| Obstructive sleep apnoea hypopnea syndrome (OSAHS) is a relatively common sleep-related breathing disorder. Upper airway(UA) lacks sufficient support of bone or cartilage tissue and is easily collapsed. Its lumen size depends on the balance between the upper airway dilator tension and upper airway negative pressure. Normally, UA muscles, especially the genioglossus, play a crucial role in maintaining the patency of the UA. Currently, we have reached a consensus in that genioglossus dysfunction may lead to OSAHS, however, with in-depth study of OSAHS, the authors found that apnoea or hypopnea will cause damage in genioglossus function, which will in turn increase the upper airway collapse, thus creating a vicious cycle and aggravating OSAHS.Treatment options for OSAHS include behavioral modification, such as weight loss, alcohol avoidance, and alteration of sleeping position, continuous positive airway pressure (CPAP), oral appliance, upper airway surgical procedures, electrical stimulation therapy, and pharmacological regimen. Oral appliance therapy has emerged as a conservative, noninvasive treatment option for patients with OSAHS. The most common type of oral appliances currently used in the treatment of OSAHS is mandibular advancement device (MAD). The American Academy of Sleep Medicine recommends oral appliances therapy for patients with mild to moderate OSAHS and for those with more severe OSAHS who are neither able nor willing to tolerate the standard continuous positive airway pressure therapy and refuse surgery. MAD has the advantages of non-invasive, low cost, perfect effect, comfortable, and easy to carry, therefore, the patients were more willing to accept and it is widely used in clinical practice. Cephalometrics, computed tomography (CT) scan, and magnetic resonance imaging were commonly used to investigate the ettects of MAD treatment on upper airway. Till now, studies on the MAD therapy for OSAHS were limited to the changes in subjective symptoms, sleep quality, as well as changes in airway structure.As mentioned above, genioglossus function associated closely with OSAHS, but now, genioglossus dysfunction in the pathogenesis of OSAHS causes most of the attention, and studies on OSAHS-induced genioglossus damage and dysfunction are still relatively limited. Two studies conducted by Carrera found that OSAHS caused genioglossus damage and dysfunction. genioglossus in patients with OSAHS showed significantly increased fatigue resistance.As we all know, chronic intermittent hypoxia (CIH) is the basic physical characteristics of OSAHS, CIH has been shown to alter upper airway muscle function and induce structural changes in upper airway muscles. Since CIH could induce functional and structural changes in upper airway muscles, this prompted us to consider whether CIH associated with OSAHS could also affect structure and function of genioglossus? Genioglossus belongs to skeletal muscle, and fiber type and energy metabolism are closely related to their function. Mitochondria, the main organelles in applying energy for muscles, can maintain the normal structure, function and energy metabolism of skeletal muscle cells. What affect does OSAHS on mitochondrial structure and function of genioglossus? What is the biological mechanism for the effect of OSAHS on genioglossus function? MAD is a preferred and popular method for treating mild to moderate OSAHS patients, however, there were no reports on improvements in the function, structure, and energy metabolism of genioglossus in the process of MAD therapy for OSAHS. Therefore, these are also the main contents of this paper intends to study.Part I Functional changes of genioglossus in a mandibular advancement device therapy for OSAHSObjective:To observe the effects of OSAHS on genioglossus contraction in vitro and mitochondrial function, as well as the changes after mandibular advancement device therapy by mandibular advancement device therapy for OSAHS animal models.Methods:1 Animal grouping and model establishmentExperiments were performed on 18 male 6-month-old New Zealand white rabbits(initial weight,3kg-3.5kg). All care and experimental procedures were approved by the Animal Care and Use Committees at Hebei Medical University (Certificate No. SCXK (J) 2013-1-003). The animals were housed under normal laboratory conditions. Food and water were available ad lib. The rabbits were randomised into 3 groups of 10 animals each:1 the control group; 2 Group OSAHS:obstructive sleep apnoea hypopnea syndrome; 3 Group MAD:mandibular advancement device.All animals in three groups were given spiral CT (USA Light Speed 16-detector spiral CT scanner) of the upper airway and polysomnography (US Bond Monet polysomnography system) in the supine position before modeling to exclude congenital airway stenosis or congenital OSAHS, while body weight and food intake were measured as a baseline value. After general anaesthesia with sodium pentobarbitone (20mg/kg-25mg/kg, intravenously), rabbits in Group OSAHS and Group MAD were injected with 2 ml of hydrophilic polyacrylamide gel, via the submucous muscular layer at the centre of the soft palate, about 1.5cm away from the junction of the hard and soft palates. The animals in the control group received no gel. Spiral computed tomography (CT) scanning and polysomnography (PSG) recordings were conducted as described above in the supine position. The sagittal and axial reconstruction by the manufacturer was conducted to evaluate the upper airway. OSAHS rabbits in Group MAD had the mandibular advancement device inserted, made from self-curing composite resin. It was positioned at 30 degrees to the upper incisors and was glued to the two upper incisors with glass ionomer, with 3mm-4mm of mandibular advancement and 2mm-3mm of jaw opening. After OSAHS rabbits were inserted with MAD, it was well tolerated. After 3 days-5 days of adaptation, no rabbits had difficulty in taking food or had signs of distress. CT and polysomnography were conducted as described above to evaluate the effectiveness of MAD.2 Sleep in supine positionOn the fifth day after the animal model was set up,no animals had difficulty in taking food or water. The body weight and food intake were recorded and they were given 10% chloral hydrate through stomach by 5ml/kg-6ml/kg. All the subjects slept 4 hours～6 hours every day in supine position for 8 weeks.Food and water were available ad lib at rest time.3 In vitro contractile propertiesAfter general anaesthesia with sodium pentobarbitone (20mg/kg-25mg/kg,intravenously),the genioglossus was separated and exposed, and then the genioglossus was immediately cut into muscle strip in 2mm of diameter, then the muscle strip was put into a tissue bath (continuously gassed with 95% O2 and 5% CO2) at 37℃ (135mM NaCl,2.5mM CaCl2,5mM KCl, 15mM NaHCO3,1mM NaH2PO4,1mM MgSO4,11mM C6H1206).The muscle strip was tied to a force transducer that connected with BL-420E+ biological and functional four-channel test system.Measurements are as follows:①The maximal twitch tension,② Contraction time,③ Half-relaxation time, ④ The force at frequencies of lOHz,20Hz,40Hz,60Hz,80Hz and 100Hz. Stimulating voltage:6V; Stimulus duration:300 ms; Adjacent stimulus interval:2min. After a 10-min rest period and the fatigability of genioglossus was tested. The fatigue test was conducted at 0.5 Hz for 5 minutes. The single switch force was recorded every one minute. At the end of the experiment, the muscle strip was removed, drained with filter, and weighed.Data processing:The tension was standardized, standardized methods:Muscle tension/Muscle cross-sectional areaCross-sectional area of the muscle strips=Quality/Optimal initial length (Lmax)/Muscle density (1.06mg/mm3)In the fatigue test, muscle tension was expressed as a percentage, ie Muscle tension at each time point/Muscle tension at baseline (muscle tension before fatigue test)Correlation between genioglossus fatigue and oxygen saturation was analyzed.4 Mitochondrial functions of genioglossusMitochondria extraction of genioglossus was in strict accordance with the mitochondria extraction kit (Nanjing Jiancheng Bioengineering Institute),the extracted mitochondrial suspension was quantificated with BCA protein assay kit. Mitochondrial membrane potential was estimated with the membrane electrical potential assay kit. After JC-1 fluorescent probe was loaded, flow cytometry analysis was conducted to quantitatively determine the relative value of the mitochondrial membrane potential. The fluorescence signals of JC-1 monomer (red fluorescence) and polymer (green fluorescence) were obtained in FL1 and FL2 detector. Flow diagram was obtained from flow cytometry and analyzed by Exp032ADC analysis software. Comparison of the fluorescence intensity of red fluorescence and green fluorescence could reflect mitochondrial membrane potential. Mitochondrial membrane potential was qualitatively determined by laser confocal microscopy. The excitation wavelength was selected at 488nm and 525nm. The red and green fluorescence signal was observed in laser confocal microscopy. Appearance of green fluorescence or decreased intensity of red fluorescence showed that mitochondrial membrane potential decreased.5 Mitochondrial Complex I activityThe quantitative detection was in strict accordance with the mitochondrial respiratory chain Complex I activity assay kit.Calculation of sample activity:Total sample activity=[(sample reading-background reading) x 1 (system capacity;ml)×sample dilution factor]÷[0.1 (sample capacity;ml)×6.2 (mmole absorption coefficient) ×1 (reaction time, minutes)]= Units/ml÷(sample protein concentration) mg/ml=Units/mg Unit=micromoles NADH/min6 Mitochondrial Complex IV activityThe quantitative detection was in strict accordance with the mitochondrial respiratory chain Complex IV activity assay kit.Calculation of sample activity:Total sample activity= [(sample reading-background reading) × 1 (system capacity; ml)×sample dilution factor]÷[0.1 (sample capacity; ml)×21.84 (mmole absorptivity)×1 (reaction time; min)]= U/ml÷(sample protein concentration) mg/ml=Units/mg Unit= micromolar cytochrome C/minResults:1 Basic health data of animalsThree days-five days after modeled, animals in each group had no difficulty in taking food or water, and they had activity freely. At baseline,2 weeks and 8 weeks post-experiment, there were no significant differences in body weight and food intake among the three groups. All animals passed the whole experiment without an accident.2 Efficacy of MAD2.1 CT scans of upper airwayCT scans of upper airway showed that cross-sectional area and the sagittal space in retropalatal upper airway decreased significantly in Group OSAHS, and there was no significant difference between Group MAD and the control group (P>0.05).2.2 PSG recordingsDuring PSG recordings, apnea or hypopnea of different degrees appeared in all animals in Group OSAHS, namely stopped airflow at mouth and nose or reduced/absent ventilation, accompanied by increased respiratory efforts, increased apnoea hypopnea index, decreased arterial oxygen saturation, and decreased sleep efficiency; While apnea or hypopnea occasionally appeared in animals in Group MAD, and there were significantly improvements in apnoea hypopnea index, arterial oxygen saturation, and sleep efficiency, similar to that in the control group.3 In vitro contractile properties of genioglossus The twitch tension in the control group, Group OSAHS, and Group MAD was 2.0±0.3g/cm2,1.8±0.2g/cm2, and 1.9±0.2 g/cm2;Contraction time in the control group, Group OSAHS, and Group MAD was 36.0±9.7ms,46.0±9.7ms, and 42.0±9.2ms;Half-relaxation time in the control group, Group OSAHS, and Group MAD was 18.0±6.8ms,22.0±5.9ms, and 18.5±4.7ms. The twitch tension decreased, contraction time and half-relaxation time increased in Group OSAHS, but there was no significant difference in three groups (P>0.05).There was no significant difference in the tension (Unit:g/cm2) at frequency of 10Hz,20Hz,40Hz,60Hz,80Hz, 100Hz among three groups (P>0.05).During fatigue test, compared with the control group, the fatigue of genioglossus increased significantly at each time point in Group OSAHS,and the difference was statistically significant (P<0.05). Compared with Group OSAHS, the fatigue of genioglossus in Group MAD decreased (P<0.05). But there was no significant difference between Group MAD and the control group (P>0.05).4Correlation analyses between genioglossus fatigue and oxygen saturationIn the fatigue test, there was positive correlation between muscle tension and oxygen saturation at each time point, especially, there was the most relevant at 2min, r=0.773 (P<0.001).5 Mitochondrial functions of genioglossus 5.1 Mitochondrial membrane potential of genioglossusFlow cytometry analysis showed the relative value of mitochondrial membrane potential was significantly lower in Group OSAHS than that in the control group (0.02±0.00 vs 10.94±3.19, P<0.05). There was no significant difference in the relative value of mitochondrial membrane potential between Group MAD and the control group (9.17±2.18 vs 10.94±3.19, P>0.05).Laser scanning confocal microscopy showed mitochondrial membrane potential of genioglossus was high in the control group, with most red fluorescence. Mitochondrial membrane potential of genioglossus decreased in Group OSAHS, with green fluorescence-based; and green fluorescence was rare in Group MAD,with red fluorescence-based. 5.2 Mitochondrial respiratory chain complexes activity of genioglossusCompared with control group, complex Ⅰ activity and complex Ⅳ activity decreased significantly in Group OSAHS (P<0.05); Compared with Group OSAHS, complex Ⅰ activity and complex Ⅳ activity in Group MAD increased (P<0.05). There was no significant difference in complex Ⅰ activity and complex Ⅳ activity between Group MAD and the control group (P>0.05).Part Ⅱ Structural changes of genioglossus in a mandibular advancement device therapy for OSAHSObjective:To observe the effect of a mandibular advancement device on structure and fiber distribution of genioglossus in OSAHS.Methods:1The experimental animals, animals grouping, animal models establishment, and sleeping in supine position were the same as that in Part one.2 Preparation of genioglossusAfter muscles of tongue bottom were separated, genioglossus was exposed and carefully cut off, and immediately fixed with 10% formalin for 24 hours, then routinely embedded in paraffin. After embedding into -20℃ refrigerator overnight, and then they underwent continuous 4μm thick paraffin sections.3 HE stainingParaffin sections obtained was followed by routine HE staining to examine the structure of genioglossus.4 Masson stainingParaffin sections obtained was followed by routine masson staining to examine the content of collagen fiber in genioglossus.5 Transmission electron microscopy (TEM)After genioglossus was exposed, it was immediately cut into a piece of 1mm× lmm×3mm3 and put into precooled 4% glutaraldehyde, and then they underwent conventional transmission electron microscopy sample preparation to examine the ultrastructure in transmission electron microscopy.6 Genioglossus fiber type determined by ATP enzyme stainingAfter consecutive frozen sections were obtained, conventional methods of ATP enzyme staining were conducted under acidic and alkaline conditions, and all the slices were observed in an optical microscope.300 adjacent fibers were counted at 100×objective and the proportion of type I fibers and type II fibers was analyzed.7 Sodium dodecyl sulfate-polyacrylamide gel electrophoresisAfter extraction of myosin, total protein concentration was assayed according to the method of BCA protein assay kit.Total 8% separating gel and 5% stacking gel were poured into glue bed. Before electrophoresis, the protein supernatants were diluted with loading buffer and were boiled for 3 min. For each sample, 100μg of protein was loaded in per lane.The gels were run at a constant voltage of 80V for 20 hours at 4℃. After electrophoresis was completed, the gel near marker of 200KD was taken and stained with Coomassie Brilliant Blue R-250 for 30 minutes, and then decolorized with Coomassie brilliant blue destaining solution. The relative amount of each myosin heavy chain isoform was determined by light intensity distribution curve with the Odyssey software.8 Real-Time PCRTotal RNA was isolated from each muscle using the Trizol technique and first-strand cDNA was synthesized. Target gene sequences were tested in Genebank, and online primer design software primer 3 was used to design and test its specificity, and then synthesized by the Biological Engineering (Shanghai) Co. Company. The total reaction system (20μl) of Real-Time PCR included 0.6μl of upstream primer (10μmol/L),0.6μ1 of downstream primers (10μmol/L),1.0l of cDNA, and 7.8μ1 of deionized water (without RNA enzyme). After the reaction was completed, the fluorescent signal generated was analyzed automaticly by the ABI 7500v 2.0.1 software, and converted it to CT value of myosin heavy chain and P-actin.Relative quantification of ΔΔCT was used to calculate:The average relative amount=2-averaseΓΔΔCT,which is the fold of gene expression in the experimental group relative to gene expression in the control sample.9 Statistical analysesResults were analysed with SPSS 13.0 analysis system. All data were expressed as Means±SD. The Chi-square test was used to compare the percentage of fibre types. The statistical significance of differences was assessed by analysis of variance (ANOVA), and LSD test was used to compare the difference between two groups. A P value<0.05 was considered statistically significant.Results:1 Morphology of genioglossus1.1 HE stainingGenioglossus fibers were regularly arranged in the control group. In Group OSAHS, varying degrees of degeneration of genioglossus fibers appeared manifested as disordered arrangement in muscle fibers, dissolved degeneration of muscle fibers, and disappearation and agglutination in normal structure; Disorders and degeneration of genioglossus fibers were at a lesser extent in Group MAD, similar to that in the control group.1.2 Masson stainingMasson staining showed that muscle fibers were red, collagen fibers were green, and blood cells were orange. In the control group, muscle fibers arranged in regular, with a small amount of collagen connective tissue cells around the muscle cells, and collagen fibers were thin and distributed in uniform. In Group OSAHS, genioglossus fiber arranged irregularly, and collagen tissue increased significantly, with uneven distribution. Genioglossus fibers arranged irregularly at a less extent, and collagen connective tissue (green matter) was significantly reduced in Group MAD, similar to that in the control group.1.3 The ultrastructural of genioglossusIn the control group, there were regular myofibrils, clearly discernible parazones, dark band, H bands, and M lines, Z lines were regularly arranged and evident in parazones; the shape of the mitochondria was regular with abundant cristae, intact inner and outer membranes. In Group OSAHS, there were disordered structure of the myofibril, mitochondria between muscle fibers with varying degrees of swelling and degeneration, a variety of sizes, irregular shapes, and fusion of cristae and membrane. Some mitochondria dissolved or even disappeared. Compared with Group OSAHS, the ultrastructural changes of genioglossus in Group MAD were less obvious, with mild disordered arrangement of partial myofibrils, and mild mitochondrial degeneration and edema. The fusion of mitochondria cristae and mitochondria membrane appeared occasionally.2 Genioglossus fiber type2.1 ATP enzyme stainingThe percentage of type Ⅱ fibers in Group OSAHS was significantly higher than that in the control group. The percentage of type Ⅰ fibers in Group OSAHS was significantly lower than that in the control group, and the difference was statistically significant (P<0.05). There was no significant difference between Group MAD and the control group (P>0.05). 2.2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresisCompared with the control group,the proportion of myosin heavy chain Ⅰand myosin heavy chain Ⅱa+Ⅱx isoforms of genioglossus decreased significantly in Group OSAHS (P<0.05); The proportion of myosin heavy chain Ⅱb isoforms of genioglossus increased significantly in Group OSAHS (P<0.05), while there was no significant difference between Group MAD and the control group (P>0.05).2.3 Real-Time PCRCompared with the control group, the mRNA gene expression of myosin heavy chain Ⅰ and Ⅱa isoforms of genioglossus decreased significantly in Group OSAHS,and the difference was statistically significant (P<0.05); The mRNA gene expression of myosin heavy chain Ⅱb and Ⅱx isoforms of genioglossus increased significantly in Group OSAHS, and the difference was statistically significant (P<0.05). There was no significant difference between Group MAD and the control group (P>0.05).Part Ⅲ Discussion on the mechanism for OSAHS-induced structural and functional changes of genioglossusObjective:Pre-established OSAHS model is used to explore whether oxidative stress pathways is the mechanisms for OSAHS-induced structural and functional disorders of genioglossus,from the view of oxidative stress.Methods:1The experimental animals, animals grouping, animal models establishment, and sleeping in supine position were the same as that in Part One.2 Eight-iso-prostagland content in plasma and genioglossus examined by enzyme-linked immunosorbent assay (ELISA).Blood obtaintion:Rabbits were anesthetized and drawn blood for 1.5ml from ear vein, and the blood obtained was centrifuged at 4000rpm/s for 5min, the supernatant was stored at -70℃.8-iso-prostagland was measured by rabbit enzyme-linked immunosorbent assay (ELISA) kit.3 Malondialdehyde measurements:Malondialdehyde content in genioglossus was determined by TBA method.4 Glutathione measurements:Glutathione content in genioglossus was determined by glutathione assay kit.5 Superoxide dismutase measurements:Tolal superoxide dismutase and CuZn-superoxide dismutase activity in genioglossus were measured by superoxide dismutase genotyping assay kit, and Mn superoxide dismutase activity was calculated by tolal superoxide dismutase and CuZn-superoxide dismutase activity.6 Catalase measurements:Catalase activity in genioglossus was deter-mined by catalase assay kit of visible method.7 Succinate dehydrogenase measurements:Succinate dehydrogenase activity in genioglossus was determined by succinate dehydrogenase assay kit.Results:1 Level of oxidative stressIn the control group,8-iso-prostagland content in plasma and genioglossus was 534.9±62.7pg/ml and 528.1±28.2pg/ml. In Group OSAHS, 8-iso-prostagland content in plasma and genioglossus was 1018.9±95.2pg/ml and 790.2±69.4pg/ml.In Group MAD,8-iso-prostagland content in plasma and genioglossus was 703.1±74.2pg/ml and 579.8±61.7pg/ml; Compared with the control group,8-iso-prostagland content in plasma and genioglossus in Group OSAHS was significantly higher (P<0.05), while there was no significant difference between Group MAD and the control group (P>0.05). The correlation analysis showed that there was positive correlation in 8-iso-prostagland content between GG and plasma, r=0.678 (P<0.05).Malondialdehyde content in genioglossus was 5.11±1.07 nmol/mgprot in the control group.Malondialdehyde content in genioglossus was 6.24±0.66 nmol/mgprot in Group OSAHS. Malondialdehyde content in genioglossus was 4.45±1.22 nmol/mgprot in Group MAD. Compared with the control group, malondialdehyde content in genioglossus in Group OSAHS increased significantly, and the difference was statistically significant (P<0.05); There was no significant difference between Group MAD and the control group (P>0.05).2 Antioxidant capacitiesTolal superoxide dismutase activity (Unit:U/mgprot) in genioglossus in the control group, Group OSAHS, and Group MAD was:3.91±1.15, 2.08±0.44, and 3.28±0.45; CuZn-superoxide dismutase activity (Unit: U/mgprot) in genioglossus in the control group, Group OSAHS,and Group MAD was:3.16±0.91,1.87±0.43,and 2.61±0.50; Mn-superoxide dismutase activity (Unit:U/mgprot) in genioglossus in the control group, Group OSAHS, and Group MAD was:0.76±0.41,0.21±0.14, and 0.68±0.37. Compared with the control group, tolal superoxide dismutase activity, CuZn-superoxide dismutase activity and Mn-superoxide dismutase activity in genioglossus in Group OSAHS decreased significantly, and the difference was statistically significant (P<0.05); There was no significant difference between Group MAD and the control group (P>0.05).Glutathione content (Unit:mgGSH/gprot) in genioglossus in the control group,Group OSAHS, and Group MAD was:9.05±2.78,4.19±0.79, and 3.15±2.23; Catalase activity (Units:U/mgprot) in genioglossus in the control group, Group OSAHS,and Group MAD was:19.15±4.92,12.76±4.55, and 17.63±5.11; Succinate dehydrogenase activity (Unit:U/mgprot) in genioglossus in the control group, Group OSAHS, and Group MAD was: 29.58±8.22,19.50±6.80, and 30.61±7.12. Compared with the control group, glutathione content, catalase activity, and succinate dehydrogenase activity in genioglossus in Group OSAHS decreased significantly, and the difference was statistically significant (P<0.05); There was no significant difference between Group MAD and the control group (P>0.05).Conclusions:1 OSAHS could cause increased genioglossus fatigue and reduced mitochondrial function, resulting in its disordered structure and abnormal fiber distribution, and oxidative stress pathway is one of the mechanisms for OSAHS-induced genioglossus damage.2 Mandibular advancement device can reverse functional and structural damage of genioglossus in OSAHS,and thus play a protective effect on the genioglossus. |
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