大鼠膜迷路积水模型的建立及评定

目的 通过腹腔注射醋酸去氨加压素(DDAVP)探讨建立大鼠膜迷路积水模型的可行性.方法 选取30只健康SD大鼠,随机分为正常组、对照组及实验组,不同时间节点分别给予不同剂量的DDAVP及生理盐水腹腔注射,17天后观察大鼠行为学变化,听觉脑干诱发电位(ABR)检测听力的变化,HE染色观察耳蜗形态结构的变化,耳蜗中阶(即蜗...     

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·kg-1·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。     

膜迷路积水产生的机制

膜迷路积水被认为是临床上梅尼埃病等耳科疾病的基本病理改变.目前关于膜迷路积水产生原因的动物模型有内淋巴囊填塞法、注射血管加压素法、免疫法、两相法、腹腔注射醛固酮法等,每种模型都有各自的特点,但不同造模方法又有其各自的局限性.有关膜迷路积水产生的原因和机制,目前仍不清楚,本文就常见的几种被认为是产生膜迷路积水的根本原因及机制综述如下.     

P38MAPK调控AQP2参与内淋巴积水的形成北大核心CSCD

目的探讨P38MAPK是否通过调控AQP2参与内淋巴积水的形成。方法筛选合格豚鼠30只,随机分为三组:空白对照组(6只):腹腔注射生理盐水;积水组(12只):腹腔注射醋酸去氨加压素;抑制剂组(12只):腹腔注射醋酸去氨加压素15min后,腹腔注射SB203580。所有组别豚鼠连续给药10天,在停止给药7天后行ABR和DPOAE检测,并在检测后同时取材,HE染色观察各组豚鼠内淋巴积水程度,免疫组化和qRT-PCR分别检测PP38MAPK、AQP2蛋白和mRNA在各组豚鼠耳蜗表达情况。结果1、行为学观察及听力检测:各组无明显差异。2、形态学观察:空白对照组积水率为0%;积水组积水率为75%;抑制剂组积水率为33%。3、免疫组化:P-P38MAPK:在各组豚鼠耳蜗中均有表达,积水组>抑制剂组>空白对照组(P<0.01)。AQP2:在各组豚鼠耳蜗中均有表达,除螺旋神经节外,积水组>抑制剂组>空白对照组(P<0.01)。4、荧光定量PCR检测结果:P38MAPK、AQP2:积水组>抑制剂组>空白对照组(P<0.01)。结论P38MAPK通过调控AQP2参与内淋巴积水的形成;P38MAPK抑制剂可减轻内淋巴积水程度。     

水通道蛋白1、2、5在膜迷路积水豚鼠耳蜗中的分布及表达研究

目的观察水通道蛋白(aquaporin,AQ)1、2、5(AQP1、AQP2、AQP5)三种蛋白在膜迷路积水豚鼠耳蜗的表达及分布情况,探讨其在内淋巴积液中可能的发生机制。方法选择健康红目豚鼠60只,随机分为空白组(30只)和模型组(30只),模型组给予醋酸去氨加压素6μg·kg-1·d-1腹腔注射10 d,空白组以1 ml生理盐水腹腔注射10 d;常规饲养7天后取出耳蜗,通过免疫组化、Western-blot方法观察AQP1、AQP2、AQP5蛋白在豚鼠耳蜗的分布及表达。结果免疫组化示空白组、模型组豚鼠耳蜗组织中AQP1表达的光密度值分别为0.134±0.021、0.273±0.053;AQP2表达分别为0.089±0.013、0.261±0.100;AQP5表达分别为0.264±0.065、0.151±0.098;与空白组比较,模型组AQP1、AQP2蛋白表达明显上调(P<0.01),AQP5蛋白表达明显下调(P<0.05)。Western-blot示空白组、模型组豚鼠耳蜗组织中AQP1表达分别为0.214±0.034、0.313±0.032;AQP2表达分别为0.283±0.050、0.544±0.042;AQP5表达分别为0.291±0.041、0.199±0.033;与空白组比较,模型组AQP1、AQP2蛋白表达上调(P<0.01),AQP5蛋白表达明显下调(P<0.05)。结论AQP1、AQP2、AQP5蛋白均可能参与了豚鼠耳蜗膜迷路积水的发生。     

单核细胞趋化蛋白-1及脂蛋白(a)与梅尼埃病关系的研究

目的探讨单核细胞趋化蛋白-1(monocyte chemotactic protein-1,MCP-1)及脂蛋白(a)[lipoprotein(a),LP(a)]与梅尼埃病的关系。方法选取30只健康SD大鼠,分成正常组、对照组及实验组,每组10只,实验组腹腔注射醋酸去氨加压素(4μg·kg^-1·d^-1连续注射7 d后改为6μg·kg^-1·d^-1连续注射3 d)行膜迷路积水造模,对照组腹腔注射等量生理盐水,正常组不作处理,停药7 d后对三组动物行ABR检测,取耳蜗中阶横截面积(SM)/(中阶+前庭阶横截面积)(SM+SV)比值评价膜迷路积水情况。造模成功后通过免疫组化染色检测耳蜗组织中MCP-1的表达,通过酶联免疫吸附实验(ELISA)法检测血清中LP(a)的水平,对LP(a)水平与内耳积水程度及大鼠ABR各指标进行相关性分析。结果实验组波Ⅱ阈值升高、波Ⅱ、Ⅴ潜伏期及Ⅲ-Ⅴ波间期较正常组和对照组均延长,差异有统计学意义(P<0.05)。实验组、对照组、正常组SM/(SM+SV)比值分别为0.45±0.04、0.34±0.02、0.35±0.01,前者显著大于后两者(P<0.05)。实验组MCP-1在耳蜗螺旋器、血管纹螺旋韧带及神经节细胞组织表达明显,前庭膜上也有表达,其它两组无表达。实验组、对照组、正常组LP(a)水平分别为4.07±0.20、1.36±0.12、1.38±0.14 mg/L,前者显著高于后两者(P<0.001),LP(a)表达水平与膜迷路积水程度及ABR各指标间均呈正相关(P<0.001)。结论膜迷路积水模型大鼠耳蜗组织中MCP-1表达明显,血清LP(a)水平明显升高,且LP(a)水平与膜迷路积水程度及大鼠听力下降呈正相关,提示MCP-1和LP(a)可能与梅尼埃病的发生有关。     

加压素对大鼠内耳细胞信号转导基因表达的影响

目的研究加压素对大鼠内耳细胞信号转导有关基因表达的影响,探讨加压素引起膜迷路积水的发生机制.方法大鼠腹腔注射精氨酸加压素50μg/kg,每天1次,共1周;取出听泡,提取内耳总RNA,逆转录成cDNA并标记;然后和大鼠cDNA芯片杂交,显示加压素注射前后大鼠内耳mRNA表达强度变化.结果筛选出和细胞信号转导有关的差异表达基因10条,Ratio＞5的上调的基因有Chnl,Pak3和Ptprc.结论加压素可能从细胞信号转导基因表达方面影响内耳的液体平衡,从而导致膜迷路积水.     

血管加压素-水通道蛋白2调控通路与内耳膜迷路积水

在肾脏集合管,血管加压素(arginine vasopressin,AVP)通过AVP-血管加压素受体2(vasopressin type 2 receptor,V2R)-环磷酸腺苷(cyclic adenosine monophosphate,cAMP)-水通道蛋白2(aquaporin2,AQP2)调控通路引起AQP2表达改变,实现对肾集合管水通透性的调节,参与人体水电解质的调节机制改变。膜迷路积水是梅尼埃病的重要病理特征,AVP-AQP2调控通路功能失常与膜迷路积水的病理生理学改变密切相关。本文综述了近年来AVP-AQP2调控通路的研究进展,并重点讨论其在梅尼埃病膜迷路积水发生发展中的作用。     

补体活化与膜迷路积水（摘要）

补体活化与膜迷路积水（摘要）王红生，赵沛英，李志光，郭敏，马大龙近年来的临床和实验研究提示，Ｍｅｎｉｅｒｅ病膜迷路积水的形成与Ⅲ型变态反应有关。本实验用酵母多糖活化豚鼠血清中的补体，将其上清液行豚鼠颈动脉注射，对受试动物进行听功能和形态学观察。一、材...     

加压素对大鼠内耳囊泡介导转运有关基因的影响

目的研究加压素对大鼠内耳囊泡介导转运有关基因的影响,探讨加压素引起膜迷路积水的发生机制。方法Wistar大鼠16只,分为实验组和对照组,每组8只,实验组大鼠腹腔注射精氨酸加压素50μg/kg,每天1次,共1周,对照组大鼠腹腔注射等量生理盐水;用药一周后,取出听泡,提取内耳总RNA,逆转录成cDNA并标记;然后和大鼠cDNA芯片杂交,显示注射加压素前后大鼠内耳mRNA表达强度变化。结果筛选出和囊泡介导转运有关的差异表达基因7条,Ratio>2的上调的基因有Ap2b1,Arf1,Arf6,Dnch1,Mlc3和Rab7,Ratio<0.5的下调的基因有Stx7。结论加压素可能从囊泡介导转运方面影响内耳的液体平衡,从而导致膜迷路积水。     

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.     

Cochlear blood flow in endolymphatic hydrops

There is no animal model for Meniere's disease but by obliteration of the endolymphatic duct, endolymphatic hydrops may be achieved in several animal species. In order to measure the cochlear blood flow in ears with endolymphatic hydrops the endolymphatic duct was obliterated in 9 guinea pigs. The blood flow was measured with the microsphere method and the cochlear histology was studied. The regional and total blood flow was determined in the serially sectioned cochleas 2, 4 and 8 months after obliteration of the endolymphatic duct. No change in regional or total cochlear blood flow was observed in the hydropic ears.     

Protective effect of edaravone against endolymphatic hydrops

CONCLUSION: Our findings suggest that edaravone prevented the production of reactive oxygen species (ROS). Edaravone also delayed the formation of endolymphatic hydrops in guinea pigs, but had no effect on endolymphatic hydrops. OBJECTIVE: To analyse the protective effect of a free radical scavenger, edaravone, on endolymphatic hydrops. MATERIALS AND METHODS: Guinea pigs were subjected to surgical obliteration of the endolymphatic duct (ED). For the detection of ROS, group 1 received intraperitoneal injections of edaravone (3 mg/kg/day) for 2 days, group 2 received edaravone for 2 weeks, group 3 saline for 2 days, and group 4 saline for 2 weeks. ROS production by the organ of Corti and stria vascularis was examined by using dihydrotetramethylrosamine. For the morphological analysis, guinea pigs were divided into five groups, i.e. 2 or 4 weeks after ED obliteration, 2 weeks with edaravone, first or last 2 weeks with edaravone and sacrificed 4 weeks after ED obliteration. Increases in the ratios of the cross-sectional area of scala media were analysed quantitatively to assess the degree of endolymphatic hydrops among the above-mentioned five groups of the hydropic cochlea. RESULTS: ROS was detected both in the organ of Corti and in the lateral wall of cochleae 2 days after ED obliteration. Edaravone prevented the production of ROS and also attenuated the formation of endolymphatic hydrops in the acute hydrops group.     

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。     

Effects of hyperbaric therapy on function and morphology of Guinea pig cochlea with endolymphatic hydrops.

OBJECTIVE: The objective of this study was to investigate the effect on experimental endolymphatic hydrops in guinea pigs after hyperbaric therapy. BACKGROUND: The histopathologic character of Ménière's disease is the presence of endolymphatic hydrops. Endolymphatic hypertension could be one of the factors resulting from endolymphatic hydrops. Some treatments of Ménière's disease are aimed toward preventing the endolymphatic hypertension. Exposure to pressure change has risen in recent years. METHODS: Thirty-two guinea pigs were operated on the right ears to induce endolymphatic hydrops by obliterating the endolymphatic sac through an extradural posterior cranial fossa approach. After 5 weeks' survival, 12 guinea pigs were put into a chamber with an absolute atmospheric pressure of 2.2 for 3 weeks (90 minutes once a day 5 times a week). We observed the morphologic and functional changes in guinea pig cochleae of the pressure group, 4-week hydrops group (n = 10), 8-week hydrops group (n = 10), and the normal group (n = 10). We measured the hearing threshold of the auditory brainstem response, the 70-dB SPL action potential (AP) latency, the ratio of 70-dB SPL summating potential magnitude to action potential magnitude (-SP/AP) of the electrocochleogram, and the maximum scala media area (SMA) ratio, respectively. RESULTS: The average 70-dB SPL-SP/AP magnitude of right ears (0.29 +/- 0.09) and the average maximum SMA ratio (2.23 +/- 0.20) in the pressure group were significantly less than that in the 8-week hydrops group (0.69 +/- 0.15 and 4.04 +/- 0.52, respectively) with the same survival time (p < 0.05). The results in the pressure group were almost as similar as that in the 4-week hydrops group (0.29 +/- 0.13 and 2.22 +/- 0.20, respectively) (p > 0.05). The average hearing threshold of ABR of right ears in the pressure group (36.67 +/- 14.30-dB SPL) was lower than that of the 8-week hydrops group (44 +/-1 4.30-dB SPL), but the difference was insignificant (p > 0.05). The average 70-dB SPL AP latency of right ears in the pressure group was not significantly different from those of the 8-week hydrops group, the 4-week hydrops group, or the normal group (p > 0.05). CONCLUSIONS: Our findings suggest hyperbaric therapy can significantly suppress the development of endolymphatic hydrops and improve cochlear function in guinea pigs. This study provided strong evidence for the development of pressure treatment of Ménière's disease without destroying the inner ear.     

Obliteration of vestibular and cochlear aqueducts in the guinea pig

The study involved 42 healthy adult guinea pigs placed into two major groups. In group A, only histologic studies were performed after obliteration of the right vestibular aqueduct, the cochlear aqueduct, or both the vestibular and cochlear aqueducts. In group B, hearing changes were monitored by repeated determination of averaged temporal response thresholds after the same obliterative operations as in group A. At the conclusion of the experiments, histologic studies of temporal bone were performed and hearing changes were correlated with the severity of disease found in the inner ear. Histologic examination of 32 temporal bones from animals in which the vestibular aqueduct or both the vestibular and cochlear aqueducts were obliterated consistently demonstrated endolymphatic hydrops. The endolymphatic hydrops was usually progressive, and its severity was proportional to the length of time from the operation. Serial determination of averaged temporal response thresholds in 18 guinea pigs after these operations demonstrated a progressive deterioration in thresholds. The hearing loss was greater at the lower frequencies. The audiometric changes correlated positively with the severity of endolymphatic hydrops. No significant audiometric or temporal bone histologic changes were found after obliteration of the cochlear aqueduct.     

Effects of a nitric oxide synthase Type II inhibitor on compound action potential thresholds in experimental endolymphatic hydrops.

HYPOTHESIS: Nitric oxide (NO) is likely to be synthesized by nitric oxide synthase Type II (NOS II) action and may partake in the origin of changes of compound action potential (CAP) threshold observed in guinea pigs with induced endolymphatic hydrops. This study aimed to assess the action of a NOS II inhibitor on CAP thresholds in these experimental samples. BACKGROUND: In guinea pigs with experimental endolymphatic hydrops, there are lesions on the cochlea and progressive increase of CAP threshold. NOS II was found in the cochlea of this animal model, and it was inferred that NO can contribute by such alterations. METHODS: The animals were divided into two groups, in which eight received an intake of a NOS II inhibitor, aminoguanidine, and another eight served as a control group. During 16 weeks, CAP thresholds at 1,000, 2,000, 4,000 and 6,000 on electrocochleography were compared between the groups. RESULTS: The group that had an intake of aminoguanidine showed a lower increase on CAP thresholds at 2,000 (p < 0.05) and 6,000 Hz (p < 0.05) at the 12th postoperative week, and at 1,000 (p < 0.05), 2,000 (p < 0.001), 4,000 (p < 0.001), > and 6,000 Hz (p < 0.001) at the 16th week. CONCLUSION: We conclude that NOS II inhibitor reduced the elevation of CAP thresholds in experimentally induced endolymphatic hydrops.     

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.     

Evaluation of the effect of dexamethasone in experimentally induced endolymphatic hydrops in guinea pigs

PURPOSE: The aim of this study was to investigate the audiological and histopathologic effects of dexamethasone in the treatment of experimentally induced endolymphatic hydrops. MATERIALS AND METHODS: Thirty mature, male guinea pigs weighing 400 +/- 50 g were operated on to induce experimental endolymphatic hydrops in their right ear. Left ear served as control. Subjects were separated into control and dexamethasone groups, with the latter receiving dexamethasone 5 mg/(kg d) intraperitoneally for 10 days. Electrocochleography and auditory brainstem response were applied to all subjects at preoperation, on the second postoperative day and also on the 15th postoperative day in animals that lived for a long time. The histopathologic examination of the inner ear in all animals was done at the end of the study. RESULTS: The summating potential and the ratio of the summating potential to the action potential measured on the second postoperative day were found to be increased in both groups, but more significantly in the control one. When the left and right ears were compared, significant difference was found in the control group; however, no significant difference was found between the ears in the dexamethasone group. Histopathologic examination revealed varying degrees of hydrops in the control group, but showed only normal findings or minor changes in the dexamethasone group. CONCLUSIONS: Dexamethasone can prevent the audiological and histopathologic findings of experimentally induced endolymphatic hydrops. However, these results must be supported by clinical and experimental studies designed with a large number of subjects.