Treatment of obstructive sleep apnoea–hypopnea syndrome by mandible advanced device reduced neuron apoptosis in frontal cortex of rabbits

Treatment of obstructive sleep apnoea–hypopnea syndrome by mandible advanced device reduced... Summary Objective To investigate effects of mandible advanced device (MAD) therapy for obstructive sleep apnoea–hypopnea syndrome (OSAHS) on the neuron apoptosis and acetylcholine esterase activity in frontal cortex. Materials and methods Thirty male New Zealand white rabbits were randomly divided into three groups (n = 10 in each group): group OSAHS, group MAD, and control group. Hydrophilic polyacrylamide gel was injected into soft palate of the animals to induce OSAHS in group OSAHS and group MAD. The group MAD animals wore MAD to relief the obstructiveness. The control group was not given any treatment. Computed tomography (CT) examination of the upper airway and polysomnography (PSG) recordings were performed in supine position. All rabbits were induced to sleep in a supine position for 4 to 6 hours every day and were observed for consecutive 8 weeks. The frontal cortices of three groups were dissected and the neuron apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and flow cytometry. Acetylcholine esterase (AchE) activity in the frontal cortex was measured by spectrophotometry. Results The group OSAHS exhibited high neuron apoptosis rate and low AchE activity than those of group MAD and control group. The blood oxygen saturation was negatively correlated with neuronal apoptosis rate and positively correlated with AchE activity. Applying MAD in OSAHS animals significantly improve the neuronal damage and function deficits by apnoea–hypoxia caused by narrowed upper airway. Conclusion This study provided evidence that MAD therapy for OSAHS can significantly decrease neuronal apoptosis and increase AchE activity in the frontal cortex. Introduction Obstructive sleep apnoea-hypopnea syndrome (OSAHS) is characterized by recurrent complete or partial airway obstruction, which leads to inspiratory flow cessation (apnoea) or airflow limitation (hypopnea) accompanied by oxygen desaturation and arousal from sleep. OSAHS has been associated with cardiovascular, pulmonary, metabolic, and neural disturbance with multiple clinical symptoms (1). Accumulated evidence has shown that OSAHS is associated with damage of the nerve system (2,3). The brain, especially within white matter of the frontal cortex, is vulnerable to repetitive hypoxia/reoxygenation. The neurons of these regions are responsible to cognitive functions, including reward, attention, short-term memory tasks et al (4). Previous studies implicated that reactive oxygen species (ROS) generation and neuron apoptosis caused by intermittent hypoxia contributed to this cognitive deficits (5–7). However, little is known about the exact mechanism underlying cognitive impairment induced by OSAHS. Evidence both from animal and human studies suggest that central cholinergic systems, including acetylcholine (Ach), acetylcholine transferase (ChAT), acetylcholine esterase (AchE), and cholinergic receptors (8–11), are important to cognitive function. Among them, Ach is one of the major neurotransmitters and widely distributed in the central nervous systems. The synthesis and hydrolysis of Ach were considered be associated with emotion change, attention, executive function, and cognitive activities (12). The neurons in cholinergic basal forebrain synthetize substantial Ach, and then project it to the cortex. Recent studies have reported that decreased Ach release is correlated to memory impairment (13,14). In addition, blocking Ach receptor activity by muscarinic receptor agonists can enhance the animal performance on learning and memory tests (15,16). Therefore, Ach level is critical to cognitive functions. As the primary hydrolase of Ach, AchE regulates the Ach and terminates the signal transmission in the post-synaptic cleft. However, the relationship between AchE activity and OSAHS is still poorly understood. Approaches to prevent repetitive hypoxia/reoxygenation are important for preventing the cerebral hypoxic damage caused by OSAHS. The basic treatment principle of OSAHS is to open the obstructed or narrowed upper airway, and then to improve the oxyhemoglobin saturation. Therapies currently used include behavioural treatment (17), continuous positive airway pressure (CPAP) (18,19), mandible advanced devices (MAD) (20,21), and surgeries aiming to enlarge the upper airway, such as uvulopalatopharyngoplast (UPPP) and maxilla-mandibular advancement osteotomy (22). Most of OSAHS patients choose CPAP and MAD due to remarkable effectiveness in reducing symptoms, less risk, and relapse. CPAP treatment delayed cognitive impairment, which suggests the relationship between OSASH and neurocognitive (23). MAD is another effective therapy of OSAHS. It has many advantages such as unobtrusive, noiseless, convenient, and less costly. There is no evidence, however, about the treatment effects of MAD in the frontal cortex. This study was designed to investigate the neuron cell apoptosis and the activity of Ach esterase in frontal cortex using our established OSAHS animal models (24), and to further evaluate the effects of MAD treatment on these events to uncover the mechanism underlying cognitive impairment by OSAHS and hoping to provide evidences for clinical treatment. Materials and methods Experimental animals Thirty male 6-month-old New Zealand white rabbits (3 to 3.5 kg) were selected from animal center of Hebei Medical University. Care and experimental protocols were approved by local Animal Care and Use Committees (Certificate No. SCXK (J) 20012-1-003). Food and water were available ad lib. Experimental animal model The animals were randomly divided into three groups: group OSAHS, group MAD, and control group, 10 animals for each group. The size of the experimental groups was decided based on the results from our previous studies and power analysis, which allowed us to detect a 20 per cent difference in the groups with a high likelihood of statistical significance. The experimental animal models were established as previously described (24,25). In brief, the animals of group OSAHS and MAD were restrained in a supine position under general anaesthesia with 1 per cent pentobarbital sodium (20 mg/kg) through auricular vein injection. Gel injection was utilized to develop OSAHS symptoms. Two millilitres of prepared gel mixture (containing 2-g hydrophilic polyacrylamide gel, 20 mg of gentamicin dissolved in saline) was injected via the submucosa muscular layer at the centre of the soft palate, approximately 1.5 cm away from the junction of the hard and soft palates. The MAD group wore MAD to alleviate the OSAHS symptoms. Animals in the control group were not given any treatment. Spiral computed tomography scanning Spiral computed tomography (CT) scanning was utilized to evaluate the upper airway obstruction by capturing the images of airway structure from the cranial crest to the tracheal opening site during sleep (GE Healthcare Technologies, Waukesha, Wisconsin, USA). Reconstruction software was used for axial and sagittal structure of the airway. Cross-section areas and sagittal diameters were measured, from the top level of soft palate to level of 1/4, 1/2, 3/4 in the posterior areas. Polysomnography Polysomnography (PSG, Rembrandt Embla Polysomnography System, Reykjavik, Iceland) was used to monitor the sleep parameters. The rabbits were equipped with surface electrodes taped on the skull, face, and chest for monitoring the electroencephalogram, right and left electrooculogram, and respiration. Nasal air flow was monitored through a nasal pressure transducer. Respiratory movements were reported by respiratory inductance plethysmography (chested and abdominal bands). Blood oxygen saturation (SaO2) was measured using an ear pulse oximeter. PSG recordings were conducted during sleeping from 09:00 to 11:30 or from 14:00 to 16:30 (26). Hypopnea was defined as a reduction between 20 per cent and 50 per cent in nasal flow for at least two breaths, which was associated with an arousal. Apnoea was defined by an absence of airflow at the nose and mouth for longer than two breaths (27). 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 five (28). MAD fabrication Impressions were then taken in maxilla of group MAD after OSAHS model is successfully established. MAD was fabricated from the casts with a 30-degree inclined plane to the long axis of upper incisor crowns using self-curing composite resin, and bonded to the upper incisors with glass ionomer. The mandible was guided forward 3–4 mm. Spiral CT scanning and PSG were used to evaluate the effectiveness of MAD treatment following the protocols described above. All the rabbits were restrained in a supine position for 4 hours per day while sleeping and were observed for consecutive 8 weeks. Basic health data including behaviour and body weight was recorded at the time point of 2 weeks and 8 weeks. Histological analyses After 8 weeks, the animals were anaesthetized with sodium pentobarbitone (20 mg/kg, intravenously). Tissues harvested from frontal cortex were dissected, fixed in 10 per cent buffered formalin for 24 hours, and embedded in paraffin. Sections were prepared for hematoxylin and eosin (H&E) for microscopic observation (Olympus, Japan). Flow cytometry A part of frontal cortex was dissected and fixed in 70 per cent cryopreservation alcohol for 24 hours. Single-cell suspension was prepared to detect the rate of apoptosis by flow cytometry. The suspension was centrifuged at 800 rpm for 2 minutes at 4°C. After removing the supernatant, the cells were incubated with 1-ml propidium iodide dye (PI: 0.05 µg/ml, Triton-X 100: 2 ml, sodium citrate: 2 mg, saline: 130 ml, double distilled water: 70 ml). The histograms and the rate of apoptosis were analyzed by flow cytometry (Beckman Coulter, USA). TUNEL assays Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Kits (Roche, Germany) was used to determine brain cell apoptosis. Briefly, sections were dewaxed and washed with phosphate buffered solution, PBS. Sections were treated with 3 per cent H2O2 in methanol for 10 minutes and incubated with proteinase K for 15 minutes. After washing with PBS for two times, sections were treated with 50-µl TUNEL reaction mixture for 1 hour at 37°C in a humidified atmosphere in the dark. Then rinsed three times with PBS. A 50 µl converter-POB was added to the sections. Slides were incubated in a humidified chamber for 30 minutes at 37°C. Diaminobenzidine tetrahydrochloride (DAB) was added for 10 minutes at room temperature. Positive cells (brown colouring) indicated DNA fragmentation during cell apoptosis. Samples were counterstained with hematoxylin and observed by microscopy at 400 magnification times (Olympus, Japan). The sections treated with DNase I were defined as positive controls, and those without TdT were defined as the negative controls. Measurement of AchE activity The frontal cortex was dissected, rinsed in saline, and dried on filter paper. Tissues were homogenized at 4°C for 1 minute in buffer (0.01 mol sucrose, 0.01 mol Tris-HCL, 0.01 ml EDTA, PH = 7.4). The homogenate was centrifuged at 3000 r at 4°C for 15 minutes. The supernatant was extracted and the AchE activity was quantified by spectrophotometry according to the manual for measurement of AchE activity (Nanjing Jiancheng biological engineering institute, China). Statistical analysis SPSS 22.0 statistical software package was used for data analysis (SPSS, Chicago, USA). All data were expressed by mean ± SD. The normality test and Levene’s variance homogeneity test showed the data were normally distributed and the variance was homogeneous. Analysis of variance (ANOVA) was used to compare the difference of three groups followed by least significant difference when appropriate. Correlation analysis was used between oxyhemoglobin saturation and apoptosis of neurons in the brain frontal cortex, or between oxyhemoglobin saturation and AchE activity. Significance level was set at P <0.05. Results Gel-injection induced OSAHS-like symptoms, and partly alleviated by MAD treatment To investigate the OSAHS and possible effects of MAD in treating OSAHS, we took advantage of previously developed gel-induced OSAHS animal model (24). In group OSAHS, it was shown that snoring and apnoea progressed with time during sleep, and intermittent awakenings and lips cyanosis were observed. Sleepiness was most obvious during daytime on tenth day. In group MAD, the aforementioned symptoms were relieved, which four animals exhibited regular rhythm of breath without snoring, and six animals showed rare snoring but no apnoea. This suggested that gel injection successfully mimicked OSAHS by inducing apnoea-like phenomena, which can be partly relieved by MAD. In control group, the breathing was normal without any snore or apnoea. There was no significant changes of body weight between the three groups during 8 weeks of experiment (Table 1). Table 1. Body weight changes of experimental animals. MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome. Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 Body weight (kg)  Baseline 3.30 ± 0.17 3.33 ± 0.12 3.31 ± 0.15 0.760 0.909 0.849  2 weeks 3.44 ± 0.15 3.52 ± 0.14 3.56 ± 0.13 0.297 0.152 0.675  8 weeks 3.70 ± 0.11 3.72 ± 0.10 3.79 ± 0.15 0.706 0.151 0.277 Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 Body weight (kg)  Baseline 3.30 ± 0.17 3.33 ± 0.12 3.31 ± 0.15 0.760 0.909 0.849  2 weeks 3.44 ± 0.15 3.52 ± 0.14 3.56 ± 0.13 0.297 0.152 0.675  8 weeks 3.70 ± 0.11 3.72 ± 0.10 3.79 ± 0.15 0.706 0.151 0.277 View Large Table 1. Body weight changes of experimental animals. MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome. Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 Body weight (kg)  Baseline 3.30 ± 0.17 3.33 ± 0.12 3.31 ± 0.15 0.760 0.909 0.849  2 weeks 3.44 ± 0.15 3.52 ± 0.14 3.56 ± 0.13 0.297 0.152 0.675  8 weeks 3.70 ± 0.11 3.72 ± 0.10 3.79 ± 0.15 0.706 0.151 0.277 Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 Body weight (kg)  Baseline 3.30 ± 0.17 3.33 ± 0.12 3.31 ± 0.15 0.760 0.909 0.849  2 weeks 3.44 ± 0.15 3.52 ± 0.14 3.56 ± 0.13 0.297 0.152 0.675  8 weeks 3.70 ± 0.11 3.72 ± 0.10 3.79 ± 0.15 0.706 0.151 0.277 View Large MAD therapy opened the upper airways to improve respiratory function and apnoea–hypoxia It is known that respiratory airway obstruction is the characteristic change in OSAHS. In our study, spiral CT scan had confirmed that the alterations of upper airway spaces by measuring the sagittal and the cross-sectional areas in this study (Table 2). Both sagittal spaces and cross-sectional areas of group OSAHS were significantly decreased at points 1/4, 1/2, and 3/4 compared to that of the control group and the group MAD (P < 0.05; Figure 1A and 1B). Comparing to group OSAHS, the reduced retropalatal spaces were significantly widened by MAD through advancing the mandible (P < 0.05), which showed no significant difference between the group MAD and the controls (P > 0.05). Therefore, the consequent respiratory functions changed accordingly. SaO2 and AHI were measured and scored for all animals. Significant increase of AHI and decrease of SaO2 were observed in the group OSAHS compared with those of the group MAD and control group (P < 0.05). No statistically significant differences were found in AHI and SaO2 levels between group MAD and control group (P > 0.05). Also, we observed nasal airflow decrease with an associated increase in respiratory effort in group OSAHS, which was consistent to our previous studies (Table 3). Table 2. The retropalatal upper airway spaces of three groups. MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome. Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 Sagittal space (mm2)  Upper point 4.53 ± 0.20 4.47 ± 0.25 4.52 ± 0.29 0.676 1.000 0.676  1/4 point 2.78 ± 0.30 3.67 ± 0.37 3.78 ± 0.39 0.001* 0.001* 0.578  1/2 point 1.60 ± 0.41 2.71 ± 0.46 2.80 ± 0.45 0.000* 0.000* 0.663  3/4 point 1.77 ± 0.35 2.76 ± 0.34 2.83 ± 0.36 0.000* 0.000* 0.751  Lower point 3.56 ± 0.42 3.82 ± 0.39 3.85 ± 0.28 0.189 0.890 0.236 Cross-sectional areas (mm2)  Upper point 22.79 ± 0.49 22.87 ± 0.20 23.08 ± 0.36 0.720 0.201 0.347  1/4 point 17.47 ± 1.22 22.41 ± 0.72 22.52 ± 1.59 0.000* 0.000* 0.890  1/2 point 13.31 ± 1.40 18.95 ± 1.96 20.15 ± 1.49 0.000* 0.000* 0.182  3/4 point 14.18 ± 2.25 20.24 ± 2.94 21.04 ± 2.55 0.000* 0.000* 0.588  Lower point 26.24 ± 2.74 27.32 ± 2.93 29.10 ± 2.92 0.679 0.147 0.286 Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 Sagittal space (mm2)  Upper point 4.53 ± 0.20 4.47 ± 0.25 4.52 ± 0.29 0.676 1.000 0.676  1/4 point 2.78 ± 0.30 3.67 ± 0.37 3.78 ± 0.39 0.001* 0.001* 0.578  1/2 point 1.60 ± 0.41 2.71 ± 0.46 2.80 ± 0.45 0.000* 0.000* 0.663  3/4 point 1.77 ± 0.35 2.76 ± 0.34 2.83 ± 0.36 0.000* 0.000* 0.751  Lower point 3.56 ± 0.42 3.82 ± 0.39 3.85 ± 0.28 0.189 0.890 0.236 Cross-sectional areas (mm2)  Upper point 22.79 ± 0.49 22.87 ± 0.20 23.08 ± 0.36 0.720 0.201 0.347  1/4 point 17.47 ± 1.22 22.41 ± 0.72 22.52 ± 1.59 0.000* 0.000* 0.890  1/2 point 13.31 ± 1.40 18.95 ± 1.96 20.15 ± 1.49 0.000* 0.000* 0.182  3/4 point 14.18 ± 2.25 20.24 ± 2.94 21.04 ± 2.55 0.000* 0.000* 0.588  Lower point 26.24 ± 2.74 27.32 ± 2.93 29.10 ± 2.92 0.679 0.147 0.286 *P < 0.01 View Large Table 2. The retropalatal upper airway spaces of three groups. MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome. Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 Sagittal space (mm2)  Upper point 4.53 ± 0.20 4.47 ± 0.25 4.52 ± 0.29 0.676 1.000 0.676  1/4 point 2.78 ± 0.30 3.67 ± 0.37 3.78 ± 0.39 0.001* 0.001* 0.578  1/2 point 1.60 ± 0.41 2.71 ± 0.46 2.80 ± 0.45 0.000* 0.000* 0.663  3/4 point 1.77 ± 0.35 2.76 ± 0.34 2.83 ± 0.36 0.000* 0.000* 0.751  Lower point 3.56 ± 0.42 3.82 ± 0.39 3.85 ± 0.28 0.189 0.890 0.236 Cross-sectional areas (mm2)  Upper point 22.79 ± 0.49 22.87 ± 0.20 23.08 ± 0.36 0.720 0.201 0.347  1/4 point 17.47 ± 1.22 22.41 ± 0.72 22.52 ± 1.59 0.000* 0.000* 0.890  1/2 point 13.31 ± 1.40 18.95 ± 1.96 20.15 ± 1.49 0.000* 0.000* 0.182  3/4 point 14.18 ± 2.25 20.24 ± 2.94 21.04 ± 2.55 0.000* 0.000* 0.588  Lower point 26.24 ± 2.74 27.32 ± 2.93 29.10 ± 2.92 0.679 0.147 0.286 Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 Sagittal space (mm2)  Upper point 4.53 ± 0.20 4.47 ± 0.25 4.52 ± 0.29 0.676 1.000 0.676  1/4 point 2.78 ± 0.30 3.67 ± 0.37 3.78 ± 0.39 0.001* 0.001* 0.578  1/2 point 1.60 ± 0.41 2.71 ± 0.46 2.80 ± 0.45 0.000* 0.000* 0.663  3/4 point 1.77 ± 0.35 2.76 ± 0.34 2.83 ± 0.36 0.000* 0.000* 0.751  Lower point 3.56 ± 0.42 3.82 ± 0.39 3.85 ± 0.28 0.189 0.890 0.236 Cross-sectional areas (mm2)  Upper point 22.79 ± 0.49 22.87 ± 0.20 23.08 ± 0.36 0.720 0.201 0.347  1/4 point 17.47 ± 1.22 22.41 ± 0.72 22.52 ± 1.59 0.000* 0.000* 0.890  1/2 point 13.31 ± 1.40 18.95 ± 1.96 20.15 ± 1.49 0.000* 0.000* 0.182  3/4 point 14.18 ± 2.25 20.24 ± 2.94 21.04 ± 2.55 0.000* 0.000* 0.588  Lower point 26.24 ± 2.74 27.32 ± 2.93 29.10 ± 2.92 0.679 0.147 0.286 *P < 0.01 View Large Table 3. Breathing parameters of three groups. AHI, apnoea–hypopnea index; MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome; SaO2, blood oxygen saturation. Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 AHI 30.85 ± 2.71 3.41 ± 0.22 2.78 ± 0.27 0.000* 0.000* 0.497 Average SaO2 (%) 80.23 ± 3.50 89.8 ± 1.33 91.8 ± 1.45 0.000* 0.000* 0.059 Min.SaO2 (%) 58.12 ± 3.02 86.0 ± 2.27 88.3 ± 1.56 0.000* 0.000* 0.111 Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 AHI 30.85 ± 2.71 3.41 ± 0.22 2.78 ± 0.27 0.000* 0.000* 0.497 Average SaO2 (%) 80.23 ± 3.50 89.8 ± 1.33 91.8 ± 1.45 0.000* 0.000* 0.059 Min.SaO2 (%) 58.12 ± 3.02 86.0 ± 2.27 88.3 ± 1.56 0.000* 0.000* 0.111 *P < 0.01 View Large Table 3. Breathing parameters of three groups. AHI, apnoea–hypopnea index; MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome; SaO2, blood oxygen saturation. Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 AHI 30.85 ± 2.71 3.41 ± 0.22 2.78 ± 0.27 0.000* 0.000* 0.497 Average SaO2 (%) 80.23 ± 3.50 89.8 ± 1.33 91.8 ± 1.45 0.000* 0.000* 0.059 Min.SaO2 (%) 58.12 ± 3.02 86.0 ± 2.27 88.3 ± 1.56 0.000* 0.000* 0.111 Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 AHI 30.85 ± 2.71 3.41 ± 0.22 2.78 ± 0.27 0.000* 0.000* 0.497 Average SaO2 (%) 80.23 ± 3.50 89.8 ± 1.33 91.8 ± 1.45 0.000* 0.000* 0.059 Min.SaO2 (%) 58.12 ± 3.02 86.0 ± 2.27 88.3 ± 1.56 0.000* 0.000* 0.111 *P < 0.01 View Large Figure 1. View largeDownload slide Spiral CT scan. A. Measurement of the sagittal diameters of the upper airway in supine position at five levels labelled. B. CT image of the cross-sectional area of the upper airway in supine position indicated by arrow. CT, computed tomography. Figure 1. View largeDownload slide Spiral CT scan. A. Measurement of the sagittal diameters of the upper airway in supine position at five levels labelled. B. CT image of the cross-sectional area of the upper airway in supine position indicated by arrow. CT, computed tomography. Frontal cortex neuronal apoptosis associated with OSAHS can be rescued by MAD treatment To explore the pathophysiological damage to the brain caused by OSAHS, histological examinations were performed (Figure 2A). In frontal cortex of group OSAHS, increased number of neurons appeared shrunken, hyperchromatic, and karyopyknosis (H&E staining). However, these were not found in group MAD and the control group. Figure 2. View largeDownload slide Histological examinations of frontal cortex neurons. A. Hematoxylin and eosin (H&E) staining. Representative apoptotic cells with karyopyknosis and hyperchromatic staining indicated with black arrow. B. TUNEL staining. The apoptotic cells were specifically stained in brown indicated by red arrows, and normal neurons were counterstained in blue indicated by black arrows. MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling. Figure 2. View largeDownload slide Histological examinations of frontal cortex neurons. A. Hematoxylin and eosin (H&E) staining. Representative apoptotic cells with karyopyknosis and hyperchromatic staining indicated with black arrow. B. TUNEL staining. The apoptotic cells were specifically stained in brown indicated by red arrows, and normal neurons were counterstained in blue indicated by black arrows. MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling. We speculated that these neuronal changes indicated possible cell death due to hypoxia. To verify this, we took advantage of TUNEL assay to investigate neuronal apoptosis in the rabbit frontal cortex regions. The apoptotic cells, specifically stained in brown, were less in group MAD and control group than that of the group OSAHS (Figure 2B). To further quantify the apoptotic cells from different groups of experiment, flow cytometry was utilized to analyze the cell viability using propidium iodide (PI). PI, permeable to the non-viable cells, bonded with double stranded DNA of dead cells. The histograms of cell counts of PI positive cells by flow cytometry in three groups were shown in Figure 3. Significant difference was observed among three groups by one-way ANOVA (F = 164.2, P = 0.000). The apoptosis rate of group OSAHS was 7.403 ± 1.256 per cent, which was higher than that of group MAD (2.259 ± 0.319%, P = 0.000) and control group (1.943 ± 0.242%, P = 0.000). The apoptosis rate of group MAD was slightly higher than the control group, but without significant different (P = 0.358; Figure 4). These results suggested that OSAHS induced frontal cortical neuron apoptosis, and treating OSAHS by MAD reduced the cell death. Figure 3. View largeDownload slide Flow cytometry histograms of apoptosis cells stained by PI. Sub-G1 peak (black arrow showed) as an indicative of apoptotic cells was induced in the group OSAHS, but not in group MAD and control group. MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome; PI, propidium iodide. Figure 3. View largeDownload slide Flow cytometry histograms of apoptosis cells stained by PI. Sub-G1 peak (black arrow showed) as an indicative of apoptotic cells was induced in the group OSAHS, but not in group MAD and control group. MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome; PI, propidium iodide. Figure 4. View largeDownload slide The oxygen saturation, AchE activity, and neuronal apoptosis rates were shown in A–C, respectively. The results expressed in mean ± SD. Statistically significant differences are indicated by asterisk (LSD test, P < 0.05). The correlation between oxyhemoglobin saturation and apoptosis of neurons showed in D. The correlation between oxyhemoglobin saturation and apoptosis of neurons showed in E. Significance level was set at P < 0.05. AchE, acetylcholine esterase; MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome; SaO2, blood oxygen saturation; SD, standard deviation. Figure 4. View largeDownload slide The oxygen saturation, AchE activity, and neuronal apoptosis rates were shown in A–C, respectively. The results expressed in mean ± SD. Statistically significant differences are indicated by asterisk (LSD test, P < 0.05). The correlation between oxyhemoglobin saturation and apoptosis of neurons showed in D. The correlation between oxyhemoglobin saturation and apoptosis of neurons showed in E. Significance level was set at P < 0.05. AchE, acetylcholine esterase; MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome; SaO2, blood oxygen saturation; SD, standard deviation. Frontal cortex neuronal impairment linked with apnoea–hypoxia cause by OSAHS Sleep apnoea caused hypoxia-ischemia, which induced cell death and further impaired neuronal functions in frontal cortex. AchE is the hydrolase catalyzing Ach and regulating the neuronal functions. To this end, we measured the AchE activity. The AchE activity of frontal cortex was 0.373 ± 0.042 U/mgprot in group OSAHS, which was lower than that in group MAD (0.450 ± 0.065 U/mgprot, P = 0.002) and in the control group (0.475 ± 0.050 U/mgprot, P = 0.000). However, there was no significant different between group MAD and the control group (P = 0.277). To examine the correlations between the frontal neuronal damages with hypoxia, we analyzed the association between neuronal apoptosis/AchE activity with blood oxygen saturation. The correlation coefficient of the neuronal apoptosis rate and blood oxygen saturation was −0.820, P <0.01. And the correlation coefficient of AchE activity and oxygen saturation was 0.442, P <0.05 (Figure 4). This suggested that blood oxygen saturation was negatively correlated with neuronal apoptosis rate and positively correlated with AchE activity. Discussion The major clinical presentations of OSAHS are frequently intermittent hypoxia during sleeping. It was reported that OSAHS triggers pathophysiological changes in multiple systems, including cardiovascular system, nerve system, genitourinary system, respiratory system, and muscular system et al (29). Experimental studies were focusing on apoptosis induced by hypoxia, which was considered to be one of the pathophysiological basis of OSHAS. In some studies using OSAHS rat model, it had been shown that chronic intermittent hypoxia increased TUNEL-positive cardiovascular myocytes, and altered expression of apoptosis genes in vascular endothelial cells. Loss of cardiomyocytes due to apoptosis can result in cardiac dysfunction and promoting development of atherosclerosis (30–32). Obstructive sleep apnoea also associated with chronic kidney disease, and the renal tubular endothelial apoptosis contributed to the pathogenesis of the hypoxia-induced renal injury (33). Recently, accumulating evidences have indicated that cognitive functions and performance deteriorate in individuals associate with OSAHS. Recurrent apnoea induces neuronal apoptosis in the guinea pig forebrain (34). Diffusion tensor imaging showed a wide range of white matter abnormalities in clinical trials, which could be associated with specific cognitive deficits (35). Most of these studies were using animal models of intermittent hypoxia induced by changing the oxygen desaturation. However, these hypoxia models could not completely simulate the status of OSAHS in human, such as lacking repeated episodes of upper airway obstruction during sleep. In our study, we used a well-established OSAHS animal model by injection gel to the soft palate to narrow the upper airway of rabbits. So, the status of apnoea or hypopnea in human with OSAHS was stimulated to the maximum limit. Utilizing our OSAHS animal model, we found the rate of neuron apoptosis in frontal cortex of group OSAHS was significantly higher than that of control group. The neuron apoptosis may result in neurocognitive impairment, although the apoptosis rate was very low. Researchers found chronic hypoxia of several hours daily could induce neuronal apoptosis and brain impairment. The number of cell apoptosis was less than that of acute hypoxia, but could result in brain cell damage and lead to neurocognitive injury further (36). The mechanism of neuron apoptosis during OSAHS probably due to intermittent hypoxia. Hypoxic injury is associated with enhanced release of glutamate, which mediated neurotoxicity (37). Oxidative stress induced by OSAHS increased generation of ROS. Free radicals of oxygen may mediate neuron apoptosis (38,39). Some reports indicated that cognitive disorder of OSAHS patients could be recovered by the CPAP (40,41). But, some researchers found the recovery of cognitive ability was not sensitive to the treatment after CPAP treatment (42,43). So, they believed that the reason of the cognitive impairment may be due to the permanent damage to the cortex caused by severe hypoxemia in the patients with OSAHS. Another study observed it could prevent or delay the cognitive disorder of the patients with OSAHS when intervened or treated at early stage of OSAHS (44). So, the study highlighted to treat OSAHS during early stage. MAD is an important method of treating mild or moderate OSAHS. The principle of the treatment is to advance the mandible which places the tongue away from the posterior pharyngeal wall, and increases the space of the oropharyngeal cavity. Thus, MAD therapy can effectively reduce snoring and correct the anoxia during sleeping, which increase the sleep quality and improve the quality of life (45). But, there is no report about effect on neurocognitive function of the patients with OSAHS early treated or prevented by MAD. This study showed the nerve cell apoptosis rate in frontal cortex of group MAD was less than that of group OSAHS, which indicated MAD could decrease the nerve cell apoptosis rate in frontal cortex in early treatment of OSAHS. The central cholinergic nerve system is widely involved in the process of learning and memory. The cholinergic neurons are mainly distributed in the brain area vulnerable to hypoxia-ischemia, such as striatum, hippocampus, and cortex et al (46). These structures and the pathway among them are important histophysiological foundation for learning and memory function. Ach is one of the important neurotransmitters of the central cholinergic nerve system, and participates in the important physiological activity of learning and memory function (47). Ach is synthesized by choline acetyl-transferase (ChAT), which is resolved and inactivation by AchE. AchE is the major bio-enzyme to catalyze Ach hydrolysis. It represents the function of the central cholinergic nerve system and capability of neurocognition. Therefore, AchE not only prevents the accumulation of Ach, but also maintains the sufficient choline to synthesize Ach. This ensures the coordination and unity of nerve excitation and inhibition. In this study, activity of AchE at frontal cortex of the rabbits with OSAHS was measured in order to study the damage of cholinergic neuro system due to the chronic intermittent hypoxia. We found activity of AchE in frontal cortex of group OSAHS was significantly decreased than that of control group, which suggested OSAHS could lead to reduction of the activity of AchE and damage the nerve cholinergic function (48). The results were consistent with previous studies, in which AchE activity decreased immediately in the model of hypoxic-ischemic injury (49,50). The decreased activity of AchE may be attributed to following reasons: First, cholinergic neurons in the cortex were impaired by repeatedly ischemia-reperfusion injury. With the level of Ach reduced, the activity of AchE decreased accordingly. Second, lipid peroxidation induced by OSAHS can change conformational state of AchE, and followed by altered physiologic functions of AchE (51). This study demonstrated that the activity of AchE in frontal cortex was significantly enhanced after early treatment of OSAHS by MAD. It is also found positive relationship between the activity of AchE and oxyhemoglobin saturation. These suggested that, by improving the oxygen saturation in blood, it could increase the activity of AchE, and lead to the high Ach in cortex. The mechanism underlying this event might be explained by reducing impairment of the tricarboxylic acid cycle and damage to the nerve cell in cortex. It should be noted that some limitations exist in this study. OSAHS patients may involve multiple sites, the animal model we used is by injection the gel only to a specific site of the soft palate. In addition, cognitive functions are generally tested on rat model by Morris water maze test, elevated plus maze test, visible platform test et al, but there is no suitable technique to assess the cognitive functions in rabbit. Furthermore, more clinical trials should be conducted to investigate the preventive effect of MAD on cognitive impairment inducing by OSAHS. Conclusion Taking together, our data demonstrate that hypoxia leads to increased neuron apoptosis and reduced activity of AchE in frontal cortex of OSAHS rabbits. Furthermore, we prove that MAD therapy for OSAHS may significantly reduce the neurons apoptosis and increase AchE activity in the brain. To our knowledge, this is the first original study providing direct evidence of histologic, pathophysiologic, and functional assessments performed in animals to demonstrate the brain injury caused by OSAHS and the therapeutic effect of MAD. 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Treatment of obstructive sleep apnoea–hypopnea syndrome by mandible advanced device reduced neuron apoptosis in frontal cortex of rabbits

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© The Author(s) 2017. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com
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Abstract

Summary Objective To investigate effects of mandible advanced device (MAD) therapy for obstructive sleep apnoea–hypopnea syndrome (OSAHS) on the neuron apoptosis and acetylcholine esterase activity in frontal cortex. Materials and methods Thirty male New Zealand white rabbits were randomly divided into three groups (n = 10 in each group): group OSAHS, group MAD, and control group. Hydrophilic polyacrylamide gel was injected into soft palate of the animals to induce OSAHS in group OSAHS and group MAD. The group MAD animals wore MAD to relief the obstructiveness. The control group was not given any treatment. Computed tomography (CT) examination of the upper airway and polysomnography (PSG) recordings were performed in supine position. All rabbits were induced to sleep in a supine position for 4 to 6 hours every day and were observed for consecutive 8 weeks. The frontal cortices of three groups were dissected and the neuron apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and flow cytometry. Acetylcholine esterase (AchE) activity in the frontal cortex was measured by spectrophotometry. Results The group OSAHS exhibited high neuron apoptosis rate and low AchE activity than those of group MAD and control group. The blood oxygen saturation was negatively correlated with neuronal apoptosis rate and positively correlated with AchE activity. Applying MAD in OSAHS animals significantly improve the neuronal damage and function deficits by apnoea–hypoxia caused by narrowed upper airway. Conclusion This study provided evidence that MAD therapy for OSAHS can significantly decrease neuronal apoptosis and increase AchE activity in the frontal cortex. Introduction Obstructive sleep apnoea-hypopnea syndrome (OSAHS) is characterized by recurrent complete or partial airway obstruction, which leads to inspiratory flow cessation (apnoea) or airflow limitation (hypopnea) accompanied by oxygen desaturation and arousal from sleep. OSAHS has been associated with cardiovascular, pulmonary, metabolic, and neural disturbance with multiple clinical symptoms (1). Accumulated evidence has shown that OSAHS is associated with damage of the nerve system (2,3). The brain, especially within white matter of the frontal cortex, is vulnerable to repetitive hypoxia/reoxygenation. The neurons of these regions are responsible to cognitive functions, including reward, attention, short-term memory tasks et al (4). Previous studies implicated that reactive oxygen species (ROS) generation and neuron apoptosis caused by intermittent hypoxia contributed to this cognitive deficits (5–7). However, little is known about the exact mechanism underlying cognitive impairment induced by OSAHS. Evidence both from animal and human studies suggest that central cholinergic systems, including acetylcholine (Ach), acetylcholine transferase (ChAT), acetylcholine esterase (AchE), and cholinergic receptors (8–11), are important to cognitive function. Among them, Ach is one of the major neurotransmitters and widely distributed in the central nervous systems. The synthesis and hydrolysis of Ach were considered be associated with emotion change, attention, executive function, and cognitive activities (12). The neurons in cholinergic basal forebrain synthetize substantial Ach, and then project it to the cortex. Recent studies have reported that decreased Ach release is correlated to memory impairment (13,14). In addition, blocking Ach receptor activity by muscarinic receptor agonists can enhance the animal performance on learning and memory tests (15,16). Therefore, Ach level is critical to cognitive functions. As the primary hydrolase of Ach, AchE regulates the Ach and terminates the signal transmission in the post-synaptic cleft. However, the relationship between AchE activity and OSAHS is still poorly understood. Approaches to prevent repetitive hypoxia/reoxygenation are important for preventing the cerebral hypoxic damage caused by OSAHS. The basic treatment principle of OSAHS is to open the obstructed or narrowed upper airway, and then to improve the oxyhemoglobin saturation. Therapies currently used include behavioural treatment (17), continuous positive airway pressure (CPAP) (18,19), mandible advanced devices (MAD) (20,21), and surgeries aiming to enlarge the upper airway, such as uvulopalatopharyngoplast (UPPP) and maxilla-mandibular advancement osteotomy (22). Most of OSAHS patients choose CPAP and MAD due to remarkable effectiveness in reducing symptoms, less risk, and relapse. CPAP treatment delayed cognitive impairment, which suggests the relationship between OSASH and neurocognitive (23). MAD is another effective therapy of OSAHS. It has many advantages such as unobtrusive, noiseless, convenient, and less costly. There is no evidence, however, about the treatment effects of MAD in the frontal cortex. This study was designed to investigate the neuron cell apoptosis and the activity of Ach esterase in frontal cortex using our established OSAHS animal models (24), and to further evaluate the effects of MAD treatment on these events to uncover the mechanism underlying cognitive impairment by OSAHS and hoping to provide evidences for clinical treatment. Materials and methods Experimental animals Thirty male 6-month-old New Zealand white rabbits (3 to 3.5 kg) were selected from animal center of Hebei Medical University. Care and experimental protocols were approved by local Animal Care and Use Committees (Certificate No. SCXK (J) 20012-1-003). Food and water were available ad lib. Experimental animal model The animals were randomly divided into three groups: group OSAHS, group MAD, and control group, 10 animals for each group. The size of the experimental groups was decided based on the results from our previous studies and power analysis, which allowed us to detect a 20 per cent difference in the groups with a high likelihood of statistical significance. The experimental animal models were established as previously described (24,25). In brief, the animals of group OSAHS and MAD were restrained in a supine position under general anaesthesia with 1 per cent pentobarbital sodium (20 mg/kg) through auricular vein injection. Gel injection was utilized to develop OSAHS symptoms. Two millilitres of prepared gel mixture (containing 2-g hydrophilic polyacrylamide gel, 20 mg of gentamicin dissolved in saline) was injected via the submucosa muscular layer at the centre of the soft palate, approximately 1.5 cm away from the junction of the hard and soft palates. The MAD group wore MAD to alleviate the OSAHS symptoms. Animals in the control group were not given any treatment. Spiral computed tomography scanning Spiral computed tomography (CT) scanning was utilized to evaluate the upper airway obstruction by capturing the images of airway structure from the cranial crest to the tracheal opening site during sleep (GE Healthcare Technologies, Waukesha, Wisconsin, USA). Reconstruction software was used for axial and sagittal structure of the airway. Cross-section areas and sagittal diameters were measured, from the top level of soft palate to level of 1/4, 1/2, 3/4 in the posterior areas. Polysomnography Polysomnography (PSG, Rembrandt Embla Polysomnography System, Reykjavik, Iceland) was used to monitor the sleep parameters. The rabbits were equipped with surface electrodes taped on the skull, face, and chest for monitoring the electroencephalogram, right and left electrooculogram, and respiration. Nasal air flow was monitored through a nasal pressure transducer. Respiratory movements were reported by respiratory inductance plethysmography (chested and abdominal bands). Blood oxygen saturation (SaO2) was measured using an ear pulse oximeter. PSG recordings were conducted during sleeping from 09:00 to 11:30 or from 14:00 to 16:30 (26). Hypopnea was defined as a reduction between 20 per cent and 50 per cent in nasal flow for at least two breaths, which was associated with an arousal. Apnoea was defined by an absence of airflow at the nose and mouth for longer than two breaths (27). 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 five (28). MAD fabrication Impressions were then taken in maxilla of group MAD after OSAHS model is successfully established. MAD was fabricated from the casts with a 30-degree inclined plane to the long axis of upper incisor crowns using self-curing composite resin, and bonded to the upper incisors with glass ionomer. The mandible was guided forward 3–4 mm. Spiral CT scanning and PSG were used to evaluate the effectiveness of MAD treatment following the protocols described above. All the rabbits were restrained in a supine position for 4 hours per day while sleeping and were observed for consecutive 8 weeks. Basic health data including behaviour and body weight was recorded at the time point of 2 weeks and 8 weeks. Histological analyses After 8 weeks, the animals were anaesthetized with sodium pentobarbitone (20 mg/kg, intravenously). Tissues harvested from frontal cortex were dissected, fixed in 10 per cent buffered formalin for 24 hours, and embedded in paraffin. Sections were prepared for hematoxylin and eosin (H&E) for microscopic observation (Olympus, Japan). Flow cytometry A part of frontal cortex was dissected and fixed in 70 per cent cryopreservation alcohol for 24 hours. Single-cell suspension was prepared to detect the rate of apoptosis by flow cytometry. The suspension was centrifuged at 800 rpm for 2 minutes at 4°C. After removing the supernatant, the cells were incubated with 1-ml propidium iodide dye (PI: 0.05 µg/ml, Triton-X 100: 2 ml, sodium citrate: 2 mg, saline: 130 ml, double distilled water: 70 ml). The histograms and the rate of apoptosis were analyzed by flow cytometry (Beckman Coulter, USA). TUNEL assays Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Kits (Roche, Germany) was used to determine brain cell apoptosis. Briefly, sections were dewaxed and washed with phosphate buffered solution, PBS. Sections were treated with 3 per cent H2O2 in methanol for 10 minutes and incubated with proteinase K for 15 minutes. After washing with PBS for two times, sections were treated with 50-µl TUNEL reaction mixture for 1 hour at 37°C in a humidified atmosphere in the dark. Then rinsed three times with PBS. A 50 µl converter-POB was added to the sections. Slides were incubated in a humidified chamber for 30 minutes at 37°C. Diaminobenzidine tetrahydrochloride (DAB) was added for 10 minutes at room temperature. Positive cells (brown colouring) indicated DNA fragmentation during cell apoptosis. Samples were counterstained with hematoxylin and observed by microscopy at 400 magnification times (Olympus, Japan). The sections treated with DNase I were defined as positive controls, and those without TdT were defined as the negative controls. Measurement of AchE activity The frontal cortex was dissected, rinsed in saline, and dried on filter paper. Tissues were homogenized at 4°C for 1 minute in buffer (0.01 mol sucrose, 0.01 mol Tris-HCL, 0.01 ml EDTA, PH = 7.4). The homogenate was centrifuged at 3000 r at 4°C for 15 minutes. The supernatant was extracted and the AchE activity was quantified by spectrophotometry according to the manual for measurement of AchE activity (Nanjing Jiancheng biological engineering institute, China). Statistical analysis SPSS 22.0 statistical software package was used for data analysis (SPSS, Chicago, USA). All data were expressed by mean ± SD. The normality test and Levene’s variance homogeneity test showed the data were normally distributed and the variance was homogeneous. Analysis of variance (ANOVA) was used to compare the difference of three groups followed by least significant difference when appropriate. Correlation analysis was used between oxyhemoglobin saturation and apoptosis of neurons in the brain frontal cortex, or between oxyhemoglobin saturation and AchE activity. Significance level was set at P <0.05. Results Gel-injection induced OSAHS-like symptoms, and partly alleviated by MAD treatment To investigate the OSAHS and possible effects of MAD in treating OSAHS, we took advantage of previously developed gel-induced OSAHS animal model (24). In group OSAHS, it was shown that snoring and apnoea progressed with time during sleep, and intermittent awakenings and lips cyanosis were observed. Sleepiness was most obvious during daytime on tenth day. In group MAD, the aforementioned symptoms were relieved, which four animals exhibited regular rhythm of breath without snoring, and six animals showed rare snoring but no apnoea. This suggested that gel injection successfully mimicked OSAHS by inducing apnoea-like phenomena, which can be partly relieved by MAD. In control group, the breathing was normal without any snore or apnoea. There was no significant changes of body weight between the three groups during 8 weeks of experiment (Table 1). Table 1. Body weight changes of experimental animals. MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome. Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 Body weight (kg)  Baseline 3.30 ± 0.17 3.33 ± 0.12 3.31 ± 0.15 0.760 0.909 0.849  2 weeks 3.44 ± 0.15 3.52 ± 0.14 3.56 ± 0.13 0.297 0.152 0.675  8 weeks 3.70 ± 0.11 3.72 ± 0.10 3.79 ± 0.15 0.706 0.151 0.277 Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 Body weight (kg)  Baseline 3.30 ± 0.17 3.33 ± 0.12 3.31 ± 0.15 0.760 0.909 0.849  2 weeks 3.44 ± 0.15 3.52 ± 0.14 3.56 ± 0.13 0.297 0.152 0.675  8 weeks 3.70 ± 0.11 3.72 ± 0.10 3.79 ± 0.15 0.706 0.151 0.277 View Large Table 1. Body weight changes of experimental animals. MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome. Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 Body weight (kg)  Baseline 3.30 ± 0.17 3.33 ± 0.12 3.31 ± 0.15 0.760 0.909 0.849  2 weeks 3.44 ± 0.15 3.52 ± 0.14 3.56 ± 0.13 0.297 0.152 0.675  8 weeks 3.70 ± 0.11 3.72 ± 0.10 3.79 ± 0.15 0.706 0.151 0.277 Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 Body weight (kg)  Baseline 3.30 ± 0.17 3.33 ± 0.12 3.31 ± 0.15 0.760 0.909 0.849  2 weeks 3.44 ± 0.15 3.52 ± 0.14 3.56 ± 0.13 0.297 0.152 0.675  8 weeks 3.70 ± 0.11 3.72 ± 0.10 3.79 ± 0.15 0.706 0.151 0.277 View Large MAD therapy opened the upper airways to improve respiratory function and apnoea–hypoxia It is known that respiratory airway obstruction is the characteristic change in OSAHS. In our study, spiral CT scan had confirmed that the alterations of upper airway spaces by measuring the sagittal and the cross-sectional areas in this study (Table 2). Both sagittal spaces and cross-sectional areas of group OSAHS were significantly decreased at points 1/4, 1/2, and 3/4 compared to that of the control group and the group MAD (P < 0.05; Figure 1A and 1B). Comparing to group OSAHS, the reduced retropalatal spaces were significantly widened by MAD through advancing the mandible (P < 0.05), which showed no significant difference between the group MAD and the controls (P > 0.05). Therefore, the consequent respiratory functions changed accordingly. SaO2 and AHI were measured and scored for all animals. Significant increase of AHI and decrease of SaO2 were observed in the group OSAHS compared with those of the group MAD and control group (P < 0.05). No statistically significant differences were found in AHI and SaO2 levels between group MAD and control group (P > 0.05). Also, we observed nasal airflow decrease with an associated increase in respiratory effort in group OSAHS, which was consistent to our previous studies (Table 3). Table 2. The retropalatal upper airway spaces of three groups. MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome. Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 Sagittal space (mm2)  Upper point 4.53 ± 0.20 4.47 ± 0.25 4.52 ± 0.29 0.676 1.000 0.676  1/4 point 2.78 ± 0.30 3.67 ± 0.37 3.78 ± 0.39 0.001* 0.001* 0.578  1/2 point 1.60 ± 0.41 2.71 ± 0.46 2.80 ± 0.45 0.000* 0.000* 0.663  3/4 point 1.77 ± 0.35 2.76 ± 0.34 2.83 ± 0.36 0.000* 0.000* 0.751  Lower point 3.56 ± 0.42 3.82 ± 0.39 3.85 ± 0.28 0.189 0.890 0.236 Cross-sectional areas (mm2)  Upper point 22.79 ± 0.49 22.87 ± 0.20 23.08 ± 0.36 0.720 0.201 0.347  1/4 point 17.47 ± 1.22 22.41 ± 0.72 22.52 ± 1.59 0.000* 0.000* 0.890  1/2 point 13.31 ± 1.40 18.95 ± 1.96 20.15 ± 1.49 0.000* 0.000* 0.182  3/4 point 14.18 ± 2.25 20.24 ± 2.94 21.04 ± 2.55 0.000* 0.000* 0.588  Lower point 26.24 ± 2.74 27.32 ± 2.93 29.10 ± 2.92 0.679 0.147 0.286 Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 Sagittal space (mm2)  Upper point 4.53 ± 0.20 4.47 ± 0.25 4.52 ± 0.29 0.676 1.000 0.676  1/4 point 2.78 ± 0.30 3.67 ± 0.37 3.78 ± 0.39 0.001* 0.001* 0.578  1/2 point 1.60 ± 0.41 2.71 ± 0.46 2.80 ± 0.45 0.000* 0.000* 0.663  3/4 point 1.77 ± 0.35 2.76 ± 0.34 2.83 ± 0.36 0.000* 0.000* 0.751  Lower point 3.56 ± 0.42 3.82 ± 0.39 3.85 ± 0.28 0.189 0.890 0.236 Cross-sectional areas (mm2)  Upper point 22.79 ± 0.49 22.87 ± 0.20 23.08 ± 0.36 0.720 0.201 0.347  1/4 point 17.47 ± 1.22 22.41 ± 0.72 22.52 ± 1.59 0.000* 0.000* 0.890  1/2 point 13.31 ± 1.40 18.95 ± 1.96 20.15 ± 1.49 0.000* 0.000* 0.182  3/4 point 14.18 ± 2.25 20.24 ± 2.94 21.04 ± 2.55 0.000* 0.000* 0.588  Lower point 26.24 ± 2.74 27.32 ± 2.93 29.10 ± 2.92 0.679 0.147 0.286 *P < 0.01 View Large Table 2. The retropalatal upper airway spaces of three groups. MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome. Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 Sagittal space (mm2)  Upper point 4.53 ± 0.20 4.47 ± 0.25 4.52 ± 0.29 0.676 1.000 0.676  1/4 point 2.78 ± 0.30 3.67 ± 0.37 3.78 ± 0.39 0.001* 0.001* 0.578  1/2 point 1.60 ± 0.41 2.71 ± 0.46 2.80 ± 0.45 0.000* 0.000* 0.663  3/4 point 1.77 ± 0.35 2.76 ± 0.34 2.83 ± 0.36 0.000* 0.000* 0.751  Lower point 3.56 ± 0.42 3.82 ± 0.39 3.85 ± 0.28 0.189 0.890 0.236 Cross-sectional areas (mm2)  Upper point 22.79 ± 0.49 22.87 ± 0.20 23.08 ± 0.36 0.720 0.201 0.347  1/4 point 17.47 ± 1.22 22.41 ± 0.72 22.52 ± 1.59 0.000* 0.000* 0.890  1/2 point 13.31 ± 1.40 18.95 ± 1.96 20.15 ± 1.49 0.000* 0.000* 0.182  3/4 point 14.18 ± 2.25 20.24 ± 2.94 21.04 ± 2.55 0.000* 0.000* 0.588  Lower point 26.24 ± 2.74 27.32 ± 2.93 29.10 ± 2.92 0.679 0.147 0.286 Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 Sagittal space (mm2)  Upper point 4.53 ± 0.20 4.47 ± 0.25 4.52 ± 0.29 0.676 1.000 0.676  1/4 point 2.78 ± 0.30 3.67 ± 0.37 3.78 ± 0.39 0.001* 0.001* 0.578  1/2 point 1.60 ± 0.41 2.71 ± 0.46 2.80 ± 0.45 0.000* 0.000* 0.663  3/4 point 1.77 ± 0.35 2.76 ± 0.34 2.83 ± 0.36 0.000* 0.000* 0.751  Lower point 3.56 ± 0.42 3.82 ± 0.39 3.85 ± 0.28 0.189 0.890 0.236 Cross-sectional areas (mm2)  Upper point 22.79 ± 0.49 22.87 ± 0.20 23.08 ± 0.36 0.720 0.201 0.347  1/4 point 17.47 ± 1.22 22.41 ± 0.72 22.52 ± 1.59 0.000* 0.000* 0.890  1/2 point 13.31 ± 1.40 18.95 ± 1.96 20.15 ± 1.49 0.000* 0.000* 0.182  3/4 point 14.18 ± 2.25 20.24 ± 2.94 21.04 ± 2.55 0.000* 0.000* 0.588  Lower point 26.24 ± 2.74 27.32 ± 2.93 29.10 ± 2.92 0.679 0.147 0.286 *P < 0.01 View Large Table 3. Breathing parameters of three groups. AHI, apnoea–hypopnea index; MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome; SaO2, blood oxygen saturation. Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 AHI 30.85 ± 2.71 3.41 ± 0.22 2.78 ± 0.27 0.000* 0.000* 0.497 Average SaO2 (%) 80.23 ± 3.50 89.8 ± 1.33 91.8 ± 1.45 0.000* 0.000* 0.059 Min.SaO2 (%) 58.12 ± 3.02 86.0 ± 2.27 88.3 ± 1.56 0.000* 0.000* 0.111 Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 AHI 30.85 ± 2.71 3.41 ± 0.22 2.78 ± 0.27 0.000* 0.000* 0.497 Average SaO2 (%) 80.23 ± 3.50 89.8 ± 1.33 91.8 ± 1.45 0.000* 0.000* 0.059 Min.SaO2 (%) 58.12 ± 3.02 86.0 ± 2.27 88.3 ± 1.56 0.000* 0.000* 0.111 *P < 0.01 View Large Table 3. Breathing parameters of three groups. AHI, apnoea–hypopnea index; MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome; SaO2, blood oxygen saturation. Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 AHI 30.85 ± 2.71 3.41 ± 0.22 2.78 ± 0.27 0.000* 0.000* 0.497 Average SaO2 (%) 80.23 ± 3.50 89.8 ± 1.33 91.8 ± 1.45 0.000* 0.000* 0.059 Min.SaO2 (%) 58.12 ± 3.02 86.0 ± 2.27 88.3 ± 1.56 0.000* 0.000* 0.111 Group OSAHS Group MAD Control group P n = 10 n = 10 n = 10 1–2 1–3 2–3 AHI 30.85 ± 2.71 3.41 ± 0.22 2.78 ± 0.27 0.000* 0.000* 0.497 Average SaO2 (%) 80.23 ± 3.50 89.8 ± 1.33 91.8 ± 1.45 0.000* 0.000* 0.059 Min.SaO2 (%) 58.12 ± 3.02 86.0 ± 2.27 88.3 ± 1.56 0.000* 0.000* 0.111 *P < 0.01 View Large Figure 1. View largeDownload slide Spiral CT scan. A. Measurement of the sagittal diameters of the upper airway in supine position at five levels labelled. B. CT image of the cross-sectional area of the upper airway in supine position indicated by arrow. CT, computed tomography. Figure 1. View largeDownload slide Spiral CT scan. A. Measurement of the sagittal diameters of the upper airway in supine position at five levels labelled. B. CT image of the cross-sectional area of the upper airway in supine position indicated by arrow. CT, computed tomography. Frontal cortex neuronal apoptosis associated with OSAHS can be rescued by MAD treatment To explore the pathophysiological damage to the brain caused by OSAHS, histological examinations were performed (Figure 2A). In frontal cortex of group OSAHS, increased number of neurons appeared shrunken, hyperchromatic, and karyopyknosis (H&E staining). However, these were not found in group MAD and the control group. Figure 2. View largeDownload slide Histological examinations of frontal cortex neurons. A. Hematoxylin and eosin (H&E) staining. Representative apoptotic cells with karyopyknosis and hyperchromatic staining indicated with black arrow. B. TUNEL staining. The apoptotic cells were specifically stained in brown indicated by red arrows, and normal neurons were counterstained in blue indicated by black arrows. MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling. Figure 2. View largeDownload slide Histological examinations of frontal cortex neurons. A. Hematoxylin and eosin (H&E) staining. Representative apoptotic cells with karyopyknosis and hyperchromatic staining indicated with black arrow. B. TUNEL staining. The apoptotic cells were specifically stained in brown indicated by red arrows, and normal neurons were counterstained in blue indicated by black arrows. MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling. We speculated that these neuronal changes indicated possible cell death due to hypoxia. To verify this, we took advantage of TUNEL assay to investigate neuronal apoptosis in the rabbit frontal cortex regions. The apoptotic cells, specifically stained in brown, were less in group MAD and control group than that of the group OSAHS (Figure 2B). To further quantify the apoptotic cells from different groups of experiment, flow cytometry was utilized to analyze the cell viability using propidium iodide (PI). PI, permeable to the non-viable cells, bonded with double stranded DNA of dead cells. The histograms of cell counts of PI positive cells by flow cytometry in three groups were shown in Figure 3. Significant difference was observed among three groups by one-way ANOVA (F = 164.2, P = 0.000). The apoptosis rate of group OSAHS was 7.403 ± 1.256 per cent, which was higher than that of group MAD (2.259 ± 0.319%, P = 0.000) and control group (1.943 ± 0.242%, P = 0.000). The apoptosis rate of group MAD was slightly higher than the control group, but without significant different (P = 0.358; Figure 4). These results suggested that OSAHS induced frontal cortical neuron apoptosis, and treating OSAHS by MAD reduced the cell death. Figure 3. View largeDownload slide Flow cytometry histograms of apoptosis cells stained by PI. Sub-G1 peak (black arrow showed) as an indicative of apoptotic cells was induced in the group OSAHS, but not in group MAD and control group. MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome; PI, propidium iodide. Figure 3. View largeDownload slide Flow cytometry histograms of apoptosis cells stained by PI. Sub-G1 peak (black arrow showed) as an indicative of apoptotic cells was induced in the group OSAHS, but not in group MAD and control group. MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome; PI, propidium iodide. Figure 4. View largeDownload slide The oxygen saturation, AchE activity, and neuronal apoptosis rates were shown in A–C, respectively. The results expressed in mean ± SD. Statistically significant differences are indicated by asterisk (LSD test, P < 0.05). The correlation between oxyhemoglobin saturation and apoptosis of neurons showed in D. The correlation between oxyhemoglobin saturation and apoptosis of neurons showed in E. Significance level was set at P < 0.05. AchE, acetylcholine esterase; MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome; SaO2, blood oxygen saturation; SD, standard deviation. Figure 4. View largeDownload slide The oxygen saturation, AchE activity, and neuronal apoptosis rates were shown in A–C, respectively. The results expressed in mean ± SD. Statistically significant differences are indicated by asterisk (LSD test, P < 0.05). The correlation between oxyhemoglobin saturation and apoptosis of neurons showed in D. The correlation between oxyhemoglobin saturation and apoptosis of neurons showed in E. Significance level was set at P < 0.05. AchE, acetylcholine esterase; MAD, mandibular advancement device; OSAHS, obstructive sleep apnoea–hypopnea syndrome; SaO2, blood oxygen saturation; SD, standard deviation. Frontal cortex neuronal impairment linked with apnoea–hypoxia cause by OSAHS Sleep apnoea caused hypoxia-ischemia, which induced cell death and further impaired neuronal functions in frontal cortex. AchE is the hydrolase catalyzing Ach and regulating the neuronal functions. To this end, we measured the AchE activity. The AchE activity of frontal cortex was 0.373 ± 0.042 U/mgprot in group OSAHS, which was lower than that in group MAD (0.450 ± 0.065 U/mgprot, P = 0.002) and in the control group (0.475 ± 0.050 U/mgprot, P = 0.000). However, there was no significant different between group MAD and the control group (P = 0.277). To examine the correlations between the frontal neuronal damages with hypoxia, we analyzed the association between neuronal apoptosis/AchE activity with blood oxygen saturation. The correlation coefficient of the neuronal apoptosis rate and blood oxygen saturation was −0.820, P <0.01. And the correlation coefficient of AchE activity and oxygen saturation was 0.442, P <0.05 (Figure 4). This suggested that blood oxygen saturation was negatively correlated with neuronal apoptosis rate and positively correlated with AchE activity. Discussion The major clinical presentations of OSAHS are frequently intermittent hypoxia during sleeping. It was reported that OSAHS triggers pathophysiological changes in multiple systems, including cardiovascular system, nerve system, genitourinary system, respiratory system, and muscular system et al (29). Experimental studies were focusing on apoptosis induced by hypoxia, which was considered to be one of the pathophysiological basis of OSHAS. In some studies using OSAHS rat model, it had been shown that chronic intermittent hypoxia increased TUNEL-positive cardiovascular myocytes, and altered expression of apoptosis genes in vascular endothelial cells. Loss of cardiomyocytes due to apoptosis can result in cardiac dysfunction and promoting development of atherosclerosis (30–32). Obstructive sleep apnoea also associated with chronic kidney disease, and the renal tubular endothelial apoptosis contributed to the pathogenesis of the hypoxia-induced renal injury (33). Recently, accumulating evidences have indicated that cognitive functions and performance deteriorate in individuals associate with OSAHS. Recurrent apnoea induces neuronal apoptosis in the guinea pig forebrain (34). Diffusion tensor imaging showed a wide range of white matter abnormalities in clinical trials, which could be associated with specific cognitive deficits (35). Most of these studies were using animal models of intermittent hypoxia induced by changing the oxygen desaturation. However, these hypoxia models could not completely simulate the status of OSAHS in human, such as lacking repeated episodes of upper airway obstruction during sleep. In our study, we used a well-established OSAHS animal model by injection gel to the soft palate to narrow the upper airway of rabbits. So, the status of apnoea or hypopnea in human with OSAHS was stimulated to the maximum limit. Utilizing our OSAHS animal model, we found the rate of neuron apoptosis in frontal cortex of group OSAHS was significantly higher than that of control group. The neuron apoptosis may result in neurocognitive impairment, although the apoptosis rate was very low. Researchers found chronic hypoxia of several hours daily could induce neuronal apoptosis and brain impairment. The number of cell apoptosis was less than that of acute hypoxia, but could result in brain cell damage and lead to neurocognitive injury further (36). The mechanism of neuron apoptosis during OSAHS probably due to intermittent hypoxia. Hypoxic injury is associated with enhanced release of glutamate, which mediated neurotoxicity (37). Oxidative stress induced by OSAHS increased generation of ROS. Free radicals of oxygen may mediate neuron apoptosis (38,39). Some reports indicated that cognitive disorder of OSAHS patients could be recovered by the CPAP (40,41). But, some researchers found the recovery of cognitive ability was not sensitive to the treatment after CPAP treatment (42,43). So, they believed that the reason of the cognitive impairment may be due to the permanent damage to the cortex caused by severe hypoxemia in the patients with OSAHS. Another study observed it could prevent or delay the cognitive disorder of the patients with OSAHS when intervened or treated at early stage of OSAHS (44). So, the study highlighted to treat OSAHS during early stage. MAD is an important method of treating mild or moderate OSAHS. The principle of the treatment is to advance the mandible which places the tongue away from the posterior pharyngeal wall, and increases the space of the oropharyngeal cavity. Thus, MAD therapy can effectively reduce snoring and correct the anoxia during sleeping, which increase the sleep quality and improve the quality of life (45). But, there is no report about effect on neurocognitive function of the patients with OSAHS early treated or prevented by MAD. This study showed the nerve cell apoptosis rate in frontal cortex of group MAD was less than that of group OSAHS, which indicated MAD could decrease the nerve cell apoptosis rate in frontal cortex in early treatment of OSAHS. The central cholinergic nerve system is widely involved in the process of learning and memory. The cholinergic neurons are mainly distributed in the brain area vulnerable to hypoxia-ischemia, such as striatum, hippocampus, and cortex et al (46). These structures and the pathway among them are important histophysiological foundation for learning and memory function. Ach is one of the important neurotransmitters of the central cholinergic nerve system, and participates in the important physiological activity of learning and memory function (47). Ach is synthesized by choline acetyl-transferase (ChAT), which is resolved and inactivation by AchE. AchE is the major bio-enzyme to catalyze Ach hydrolysis. It represents the function of the central cholinergic nerve system and capability of neurocognition. Therefore, AchE not only prevents the accumulation of Ach, but also maintains the sufficient choline to synthesize Ach. This ensures the coordination and unity of nerve excitation and inhibition. In this study, activity of AchE at frontal cortex of the rabbits with OSAHS was measured in order to study the damage of cholinergic neuro system due to the chronic intermittent hypoxia. We found activity of AchE in frontal cortex of group OSAHS was significantly decreased than that of control group, which suggested OSAHS could lead to reduction of the activity of AchE and damage the nerve cholinergic function (48). The results were consistent with previous studies, in which AchE activity decreased immediately in the model of hypoxic-ischemic injury (49,50). The decreased activity of AchE may be attributed to following reasons: First, cholinergic neurons in the cortex were impaired by repeatedly ischemia-reperfusion injury. With the level of Ach reduced, the activity of AchE decreased accordingly. Second, lipid peroxidation induced by OSAHS can change conformational state of AchE, and followed by altered physiologic functions of AchE (51). This study demonstrated that the activity of AchE in frontal cortex was significantly enhanced after early treatment of OSAHS by MAD. It is also found positive relationship between the activity of AchE and oxyhemoglobin saturation. These suggested that, by improving the oxygen saturation in blood, it could increase the activity of AchE, and lead to the high Ach in cortex. The mechanism underlying this event might be explained by reducing impairment of the tricarboxylic acid cycle and damage to the nerve cell in cortex. It should be noted that some limitations exist in this study. OSAHS patients may involve multiple sites, the animal model we used is by injection the gel only to a specific site of the soft palate. In addition, cognitive functions are generally tested on rat model by Morris water maze test, elevated plus maze test, visible platform test et al, but there is no suitable technique to assess the cognitive functions in rabbit. Furthermore, more clinical trials should be conducted to investigate the preventive effect of MAD on cognitive impairment inducing by OSAHS. Conclusion Taking together, our data demonstrate that hypoxia leads to increased neuron apoptosis and reduced activity of AchE in frontal cortex of OSAHS rabbits. Furthermore, we prove that MAD therapy for OSAHS may significantly reduce the neurons apoptosis and increase AchE activity in the brain. To our knowledge, this is the first original study providing direct evidence of histologic, pathophysiologic, and functional assessments performed in animals to demonstrate the brain injury caused by OSAHS and the therapeutic effect of MAD. 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The European Journal of OrthodonticsOxford University Press

Published: Sep 23, 2017

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