Designer Receptors Exclusively Activated by Designer Drugs Approach to Treatment of Sleep-disordered Breathing.

Rationale: Obstructive sleep apnea is recurrent upper airway obstruction caused by a loss of upper airway muscle tone during sleep. The main goal of our study was to determine if designer receptors exclusively activated by designer drugs (DREADD) could be used to activate the genioglossus muscle as a potential novel treatment strategy for sleep apnea. We have previously shown that the prototypical DREADD ligand clozapine-N-oxide increased pharyngeal diameter in mice expressing DREADD in the hypoglossal nucleus. However, the need for direct brainstem viral injections and clozapine-N-oxide toxicity diminished translational potential of this approach, and breathing during sleep was not examined.Objectives: Here, we took advantage of our model of sleep-disordered breathing in diet-induced obese mice, retrograde properties of the adeno-associated virus serotype 9 (AAV9) viral vector, and the novel DREADD ligand J60.Methods: We administered AAV9-hSyn-hM3(Gq)-mCherry or control AAV9 into the genioglossus muscle of diet-induced obese mice and examined the effect of J60 on genioglossus activity, pharyngeal patency, and breathing during sleep.Measurements and Main Results: Compared with control, J60 increased genioglossus tonic activity by greater than sixfold and tongue uptake of 2-deoxy-2-[18F]fluoro-d-glucose by 1.5-fold. J60 increased pharyngeal patency and relieved upper airway obstruction during non-REM sleep.Conclusions: We conclude that following intralingual administration of AAV9-DREADD, J60 can activate the genioglossus muscle and improve pharyngeal patency and breathing during sleep.

Chemogenetic stimulation of the hypoglossal neurons improves upper airway patency.

Obstructive sleep apnea (OSA) is characterized by recurrent upper airway obstruction during sleep. OSA leads to high cardiovascular morbidity and mortality. The pathogenesis of OSA has been linked to a defect in neuromuscular control of the pharynx. There is no effective pharmacotherapy for OSA. The objective of this study was to determine whether upper airway patency can be improved using chemogenetic approach by deploying designer receptors exclusively activated by designer drug (DREADD) in the hypoglossal motorneurons. DREADD (rAAV5-hSyn-hM3(Gq)-mCherry) and control virus (rAAV5-hSyn-EGFP) were stereotactically administered to the hypoglossal nucleus of C57BL/6J mice. In 6-8 weeks genioglossus EMG and dynamic MRI of the upper airway were performed before and after administration of the DREADD ligand clozapine-N-oxide (CNO) or vehicle (saline). In DREADD-treated mice, CNO activated the genioglossus muscle and markedly dilated the pharynx, whereas saline had no effect. Control virus treated mice showed no effect of CNO. Our results suggest that chemogenetic approach can be considered as a treatment option for OSA and other motorneuron disorders.

Tongue fat infiltration in obese versus lean Zucker rats.

STUDY OBJECTIVES: Obesity is the most important risk factor for obstructive sleep apnea (OSA), and the effects of obesity may be mediated by tongue fat. Our objective was to examine the effects of obesity on upper airway structures in obese (OBZ) and non-obese (NBZ) Zucker rats. DESIGN: Animal study. SETTING: Academic Medical Center. PARTICIPANTS: OBZ (638.2 ± 39 g; 14.9 ± 1.1 w) and age-matched NBZ Zucker (442.6 ± 37 g, 15.1 ± 1.5 w) rats. INTERVENTIONS: TONGUE FAT AND VOLUME AND WERE ASSESSED USING: in vivo magnetic resonance spectroscopy (MRS), magnetic resonance imaging including Dixon imaging for tongue fat volume, ex vivo biochemistry (fat quantification; triglyceride (mg)/tissue (g), and histology (Oil Red O stain). MEASUREMENTS AND RESULTS: MRS: overall OBZ tongue fat/water ratio was 2.9 times greater than NBZ (P < 0.002) with the anterior OBZ tongue up to 3.3 times greater than NBZ (P < 0.002). Biochemistry: Triglyceride (TG) in the tongue was 4.4 times greater in OBZ versus NBZ (P < 0.0006). TG was greater in OBZ tongue (3.57 ± 1.7 mg/g) than OBZ masseter muscle (0.28 ± 0.1; P < 0.0001) but tongue and masseter TG were not different in NBZ rats (0.82 ± 0.3 versus 0.28 ± 0.1 mg/g, P = 0.67). Dixon fat volume was significantly increased in OBZ (56 ± 15 mm3) versus NBZ (34 ± 5 mm3, P < 0.004). Histology demonstrated a greater degree of intracellular muscle fat and extramuscular fat infiltration in OBZ versus NBZ rats. CONCLUSIONS: Genetically obese rats had a large degree of fat infiltration in the tongue compared to both skeletal muscle and tongue tissues of the non-obese age-matched littermates. The significant fat increase and sequestration in the obese tongue may play a role in altered tongue neuromuscular function, tongue stiffness or metabolic function.

Respiratory modulation of the pharyngeal airway in lean and obese mice.

UNLABELLED: Obesity is an important risk factor for pharyngeal airway collapse in obstructive sleep apnea (OSA). To examine the effect of obesity on pharyngeal airway size on inspiration and expiration, respiratory-gated MRI of the pharynx was compared in New Zealand obese (NZO) and New Zealand white (NZW) mice (weights: 50.4g vs. 34.7g, p<0.0001). RESULTS: (1) pharyngeal airway cross-sectional area was greater during inspiration than expiration in NZO mice, but in NZW mice airway area was greater in expiration than inspiration; (2) inspiratory-to-expiratory changes in both mouse strains were largest in the caudal pharynx; and (3) during expiration, airway size tended to be larger, though non-significantly, in NZW than NZO mice. The respiratory pattern differences are likely attributable to obesity that is the main difference between NZO and NZW mice. The data support an hypothesis that pharyngeal airway patency in obesity is dependent on inspiratory dilation and may be vulnerable to loss of neuromuscular pharyngeal activation.

Modeling upper airway collapse by a finite element model with regional tissue properties.

This study presents a new computational system for modeling the upper airway in rats that combines tagged magnetic resonance imaging (MRI) with tissue material properties to predict three-dimensional (3D) airway motion. The model is capable of predicting airway wall and tissue deformation under airway pressure loading up to airway collapse. The model demonstrates that oropharynx collapse pressure depends primarily on ventral wall (tongue muscle) elastic modulus and airway architecture. An iterative approach that involves substituting alternative possible tissue elastic moduli was used to improve model precision. The proposed 3D model accounts for stress-strain relationships in the complex upper airway that should present new opportunities for understanding pathogenesis of airway collapse, improving diagnosis and developing treatments.

Altered upper airway and soft tissue structures in the New Zealand Obese mouse.

RATIONALE: The effect of obesity on upper airway soft tissue structure and size was examined in the New Zealand Obese (NZO) mouse and in a control lean mouse, the New Zealand White (NZW). OBJECTIVES: We hypothesized that the NZO mouse has increased volume of neck fat and upper airway soft tissues and decreased pharyngeal airway caliber. METHODS: Pharyngeal airway size, volume of the upper airway soft tissue structures, and distribution of fat in the neck and body were measured using magnetic resonance imaging (MRI). Dynamic MRI was used to examine the differences in upper airway caliber between inspiration and expiration in NZO versus NZW mice. MEASUREMENTS AND MAIN RESULTS: The data support the hypothesis that, in obese NZO versus lean NZW mice, airway caliber was significantly smaller (P < 0.03), with greater parapharyngeal fat pad volumes (P < 0.0001) and a greater volume of other upper airway soft tissue structures (tongue, P = 0.003; lateral pharyngeal walls, P = 0.01; soft palate, P = 0.02). Dynamic MRI showed that the airway of the obese NZO mouse dilated during inspiration, whereas in the lean NZW mouse, the upper airway was reduced in size during inspiration. CONCLUSIONS: In addition to the increased volume of pharyngeal soft tissue structures, direct fat deposits within the tongue may contribute to airway compromise in obesity. Pharyngeal airway dilation during inspiration in NZO mice compared with narrowing in NZW mice suggests that airway compromise in obese mice may lead to muscle activation to defend upper airway patency during inspiration.

Mechanical effects of genioglossus muscle stimulation on the pharyngeal airway by MRI in cats.

To examine the regional mechanical effects of selective genioglossus muscle activation on pharyngeal airway size and function, magnetic resonance images of the pharyngeal airway were obtained in five paralyzed, anesthetized cats over a range of positive and negative pressures in an isolated, sealed upper airway. When all results across pressure levels and pharyngeal regions were analyzed, genioglossus stimulation significantly increased the cross-sectional area (CSA) of the nasopharyngeal airway. Within specific regions, stimulation tended toward significantly increasing cross-sectional airway area in the mid-nasopharynx. Despite its dilating effect, genioglossus muscle stimulation did not alter compliance in the nasopharyngeal airway, as evidenced by the similar slopes of the pressure versus cross-sectional area relationships with and without stimulation. Finally, airway shape in the mid pharynx became more circular with either increased airway pressure or genioglossus stimulation. The results indicate that selective stimulation of the genioglossus muscle dilates the nasopharynx and provide evidence that stimulation of the genioglossus alone does not alter airway compliance.

Phasic respiratory pharyngeal mechanics by magnetic resonance imaging in lean and obese zucker rats.

RATIONALE: Although obstructive sleep apnea is strongly associated with obesity, we have little understanding of how obesity may alter the mechanical properties of the pharynx and the role of obesity in the pathogenesis of sleep apnea. OBJECTIVES: The overall objective of this study was to determine the effect of obesity on pharyngeal airway size and pharyngeal wall tissue strain in lean and obese Zucker rats. METHODS: Respiratory-gated magnetic resonance imaging with noninvasive tissue tagging was performed in anesthetized, spontaneously breathing lean (n = 9) and obese (n = 9) Zucker rats. Images acquired during expiration and inspiration of the rostral, mid-, and caudal pharynx were analyzed for airway size and pharyngeal wall tissue strain, using planimetry, optical flow, and finite element analyses. Differences in cross-sectional airway area, lateral and anteroposterior airway diameters, and tissue strain (stretch, compression, and direction of stretch) in the lateral and ventral pharyngeal walls were compared by analysis of variance (significance at p < 0.05). MEASUREMENTS AND MAIN RESULTS: Compared with their lean littermates, obese rats had the following significant findings: reduced pharyngeal airway cross-sectional area during inspiration and expiration, smaller increases in airway area during inspiration, and decreased lateral airway dilation during inspiration. Tissue strain in the pharyngeal walls showed no significant differences between obese and lean rats. CONCLUSIONS: These findings suggest that obesity results in a mechanical abnormality that decreases pharyngeal airway size and prevents a normal airway response to a given change in pharyngeal wall tissue strain.

Pharyngeal airway wall mechanics using tagged magnetic resonance imaging during medial hypoglossal nerve stimulation in rats.

To better understand pharyngeal airway mechanics as it relates to the pathogenesis and treatment of obstructive sleep apnoea, we have developed a novel application of magnetic resonance imaging (MRI) with non-invasive tissue tagging to measure pharyngeal wall tissue motion during active dilatation of the airway. Eleven anaesthetized Sprague-Dawley rats were surgically prepared with platinum electrodes for bilateral stimulation of the medial branch of the hypoglossus nerve that supplies motor output to the protrudor and intrinsic tongue muscles. Images of the pharyngeal airway were acquired before and during stimulation using a gated multislice, spoiled gradient recalled (SPGR) imaging protocol in a 4.7 T magnet. The tag pulses, applied before stimulation, created a grid pattern of magnetically imbedded dark lines that revealed tissue motion in images acquired during stimulation. Stimulation significantly increased cross-sectional area, and anteroposterior and lateral dimensions in the oropharyngeal and velopharyngeal airways when results were averaged across the rostral, mid- and caudal pharynx (P < 0.001). Customized software for tissue motion-tracking and finite element-analysis showed that changes in airway size were associated with ventral displacement of tissues in the ventral pharyngeal wall in the rostral, mid- and caudal pharyngeal regions (P < 0.0032) and ventral displacement of the lateral walls in the mid- and caudal regions (P < 0.0001). In addition, principal maximum stretch was significantly increased in the lateral walls (P < 0.023) in a ventral-lateral direction in the mid- and caudal pharyngeal regions and principal maximum compression (perpendicular to stretch) was significantly increased in the ventral walls in all regions (P < 0.0001). Stimulation did not cause lateral displacement of the lateral pharyngeal walls at any level. The results reveal that the increase in pharyngeal airway size resulting from stimulation of the medial branch of the hypoglossal nerve is predominantly due to ventral displacement of the ventral and lateral pharyngeal walls.

Effects of pharyngeal muscle activation on airway pressure-area relationships.

Fiberoptic imaging in an isolated, sealed upper airway was performed in 10 decerebrate cats to determine the effect of pharyngeal muscle activation on airway pressure-area relationships. Bilateral cuff electrodes stimulated the distal cut ends of the following nerves: medial and lateral hypoglossus, glossopharyngeus, and pharyngeal branch of vagus. At given intraluminal pressures ranging from +6 to -6 cm H2O, cross-sectional area was measured in the rostral oropharynx, velopharynx, and caudal oropharynx, with and without nerve stimulation. A mixed model analysis of variance indicated a relatively constant increase in area across the pressure range with glossopharyngeal stimulation at any given level. Significant interactions between pressure and stimulation were present in the rostral oropharynx with medial hypoglossus stimulation and in the caudal oropharynx with independent and combined hypoglossal branch stimulation and pharyngeal branch of vagus stimulation. With stimulation of the hypoglossal nerves, greater increases in area in these regions occurred in the lower pressure ranges. Stimulation of the pharyngeal branch of the vagus caused a greater decrease in area at the higher pressure ranges in the caudal oropharynx and velopharynx. The results indicate that the mechanical effects of pharyngeal muscle activation depend not only on the region and muscles activated but also on the intraluminal pressure.