豚鼠耳蜗一氧化氮合酶异型体的定位

目的 研究一氧化氮合酶(NOS)的异型体在豚鼠耳蜗的定位分布，以探讨一氧化氮(NO)在内耳听觉生理和病理生理机制中的作用。方法 使用特异性NOS异型体抗体，采用ABC免疫组化染色法，观察NOS异型体在正常豚鼠耳蜗的定位表达。结果 NOS Ⅰ主要分布在内骨膜、螺旋神经节的核周体、螺旋韧带和Corti’s器的细胞。NOS Ⅲ是耳蜗的主要NOS异型体免疫染色，其主要免疫染色分布于耳蜗神经、螺旋神经节核周体、螺旋韧带和耳蜗毛细血管球的内皮细胞，也见于Corti’s器的细胞和神经纤维。NOS Ⅱ在正常豚鼠耳蜗内不表达。结论 结构型NOS(cNOS)表达在耳蜗的多个部位，表明NO参与内耳的正常生理功能，包括神经突触的神经传导、耳蜗血流的调节和耳蜗的骨代谢。     

豚鼠耳蜗一氧化氮合酶的分布

目的 用组织化学法,通过观察NADPH-黄递酶的活性了解一氧化氮合酶在豚鼠耳蜗内分布。方法 经4％多聚甲醛心脏灌注固定后,取出耳蜗,经3％依地酸脱钙后,作厚10μm冰冻切片,用辅酶Ⅱ孵育液在37℃条件下孵育l小时。结果 发现在耳蜗内、外毛细胞底部与耳蜗神经末梢接头处及毛细血管球内皮细胞有明显NADPH-黄递酶活性,此外在内、外柱细胞、支持细胞、血管纹及螺旋神经节细胞也有NADPH-黄递酶活性反应。结论 NO在维持耳蜗正常神经传导及毛细血管张力中起着重要作用。     

一氧化氮合酶和心钠素在豚鼠耳蜗的分布

目的 观察豚鼠耳蜗局部心钠素（atral natruretc peptde，ANP）和一氧化氮合酶（nitric oxide synthase，NOS）免疫组化反应产物的分布，为研究ANP和NOS在豚鼠耳蜗局部血流、淋巴以及神经调节中的相互作用提供形态学依据。方法 采用免疫组织化学双标法检测ANP和NOS在正常豚鼠耳蜗的分布特征。结果 在耳蜗各转螺旋动脉和血管纹．螺旋缘、螺旋韧带和Corti器显示双阳性染色，螺旋神经节细胞及囊斑神经上皮细胞膜及轴突NOS阳性染色，胞质ANP阳性染色；盖膜、前庭膜阴性染色。结论 ANP和NOS在内耳血 流调节，内、外淋巴平衡调节以及神经信号传递等方面可能具有重要作用，二者之间可能存在密切的相互作用机制，其分布特点与功能密切相关。     

噪声刺激对耳蜗一氧化氮合酶的影响

目的：探讨一氧化氮（ＮＯ）在噪声性聋发病中的作用。方法：用中高频连续稳态噪声制作噪声性聋的动物模型,用ＮＡＤＰＨ－黄递酶组织化学、原位杂效和Ｎｏｒｔｈｅｒｎ印迹法,观察噪声刺激对耳蜗一氧化氮合酶（ＮＯＳ）表达的影响。结果：组织化学法显示ＮＯＳ主要分布于内外毛细胞、螺旋神经节细胞和血管纹边缘细胞;原位杂效法发现ＮＯＳｍＲＮＡ在内外毛细胞、螺旋神经节细胞胞浆内均可见阳性染色,但血管纹边缘细胞无阳性染色     

大鼠耳蜗一氧化氮合酶的分布和表达

目的 本实验先用组织化学法，通过观察还原型辅酶Ⅱ－黄递酶（NADPH－黄递酶）的性了解一氧化氮合酶（nitric oxide synthase,NOS）在大鼠耳蜗内分布。再用亲合免疫细胞组织化学技术，研究大鼠耳蜗内神经元型NOS（neuronal NOS,nNOS）与内皮型NOS（endthelial NOS,eNOS）的表达。方法 组化组大鼠耳蜗切片用辅酶Ⅱ孵育液在37℃条件下孵育1小时。免疫组化组大鼠耳蜗切片经消除内源性过氧化物酶，3％山羊正常血清封闭非正常结合点后，用兔抗nNOS抗体、兔抗eNOS抗体，室温下孵60发钟，再用生物素标记的山羊抗兔第二抗体孵育、滴加ABC试剂，以DAB试剂显色。结果 大鼠耳蜗血管球内皮细胞有明显NADPH－黄递酶活性，血管纹及螺旋神经节细胞也有NADPH－黄递酶活性反应。大鼠耳蜗内、外毛细胞、螺旋神经节细胞nNOS、eNOS的表达呈阳性。血管纹细胞处有阳性nNOS、eNOS的表达。耳蜗血管球的内皮细胞无nNOS的表达，但eNOS的表达呈强阳性。结论 由nNOS及eNOS合成的NO在维持耳蜗正常神经传导及耳蜗毛细血管张力和正常血液供应中起着重要作用。     

白噪声对豚鼠耳蜗核一氧化氮合酶活性的影响

采用硫辛酰胺脱氢酶组织化学方法及图象分析技术，研究白噪声暴露后豚鼠耳蜗核一氧化氮合酶（ＮＯＳ）神经元及ＮＯＳ活性的变化与听阈的关系，探讨豚鼠耳蜗核ＮＯＳ神经元在白噪声损伤过程中可能的作用。结果表明，白噪声暴露后耳蜗核ＮＯＳ阳性神经元的数量及染色强度明显增加，２周达到高峰，３￣４周持续高表达，至５周有所恢复，仍高于正常水平。白噪声暴露后７ｄ以内，耳蜗核ＮＯＳ活性与ＡＢＲ阈值有分离现象，７ｄ后，ＮＯＳ     

蒙古沙鼠耳蜗一氧化氮合酶的胆碱能调节

目的　通过检测乙酰胆碱对蒙古沙鼠耳蜗中一氧化氮合酶 (nitricoxidesynthase ,NOS)的影响 ,初步探讨耳蜗中NOS的调节机制。方法　蒙古沙鼠耳蜗经还原型辅酶Ⅱ -黄递酶 (NADPH -黄递酶 )染色后行耳蜗铺片 ,分别以同一动物两侧耳蜗为实验组和对照组进行对比观察 ,检测乙酰胆碱 (ACh)对耳蜗NOS活性的影响 ,其染色深度的差异通过HPIAS - 10 0 0高清晰度彩色病理图像免疫组化测量系统判断。结果　蒙古沙鼠耳蜗外毛细胞基底部传出神经末梢呈现NADPH -黄递酶染色阳性反应 ,ACh处理后染色深度明显增强 ,其平均灰度值从 74.2 8± 11.71减低至 46 .5± 7.78(P <0 .0 1)。结论　蒙古沙鼠耳蜗外毛细胞基底部的传出神经末梢可见NOS分布 ,ACh能增强其活性 ,提示ACh通过第二信使分子一氧化氮在耳蜗生理调控机制中起重要作用     

一氧化氮-环磷酸鸟苷通路对耳蜗灵敏度的调节

目的探讨一氧化氮（NO，nitric oxide）-环磷酸鸟苷（cyclic guanosine monophosphate，cGMP）通路对耳蜗功能的调节。方法健康杂色豚鼠100只，雌雄不限，用随机数字表法随机分为10组，每组10只：①第1组：人工外淋巴液组；②第2组：L-精氨酸组；③第3组：Ca^2＋-ATP酶抑制剂组；④第4组：Ca^2＋-ATP酶抑制剂＋L-精氨酸组；⑤第5组：Ca^2＋-ATP酶抑制剂＋cGMP；⑥第6组：Ca^2＋-ATP酶抑制刺＋L．精氨酸＋非选择性一氧化氮合酶（NOS）抑制剂组；⑦第7组：血管内皮性一氧化氮合酶（eNOS）抑制剂组；⑧第8组：Ca^2＋-ATP酶抑制剂＋eNOS抑制剂组；⑨第9组：Ca^2＋-ATP酶抑制剂＋eNOS抑制剂＋L．精氨酸组；⑩第10组：Ca^2＋-ATP酶抑制剂＋eNOS抑制剂＋L-精氨酸＋神经元性一氧化氮合酶（nNOS）抑制剂组。分别全耳蜗灌流以上各组药物120min，由圆窗龛每隔30min测1次耳蜗微音器电位（cochlear microphonic，CM）和耳蜗听神经复合动作电位（compound action potential，CAP）。第3，4组灌流后留置标本做透射电镜的标本固定。结果第3组灌流Ca^2＋-ATP酶抑制刺前后CAP阈移为28．5dB，第4组在此基础上加入L．精氨酸可使CAP阈移改善9dB，且与加入cGMP后作用相似。第8组多加入eNOS抑制剂抑制血管纹功能后CAP阈移为42．5dB，再加入L-精氨酸可使CAP阈移改善7dB，而第10组加入nNOS抑制剂后CAP阈移较第9组增加了6、5dB，与第8组无明显差异。提示L-精氨酸在nNOS作用下可通过NO-cGMP通路来拮抗Ca^2＋-ATP酶抑制剂引起的胞内Ca^2＋-升高。透射电镜的结果显示：在Ca^2＋-ATP酶抑制剂的基础上加入L-精氨酸减轻了由Ca^2＋-ATP酶抑制剂所造成的外毛细胞的空泡化。结论NO-cGMP通路可调节耳蜗电位，L-精氨酸通过nNOS改善Corti器的功能。     

钙蛋白酶在卡那霉素致毒豚鼠耳蜗中的表达

目的探讨钙蛋白酶(calpain)在卡那霉素(kanamycin,KM)致耳中毒豚鼠耳蜗的表达。方法将豚鼠随机分成对照组、KM 3d组、KM 7d组和KM1 4d组,应用免疫组织化学SABC(streptavidin-biotin peroxidae complex,链霉亲合素-生物素过氧化物酶复合物)法和显微图像分析技术检测耳蜗中钙蛋白酶的表达,用药前后给予短纯音刺激检测听性脑干反应阈值,观察豚鼠听力的变化。结果对照组calpain 1阳性免疫反应主要见于耳蜗毛细胞、螺旋神经节、血管纹和螺旋韧带,以螺旋神经节的染色较深,而其它部位均呈阴性。肌肉注射KM后,calpain 1在耳蜗中的阳性反应部位与对照组大致相同,显微图像分析结果表明,随着给药天数的增加,calpain 1在耳蜗上述部位的阳性反应逐渐减弱。Calpain 2在各组豚鼠耳蜗中的表达部位与calpain 1的相同,显微图像分析结果提示,随着给药天数的增加,calpain 2在耳蜗上述部位的阳性反应逐渐增强。结论正常豚鼠耳蜗中有calpain 1和calpain 2的表达。注射KM后,随着给药天数的增加,calpain 1在耳蜗的表达逐渐减弱,而calpain 2的表达则逐渐增强,提示calpain 2可能参与了卡那霉素致耳中毒的过程。     

Immunocytochemical detection of peptides in the guinea pig cochlea

Summary The cochleae of juvenile guinea pigs were investigated for the presence of several neuropeptides. Glucagon, insulin, CCK and -endorphin immunoreactive neurons and nerve fibers as well as hair cells were demonstrated by the peroxidase antiperoxidase technique. Small amounts of substance P were also found in different sites in the inner ear. In contrast, prolactin-like material could not be found at all. These findings have significance with regard to the putative role of neuropeptides in neuromodulation.     

An anionic charge barrier in the guinea pig cochlea

Summary A charge barrier has been found in the the glomerular basement membrane of the kidney and plays an important role in the filtration of solutes. In the present study, we used electron microscopy to localize anionic sites of a similar charge barrier in the guinea pig cochlea. Polyethyleneimine (PEI) was used as a cationic marker to detect anionic sites. Our results showed a localization of PEI with regular interspaces, indicating the anionic sites to the charge in the capillary basement membrane of the stria vascularis and the spiral ligament, and in the basal lamina of Reissner's membrane and the spiral prominence. This charge barrier, as well as structural size barrier, may play an important role in the maintenance of normal inner ear functions.     

Lipopolysaccharide-induced expression of nitric oxide synthase II in the guinea pig vestibular end organ

The purpose of the investigation was to ascertain whether inoculation of bacterial lipopolysaccharide (LPS) into the vestibular organ of the guinea pig might induce formation of nitric oxide synthase (NOS) II. Forty-eight hours after the animals were injected with 1 mg transtympanic LPS, varying degrees of impaired caloric responses were observed with similar degeneration of vestibular hair cells. These effects could be blocked with N-nitro-l-arginine methylester, a competitive inhibitor of NOS. Findings suggested that NOS II, which was not normally detectable in the guinea pig vestibular organ but was present following inoculation of LPS, produced the nitric oxide as the toxic factor causing cell damage. If true, LPS may represent a reproducible method for studying the vestibular pathogenesis of inner ear disease. Received: 22 July 1997 / Accepted: 18 September 1997     

雏鸡耳蜗中的一氧化氮合成酶分布

应用冰冻切片组织化学技术，以还原型尼克酰胺腺嘌呤二核苷酸磷酸-黄递酶特异性地确定一氧化氮合成酶（NOS）在小鸡耳蜗中存在，观察其分布情况，发现听毛细胞底部颗粒较集中，染色深，毛细胞周围亦有散在颗粒，基底乳头近端与远端染色强弱相似。阳性反应神经纤维连接毛细胞底部。螺旋神经节细胞胞浆中有大量的蓝色颗粒，细胞核无着色，这些细胞大小均匀，呈圆形或椭圆形，周围有多量阳性神经纤维，提示可能为神经性NOS。血管盖内皮细胞胞浆中酶活性较强。对这些分布特点和意义进行了讨论。     

Immunoelectron microscopic localization of nitric oxide synthase III in the guinea pig organ of Corti

Nitric oxide synthase III (NOS III) was identified in the guinea pig cochlea on an ultrastructural level using a post-embedding immunolabeling procedure. Ultrathin sections of London Resin (LR) White-embedded specimens were incubated with various concentrations of a commercially available antibody to NOS III and the immunoreactivity visualized by a gold-labeled secondary antibody. Analysis of ultrathin sections of the organ of Corti in the second turn of the cochlea showed that NOS III could be localized in the endothelial cells of the blood vessels under the basilar membrane, which was comparable to its location in similar cells types in various biological systems. Besides this, NOS III was also found in the cytoplasm and in the nuclei of inner and outer hair cells. Immunoreactivity was not distributed homogeneously within receptor cells. Numerous gold particles could be identified at the border of the cuticular plates, in the middle parts of the stereocilia and in the cytoplasm. Gold-labeled anti-NOS III antibodies in these sites were seen mostly on the cytoplasmic side of the submembranous cisterns in the vicinity of mitochondria and in the central parts of the hair cells, whereas the cisterns were nearly free from any immunoreactivity. NOS III was also detected in the efferent and afferent nerve endings that were located at the basal and basolateral side of the outer hair cells. Some immunoreactivity was visible in different nerve fibers of the inner and outer spiral tunnels. Besides this, gold-labeled antibodies were also present in the cuticular plate of inner and outer pillar cells, in the cytoskeletal elements located in the apical parts of Deiters cells, forming the lamina reticularis, and in the cytoskeletal-containing region of the cytoplasm of those Deiters cells located at the basal side of the outer hair cells. The role of the NOS III immunoreactivity identified in the organ of Corti was consistent with respect to hair cell and tissue modulation. Received: 24 September 1997 / Accepted: 26 June 1998     

血管内皮舒张因子对豚鼠耳蜗功能的影响

目的 探讨血管内皮性一氧化氮合酶(endothelial nitric oxide synthase,eNOS)对耳蜗电位的影响.方法 健康豚鼠100只,随机等分为10组:①人工外淋巴液组;②L-精氨酸组;③Ca2+-ATP酶抑制剂组;④Ca2+-ATP酶抑制剂+L-精氨酸组;⑤Ca2+-ATP酶抑制剂+环磷酸鸟苷(c...     

Lipopolysaccharide-induced expression of inducible nitric oxide synthase in the guinea pig organ of Corti

The purpose of the investigation was to ascertain whether inoculation of bacterial lipopolysaccharide (LPS) into the cochlea of the guinea pig could elicit formation of inducible nitric oxide synthase (iNOS). Immunohistochemical study revealed that immunoreactivity to iNOS was seen below outer hair cells representing nerve fibers and synaptic nerve endings. iNOS-staining could also be observed in phalangeal dendrites of Deiter’s cells pointing to the cuticular membrane, Hensen’s cells and on stria vascularis 48 h after inoculation with LPS. Immunohistochemical investigation with a specific anti-nitrotyrosine antibody also revealed intense immunoreactivity identical to that of iNOS, suggesting formation of peroxynitrite in the organ of Corti by the reaction of NO with O₂⁻. On the basis of these findings, it can be concluded that NO together with O₂⁻, which form the more reactive peroxynitrite, are the most important pathogenic agents in LPS-induced damage of cochlea in the guinea pig.     

一种豚鼠耳蜗内铅含量的检测方法

摘要：目的介绍一种豚鼠耳蜗内铅含量的检测方法。方法将10只成年豚鼠按数字随机法分成实验组及对照组(每组5只动物),实验组用含浓度为2 mmol/L醋酸铅的纯净水喂养1个月,对照组用不含醋酸铅的纯净水喂养1个月。实验结束时用10%水含氯醛溶液麻醉豚鼠后采血2 ml送检,并在显微镜下解剖出耳蜗基底膜及螺旋韧带并用65%~68%浓硝酸溶解,在原子吸收光谱仪中分别检测血铅及耳蜗组织中铅含量,然后对两组结果进行对比分析。结果实验组与对照组的血铅浓度分别为（73.26±12.06）、（5.53±1.25）μg/dL,实验组血铅浓度明显增高,与对照组比较其差异具有统计学意义(t=12.49,P<0.001);实验组与对照组耳蜗内铅含量分别为（25.87±14.60）、（29.31±11.70）μg/g,两组之间比较差异无统计学意义（t=0.74,P>0.05）。结论通过原子吸收光谱法检测豚鼠耳蜗内铅含量是一种简单有效的定量检测耳蜗内铅含量的方法。