02-0.05mm.However, the ranges of deformational changes were more than two times larger in obese/OSA Yucatan minipigs (0.09-0.17 mm), and more than four times larger in obese/OSA Panepinto minipigs (0.32-0.81 mm, p < .05).
Although the deformational patterns in the tongue base and soft palate were similar across the three groups of minipigs, the differences were seen in the extents of the distance change during respiration in each dimension between these two oropharyngeal structures.During inspiration, both anterior and posterior distances between the dorsal tongue base and soft palate were increased (0.09-0.12 mm) in normal Yucatan minipigs, indicating the separation of these two key structures.However, the anterior distance of these two key structures in obese/OSA Yucatan and Panepinto/OSA minipigs was shortened (0.42-1.60 mm), indicating the possible closure between these two key structures.
The correlation analysis showed that neither AHI, nor BMI was associated with the ranges of distance changes in each dimension (a given crystal pair) during respiration in both the tongue base and soft palate.

| DISCUSSION
Both the tongue and soft palate are muscular structures, so the muscular hydrostat theory applies (Napadow et al., 2002).However, as explored and published previously, this theory may not be applied to regional deformational changes of the tongue (and soft plate) as the regional volume changes have been verified (Liu et al., 2008).A tongue swallowing kinematics study using X-ray Reconstruction of Moving Morphology has also challenged this hydrostat theory in the tongue (Orsbon et al., 2020).The present study further demonstrated that the increase and decrease of each measured deformational change do not compensate for each other to maintain the volume constant in the region circumscribed by the ultrasonic crystals.More importantly, the present study revealed that the elongation and widening of both the tongue base and soft palate may be the key players in leading or guiding the airflow into the oropharyngeal airway during respiration.At the same time, while both the dorsal tongue base and the soft palate widened, the ventral tongue base narrowed instead, along with anterior thinning and posterior thickening of the tongue base.Therefore, during inspiration, the cubic shape circumscribed by the eight implanted ultrasound crystals (Figure 6a) becomes an irregular trapezoid-like shape (Figure 6b).
Along with this deformational or shape change in the tongue base, the soft palate extends in both length and width.These dynamic deformational changes in the shapes of the tongue base and soft palate expand the lumen of velar and oral pharynx thus increase the volume of the oropharyngeal airway for its patency, as our findings in the airflow dynamics in the same minipig models previously published (Liu et al., 2021).Thus, it is reasonable to speculate that an enlarged tongue base and/or soft palate due to obesity or other pathological conditions are predisposing factors of airway obstruction, particularly at the status of the decreased tone of the tongue and soft palate muscles during sleep (Kim et al., 2014).
Respiration is a complex process divided into three phases: inspiration, postinspiration, and active expiration (Malheiros-Lima et al., 2020).During respiration, the morphologies of oropharyngeal structures and the volume of airway spaces continuously change from the anterior nostril to pulmonary alveoli.In addition, the luminal pressure in certain airway segments is proportionally distributed, and the airflow patterns and resistance may therefore be determined by the regional morphological characteristics.A previous study has shown that during oronasal breathing (as during exercise, speech, or smoking), the impedances of the nasal and oral pharynx are determined by the position of the soft palate (Kurimoto, 1989;Stănescu & Rodenstein, 1988).Therefore, the observed respiratory deformational changes in the tongue base and soft palate would certainly alter the morphology of the velar and oral pharyngeal airway which in turn modify the inspiratory and expiratory airflows as we found in the computational fluid dynamic modeling in these same minipig models (Liu et al., 2021).
The present study of the internal kinematics of the tongue base and soft palate in OSA minipig model is particularly relevant to the human OSA condition.A most recent clinical survey indicated that the prevalence of snoring in obese individuals is almost 100% among whom 58% presents severe degree of OSA, and have their airway obstructions, and these obstructions most often occur at the retro-palatal and retro-glossal levels (da Silva et al., 2022).A recent meta-analysis of 2950 patients from 19 studies also showed the soft palate and tongue base were the two most common sites of airway obstruction.This meta-analysis also showed that the degree of tongue base obstruction was associated with the severity of OSA (Lee & Cho, 2019).
In the present study, both heavy snoring and severe OSA were  There are several limitations in the present study.The first is the small sample sizes in each type of minipigs, particularly the lack of the same aged controls of Panepinto minipigs due to the source unavailability.Therefore, the observed differences in Panepinto obese/OSA minipigs could be derived from the different breeds of minipigs.However, given the fact that Panepinto minipigs are a Yucatan crossbreed (Panepinto et al., 1978), and obese Panepinto had similar BMI to obese Yucatan minipigs, it could be reasonably speculated that the observed differences between normal Yucatan and obese/OSA Panepinto minipigs were most likely resulted from obesity and/or OSA.The second is that not all crystal recordings were successful due to the vulnerability of the implanted ultrasound crystals.Despite these, the results clearly reveal the respiratory characteristics of the internal kinematics in the tongue base and soft palate in normal minipigs and the differences in obese minipigs with OSA.As described in the methods, the recordings were performed under sedated sleep, instead of natural respiration in consciousness or sleep.Fortunately, the confounding effect of sedation and anesthesia on respiration has been proven to be minor (Mak et al., 1993;Oliven et al., 2003Oliven et al., , 2007)), and the physical parameters between sedated and natural sleep present clear similarity in these normal and obese minipigs (Deng et al., 2020).Therefore, this limitation could be considered minor but still needs to be confirmed during natural respiration when the technique becomes available.The last limitation is that although there are great similarities in the oropharyngeal morphology and function, the differences are also obvious, particularly the retroglossal area.In pigs, this area is sealed by the soft palate, dorsal surfaces of the tongue base, and epiglottis as compared to the open area in humans as the epiglottis descend.Another major difference is the descending pharyngeal airway as compared with that of human, although the orientation of pharyngeal airway is similar when humans and pigs on sleep position (Rosero-Salazar et al., 2024).These limitations may decrease the value of direct translation from this study to humans.

| CONCLUSIONS
The present study suggests that the greater respiratory internal kinematics in the tongue base and soft palate are related to the presence of obesity/OSA, and these increases in the deformational changes in the tongue base may have compensatory effects on the potential oropharyngeal airway restriction or collapse.
Illustration of the configuration of eight implanted ultrasound crystals in the tongue base to circumscribe a cubic region.Crystals #1 and #2 were implanted 2 mm posterior to the two circumvallate papillae and 3 mm underneath the dorsal mucosa; crystal #3 and #4 crystals were 20 mm posterior to crystal #1 and #2 crystals, respectively, and located in the dorsal area.Crystals #5, 6, 7, 8 were placed in the ventral region with a distance of 20 mm to crystals #1, 2, 3, 4, respectively.(b) 2 mm barbed ultrasound crystals.(c) Illustration of the four crystals (green dots #9-#12) in the soft plate to circumscribe a square area.Note that white dots (#1-#4) indicate the four crystals implanted in the dorsal region of the tongue base.So, the change in distance changes among these two groups of crystals indicate the separation of the dorsal surface of the tongue base and ventral surface of the soft palate during respiration.F I G U R E 2 Raw tracings of synchronized electromyographic activity and ultrasound crystals during respiration from in a control minipig.Highlighted region indicates the inspiratory phase.LAT, left anterior thickness; PVW, posterior ventral width; RDL, right dorsal length; RPT, right posterior thickness; RTH, right thyrohyoid muscle; Refer to Figure 1a for actual crystal pairs.The red arrow and highlighted region indicate the activity burst of RTH and inspiratory phase, respectively.F I G U R E 3 Three-dimensional raw tracings of ultrasound crystals in the length, width, and thickness of the tongue base during respiration.Each tracing indicates a given crystal pair.ADW and PDW, anterior and posterior dorsal widths; AVW and PVW, anterior and posterior ventral widths; RAT, right anterior thickness; RDL, right dorsal length; RPT and LPT, right and left posterior thickness; RVL and LVL, right and left ventral lengths.Red highlight indicates the inspiratory phase.The red and green arrows indicate distance increase and decrease of a given crystal pair respectively.

F
I G U R E 4 Comparison of three-dimensional raw tracings of ultrasound crystals in the length, width, and thickness of the tongue base during respiration between a control (a) and obese obstructive sleep apnea (b) minipigs.Each tracing indicates a given crystal pair.Refer to Figures 2 and 3 for all captions.

F
I G U R E 5 Bar graph showing inspiratory dimensional changes from the initial distance at rest (0.00) in dorsal/ventral lengths (DL and VL), anterior and posterior thicknesses (AT and PT), and anterior dorsal/ventral (ADW and AVW) and posterior dorsal/ventral (PDW and PVW) widths among three types of minipigs.*Significant difference.F I G U R E 6 The illustration of the shape changes in the region of the tongue base circumscribed by the 8 ultrasonic crystals.(a) Initial shape at the crystal implantation; (b) the shape during inspiratory phase.
identified in obese minipigs although snoring did not occur in young obese Yucatan minipigs during the implanted ultrasound crystal recording.Nevertheless, the greater internal kinematics and altered spatial relationship in the tongue base and soft palate during respiration is related to the presence of obesity/OSA, and these increases in the deformational changes in the tongue base may have a compensatory effect on the potential oropharyngeal airway restriction or collapse.

F
I G U R E 7 Raw tracing of synchronized electromyographic activity and ultrasound crystals in the soft palate from a control minipig.AW and PW, anterior and posterior widths; RL and LL, right and left length; RTH, right thyrohyoid muscle.Highlighted region indicates inspiratory phase.LIU ET AL. | 7 of 9