European Journal of Orthodontics, 2015, 284–289 doi:10.1093/ejo/cju041 Advance Access publication 22 September 2014
An animal model of obstructive sleep apnoea– hypopnea syndrome corrected by mandibular advancement device Hai-yan Lu*,**, Fusheng Dong**,***, Chun-yan Liu*,**, Jie Wang**,****, Ye Liu*,**, and Wei Xiao*,*** *Department of Orthodontics, College of Stomatology, Hebei Medical University, Shijiazhuang, **The Key Laboratory of Stomatology, Shijiazhuang, Hebei, Departments of ***Oral & Maxillofacial Surgery and ****Oral Pathology, College of Stomatology, Hebei Medical University, Shijiazhuang, China Correspondence to: Jie Wang, Department of Oral Pathology, College of Stomatology, Hebei Medical University, No. 383, East Zhongshan Road, Shijiazhuang, Hebei 050017, P.R. of China. E-mail: [email protected]
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Summary Objective: The aim of the study is to establish a stable animal model of obstructive sleep apnoea– hypopnea syndrome (OSAHS) and assess the effectiveness of a mandibular advancement device (MAD). Materials and methods: Eighteen 6-month-old male New Zealand white rabbits were randomized into three groups according to intervention: Group OSAHS, Group MAD, and a control group (n = 6 for each group). Rabbits in Group OSAHS and Group MAD were established as OSAHS model by injection, at a dose of 2 ml hydrophilic polyacrylamide gel, in the submucous muscular layer of the soft palate. Computed tomography (CT) and polysomnography (PSG) showed that OSAHS was developed successfully, the rabbits in Group MAD were fitted with the MAD and CT of the upper airway and PSG evaluated its effectiveness. Histological observation of the injection sites was conducted. Results: CT scans showed the reduced sagittal space and cross-sectional areas of retropalatal upper airway in Group OSAHS were corrected by MAD (upper airway space in Group MAD was similar to that in the control group). The rabbits in Group OSAHS developed obvious sleep apnoea and hypopnea in supine position, with increased apnoea–hypopnea index and decreased oxygen saturation (SaO2). These were significantly improved by MAD and apnoea and hypopnea were not observed. Histology of the soft palate showed that the injected gel was entirely surrounded with connective tissues. Conclusion: We primarily developed an OSAHS and MAD therapy animal model with narrow oropharynx in upper airway which could be further available for OSAHS analysis.
Introduction Obstructive sleep apnoea–hypopnea syndrome (OSAHS) is a common chronic sleep disorder (1). It is associated with a series of health problems such as metabolic dysfunction, cardiovascular disease, neurocognitive deficits, and the risk of traffic accidents (2). Prevalence of OSAHS ranges from 2 to 4%, whereas it is as high
as 20–40% in 65-year olds and over (3). Morphological features of the craniofacial complex in OSAHS patients suggest inharmonious craniofacial structure, such as short and retrusive mandible, hypertrophic tongue and soft palate, may cause a structural alteration of the upper airway and lead to different levels of obstruction (4). Several OSAHS animal models have been described, including tracheostomized models in cats (2), dogs (5, 6), rats (7), lambs (8), and
© The Author 2014. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: [email protected]
H.-y. Lu et al. a primate model with injection of small amounts of liquid collagen into tissues surrounding the upper airway (9). Spontaneously occurring OSAHS has been documented in the English bulldog (10) and obese miniature pig (11), but studies using these models are difficult due to limited availability of the animals and their relatively large size and cost. Lately, a new animal model of OSA has been successfully developed in rabbits by injecting liquid silicone into the base of the tongue (12) or paralyzing the genioglossus (13). In addition, the mandibular advancement device (MAD) has been applied to treat OSAHS in clinical practice. Previous studies (14–17) used cephalometrics, computed tomography (CT) scan, and magnetic resonance imaging to investigate the effects of MAD treatment. However, very little is known about the mechanics of MAD therapy for OSAHS and there is no reports concerning alterations in heart, brain, or kidney following MAD therapy for OSAHS. Because the data were extrapolated from studies in vitro or in animal models, thus there is a need for suitable animal models of MAD therapy of OSAHS. The purpose of this study is to establish a narrowed oropharynx in upper airway model of OSAHS and evaluate treatment with MAD in rabbits.
Materials and methods Animals
All the animals were trained to sleep in a supine position by an animal expert with petting skills. They were then restrained in a supine position by fixing their limbs with a soft and light force strip to a preformed wood-plate to avoid unexpected movement during CT scanning and PSG. The head was positioned to make the airway analysis comparable for control and experimental groups, keeping the forehead and the wood-plate at 60°.
Spiral CT scanning The images of airway structure from the cranial crest to the tracheal opening site during sleep were captured using a GE Light Speed 16-detector spiral CT scanner (GE Healthcare Technologies, Waukesha, Wisconsin, USA). Settings of CT scan were maintained the same: e.g. spiral continuous scanning with 16-dector, 0.625 mm of layer thickness, pitch at 1.375: 1, bed speed at 13.75 mm/circle, window width 250 HU, window level 40 HU, with 80 kV and 30 mA scan for 5 seconds. Reconstruction software was used for sagittal reconstruction of the data based on the centre of the reconstructing sagittal image. Axial reconstruction perpendicular to the pharynx wall was also done. Crosssectional areas and sagittal diameter at the top level of the soft palate down to level of 1/4, 2/4, 3/4 in the posterior areas were measured (Figure 2).
The rabbits were randomly assigned to two groups, with 6 animals in the control group and 12 animals in experimental group. They were acclimatized for at least 1 week before the start of the experiment and were housed under normal laboratory conditions. On the day before experiment, the rabbits in the experimental group experienced fasting and water-deprivation for about 3 hours. After a successful anaesthesia with 1% pentobarbital sodium injection at a dose of 20 mg/kg via the auricular vein, the rabbit was then restrained in a supine position. Two milliliters of the prepared gel mixture, including 2 g hydrophilic polyacrylamide gel, 20 mg of gentamicin, and 1.5 ml of physiological saline 0.9%, was injected via the submucous muscular layer at the centre of the soft palate about 1.5 cm away from the junction of the hard and soft palates(Figure 1). Control group animals received no gel. Experimental animals were later confirmed to have OSAHS by spiral CT scanning and polysomnography (PSG).
All animals were induced to sleep in the supine position and gently restrained on the wood-plate as described. Breathing and sleep were monitored via PSG. The rabbits were equipped with surface electrodes taped on the skull, face, and chest for monitoring of the electroencephalogram (C3/A2, C4/A1, O2/A1), right and left electrooculogram, and respiration. Nasal airflow was monitored through a nasal pressure transducer. Respiratory movements were monitored by respiratory inductance plethysmography (chested and abdominal bands). Blood oxygen saturation (SaO2) was measured using an ear pulse oximeter. PSG recordings were performed during the day, e.g. from 09:00 to 11:30 in the morning or from 14:00 to 16:30 in the afternoon (10). Hypopneas were defined as a reduction between 20 and 50% in nasal flow for at least two breaths; their occurrence was associated with an arousal. Apnoeas were defined by an absence of airflow at the nose and mouth for longer than two breaths (7). Apnoea–hypopnea index (AHI), the average number of episodes of apnoea and hypopnea per hour of sleep, was scored for all animals. OSAHS was confirmed if the AHI was larger than 5 (18). The AHI suggested OSAHS model was successfully established. These rabbits were randomly divided into Group OSAHS (n = 6) and Group MAD (n = 6).
Figure 1. The arrow points to the injection site of soft palate, about 1.5 cm to the junction of the hard and soft palate. A: tongue; B: the junction of the hard and soft palate; C: maxillary incisors.
Figure 2. The upper airway in the posterior soft palate was equally divided into five levels from upper to the lower point, and the cross-sectional areas were measured at these levels.
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Experiments were performed on 18 male 6-month-old New Zealand white rabbits (initial weight, 3–3.5 kg). Care and experimental procedures were approved by the Animal Care Committee (Certificate No. SCXK (J) 2008-1-003). Food and water were available ad lib.
Mandibular advancement device Animals in Group MAD were cemented with a modified MAD. This MAD was made of self-curing composite resin at the upper denture model, with a 30° inclined plane to the crown axis of upper incisors, which was adhered to the two upper incisors with glass ionomer (3M ESPE AG, Seefeld, Germany) (Figure 3). The mandible was guided forward 3–4 mm. After 3 days of adaptation, the rabbits had flexible movement with no difficulty in taking food, and then the effectiveness of MAD treatment was assessed through spiral CT scanning and PSG, with the protocols described. After 2 weeks, body weight and food intake were recorded. Then, all the rabbits in three groups were housed under normal laboratory conditions.
Histology After 8 weeks, the rabbits were sacrificed and the soft palates in the Group OSAHS and Group MAD were collected. The samples were fixed with 10% formalin and processed for paraffin sectioning. Following haematoxylin and eosin (HE) staining, the sections were observed under an Olympus AX80 Provis Research Microscope System (Olympus Corporation, Tokyo, Japan).
OSAHS showed that the sagittal spaces were significantly (P