Effect Of Mandible Advanced Device Upon Genioglossus Function And Structure Of Rabbit In OSAHS

Objectives: By setting up the animal model of New-Zealand white rabbits with obstructive sleep apnea-hypopnea Syndrome (OSAHS) , to study the effects of OSAHS upon the function and structure of the genioglossus(GG), and to investigate the changes of GG of OSAHS rabbits after treatment with mandible advanced device(MAD), which provided the theoretical base for further study the effect of OSAHS on GG in clinical patients treated with MAD.Methods:Animals grouped: Thirty male New-Zealand white rabbits in six months old were randomly divided into three groups and there were ten rabbits in each group. Three groups were obstructive sleep apnea-hypopnea syndrome group(Group OSAHS),mandible advanced device group(Group MAD) and control group respectively.Animal model established : a total of 2ml polyacrylamide hydrogel were injected into soft palate of Group OSAHS and Group MAD. After the polyacrylamide hydrogel was injected, the animals were snored with intermittent apnea. Cephalometrical data of the upper airway width at upper 1/4 point, 1/2 point and 3/4 point of soft palate in supine position were significantly decreased compared with control group, and the blood oxygen saturation decreased by 22% compared with control group, which indicated OSAHS animal model was feasible.The animals of Group MAD were made mandible advanced device after the animal model was set up successfully, and the animals of control group did not receive any treatment.Sleep observed: On the second day after the animal model was set up, all animals were given 10% chloral hydrate through stomach by 5⁶ml/ kg. All the subjects slept 4⁶ hours every day in supine position for 8 weeks.Upper airway measured: The cephalometry of upper airway of three groups in supine position was made and measured. The upper airway width was measured at upper point, 1/4 point, 1/2 point 3/4 point and lower point of soft palate.Arterial blood gas analysis was performed on the first day when the subjects slept in supine position. One milliliter auricle arterial blood was collected when the animals were apnea and blood gas analysis was performed.In vitro contractile properties measured: 8 weeks later, all the animals were anaesthetized with 1% pentobarbital sodium and genioglossus (GG) was exposed and separated, the GG was immediately made into muscle strip in 2mm diameter, then the muscle strip was mounted vertically in a tissue bath. One end of the strip was tied to an immobile hook at the bottom of tissue bath and the other end was tied to a high-sensitivity force transducer. The salt solution was continuously gassed with 95% O₂ and 5% CO₂, its temperature was kept constant at 37℃through the water chamber and its pH was adjusted to 7.40. Muscles were allowed to equilibrate with the incubation solution for 30 min. Muscle strips were always stimulated through platinum electrodes and obtained the maximal twitch tension, the contraction time, the half-relaxation time, and the force-frequency relationship in response to stimulation frequencies of 10 Hz, 20 Hz, 40 Hz, 60 Hz, 80 Hz and 100 Hz. A 10-min rest period was allowed and the fatigability of each muscle was tested. The fatigue protocol was performed at 0.5 Hz/s for 5 minutes. The single switch force that was stimulated for 300ms was recorded every one minute for five minutes.Fibers type identified: GG samples were cut into a piece of 5mm by 5mm by 3mm and were frozen in isopentane, cooled with liquid nitrogen, and then stored at -80℃. Type I (slow twitch) and type II (fast twitch) fibers were identified by the standard ATPase staining.Hematoxylin-eosin staining: the soft palate from the injection site was obtained and fixed in 10% formalin, and then was performed with paraffin section, hematoxylin-eosin staining in routine method to be observed in light microscope.Data analyzed:the statistical analyses of all the data were performed by SPSS13.0 statistical program, 0.05 was selected as the standard.Results:1 Cephalometric data of the upper airway width at upper 1/4 point, 1/2 point and 3/4 point of soft palate in Group OSAHS were significantly decreased compared with both Group MAD and the control group after the animal model was set-up(P<0.05). There was no difference between Group MAD and the control group(P>0.05).2 The blood gas analysis showed that after the animal model was set up, blood oxygen saturation, oxygen partial pressure and PH value of Group OSAHS were significantly decreased compared with Group MAD and the control group(P<0.05). Carbon dioxide partial pressure of Group OSAHS was significantly increased compared with the control group (P<0.05). There was no difference between Group MAD and the control group (P>0.05).3 In vitro contractile properties of the GG: There was no difference in three groups in the maximal twitch tension, the contraction time, the half-relaxation time and the force-frequency relationship (P>0.05); But during the fatigue protocol, force was normalized to that produced during the first stimulation train, the value in Group OSAHS at each minute was 88.20±4.80%, 64.80±6.58% , 57.30±4.24%, 37.10±3.38% and 28.30±3.50% respectively, which was enhanced compared with the control group (98.80±7.33%, 87.70±4.50%, 74.20±4.99%, 52.00±5.10% and 48.20±4.10%) (P<0.05), but there was no difference between Group MAD(94.30±3.33%, 83.10±3.51%, 71.60±4.01%, 55.40±2.95% and 45.70±6.27%) and the control group (P>0.05).4 Fiber type distribution: In light microscope, fibers were counted in 100 times objective lens. Group OSAHS showed a significantly higher percentage of type II fibers (85.64±3.03%) than the control group (78.70±3.34%)(P<0.05), but there was no difference between Group MAD (80.21±2.07%) and the control group (P>0.05). 5 The correlation analysis of the saturation of blood oxygen and the genioglossus fatigue: There was significantly positive correlation at each minute, but the maximum coefficient correlation was 0.773 at the second minute, which indicated statistical significance( P<0.001).6 Histological observation of soft palate from the injection site: Hyalo-white gel was invested integrally in the soft palate, which was tenacious and flexible. In light microscope, polyacrylamide hydrogel was dyed with blue, which was completely surrounded by integrated fiber integument. And no tissue damage and necrosis were observed.Conclusions:Fatigability of GG was enhanced in OSAHS. Both functional and structural damage of GG could be prevented in the treatment of OSAHS with MAD.