【作者】 卢海燕；

【导师】 王洁； 董福生；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 目的：阻塞性睡眠呼吸暂停低通气综合征(obstructive sleep apnea-hypopnea syndrome, OSAHS)是临床常见的慢性睡眠障碍性疾病,多见于体重偏肥的患者。由于睡眠时下颌后缩、肌肉松弛、舌骨位置改变等原因反复发生的上气道阻塞,引起呼吸暂停和通气不足,导致白天嗜睡,并发心、脑肺、肾等多脏器损害,严重影响患者的生命和生存质量。该疾病发病率高、危害性大,近年来成为临床医学和口腔医学共同关注的边缘学科,对OSAHS的治疗引起了医患的高度重视。迄今,临床上对OSAHS的治疗方法有行为治疗,如降低体重和侧卧位睡眠等；持续气道正压通气(Continue positive airway pressure, CPAP)疗法；口腔矫治器治疗；手术治疗等。CPAP为无创性治疗,通过呼吸机持续正气压向上气道通气,打开阻塞部位。这种方法的缺点是携带不方便,由于向上气道加压,使通气患者口咽干燥不适、影响睡眠,很大一部分病人不能耐受而放弃该方法。手术治疗风险大,疗效不确定,容易复发。因此,非手术的、安全的、容易被患者接受的口腔矫治器治疗,成为医患关注的治疗方法,矫治器及其疗效的研究成为目前OSAHS非手术治疗研究的热点。本研究通过建立OSAHS兔动物模型,观察OSAHS对外周血内皮素-1 (Endothelin-1, ET-1)、血管紧张素II (AngiotensinⅡ, AngⅡ)含量及心脏组织中ET-1 mRNA表达的影响；观察OSAHS对兔额叶皮层神经元及乙酰胆碱酯酶活性(acetylcholin-esterase, AchE)的影响；探讨OSAHS对心尖区组织及心肌内毛细血管的损伤。通过建立OSAHS兔配戴下颌前移矫治器的动物模型,观察下颌前移矫治器(Mandibular advancement device, MAD)治疗OSAHS后,对软腭后上气道间隙、血氧饱和度、氧分压、二氧化碳分压及pH值的改变；观察MAD治疗OSAHS对外周血ET-1、AngII及心脏组织中ET-1 mRNA表达的影响；观察MAD早期治疗OSAHS兔对大脑神经元凋亡及AchE活性的影响。探讨配戴下颌前移矫治器对OSAHS兔心脏损伤的影响,为临床研究MAD治疗OSAHS对心脏和大脑的影响提供理论依据和实验室数据。通过改进临床应用的下颌前移矫治器,研制下颌活动可调式下颌前移矫治器；通过观察OSAHS患者配戴MAD 6个月前后上气道直径变化、睡眠呼吸紊乱指数变化、血氧饱和度变化、Epworth值变化、外周血中ET-1、AngⅡ含量变化及血压、心率等变化,评价MAD治疗OSAHS的疗效,探讨下颌活动可调式MAD对OSAHS的治疗作用。方法：1 OSAHS动物模型建立1.1动物分组：30只6月龄雄性新西兰兔随机分为2组：实验组,即OSAHS组和正常对照组,每组15只。1.2模型建立前检查1.2.1 CT检查：两组实验前仰卧位上气道CT检测实验后两组软腭后缘上1/4点、1/2点、3/4点、尖端处上气道宽度、横截面积。1.2.2动脉血气分析：检测两组耳缘动脉血氧饱和度、氧分压、二氧化碳分压和pH值。1.3模型建立：OSAHS组以1%戊巴比妥钠全身麻醉,于软腭中间距软硬腭交界0.5cm处,粘膜下肌层注射聚丙烯酰胺水凝胶2ml,动物出现明显打鼾和间断的呼吸暂停,停止注射。对照组不作任何处理。1.4睡眠观察：两组动物每天以5-6ml/kg经口灌注10%水合氯醛后,仰卧位睡眠4-6小时/天,持续8周,累计记录49天睡眠期出现呼吸暂停的时间及次数。1.5实验后检查：1.5.1 CT检查：拍摄两组实验后仰卧位上气道CT。1.5.2血气分析：做两组实验动物睡眠呼吸暂停时动脉血气分析。1.5.3 HE染色：软腭HE染色检测注射凝胶的稳定性。1.6 OSAHS动物模型鉴定标准：(1)临床出现睡眠时打鼾或呼吸暂停,呼吸暂停指数AHI>5次/小时。(2)CT示上气道狭窄。(3)呼吸暂停时动脉血氧饱和度降低>4%。(4)氧分压、pH值降低,二氧化碳分压升高。(5)软腭局部注射物稳定,无移位,无扩散。1.7SPSS13.0统计软件进行统计学分析,以P<0.05作为显著性检验标准。2下颌前移矫治器治疗OSAHS兔的实验研究动物分组：30只6月龄雄性新西兰兔随机分为3组：阻塞性睡眠呼吸暂停低通气综合征组(OSAHS组)、OSAHS兔配戴下颌前移矫治器组(MAD组)和正常对照组,每组10只。下颌前移矫治器模型建立：OSAHS组和MAD组共20只兔,通过软腭注射凝胶建立OSAHS动物模型后,随机选10只兔作为MAD组,取印模制作连冠式斜面导板矫治器,粘接于上前牙,下前牙咬合在斜面上导下颌向前3-4mm,前牙咬合打开2-3mm,斜面与牙长轴成30℃。CT检查：建模后第3天,OSAHS组、MAD组和对照组动物均在全麻下做仰卧位上气道CT检查。睡眠观察：每天上午以5-6ml/kg经口灌注10%水合氯醛,仰卧位睡眠4-6小时/天,连续8周,累计49天,记录睡眠期出现呼吸暂停的时间及次数。血气分析：取睡眠中出现呼吸暂停时,兔耳缘动脉血做血气分析,检测血氧饱和度、氧分压、二氧化碳分压和pH值。HE染色：软腭注射凝胶处的组织取材,进行HE染色检测其稳定性。MAD治疗OSAHS的动物模型鉴定标准：(1)戴用MAD后2-3天能适应,能正常进食。(2)戴用MAD后仰卧位睡眠能够消除或减轻呼吸暂停。(3)戴用MAD后上气道打开,无狭窄及阻塞。(4)戴用MAD后动脉血气分析显示,血氧饱和度恢复正常。(5)模型稳定能够重复。SPSS13.0统计软件进行统计学分析,以P<0.05作为显著性检验标准。3配戴下颌前移矫治器对OSAHS兔心脏损伤的影响3.1动物分组、模型建立及睡眠观察：同第二部分3.2标本留取：建模8周后,OSAHS组、MAD组和对照组动物在1%戊巴比妥钠20mg/kg全麻下,颈外静脉采血4ml,用于检测血浆中ET-1、AngⅡ含量；留取心尖区标本进行光镜及电镜观察,以及心脏组织中ET-1mRNA表达的检测。3.3测定血浆中ET-1和AngⅡ含量：用酶联免疫吸附测定法(Enzyme-linked Immunosorbent Assay, ELISA)检测血浆中ET-1、AngⅡ含量。3.4 RT-PCR检测心脏组织中ET-1 mRNA的表达：一部分心尖组织放入液氮中冷冻,随后转存-80℃冰柜,进行RT-PCR检测心脏组织中ET-1mRNA的表达。3.5心尖组织HE染色：一部分心尖组织立即放入10%福尔马林中固定,常规石蜡包埋,HE染色,AX-80多功能显微镜下,观察心肌纤维的组织学结构改变。3.6心尖组织透射电镜观察：在同一麻醉状态下,打开动物的胸腔,暴露心脏。从距离心尖5mm处切下心尖区组织,立即取1×1×3mm3放入预冷的4%戊二醛中,常规透射电镜标本处理,Epon 812包埋,Hitach-7500透射电镜下,观察心肌纤维及纤维间毛细血管变化。3.7 SPSS13.0统计软件进行统计学分析,以P<0.05作为显著性检验标准。4配戴下颌前移矫治器对OSAHS兔大脑损伤的影响。4.1动物分组、模型建立及睡眠观察：同第二部分4.2标本留取：建模8周后,以20mg/kg的1%戊巴比妥钠全麻三组动物,完整取出兔大脑。4.3脑额叶HE染色：部分右脑额叶皮层固定于10%福尔马林24小时,常规石蜡包埋,切片厚度4μm, HE染色,AX-80万能显微镜下观察大脑神经元改变。4.4 TUNEL染色：部分右脑额叶皮层固定于10%福尔马林中24小时后,常规石蜡包埋、切片厚度4μm, TUNEL染色,观察额叶神经元凋亡情况。4.5流式细胞分析：部分右脑额叶皮层固定于预冷的70%乙醇中24小时,制备成单细胞悬液,进行流式细胞分析,检测细胞凋亡率。4.6比色法检测乙酰胆碱酯酶活性：左脑额叶皮层称重,置于相当于组织重量9倍的匀浆介质中,将额叶皮层制备成匀浆液,4℃下3000转/分离心15min,取上清,-80℃保存。Bardford法测样品蛋白含量,比色法检测样品反应后各管吸光度,利用公式计算额叶皮层中AchE活性。4.7应用SPSS13.0统计软件进行统计学分析,以P<0.05作为显著性检验标准。5可调式下颌前移矫治器治疗OSAHS的临床研究5.1患者基本资料：由河北医科大学口腔医院阻塞性睡眠呼吸障碍中心选择OSAHS患者20例,男性19人,女性1人,年龄32-72岁,平均体重83.72±13(68-95)kg,肥胖指数(BMI)28.97±2.35kg/m2,平均颈围41.92±2.12cm。5.2治疗前检查5.2.1多导睡眠监测(Polysomnograph,PSG):治疗前做PSG,确定为阻塞性睡眠呼吸紊乱。5.2.2 X线头颅侧位片：拍摄治疗前X线头颅侧位片,定点、测量上气道大小及舌骨位置。5.2.3 Epworth测量表评估。5.2.4测量晨起血压。5.2.5放射免疫法测定治疗前血浆中ET-1.AngII含量。5.3制戴矫治器：患者在知情情况下,同意咬取印模,咬蜡牙合记录。拍摄蜡牙合记录时的头颅侧位片确定上气道是否打开,制戴下颌可活动式MAD。戴用后下颌前伸量为最大前伸量的75%,前牙垂直向打开1-2mm,一般3-7天适应。5.4复诊检查：制戴矫治器后6个月,再次做多导睡眠呼吸监测；Epworth测量表评估；测量晨起血压；放射免疫法测定血浆中ET-1.AngII含量。5.5应用SPSS13.0统计软件进行配对t检验,以P<0.05作为显著性检验标准。结果：1 OSAHS动物模型建立1.1上气道CT结果实验前OSAHS组软腭1/4点、1/2点、3/4点及最下点处上气道矢状向间隙分别为：4.14±0.74mm.3.86±0.58mm.3.89±0.72mm.4.86±1.20mm；横截面积分别为：22.60±3.91 mm2、20.20±2.59 mm2、20.40±3.05 mm2、30.00±3.94 mm2,和正常对照组相比,P>0.05,无统计学差异。实验后OSAHS组仰卧位上气道软腭1/4、1/2、3/4点、最下点处矢状向直径分别为3.38±0.75mm.2.59±0.69mm.2.92±0.70mm:横断面积分别为17.50±1.0mm2、13.33±1.97 mm2、14.00±2.74 mm2,均较对照组减小,P<0.05,有统计学意义。1.2血气分析实验前OSAHS组血氧饱和度(SaO2)、氧分压(PO2)、二氧化碳分压(Pco2)、pH值分别为：96.58±1.97%、90.10±17.26mmHg.37.10±5.20 mmHg.7.43±0.14,与正常对照组相比无差异,P>0.05。实验后OSAHS组睡眠呼吸暂停时的SaO2、Po2、pH值分别为77.94±13.83%、51.20±13.23mmHg、7.31±0.11低于对照组,有统计学意义,P<0.05；Pco2为51.55±13.64 mmHg高于对照组,P<0.05,有统计学意义。1.3睡眠观察OSAHS组出现打鼾和间断的呼吸暂停；连续8周,累计49天的观察记录：呼吸紊乱指数为16±4次/小时,呼吸暂停最长时间为27秒。呼吸暂停时发现耳缘动脉颜色变暗,唇粘膜发绀,时常因憋气而觉醒。在注射后10天左右出现嗜睡症状。1.4OSAHS组注射后8周,软腭组织切片光镜观察发现,注射凝胶周围被纤维结缔组织完整包裹,没有扩散。2下颌前移矫治器对OSAHS兔治疗作用的实验研究2.1 CT上气道矢状向间隙变化的测量结果MAD组软腭1/4点、1/2点和3/4点上气道矢状向间隙分别为3.67±0.45mm.2.71±0.44mm和2.77±0.43mm,与0SAHS组相比软腭后气道间隙增加,有统计学意义,P<0.05；和正常对照组比减小,但没有统计学意义,P>0.05。2.2 CT上气道横截面积变化的测量结果MAD组的软腭1/4点、1/2点和3/4点处上气道横截面积分别为22.45±2.13 mm2、18.97±1.65 mm2、20.27±3.41 mm2,和0SAHS组相比软腭后上气道横截面积增加,有统计学意义,尸<0.05.MAD组较对照组上气道横截面积均减小,但无统计学意义,P>0.05。2.3动脉血气分析的测定结果MAD组SaO2(%)、Po2、Pco2、pH分别为92.40±3.13%、91.20±3.15mmHg.39.26±2.93mmHg.7.41±0.07,和OSAHS相比SaO2(%)、P02和pH值增加,PcO2降低,P<0.05,均有统计学意义；MAD组和正常对照组相比各项指标,P>0.05,均无统计学意义。2.4实验动物的一般情况OSAHS组及MAD组动物1-2天适应后无进食、进水困难,注射伤口愈合良好无感染,仰卧位睡眠时,MAD组4只呼吸均匀平稳无打鼾,6只轻微打鼾,未见呼吸暂停；对照组呼吸均匀平稳,无打鼾和呼吸暂停；OSAHS组有打鼾和呼吸暂停。2.5 MAD组戴矫治器8周后,软腭组织切片光镜观察发现,注射凝胶周围被纤维结缔组织完整包裹,没有引起组织损伤、炎症或坏死。3下颌前移矫治器对OSAHS兔心脏损伤的影响3.1血浆中ET-1.AngII含量测定3.1.1 ET-1在三组分别为OSAHS(7.93±1.18 pk/ml)；MAD(2.80±1.41 pk/ml)；对照组(2.11±0.80 pk/ml).3.1.2 AngⅡ在三组分别为OSAHS(13.18±3.64 pk/ml)；MAD(8.80±1.70 pk/ml)；对照组(7.19±1.92pk/ml).两项指标中,OSAHS与MAD、对照组相比均有增加,P<0.05；而MAD与对照组无差别,P>0.05。3.2血浆中ET-1.AngII与血气分析的相关分析结果ET-1与SaO2、Po2呈负相关,相关系数分别为-0.64、-0.52,P<0.05；与Pco2呈正相关,相关系数为0.53,P<0.05。AngII与SaO2、Po2呈负相关,相关系数分别为-0.66、-0.53,P<0.05；与Pco2呈正相关,相关系数为0.61,P<0.05。3.3 RT-PCR结果显示：正常对照组ET-1/GAPDH之比为1.78±0.75；MAD组ET-1/GAPDH之比为1.98±0.59；OSAHS组ET-1/GAPDH之比为2.47±0.58。OSAHS组较正常对照组及MAD组心肌细胞ET-1 mRNA表达明显升高,P<0.05；正常对照组与MAD组比较无差异,P>0.05。3.4心肌纤维的改变3.4.1肉眼观察：三组动物心尖外形无明显改变。3.4.2光镜观察OSAHS组中大量心肌纤维断裂、消失、形成裂隙,纤维排列紊乱。心肌组织出现液化,形成空泡,心肌结构严重破坏,未见清晰闰盘结构。MAD组中少量心肌纤维排列紊乱,结构疏松,偶有纤维断裂,见清晰闰盘结构。对照组中心肌纤维完整,排列整齐,闰盘清晰可见。3.4.3电镜观察OSAHS组肌原纤维排列紊乱,结构模糊,大量肌原纤维溶解、破坏消失,形成许多大小的间隙,肌节不完整。线粒体大部分嵴和膜有融合和消失现象,有的线粒体有致密化。MAD组肌原纤维排列稍紊乱,有少量心肌纤维断裂消失,部分肌节结构不清,线粒体部分嵴和膜有融合和消失现象。对照组肌原纤维排列整齐,横纹清晰可见,闰盘结构清晰。3.5心肌纤维间毛细血管的改变电镜观察发现：OSAHS组毛细血管管腔无明显扩张或狭窄。内皮细胞表面有微绒毛突起,内皮细胞胞质内局部有肿胀,线粒体的嵴和膜有融合和消失现象,核周间隙增宽,核染色质固缩,有核膜下边集现象。毛细血管基底膜增厚。MAD组内皮细胞胞质内可见吞饮小泡,细胞间可见紧密连接,线粒体的嵴和膜有部分融合和消失现象,有的线粒体出现致密化和髓样化改变,核染色质有固缩和核膜下边集。基底膜稍增厚。对照组内皮细胞胞质内可见吞饮小泡,微丝,线粒体和粗面内质网。内皮细胞间有紧密连接,基底膜完整。4配戴下颌前移矫治器对OSAHS兔大脑损伤的影响4.1 HE染色显示,OSAHS组额叶皮层可见神经元胞体缩小,核固缩、深染。MAD组和对照组额叶皮层神经元未发现或偶见核固缩。4.2 TUNEL染色显示,OSAHS组额叶皮层可见核染色呈棕黄色凋亡神经元。MAD组和对照组未发现或偶见凋亡。4.3流式细胞仪检测发现,OSAHS组凋亡率为7.40±1.26%,较MAD组(2.26±0.38%)和对照组(1.94±0.24%)明显升高,有统计学意义,P<0.05。4.4 AchE活性：OSAHS组为0.08±0.15U/mgprot,较MAD组(0.12±0.13 U/mgprot)和对照组(0.13±0.13U/mgprot)明显降低,有统计学意义,P<0.05。4.5大脑额叶神经元凋亡率与血氧饱和度相关系数为-0.78,有统计学意义,P<0.05、乙酰胆碱酯酶活性与血氧饱和度的相关系数为0.639,有统计学意义,P<0.05。5可调式下颌前移矫治器治疗OSAHS的临床研究5.1多导睡眠监测结果5.1.1治疗后AI、AHI、RAI、低氧指数较治疗前显著减小,P<0.05；呼吸暂停平均时间及最长时间均较治疗前有显著减少,P<0.05。5.1.2平均呼吸紊乱指数降低百分数△AHI(%)为43.69±14.56%,根据疗效鉴定标准为有效；20例中AAHI(%)<25%的有一例为无效；△AHI(%)>50%的有9例为显效；其余10例△AHI(%)在25%-50%均为有效。5.1.3平均SaO2治疗前为81.92±3.93%、治疗后为90.33±2.50%；最低SaO2治疗前为71.20±11.46%、治疗后为80.34±12.19%,均显著增加,P<0.05。低氧指数由46.82±13.21降低为29.50±9.87,P<0.05。5.1.4睡眠效率治疗前为82.78±13.21%,治疗后为92.45±17.21%,P<0.05。NREM治疗前占73.24±11.91%,治疗后占78.95±17.2%,P<0.05。REM治疗前占27.03±3.23%,治疗后占21.04±2.10%,P<0.05。5.2 X线头颅侧位片戴用MAD后上气道软腭后间隙及舌根后气道间隙均有显著增加,P<0.05；鼻咽直径、硬腭后气道间隙及喉咽间隙未见改变,P>0.05；舌骨到MP平面距离减小,P<0.05；舌骨到颈椎前平面距离增加,P<0.05。5.3Epworth测量表比较20例治疗前平均16±5分,最低为9分,最高为23分。20例治疗满半年后7±4分,最低为4分,最高为16分,P<0.05。5.4 ET-1、Angll测量结果ET-1矫治前为63.90±11.07pk/ml,矫治后为57.76±9.36pk/ml,矫治半年后明显降低,P<0.05。Angll治疗前为94.26±16.38pk/ml,治疗后为85.29±13.52pk/ml,矫治半年后明显降低,P<0.05。5.5 ET-1与呼吸紊乱指数成正相关,相关系数为0.58,P<0.05,与平均血氧饱和度成负相关,相关系数为-0.69,P<0.05。Angll与呼吸紊乱指数呈正相关,相关系数为0.60,P<0.05,与平均血氧饱和度成负相关,相关系数为-0.74,P<0.05。5.6早晨收缩压治疗前为135.25±15.98mmHg;治疗后为120±8.95mmHg, P<0.05。5.7心率改变情况治疗前睡眠期间最快心率为121±12次/分,治疗后为98±9次/分,P<0.05；治疗前睡眠期间最低心率为40±15次/分,治疗后为50±10次/分,P<0.05；治疗前睡眠期间平均心率为72.44±6.8次/分,治疗后为66.23±7.32次/分,P<0.05；治疗前清醒期间平均心率为78.47±9.60次/分,治疗后为71.29±8.67次/分,P<0.05。结论：1成功建立OSAHS兔动物模型；2 OSAHS可以引起心肌纤维和心肌纤维间毛细血管内皮的损伤；3 OSAHS可以增加额叶皮层神经元的凋亡,降低乙酰胆碱酯酶活性；4成功建立OSAHS兔配戴下颌前移矫治器动物模型；5下颌前移矫治器能有效治疗OSAHS兔；6下颌前移矫治器早期治疗OSAHS兔可以预防心肌纤维和心肌纤维中毛细血管内皮的损伤；7下颌前移矫治器治疗OSAHS兔可减少额叶皮层神经元的凋亡、预防乙酰胆碱酯酶活性降低；8临床下颌活动可调式MAD治疗OSAHS患者,可以打开口咽部上气道间隙,舌骨前上移位,AHI提高43.69±14.56%,达到有效治疗目的；9临床下颌活动可调式MAD治疗OSAHS患者,可以改善睡眠状态,提高睡眠效率；10临床下颌活动可调式MAD治疗OSAHS患者,可以降低外周血中ET-1、AngⅡ的含量,预防OSAHS对心脏及血管内皮细胞的损伤。更多还原

【Abstract】 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.更多还原