用4．7特斯拉磁共振成像活体内观测豚鼠实验性内淋巴积水

目的:探讨用4．7特斯拉试验用磁共振成像系统能否在豚鼠中检测内淋巴积水。方法:20只白色或者杂色豚鼠用于该研究。5只正常豚鼠作为对照组,15只豚鼠用于制作内淋巴积水模型。9只内淋巴囊破坏组中的5只和6只内淋巴囊完整组(与乙状窦游离)动物采用 gadolinium(Gd)-DTPA-BMA增强MRI检测内淋巴积水。结果:由于 Gd-DTPA-BMA主要进入鼓阶和前庭阶,耳蜗的三个阶可在所有动物中由MRI清晰显示。在内淋巴囊完整组,内淋巴囊手术后6天MRI即可检测到内淋巴积水,并且由组织学证实。在内淋巴囊破坏组中的1 只动物,因内耳屏障的严重破坏而使Gd-DTPA-BMA快速漏入中阶, MRI可检测到该变化,其听力损失为60dB。结论:用Gd-DTPA-BMA 增强的高分辨MRI可检测出内淋巴积水,有可能对积水程度进行定量测试。在Gd-DTPA-BMA的帮助下,内耳屏障损伤或可能的膜破裂可以被检出。图4表4参12     

用MRI体内观测钥孔嘁血蓝素中耳免疫诱导的内淋巴积水的初步探讨

目的用磁共振成像方法研究中耳免疫反应诱导的动物内淋巴积水.方法将9只豚鼠分为2组,中耳免疫组(4只)和磷酸盐缓冲液(Phosphate buffered saline,PBS)对照组(5只).先用KLH加福氏完全佐剂行四肢趾蹼免疫,2周后将KLH投放至中耳进行局部免疫.另外5只豚鼠中耳放置PBS作为对照组.用gadolinium增强的磁共振成像观察内耳淋巴液的动态变化,用耳蜗电图评估听功能改变.结果在KLH中耳免疫的动物中,2只发生内淋巴积水,3只血迷路屏障通透性增加,2只听力损失大于10dB.结论磁共振成像可以显示KLH中耳免疫反应的耳蜗改变,内淋巴积水和血迷路屏障通透性的增加提示存在着一个"漏的迷路",将有可能为内耳疾病的诊断提供更加精确的依据.     

同种异体内耳组织免疫后豚鼠内耳形态学变化

目的建立一高阳性率、可供圆窗给药治疗内耳病研究用的自身免疫性内耳病动物模型。方法实验动物为86只豚鼠,其中32只用于制备粗制内耳抗原。其余动物随机分为实验组42只,对照组1、2各6只。将实验组动物腹腔注射环磷酰胺进行预处理,2d后用粗制内耳抗原行皮下多点接种,分别于接种后4、6、8、10、12、14、20d时用光镜观察动物内耳形态学变化,并检测其血清中IgG、IgM、C3、CH50、循环免疫复合物(CIC)水平。对照组1不行任何处理,对照组2不接种内耳抗原,余同试验组。结果接种后豚鼠耳蜗、前庭、内淋巴囊等部位出现以淋巴细胞为主的炎性细胞浸润,尤以前庭阶、鼓阶、螺旋神经节区域及耳蜗底圈等部位为重。接种后4d时,67%动物内耳有炎性细胞浸润;8~12d时,100%动物出现炎性细胞浸润;接种14d后,炎性细胞浸润明显减少。接种6~14d时,内耳尚有血管周围炎、螺旋神经节细胞变性、内淋巴积水等改变。结论将豚鼠采用环磷酰胺预处理及同种异体内耳抗原接种,可建立一高阳性率的自身免疫性内耳病动物模型。     

内淋巴积水程度的定量分析

目的　探讨实验性内淋巴积水动物模型积水程度的定量分析方法。方法　 30只豚鼠随机分为 3组 :对照组 10只 ,双耳未手术 ;2 0只豚鼠用右侧枕后硬脑膜外进路阻塞内淋巴囊的方法制备内淋巴积水的动物模型 ,其中术后 4、8周 2组各 10只。应用计算机辅助设计软件 (AutoCADR14 )分别测量双侧耳蜗中轴左右两侧 1～ 4回前庭阶 (scalavestibuli,SV)和中阶面积 ,计算最大中阶面积比率 ,并作比较。结果　所有未手术耳均无内淋巴积水 ,所有手术耳有不同程度的内淋巴积水。最大中阶面积比率 :术后 4周组 (2 2 2 31± 0 1996 )比对照组 (1 0 971± 0 0 6 4 4 )显著增大 ,术后 8周组 (4 0 14 2± 0 5 2 18)比术后 4周组更大 ,差异均有统计学意义 (P <0 0 5 )。结论　此测量方法方便可靠 ,可应用于定量分析内淋巴积水程度。     

施用耳毒性药物于内淋巴囊的作用

作者用豚鼠做实验,探讨为治疗梅尼埃病施行内淋巴囊减压术的同时,把氨基糖甙类药物施用于内淋巴囊,观察其对内耳感受器的影响,以及药物从内淋巴囊向前庭和耳蜗的扩散机制。33只豚鼠,在全麻无菌条件下,经硬膜外颅后凹进路到达内淋巴囊。第一组把10μl庆大霉素(40mg/ml)放在8只豚鼠的内淋巴囊壁表面。第2组将10μl庆大霉素注入8只豚鼠的内淋巴囊内。第三组在17只豚鼠的前庭导水管裂隙周围做一小孔,把4～5粒链霉素粉球放人内淋巴囊腔内,总药量16～20μg,然后修复术创。一月后,无痛处死动物,取出颞骨做切片染色,在光镜下观察,结果发现,第一组对内耳感受器损害不明显,仅4只豚鼠(其中     

正常和膜迷路积水豚鼠内耳水通道蛋白的表达及意义

目的检测正常和膜迷路积水豚鼠内耳水通道蛋白（aquaporins,AQPs）的表达,探讨其机理和意义。方法 20只健康豚鼠随机分为实验组和对照组,每组10只,实验组（膜迷路积水模型组）豚鼠腹腔注射醋酸去氨加压素,4μg&#183;kg-1&#183;d-1,共1周,诱导其内耳膜迷路积水,对照组豚鼠腹腔注射等量生理盐水。用免疫组化方法检测两组豚鼠内耳水通道蛋白的表达,并进行比较。结果对照组豚鼠内耳中有AQP0、1、2、3、5、7、8的表达,其中AQP0仅在血管纹和螺旋神经节细胞有较弱的表达;AQP1分布于包绕骨迷路、内淋巴囊、内淋巴管的纤维细胞,基底膜鼓阶面细胞、螺旋韧带纤维细胞、螺旋缘纤维细胞、Corti’s器、内外螺旋沟、血管纹、椭圆囊壁、球囊壁、螺旋神经节等;AQP2表达在血管纹、Corti’s器、螺旋神经节细胞和内淋巴囊中;AQP3、7、8三者的分布类似,在螺旋神经节和包绕膜迷路的组织中表达,包括Corti’s器、内外螺旋沟、血管纹、螺旋韧带纤维细胞、螺旋缘、椭圆囊壁、球囊壁、内淋巴囊等;AQP5则表达在Corti’s器、内外螺旋沟、螺旋神经节、螺旋韧带纤维细胞。实验组豚鼠内耳中,血管纹AQP2的表达与对照组较正常豚鼠表达增加,其它亚型AQPs的表达与对照组无明显差异。结论正常豚鼠内耳中有多种AQPs表达,膜迷路积水后豚鼠内耳中AQP2的表达增强,提示膜迷路积水可能与AQP2表达上调有关。     

内淋巴囊远侧端全切或烧灼后的内淋巴积水

传统的实验性内淋巴积水是将大部分内淋巴囊和，前庭导水管破坏，使内淋巴囊内淋巴管广泛纤维化或完全闭锁致内淋巴管囊功能严重障碍，这与临床梅尼埃病患者的内淋巴囊及内淋巴管仍为开放状态大相径庭。该作者通过全切或烧灼邻近乙状窦的内淋巴囊骨外部分，成功建立了更接近人类梅尼埃病的中度到重度的内淋巴积水的动物模型。实验选用健康雌性豚鼠17只，平均体重25Og，分为两组，第一组12只，手术全切同乙状窦相邻的内淋巴囊远侧端；第H组5只，用硝酸银不同程度地烧灼内淋巴囊的远侧端。每只豚鼠一耳用作试验，另一耳作对照。经上述处理3周…     

耳蜗振动创伤后血迷路屏障变化的磁共振成像表现

目的 在动物模型中探讨电钻振动诱导听力减退的机制和可能的血迷路屏障变化。方法 用电磁振荡器在5只豚鼠听泡上产生可重复的振动，其振动频率为250Hz，用复合动作电位评估其听功能，振动后3小时至4天用4．7特斯拉磁共振成像分析其血迷路屏障变化。静脉注射T1增强剂Gadolinium-diethylenetriaminepentaacetate-bismethylamide （Gd-DTPA-BMA）作为血迷路屏障的示踪剂。结果 振动诱导平均听力损失40dBHL，在振动后立即测试的耳蜗中．其中阶摄取Gd-DTPA-BMA，提示血迷路屏障通透性增加。在振动后2天至4天的动物中，其中阶内无Gd-DTPA-BMA摄取。结论 250Hz振动使血路屏障通透性增加是使导致听力减退的原因之一。其通透性的变化为一个可逆的过程。     

两种内淋巴积水动物模型中水通道蛋白1的表达

目的了解水通道蛋白1（aquaporin 1，AQP1）在手术及醛固酮引起的内淋巴积水豚鼠耳蜗及内淋巴囊中的表达情况。方法30只健康豚鼠随机分为手术组、醛固酮注射组和对照组，每组10只。通过手术阻塞内淋巴囊和腹腔注射醛固酮的方法制造出两种内淋巴积水动物模型，采用免疫组化染色及蛋白质印迹法检测豚鼠耳蜗及内淋巴囊中AQP1的表达，通过图像处理软件进行半定量分析。结果手术组出现中、重度内淋巴积水，以顶回最为明显，自顶回向底回减轻；醛固酮注射组出现轻、中度积水，积水多位于底回。两组积水动物模型中AQP1表达的部位与对照组相同，手术组耳蜗与对照组相比AQP1表达差异无统计学意义（t=0．718，P〉0．05）；醛固酮组耳蜗AQP1表达低于对照组（t=6．609，P〈0．01）；对照组与醛固酮组内淋巴囊AQP1表达差异无统计学意义（t=0．998，P〉0．05）。醛固酮组耳蜗外侧壁组织中AQP1蛋白定量低于对照组（t=13．626，P〈0．01）。结论AQP1在手术引起的内淋巴积水中表达没有改变，在醛固酮引起的内淋巴积水中表达下降。AQP1在豚鼠内耳中的表达可能受离子浓度的调节。     

豚鼠内耳钆喷酸葡胺MRI显像的初步探讨

目的 通过MRI观察豚鼠鼓膜穿刺听泡内注射钆喷酸葡胺后内耳增强显影的特征,观察不同时间点造影剂在内耳的分布,找出内耳增强显影的最佳时间,同时了解钆喷酸葡胺在内耳的药代动力学特点,探讨在现有实验条件下内、外淋巴区分显影的可行性.方法 65只豚鼠随机数字表法分为13组,每组5只.豚鼠鼓膜穿刺听泡内注射生理盐水稀释8倍的钆喷酸葡胺,分别在注射前、注射后0.5、1、2、4、6、8、10、12、24、48、72及96 h行内耳MRI扫描(3D-T1 FSE序列).使用e-Film软件对内耳各部位MRI图像灰度值进行提取,应用体外试验获得的灰度值-造影剂浓度关系将灰度值转化成浓度.分别测量注射前、注射后1 d及7 d豚鼠左耳(生理盐水)和右耳(稀释造影剂)的听性脑干反应(ABR)阈值并进行对比.结果 注射后6 h为造影剂扩散至全内耳并达到较好显影条件的时间点,也即造影剂在内耳各部分达到较高浓度的时间,此时造影剂在前庭、底转鼓阶、底转前庭阶、第二转、第三转、顶转的浓度分别为589.29、552.54、570.17、255.08、107.09、139.18 μmol/L;造影剂选择性进入外淋巴,未见内淋巴增强显影.造影剂注射后1 d及7 d豚鼠左、右耳ABR阈值相比,差异无统计学意义(P值均＞0.05).结论 豚鼠听泡内注射钆喷酸葡胺后6 h为MRI内耳增强显影的最佳观察时间.造影剂可选择性显影外淋巴,对豚鼠ABR阈值无明显影响.通过MRI可以间接研究钆喷酸葡胺在内耳的代谢特点.     

In vivo visualization of endolymphatic hydrops in guinea pigs: magnetic resonance imaging evaluation at 4.7 tesla

In order to find out whether it is possible to visualize experimental endolymphatic hydrops in the cochlea with magnetic resonance imaging (MRI) at 4.7 T, we used 11 guinea pigs. Five normal guinea pigs were used as controls. Early manifestation of endolymphatic hydrops was evaluated in endolymphatic sac (ES)-intact animals (n = 6), and advanced manifestation in ES-damaged animals (n = 5) by means of MRI with gadolinium-diethylenetriaminepentaacetate-bismethylamide (Gd-DTPA-BMA) contrast agent. Hearing was tested with electrocochleography. The surface area of 3 partitions of the cochlea was used to quantify endolymphatic hydrops. The fine structure of the 3 partitions of the cochlea was visualized with MRI in all animals, as Gd-DTPA-BMA appeared mainly in the scala tympani and scala vestibuli. As early as 5 days after endolymphatic sac surgery, endolymphatic hydrops started to appear as visualized by MRI and also verified with histology. Severe damage to the inner ear barrier with Gd-DTPA-BMA leakage into the scala media was detected with MRI in 1 ES-damaged animal that had a 60-dB hearing loss. To conclude, endolymphatic hydrops can be visualized with high-resolution MRI by means of Gd-DTPA-BMA, and it is possible to quantify the extent of endolymphatic hydrops. Damage to the inner ear barrier or possible rupture of membranes can be shown with the assistance of Gd-DTPA-BMA.     

The quantification of endolymphatic hydrops in an experimental animal model with guinea pigs

To better quantify endolymphatic hydrops in experimental guinea pigs, endolymphatic hydrops was induced by endolymphatic sac obliteration through an extradural posterior cranial fossa approach in the right ear. The area of the scala vestibuli and scala media of each turn on both cochlear midmodiolar sections was measured, using an automated computer-aided design (AutoCAD R14) software combined with a digital camera. No endolymphatic hydrops was observed in all nonoperated ears; however, various degrees of hydrops were present in all operated ears. The average maximum SMA (scala media area) ratio in the 4-week group (2.22 +/- 0.20) was greater than that in the control group (1.10 +/- 0.06). The average maximum SMA ratio of the 8-week group (4.04 +/- 0.52) was greater than that of the 4-week group (p < 0.05). This study provides a reliable methodologic base for the experimental study of Ménière's disease.     

Expression of inducible nitric oxide synthase in the cochlea following immune response in the endolymphatic sac of guinea pigs

Immunohistochemical study for inducible nitric oxide synthase (iNOS or NOS II) in the cochlea of guinea pigs was performed after the injection of keyhole limpet hemocyanin (KLH) into the endolymphatic sac. Morphological changes were observed in the cochlea of all animals after the injection of KLH. Increased iNOS expression was detected in the lateral wall, organ of Corti and ganglion cells. It is known that high levels of nitric oxide can lead to inner ear dysfunction. Our results suggest that iNOS may mediate the inner ear disturbance as seen in endolymphatic hydrops.     

内淋巴积水程度的定量分析

OBJECTIVE: To investigate a method of quantification of the endolymphatic hydrops in experimental animal model. METHODS: Thirty guinea pigs were divided into three groups at random. In control group, there were ten guinea pigs without operation on both ears. Endolymphatic hydrops was induced by endolymphatic sac obliteration through extradural posterior cranial fossa approach in right ear, including 4-week postoperative group(n = 10) and 8-week postoperative group(n = 10). The area of scala vestibuli (SV) and scala media(SM) of each turn on both cochlear midmodiolar sections was measured, respectively, using auto computer aided design(AutoCAD R14) software combined with digital camera, and then the maximum scala media area(SMA) ratio was calculated and compared. RESULTS: No endolymphatic hydrops was observed in all non-operated ears, however, variety degree of hydrops was present in all operated ears. The average maximum SMA ratio in the 4-week group (2.2231 +/- 0.1996) was greater than that in the control group (1.0971 +/- 0.0644). The average maximum SMA ratio of the 8-week group (4.0142 +/- 0.5218) was greater than that in the 4-weekgroup. There was significant difference between the two groups(P < 0.05). CONCLUSION: It is convenience and reliable to be used to quantify the experimental endolymphatic hydrops with the present method. This study provides a reliable methodological base for the experimental study of Meniere's disease.     

The effect of glycerol on short-term experimental endolymphatic hydrops

The aim of this paper was to evaluate the effect of one month of treatment with different dosages of glycerol on experimental endolymphatic hydrops produced by obliteration of endolymphatic sac and duct through an extradural approach. Forty-two guinea pigs were used. The animals, divided into six groups, received 0.5-2 gr/kg body weight of the drug per day for one month. The activity of glycerol was determined by statistical analysis of volumetric changes of scala media. Glycerol demonstrated the effect of reducing endolymphatic hydrops. The decrease in hydrops was influenced by the dosage suggesting a stria metabolic response.     

Antigen Diffusion from the perilymphatic space of the cochlea

The diffusion pattern of horseradish peroxidase (HEP) injected into the scala tympani of the cochlear basal turn of guinea pigs was studied to test whether antigen presented in this manner can gain access to the endolymphatic sac. By two hours, HEP reaction product was found throughout the cochlea, with the greatest amounts in the spiral ligament, spiral lim-bus, basilar membrane, and organ of Corti. In several cochleas, very weak labeling was seen in the stria vascularis. HEP reaction product was maximal in the basal turn. By two hours, HEP reaction product was also observed in the endolymphatic sac lumen, epithelial cells, subepithelial tissue, and perisaccular connective tissue. It was more common in the proximal portion. At this time, macrophages within the lumen already appeared to have phagocytosed the HEP. By 72 hours after injection, the inner ear was cleared of HEP. The results of this study support the hypothesis that antigen in the scala tympani gains access to the endolymphatic sac lumen, where it may be presented by macrophages to the systemic immune system. Antigen most likely does not gain access to the endolymphatic space in the cochlea, but it gets to the endolymphatic sac through the perilymph and the Derisaccular tissue.     

Morphology of the endolymphatic sac in the guinea pig after an acute endolymphatic hydrops

The role of the endolymphatic sac (ES) in endolymph volume homeostasis is speculative. The present study investigates changes of the ES's epithelia and luminal filling after induction of an acute endolymphatic hydrops. After microinjection of 1.1 mul artificial endolymph into scala media of the cochlea, guinea pigs were terminated immediately (n = 6) or after different time intervals ; 1/2 h (n = 3), 1 h (n = 4) and 2 h (n = 4). Inner ear specimens were processed for light and/or transmission electron microscopy. The non-injected contralateral ear served as a histological control. Correct injection was confirmed by detection of microspheres in the endolymphatic compartment after the same microinjection procedure. In all specimens, ribosome rich cells and intraluminal macrophages appeared to be actively involved in degradation of homogeneous substance (HS) by secreting lytic enzymes and digestion, respectively. Amazingly, in our study no ES differences were found between injected and non-injected ears and no distinct changes were observed in guinea pigs terminated after different time intervals. The ES's luminal HS was always present and often to a large extent. This is in contrast with [Hear. Res. 138, 81] dramatic changes were observed. Endolymph volume homeostasis is a complex mechanism, in which the role of HS remains obscure.     

Perilymphatic and endolymphatic pressures during endolymphatic hydrops

We analysed the fluid pressure in the perilymphatic and endolymphatic spaces of the cochlea in eight guinea pigs after endolymphatic hydrops (EH) was surgically created. The control experiments were done in the opposite ears. EH was histologically confirmed after the conclusion of the pressure measurements. No statistically significant pressure difference between the scala media and the scala tympani was found, either in ears with EH or the control ears. However, the endocochlear potential, which we evaluated for position verification of the measurement pipette in the cochlea, was statistically significantly decreased in ears with EH.