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CO2激光镫骨足板造孔术后内耳超微结构的实验研究
http://www.100md.com 《中国激光医学杂志》 2000年第2期
     作者:康梦奎 史晓瑞 龙顺波 孙建和

    单位:康梦奎 史晓瑞 龙顺波(中国人民武装警察部队总医院耳鼻喉科,北京市,100039);孙建和(解放军总医院耳鼻喉科研究所)

    关键词:镫骨手术;激光手术;显微镜检查,电子,扫描

    中华激光医学杂志000205摘要 目的 探讨以CO2激光行镫骨足板手术对内耳感受器超微结构的影响。方法 9只白色红目豚鼠分3组,每组3只,分别以功率2、4、6 W的CO2激光对左耳进行内耳镫骨足板造孔,以右耳为对照。术后动物断头处死,利用扫描电镜技术观察激光照射对内耳感受器毛细胞的影响。结果 以功率2 W的CO2激光行内耳镫骨足板造孔术时,豚鼠内耳毛细胞未见损伤;功率4 W时,外毛细胞之间出现云雾状渗出物,并有散在的外毛细胞倒伏,内毛细胞部分融合,表面有球状物附着;功率6 W时,内耳毛细胞及壶腹嵴毛细胞均被损伤,并出现外毛细胞严重缺失、破坏,壶腹嵴纤毛广泛损伤、脱落,失去原有的形态。结论 应用CO2激光行镫骨足板手术应注意功率的选择,以免造成内耳感受器的损伤。所得实验数据可供激光治疗耳硬化症选择适当的功率参考。
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    中图分类号: TN249;R764.32 文献标识码: A

    文章编号: 1003-9430(2000)02-0085-04

    Scanning Electron Microscopic Observation of Inner Receptor Organ after

    CO2 Laser Stapedostomy in Guinea Pigs

    KANG Mengkui, SHI Xiaorui, LONG Shunbo

    (Department of Otolaryngology, General Hospital of the Armed Policeforce Army, Beijing 100039, China)
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    SUN Jianhe

    (Institute of Otolaryngology,Chinese PLA General Hospital)

    ABSTRACT Objective CO2 laser stapedotomy was performed in 9 guinea pigs to investigate the influence of varying dosage of CO2 laser irradiation on inner receptor organ.Method Nine guinea pigs after stapedostomy on the left ear were equally divided into 3 groups, which were irradiated with CO2 laser with 2 W, 4 W and 6 W respectively, using the right ear as the control. After irradiation the animals were decapitated. The effect of laser treatment on the hair cells of inner ear was observed by scanning electron microscopy.Results It was found that cochlear hair cells were not affected by low power(2 W) CO2 laser irradiation. However, with 4 W CO2 irradiation, there were cloud effusion on the surface of outer hair cells, collapsing hair cells were also observed. With 6 W CO2 irradiation, serious loss of outer hair cells and disappearance of microvilli of ampulate crypt were observed.Conclusions Our findings indicate that careful selection of appropriate dosage of laser irradiation is of essential significance in stapes surgery.The experimental data obtained may be used as a reference for the treatment of otosclerosis with laser of appropriate power density.
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    Key words Stapes surgery; Laser surgery; Microscopy, electron, scanning

    耳硬化症是骨迷路原发性病变,硬化阶段出现陷窝性吸收、排列不规则的板状骨,使镫骨固定,导致传音性耳聋。临床上多采用镫骨切除术或活塞术来提高听力,但可能会因术中操作不当,而引起迷路和面神经等重要结构的损伤。近年来激光技术发展迅速,不断有新的激光器问世,普遍认为激光用于镫骨手术,有比手持工具创伤小、精确度高、手术时间短等优点[1,2],但剂量选择不当,则易造成内耳损伤[3]。为给临床合理应用激光治疗耳硬化症提供参考,我们以豚鼠为实验对象,探讨了以不同剂量CO2激光行镫骨足板手术,对其耳蜗毛细胞及前庭毛细胞超微结构的影响。

    材料与方法

    一、实验动物及分组
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    取9只白色红目豚鼠(解放军总医院动物实验中心提供),体重250~450 g,耳廓反射正常。随机分成A、B、C组(每组3只),激光功率依次为2、4、6 W,功率密度依次为110、220、330 W/cm2 。均行左耳照射,右耳为对照。

    二、镫骨造孔方法

    每只豚鼠腹腔注射安定5 mg/kg,15 min后肌内注射枸缘酸芬太尼0.32 mg/kg。待麻醉后行气管切开、插管,以保持呼吸通畅。保持豚鼠体温于38℃±1℃。手术采用腹侧进路,打开左侧听泡,保持听骨链的完整,暴露耳蜗。在放大35倍的体视显微镜下,用CO2激光先将镫骨肌腱汽化断除,然后于镫骨后脚拱起处开始继续照射,渐向足板方向,直到后脚几乎完全汽化切除。连续吸引烟雾后,汽化区可看到白色斑点,用小针和吸引器去除汽化斑点,分开砧镫关节,取出镫骨脚,将CO2激光直接射向足板中央,形成直径0.6 mm的小窗,完成镫骨足板造孔。
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    三、扫描电镜样品制备技术

    动物断头处死后,迅速取出颞骨,从蜗尖和前庭窗灌入2%戊二醛固定液。分离取出螺旋韧带,1%锇酸固定液固定,临界点干燥法制备标本,观察耳蜗毛细胞及前庭毛细胞超微结构的变化。

    结 果

    一、CO2激光镫骨造孔后3组豚鼠耳蜗底周毛细胞的扫描电镜观察

    3组豚鼠非激光照射耳蜗均表现正常的形态结构(图1)。A组豚鼠术耳在功率2 W激光照射后,耳蜗底周内,外毛细胞排列规则(图2)。B组豚鼠术耳在功率4 W激光照射后,耳蜗底周外毛细胞间有云雾状渗出物,部分外毛细胞倒伏,内毛细胞可见融合,表面有球状物附着(图3)。C组豚鼠术耳在功率6 W激光照射后,耳蜗底周内、外毛细胞纤毛融合、倒伏、脱落,螺旋器皮板呈剥脱改变(图4)。t8601.gif (6105 bytes)
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    图1 扫描电镜观察示豚鼠未接受激光照射耳耳蜗底周毛细胞形态结构正常 ×4 000

    Fig.1 Structure of hair cell of cochlear based circle in normal guinea pig under scanning electron microscope. SEM ×4 000 t8602.gif (7323 bytes)

    图2 A组豚鼠2 W功率CO2激光镫骨造孔术耳耳蜗底周毛细胞的扫描电镜观察,外毛细胞(OHC)和内毛细胞(IHC)无损伤,纤毛显示良好 ×4 000

    Fig.2 SEM image of inner hair cells(IHC) and outer hair cells(OHC) in cochlear of the based circle irradiated with 2 W CO2 laser. Both hairs and cells are normal. SEM ×4 000 t8701.gif (9887 bytes)
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    图3 B组豚鼠4 W功率CO2激光镫骨造孔术耳耳蜗底周外毛细胞间可见云雾状渗出物(E),散在倒伏(C),内毛细胞可见融合、球状物附着(M) 扫描电镜×4 000

    Fig.3 Cochlea of B group guinea pig irradiated with 4 W CO2 laser. E:cloudy exudate among OHC;C:scattered OHC;M: IHC fused and covered by spherical secretion materials. SEM

    ×4 000 t8702.gif (7396 bytes)

    图4 C组豚鼠6 W功率CO2激光镫骨造孔术耳耳蜗底周内、外毛细胞纤毛倒状、脱落,螺旋器皮板呈剥脱改变(箭头) 扫描电镜 ×4 000
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    Fig.4 Cochlea of C group irradiated with 6 W CO2 laser. Hairs were bend and sloughed off, and the Corti skinboard was peeled off. SEM ×4 000

    二、CO2激光镫骨造孔术后3组豚鼠内耳前庭毛细胞的扫描电镜观察

    A组动物术耳内耳前庭及上、外半规管壶腹嵴毛细胞正常(图5)。B组动物术耳内耳前庭、后壶腹嵴毛细胞纤毛片状缺失、局限缺损(图6)。C组动物术耳前庭上、外壶腹嵴毛细胞纤毛广泛损伤、脱落,失去原有的形态(图7)。非激光照射耳前庭形态结构正常。t8703.gif (8228 bytes)
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    图5 A组豚鼠2 W功率CO2激光镫骨造孔术耳前庭及上、外半规管壶腹嵴毛细胞正常 扫描电镜 ×4 000

    Fig.5 Group A irradiated with 2 W CO2 laser. Hairs of vestibule and crista ampullaris in superior semicircular canal cells are normal. SEM ×4 000 t8704.gif (8099 bytes)

    图6 B组豚鼠4 W功率CO2激光镫骨造孔术耳前庭、后壶腹嵴毛细胞纤毛呈片状损伤(箭头) 扫描电镜 ×4 000

    Fig.6 After 4 W CO2 laser irradiation, the hairs of vestibule and posterior crista ampullaris cells in B group were injured. SEM ×4 000 t8705.gif (7974 bytes)
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    图7 C组豚鼠6 W功率CO2激光镫骨造孔术耳前庭及上、外壶腹嵴毛细胞广泛损伤,失去原有的形态(箭头) 扫描电镜 ×4 000

    Fig.7 Group C irradiated with 6 W CO2 laser. Hair cells of vestibule, superior and lateral crista ampullaris were extensively injured and showed abnormal appearance. SEM ×4 000

    讨 论

    1985年Coker等[4]首先利用CO2激光对猫耳行镫骨足板切除术,并详细记录了所用激光的辐射能量、输出方式及作用时间。但1998年Jovanovic等报道,部分患者在接受激光镫骨足板手术后出现眩晕、头痛及高频区听力下降等症状。本实验就临床常用剂量模拟镫骨手术,观察了不同剂量的CO2激光对耳蜗感受器的影响,结果发现以小剂量低功率CO2激光行镫骨足板手术时,感受器的结构不被损伤,临床应用是安全的;高功率的CO2激光则影响内耳的超微结构,表现为毛细胞融合、倒伏、缺失及表面渗出物黏附毛细胞表面,同时前庭感受器壶腹嵴纤毛也发生病理变化,出现纤毛缺失、结构紊乱等现象。造成毛细胞损伤的机制可能有:(1)热效应。在激光镫骨足板造孔时,因需反复照射,热能的积聚易引起耳蜗局部温度的骤然上升[5],而前庭壶腹嵴对热尤为敏感,很容易受热致伤。1981年德国Thoma等[6]曾观察了以氩激光照射豚鼠镫骨足板时内耳温度与激光束能量之间的关系,以及脉冲频率与突然局部温度升起之间的关系,发现脉冲频率与被照射局部温度呈线性关系,外淋巴的温度升高与光束的能量呈明显的正相关关系。从我们的实验中可以看出,低功率的CO2激光照射对壶腹嵴纤毛没有明显影响,但随着功率的加大,热损伤加重,前庭壶腹嵴纤毛损伤加重。这些结果也证实了1997年Jovanovic等[7]的发现,他们认为以不同脉冲模式的CO2激光进行镫骨足板手术,热压力可能导致内耳结构的损伤。(2)压力效应。本实验中我们主要观察了耳蜗离镫骨足板比较近的耳蜗底周的内、外毛细胞形态变化,而扫描电镜能较好地反映细胞表面的超微结构。实验发现,激光功率越大,耳蜗底周毛细胞的损伤越重,而且大部分毛细胞表现为倒伏、缺失,这一现象很可能与激光的压力波作用有关。1996年Pratisto等[5]在用铒激光行镫骨手术中,发现2.79 nm波长的激光由于压力波的作用而导致10 nm范围毛细胞的损伤,因此,激光镫骨手术后的前庭反应及高频听力下降,是否与激光的物理效应有关,尚需进一步的探讨。
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    激光手术是一种较为先进的治疗方法,在进行镫骨手术时有独特的优点:(1)可精确定位,避免了以力学手段作镫骨切除时可能遇到的困难;(2)手术创伤小,术后并发症少。但激光的热效应和压力效应,同时也可能带来一些有害的生物学影响。在进行镫骨足板手术时,必须选择适宜的、安全的应用剂量,以达到快速、安全、有效的目的。目前我们仅仅观察了不同功率的激光对内耳感受器结构的影响,有关结构变化与听力损伤的动态关系,还需进一步的探讨。

    作者简介:康梦奎(1941-),女,北京人,中国人民武装警察部队总医院主任医师,主要从事激光医学研究特别是低功率激光在耳鼻喉科应用的基础研究。

    参考文献

    [1] Segas J, Georgiadis A, Christodoulou P, et al. Use of the excimer laser in stapes surgery and ossiculoplasty of middle ear ossicles: preliminary report of an experimental approach[J]. Laryngoscope, 1991, 101:186-191.
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    [2] Kautzky M, Trodhan A, Susani M, et al. Infrared laser stapedotomy[J]. Eur Arch Otorhinolaryngol, 1991, 248:449-451.

    [3] Jovanovic S, Schonfeld U, Fischer R, et al. Thermal stress of the inner ear during laser stapedotomy. 1.Continuous-wave laser[J]. HNO, 1995,43:702-709.

    [4] Coker NJ, Ator GA, Jenkin HA, et al. Carbon dioxide laser stapedotomy. Thermal effects in the vestibule[J]. Arch Otolaryngol, 1985, 111:601-605.
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    [5] Pratisto H, Frenz M, Ith M, et al. Temperature and pressure effects during erbium laser stapedotomy[J]. Lasers Surg Med, 1996, 18:100-108.

    [6] Thoma J, Unger V, Kastenbauer E. Measurement of temperature and sound pressure in the inner ear during application of the argon-laser[J]. Laryngol Rhinol Otol, 1981, 60:587-90.

    [7] Jovanovic S, Schonfeld U, Prapavat V, et al. Effects of pulsed laser systems on stapes footplate[J]. Lasers Surg Med, 1997, 21:341-350.

    (收稿日期:1999-08-17), 百拇医药