The Animal Experiment And Clinical Application Of OSAHS Treated By Mandibular Advancment Device

Objective:Obstructive sleep apnea-hypopnea syndrome (OSAHS) is a common chronic sleep disorder. There was high (Body mass index) BMI in patients with OSAHS. The upper airway was repetitive obstruction because of the mandibular retraction, muscle relaxation of the upper airway and the change of hyoid position, which causes apnea, hypoxia, drowsiness in daytime and results in cardiovascular complications even multiple organs damaged, and it affects the quality of patient life seriously. It became a hotspot of medical multidisciplinary research nowdays.OSAHS is associated with great harmfulness and morbidity. In recent years, sleep medicine has been concerned by many scholars and become the focus of multiple disciplines such as respiratory physicians, otorhinolaryngo-logy, stomatology, neurology, cardiology and paediatrics. It becames a new interdisciplinary subject. Medical workers have payed more and more attention to the treatment measures of OSAHS. So far, the treatment measures of OSAHS include behavioral therapy (weight loss, lateral position sleep, etc.), nasal continuous positive airway pressure (CPAP), oral appliance, surgical treatment etc. As a' non-invasive therapy, CPAP is an effective treatment. While there are some shortcomings:some patients feel uncomfortable and inconvenient during used it. Therefore a large part of the patient can't tolerate this method and give it up. The effect of surgical treatment is unobvious, the high risk and easily relapse. As a result, the orthodontic treatment is an important part of treating OSAHS. The mandibular advancement device (MAD) is gradually accepted and applied in the clinical as its advantages such as low cost, no trauma, good effect and easy acceptance. The rabbits model of OSAHS were established in this study, the influence of OSAHS on ET-1,AngⅡin peripheral blood and the ET expression in heart tissue was researched, the effect of OSAHS in cardiac apex structure and the capillary within myocardial was observed. By the MAD treatment, the influence in ET-1 and AngⅡexpression in peripheral blood and the ET-1 expression in heart tissue was studied, the changes of myocardial and the capillary within myocardia were explored. All data of this study provide theoretical basis for using MAD to prevent clinical heart disease which caused by OSAHS.Through the rabbit model of OSAHS established, the neurons in frontal lobe cortex of rabbits and the acivity of AchE was observed. After OSAHS treated by using MAD, the changes of the neurons in frontal lobe cortex of rabbits and the acivity of AchE were studied and providing laboratory data for clinical research on cognition impaired induced by OSAHS.This study intended to provide the mandibular advancement device (MAD) for clinical application which could make mandible action. By using the active MAD to treat OSAHS and compare the changes of upper airway, polysomnography, Epworth, ET-1,AngⅡin peripheral blood and the heart rate before and after treatment were checked and the efficacy of MAD to treat moderate and severe OSAHS was evaluated.Method:1 Model of obstructive sleep apnea hypoxia syndrome (OSAHS) was establishedThirty rabbits were divided into two groups at random,15 rabbits were of OSAHS group and 15 rabbits were of control group.The examinations before model established were carried on.CT examination was performed through the upper airway in supine position, compared cross section areas and sagittal diameters of the upper airway in soft palate 1/4,1/2,3/4 and tip of the two groups.Arterial blood gas analysis was done for two groups, blood oxygen saturation, oxygen partial pressure, partial pressure of carbon dioxide and pH were measured. Histological change of injection site was observed by light microscope. There was no difference between two groups by the upper airway CT in supine position and arterial blood gas analysis. Animals of group OSAHS received 1% sodium pentobarbital injection into conducting general anesthesia and 2ml mixed colloid was injected into muscularis mucosa of soft palate at the point of 0.5cm far from the part of soft-hard palate. The gel was stopped injecting until the subjects showed obvious snoring and interrupted apnea. There was no any treatment for the normal control group.Sleeping situation was observed for 3 groups, all rabbits were given 10% chloral hydrate orally 5-6ml/kg. The subjects sleeped 4-6 hours in supine position every morning for 8 weeks.The examinations after modeling were carried on:The upper airway CT was examed after models established for two groups.Arterial blood gas analysis of two groups was checked.Histological change of injection site was observed by light microscope.The criteria of model established was identified:(1) Clinical snoring or apnea occured when sleeping, AHI>5/h. (2) Narrow upper airway. (3) Arterial oxygen saturation decreased to 4% when apnea. (4) Stable injection in soft palate without inflammation and diffusion.All data were statistical analyzed by SPSS 13.0, significance test was P< 0.05.2 The observation of OSAHS rabbit treated by MAD2.1 Animal groups were comp6sed of 30 male rabbits with 6 month-old, were divided into three groups at random. They were OSAHS group, MAD group and control group and 10 rabbits each.2.2 Twenty rabbits were injected by 2ml mixed colloid into muscularis mucosa of soft palate to establishe OSAHS animal models and then devided into two groups (OSAHS group and MAD group) of 10 rabbits each. Group MAD was worn appliance and group OSAHS did not been worn. The specific tray was made by two-layer wax slice and anhydrite of lower-upper dentin impression of MAD group was obtained. Maxillary inclined plane appliance was made of self-freezed composite resin with a 30 angle to the upper incision. Mandible was pulled forward 3-4 mm until the anterior teeth incision was in the state of crossbite. MAD was adhereded with 3M Glass Ionomer Luting Cement on the upper anterior teeth.2.3 The upper airway CT of three groups was taken in anesthesia state.2.4 Sleeping situation was observed, every morning all animals of 3 group were given 10% chloral hydrate through orally 5-6ml/kg. The subjects sleeped 4-6 hours in supine position every day for 8 weeks.2.5 Blood gas analysis of 3 groups was performed, blood was drawn from the ear arterial of the animal subjects to do blood gas analysis (including blood oxygen saturation, oxygen partial pressure partial pressure of carbon dioxide and pH) while they were in sleep apnea.2.6 Histological change of injection site of MAD was observed by light microscope.2.7 The criteria of model established was identified:(1) The MAD group could accommodate and could normally take food. (2)The snoring or apnea disappeared with MAD when they were sleeping. (3)There was no obstruction and narrow upper airway. (4) Blood gas analysis showed arterial oxygen saturation increased. (5) Stable injection in soft palate was free of inflammation and diffusion.2.8 All the data were statistic analyzed by SPSS 13.0, significance test was P <0.05.3 The effect of heart injury of OSAHS rabbits treated by MAD3.1 Animal groups and sleeping observation were the same as the second part.3.2 ELISA determination of ET-1 and AngⅡwas performed after 8 weeks of animal model established,1% sodium pentobarbital was injected into all animals of 3 groups. Two months after experiment,2ml blood drawn from external jugular vein was poured immediately into a one-off blood tube, mixed at 4℃, and centrifuged at 3000 rpm for 10 minutes. The plasma was collected and preserved in EP tube at-80℃. The level of ET-Ⅰand AT II was measured by ELISE. 3.3 The expression of ET-1mRNA in heart tissue was detected by RT-PCR.3.4 The parts of cardiac apex tissue was fixed by 10% formalin and stained with hematoxylin and eosin (HE staining). Changes were observed after gel-injection under optical microscope.3.5 Myocardium (1X1 X3mm3) was obtained from cardiac apex and fixed in the 4% cryopreservation glutaraldehyde solution and then was put at 4℃. By means of routine methods, Epon812 embedding and observed under Hitachi Hu-12 A transmission electron microscope (TEM).3.6 SPSS13.0 soft ware was used to analyze the data. The test standard was a=0.05.4 The treatment of brain injured of OSAHS rabbits by MAD4.1 Animal groups and sleeping observation were the same as the second part.4.2 Specimen obtained at the 8th week after the model established,1% sodium pentobarbital was injected into all animals of 3 groups and the brain completely was obtained.4.3 The parts of brain was fixed with 10% formalin and stained with hematoxylin and eosin (HE staining). Changes were observed after gel-injection under optical microscope.4.4 The part of frontal lobe cortex was fixed with 10% formalin for 24 hours, then conducted conventional paraffin embedding. The frontal lobe cortex in neurons apoptosis was observed by TUNEL staining.4.5 The part of right frontal lobe cortex'section was fixed with 70% cryopreservation alcohol for 24 hours. Single-celled suspension liquid was prepared to detect the rate of apoptosis by FCM.4.6 The part of frontal lobe cortex was weighed, put in homogenate medium equivalent to 9 times of organization, then fully mixed at 4℃and isolated at 3000 r.p.m for 15 minutes. The supernatant was taken and stored at-80℃. Sample protein content was tested by Bardford method. OD was tested by colorimetric method. AchE activity was tested by formula calculating.4.7 SPSS13.0 soft ware was used to analyze the data. P<0.05 was considered to be statistically significant. 5 Clinical evaluation of OSAHS treated by MAD5.1 The patients with OSAHS were chosen from the center of Obstructive sleep disordered breathing, College of Stomatology, Hebei Medical University. Twenty patients with moderate or severe OSAHS, including 19 male and 1 female, aged from 32 to 72 year. The average weight was 83.72±13 (68-95)kg, BMI was 28.97±2.35kg/m2. The average neck circumference was 41.92±2.12cm.5.2 The examination before the clinical treatment was performed:5.2.1 The all patients were examed by polysomnography, final diagnosis was obstructive sleep apnea.5.2.2 Professional staffs in radiology department shot the cephalometric at the end of the expiratory using digital X-ray machines made in Germany. The magnification was 11.5%. The meansure point of the upper airway and hyoid bone was fixed, measured.5.2.3 Epworth evaluation was done.5.2.4 The blood pressure in the morning was measured.5.2.5 The contents of ET-1 and AngⅡin blood plasm before treatment were examed by radioimmunity.5.3 All patients consented to wear MAD. After MAD wore, the mandible protrusion was 75% of maximum amount, the front teeth were opened 1-2mm vertically, patients would be adapted from 3 to 7 days generally.5.4 Followed up by 6 months, the patients were examed by PSG, Epworth evaluation and cephalometric. They were measured the blood pressure in the morning and determined the contents of ET-1 and AngⅡ.5.5 SPSS 13.0 soft ware was used to analyze the data. The paired t test was utilized. P<0.05 was considered to be statistically significant.Results:1 Model of obstructive sleep apnea hypoxia syndrome (OSAHS) was established.1.1 Before the tset, compared with the normal control group, the upper airway CT of the soft palate was no change in the experimental group, P> 0.05. After injection in soft palate, the upper airway of sagittal diameter and cross-sectional area of soft palate in experiment group were significantly reduced compared with the control group, P<0.05.1.2 The results of blood gas analysis showed that before the experiment there was no difference between experimental group and the control group in oxygen saturation, oxygen pressure, carbon dioxide pressure, pH value, P> 0.05. After injection during sleep apnea, oxygen pressure (Po2), oxygen saturation, pH value of the experimental group was significantly lower than that of the control group, P<0.05. Carbon dioxide pressure (Pco2) was significantly higher than that of the control group. P<0.05.1.3 The observation of sleeping situation found that 8 weeks after the experiment, the animals of group OSAHS appeared significant intermittent snoring and apnea during sleep in supine position; the longest apnea time was 27 seconds, the AHI was 16±4 times every hour. When apnea, ear artery draken, oral mucosa cyanosised were found and often awaked due to suffocation. About 10 days after injection, significant sleepiness was observed.1.4 The observation of HE staining at 8 weeks after injection showed that the injected gel in the soft palate of group OSAHS was completely surrounded by fibrous connective tissue, free of the tissue damage, inflammation or necrosis.2 The study of OSAHS rabbit treated by MAD2.1 The upper airway anteroposterio CT examination found that the upper airway spade of 1/4 point,1/2 point and 3/4 point of the soft palate in CT of group MAD was significantly increased than that of group OSAHS, P<0.05. Group MAD was narrower than the control group, but there was no significant difference, P>0.05.2.2 The cross-section area of upper airway CT examination showed that in group MAD, the cross-section area of upper airway of 1/4 point,1/2 point and 3/4 point of the soft palate was 22.45±2.13 mm2,18.97±1.65 mm2 and 20.27±3.41 mm2, compared with group OSAHS, there was significantly increased, P<0.05. Compared with the normal control group, there was no significant difference, P>0.05. 2.3 The results of blood gas analysis releaved that oxygen pressure, oxygen saturation and pH values of group MAD was 91.20±3.15mmHg,92.40±3.13% and 7.41±0.07, compared with group OSAHS, there was significantly increased, P<0.05. Compared with the normal control group, there was no significant difference, P>0.05. Carbon dioxide pressure of group MAD was 39.26±2.93mmHg, compared with group OSAHS, there was significantly decreased, P<0.05. Compared with the normal control group, there was no significant difference, P>0.05.2.4 The observation of sleeping situation found that there was no difficulties to diet and drink for the animals of both group OSAHS and group MAD. When dorsal position slept, there was no apnea in group MAD and the normal control group, there was snore for six rabbits of group MAD.2.5 The observation of HE stain at the 8th week after injection showed that the injected gel of the soft palate of group OSAHS was completely surrounded by fibrous connective tissue, free of the tissue damage, inflammation or necrosis.3 The effect of heart injury of OSAHS rabbits treated by MAD3.1 The contents of both ET-1 and AngⅡin plasma were assayed.3.1.1 The content of ET-1 in 3 groups was 7.93±1.18 pk/ml (group OSAHS); 2.80±1.41 pk/ml (group MAD); 2.11±0.80 pk/ml (the normal control group). There was significantly increased in group OSAHS than in group MAD and the control group, P<0.05, there was no significantly differences between in group MAD and the control group, P>0.05.3.1.2 The content of AngⅡin 3 groups was 13.18±3.64 pk/ml (group OSAHS); 8.80±1.70 pk/ml (group MAD); 7.19±1.92 pk/ml (the control group) respectively. There was significantly increased in group OSAHS than in group MAD and the control group, P<0.05, there was no significantly differences in group MAD and the control group, P>0.05.3.2 The correlation analysis results of ET-1,AngⅡand Blood gas analysis revealed that ET-1 was negative correlation with SaO2,Po2, the correlation coefficient was-0.64,-0.52, P<0.05. ET-1 was positive correlation with Pco2, the correlation coefficient was 0.53, P<0.05. AngⅡwas negative correlation with SaO2,Po2, the correlation coefficient was-0.66,-0.53, P<0.05. Angll was positive correlation with Pco2, the correlation coefficient was 0.61, P< 0.05.3.3 The result of ET-1 mRNA in heart tissue showed that ET-1/GAPDH of 3 groups was respectively 2.47±0.58 (group OSAHS); 1.98±0.59 (group MAD); 1.78±0.75 (the control group). There was significantly increased in group OSAHS than in group MAD and the control group, P<0.05, there was no significantly differences in group MAD and the control group, P>0.05.3.4 The changes of cardiac apex tissue showed that there was no visible change of cardiac apex of 3 groups.Observation of myocardium by the light microscope found that in group OSAHS, massive cardiac muscle fibers were collapsed, and fissures were observed. The arrangement of cardiac muscle fibers was disorder. The structure of myocardium was destroyed severely and there was no clearly integrate intercalated disk structure. In group MAD, the arrangement of cardiac muscle fibers was somewhat disorder and a few of muscle fibers were broken. There was clear integrated intercalated disk structure. In control group, Cardiac muscle fibers were integrate and arranged orderly, and intercalated disks were clear.In group OSAHS, the cardiac muscle fibers were disarranged and the structure was vague. A number of myofibrils fused and disappeared which formed a lot of spaces. The myocomma was incomplete. The whole structure of cardiac muscle fibers was destroyed. There was confluence and disappear of a lot of cristae and membrane of chondrosome. There was pyknosis of chondrosome. In group MAD, the arrangement of myfibrils was somewhat disorder. A few of cardiac muscle fibers was broken and disappeared. The structure of a few myocomma was vague. The whole structure of myocardial fibers was more integrate. There was confluence and disappear of a few of cristae and membrane of chondrosome. In control group, the arrangement of myfibrils was orderly. The transverse striation could be seen and the structure of intercalated disc was clear.3.5 The changes of blood capillary among cardiac muscle fibers showed that the wall was thicked in group OSAHS. There was microvilli pustute of endotheliocyte surface. There was swelling in endotheliocyte intracytoplasm. There was confluence and disappear of cristae and membrane of chondrosome. There was perinuclear space widen, chromatinic pyknosis and basal membrane thicken. In group MAD, there was pinocytosis bullule in endotheliocyte intracytoplasm, there was tight junction between cells, there was a few confluence and disappear of cristae and membrane of chondrosome, there was slightly chromatinic pyknosis and basal membrane thicken. In normal control, there was pinocytosis bullule, microfilament, chondrosome, rough endoplasmic reticulum in endotheliocyte intracytoplasm, there was tight junction between cell and integrity basal membrane.4 The influence on brain injury of OSAHS rabbits treated by MAD4.1 In group OSAHS the frontal cortex neurons were deflated, karyopycnosis and anachromasis observed by HE staining. There was no or a few of these phenomena in group MAD and the control group.4.2 TUNEL staining found that nuclear staining brown apoptotic neurons in frontal cortex was seen in group OSAHS. It was no or occasionally observed in group MAD and the control group.4.3 Flow cytometry revealed that apoptosis rate was 7.40±1.26% in group OSAHS, it was higher than that in group MAD (2.26±0.38%) and in the' control group (1.94±0.24%) (P<0.05)4.4 AchE activity revealed that it was 0.08±0.15 U/mgprot in group OSAHS, it was lower than that in group MAD (0.12±0.13 U/mgprot) and in the control group (0.13±0.13 U/mgprot).4.5 The correlation coefficient of the neurons apoptosis rate in frontal lobe cortex and blood oxygen saturation was-0.788, P<0.05, the correlation coefficient of AchE activity and oxygen saturation was 0.639, P<0.05.5 Clinical evaluation of OSAHS treated by MAD5.1 The results of polysomnography (PSG) 5.1.1 After treatment, AI, AHI, RAI, oxygen index decreased significantly than that before treatment, P<0.05; the average time and the longest time of apnea was significantly reduced than before treatment, P<0.05.5.1.2 The decreased percentage ofAAHI (%) was 43.69±14.56%, it was effective according to identification standards of efficacy; one case of△AHI (%)<25% in 20 cases was void; 9 cases△AHI (%)>50% were markedly effective; the other 10 cases AAHI (%) 25%-50% are valid.5.1.3 Average SaO2 was 81.92±3.93% before treatment,90.33±2.50%% after treatment; the minimum SaO2 was 71.20±11.46% before treatment, 80.34±12.19% after treatment, they were both significantly increased, P< 0.05. Hypoxia index decreased from 46.82±13.21 to 29.50±9.87, P<0.05.5.1.4 Sleep efficiency was 82.78±13.21% before treatment,92.45±17.21% after treatment, P<0.05; NREM accounted for 73.24±11.91% before treatment, accounting for 78.95±17.2% after treatment, P<0.05; REM accounted for 27.03±3.23% before treatment, accounting for 21.04±2.10% after treatment, P< 0.05.5.2 Cephalometric results showed that the upper airway of soft palate and tongue base after wore MAD were significantly increased, P< 0.05; Nasopharyngeal diameter, the airway space after hard palate and hypopharyngeal space didn't change, P>0.05. The distance of hyoid bone to MP plane decreased, P< 0.05.The distance of the hyoid bone to the cervical plane increased, P<0.05.5.3 Epworth measurement table comparison of 20 cases before treatment showed 16±5 points, a minimum of 9 points, a maximum of 23 points, while 20 cases after treatment over six months, the mean was 9±4 points, a minimum of 4 points and a maximum of 16 points, P<0.05.5.4 ET-1 and AngⅡmeasurements results found that ET-1 was 63.90±11.07pk/ml before treatment,57.76+9.36pk/ml after treatment, it was significantly reduced after six months treatment, P<0.05. AngⅡwas 94.26±16.38pk/ml before treatment,85.29±13.52pk/ml after treatment, it was significantly decreased after six months treatment, P<0.05. 5.5 ET-1 and respiratory disorder index were positively correlated. The correlation coefficient was 0.58, P<0.05, ET-1 and average oxygen saturation were negatively correlated with correlation coefficient-0.69, P<0.05. AngⅡand the respiratory disturbance index were positively correlated, with correlation coefficient 0.60, P<0.05, AngⅡand the average oxygen saturation were negative correlated. The correlation coefficient was-0.74, P<0.05.5.6 Systolic blood pressure was 135.25±15.98mmHg before treatment; 120±8.95mmHg after treatment, P<0.05.5.7 The fastest heart rate during sleep was 121±12 times/min before treatment,98±9 times/min after treatment, P<0.05; the lowest heart rate during sleep was 40±15 times/min before treatment,50±10 times/min after treatment, P<0.05; average heart rate during sleep was 72.44±6.8 times/min before treatment,66.23±7.32times/min after treatment, P<0.05; an average heart rate during the awake was 78.47±9.60 times/min before treatment, 71.29±8.67 times/min after treatment, P<0.05.Conclusions:1 An animal model of OSAHS rabbits was successfully established2 OSAHS may result in cardiac structure and myocardial capillary endothelium damaged.3 OSAHS could increase neuronal apoptosis in frontal cortex and decrease AchE activity significantly4 An animal model of OSAHS rabbits treated by MAD was successfully established5 There was effective treatment of OSAHS rabbits by MAD.6 OSAHS treated by MAD could decrease the damage in cardiac structure and myocardial capillary endothelium.7 OSAHS treated by MAD could reduce neuronal apoptosis in frontal cortex, and prevent AchE activity from decreasing.8 The patients with OSAHS treated by MAD could open upper airway space with oropharyngeal, hyoid anterior removed and AHI increased by 25%-50% getting effective treatment purposes. 9 The patients with OSAHS could improve sleep state and increase sleep efficiency by wearing MAD.10 The patients with OSAHD could reduce the peripheral blood ET-1, AngⅡlevels and lower systolic blood pressure by using MAD.