一氧化氮合酶在雏鸡外周前庭系统中的分布

目的 :观察一氧化氮合酶 (NOS)在雏鸡外周前庭系统中的分布。方法 :用冰冻切片、还原型尼克酰胺腺嘌呤二核苷酸脱氢酶组织化学技术 ,观察 12只雏鸡外周前庭系统中NOS阳性反应神经元和神经纤维的分布。结果 :壶腹嵴、椭圆囊、球囊的感觉毛细胞周围及底部均有阳性反应产物 ,阳性神经纤维分布于感觉上皮中 ,前庭神经节NOS阳性细胞呈圆形或椭圆形 ,着色深浅不一 ,周围有较多的阳性神经纤维围绕。结论 :NO可能在内耳前庭功能信息的传递和调控中起重要作用     

大鼠耳蜗器官培养及其组织学检查技术

目的探讨大鼠耳蜗器官体外培养的方法和观察毛细胞、神经纤维及螺旋神经节细胞的组织学检查技术。方法将出生3天的SASCO Sprague Dawley大鼠耳蜗取出平铺培养。应用神经丝免疫组织化学方法对螺旋神经节细胞及神经纤维染色,同时应用鬼笔环肽染色特异性显示耳蜗毛细胞的静纤毛和表皮板。在共聚焦显微镜下应用不同的激发荧光分别显示耳蜗毛细胞、螺旋神经节和神经纤维。结果耳蜗器官经1～3天离体培养,内、外毛细胞生长良好,无衰亡和缺损;神经纤维排列有序;螺旋神经节细胞形态正常。结论本文介绍的耳蜗器官体外培养方法和组织学检查指标,将在常规评估耳蜗毛细胞、螺旋神经节细胞和神经纤维的离体培养损害实验模型中,有一定应用价值。     

豚鼠内毛细胞传出神经介质与细胞内Ca~(++)浓度的关系

据文献报道耳蜗感觉毛细胞底部的传出突触和听觉旁体系统的传入终末均有调节这些系统的感觉能量转换功能。因为内毛细胞在感觉转能系统中具有主导的作用,故虽传出神经纤维间接与内毛细胞接触,但内毛细胞的传出抑制机制仍不容忽视。以往的研究发现传出神经纤维主要与传入神经纤维间构成一种轴树突     

慢性鼻炎鼻黏膜中一氧化氮合酶的表达

目的比较正常鼻黏膜和慢性鼻炎鼻黏膜中一氧化氮合酶(NOS)的分布,探讨NOS与慢性鼻炎的关系.方法应用还原型尼克酰胺嘌呤二核苷酸磷酸-黄递酶(NADPH-d)组织化学染色与免疫组织化学技术,测定内皮型NOS(eNOS)和诱导型NOS (iNOS) 在正常组和慢性鼻炎组鼻黏膜中的分布和表达.结果eNOS免疫组化显示正常鼻黏膜和慢性鼻炎鼻黏膜NOS均呈阳性反应,主要分布于表层上皮细胞,血管内皮细胞胞浆;iNOS免疫组化则显示慢性鼻炎表层上皮细胞,血管内皮细胞胞浆NOS阳性,部分炎性细胞亦呈阳性反应;而正常鼻黏膜NOS呈阴性.酶组织化学染色显示,NOS有同样的分布部位.结论正常鼻黏膜存在NOS分布,慢性鼻炎鼻黏膜中iNOS活性明显高于正常,由iNOS产生的NO和慢性鼻炎的鼻分泌物增多和黏膜水肿有关.     

正常雏鸡耳蜗中谷氨酸免疫反应分布

目的：证实谷氨酸在耳蜗毛细胞－听神经传入过程中的递质作用。方法：用半薄切片单克隆抗体免疫组织化学技术，研究正常雏鸡基底乳头及螺旋神经节的谷氨酸免疫反应。结果：高、矮毛细胞浆均呈Ｇｌｕ－ＩＲ阳性，毛细胞下神经纤维着色较淡，支持细胞的反应阴性，高毛细胞反应较矮毛细胞反应强；螺旋神要节细胞呈圆形或椭圆形，胞浆Ｇｌｕ－ＩＲ阳性，胞核不着色，结论：Ｇｌｕ可能作为小鸡耳蜗传入神经递质（或调质）而起作用。     

听力学

201856正常雏鸡耳蜗中谷氨酸免疫反应分布/聂国辉…//临床耳鼻咽喉科杂志一2000.14(4)一1 75一176 目的:证实谷氨酸在耳蜗毛细胞一听神经传入过程中的递质作用。方法:用半薄切片单克隆抗体免疫组织化学技术,研究正常雏鸡基底乳头及螺旋神经节的谷氨酸免疫反应(Glu-IR)。结果:高、矮毛细胞胞浆均呈Glu4R阳性,毛细胞下神经纤维着色较淡,支持细胞反应阴性;高毛细胞反应较矮毛细胞反应强;螺旋神经节细胞呈圆形或椭圆形,胞浆Glu~IR阳性;胞核不着色。结论:Glu一IB可能作为小鸡耳蜗传入神经递质(或调质)而起作用。图2参7(校超)201857瞬态诱发…     

锰诱导体外培养耳蜗毛细胞的损伤与凋亡研究

目的探讨锰诱导体外培养耳蜗毛细胞损伤与凋亡的关系。方法将出生3天的新生Sprague Dawley大鼠的耳蜗器官体外培养,将贮存浓度为100m M氯化锰用SFM稀释到终浓度为1m M,3m M,5m M后进行体外染毒处理耳蜗基底膜。应用鬼笔环肽染色特异性显示耳蜗毛细胞的静纤毛,同时用β-Tubulin对神经纤维进行染色,用TUNEL试剂盒;Cleaved caspase-3抗体对耳蜗基底膜进行染色,在激光共聚焦显微镜下分别观察耳蜗基底膜的毛细胞,神经纤维。结果耳蜗基底膜培养24小时,1m M氯化锰对耳蜗毛细胞及神经纤维损伤甚微,差异无统计学意义(P>0.05),内,外毛细胞排列整齐规律,听神经纤维分布均匀,成束排列。最高浓度5m M氯化锰处理24小时后对神经纤维造成明显的损害,与对照组相比,差异有统计学意义(P<0.05)。当浓度逐渐增加,损伤愈加显著并呈浓度依赖性。2m M氯化锰处理,对耳蜗基底膜底转损伤较中转、顶转明显。不同浓度氯化锰处理24小时TUNEL染色阳性细胞随着氯化锰浓度的增加而增多;Cleaved caspase-3染色阳性细胞亦随着氯化锰浓度的增加而增多。结论通过大鼠耳蜗器官体外培养方法,发现氯化锰会造成毛细胞缺失,神经纤维变细变少,而这种损伤的机制与细胞凋亡密切相关。     

大鼠内淋巴囊与肾脏集合管神经分布研究

目的从形态学上验证内淋巴囊上存在神经分布，并将这些神经纤维的分布与肾脏集合管的神经分布进行比较，为梅尼埃病的病因病理学研究及耳肾相关性研究提供新的思路。方法成年大鼠15只，经活体心脏灌注，取双侧颞骨和肾脏，常规石蜡包埋切片。利用免疫组织化学方法观察神经特异性烯醇化酶（neuronal specific enolase，NSE）、胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）及神经微丝（neurofilament，NF）蛋白在内淋巴囊和肾脏集合管上的表达，通过图像分析仪对切片阳性染色的分布密度和灰度进行分析，比较二者神经纤维有无类似分布。结果光镜下NSE、GFAP、NF在内淋巴囊上皮及上皮下表达为棕黄色颗粒；肾脏集合管的主细胞胞膜、胞质有稳定、清晰的阳性染色反应，但在肾脏阳性染色并非表现为神经纤维染色，而更像是某种神经物质的染色。内淋巴囊和肾脏集合管在GFAP、NSE、NF的阳性表达上，只有NSE的分布密度差异有统计学意义（P〈0．05），其余指标在灰度和分布密度上差异无统计学意义（P值均〉0．05）。结论内淋巴囊近侧段、中间段和远侧段均有神经纤维分布，以中间段最为丰富；其分布与肾脏集合管上皮主细胞存在的神经染色在灰度和分布密度上并无差别。     

谷氨酸-天冬氨酸转运体在豚鼠耳蜗的分布

目的研究谷氨酸-天冬氨酸转运体(glutamate—aspartate transporters，GLAST)在正常豚鼠耳蜗内的分布，为探讨GLAST在防止耳蜗谷氨酸(Glu)神经毒性中的作用提供形态学基础。方法选取健康红目豚鼠6只，采用免疫组织化学方法，以山羊抗GLAST抗体为标记物，观察正常豚鼠耳蜗中GLAST的表达及分布。结果在正常豚鼠耳蜗的内、外毛细胞，内、外毛细胞周围的支持细胞，螺旋神经节细胞，血管纹边缘细胞和螺旋缘上皮，均有GLAST阳性表达。结论GLAST在正常豚鼠耳蜗内主要分布于内、外毛细胞，内、外毛细胞周围的支持细胞，螺旋神经节细胞、血管纹边缘细胞和螺旋缘上皮，其功能尚需进一步的研究。     

酪氨酸硝基化在分泌性中耳炎所致耳蜗损伤作用实验观察

目的通过观察3-硝基酪氨酸(3-nitrotyrosine,3-NT)在分泌性中耳炎(Otitis Media with Effusion,OME)大鼠内耳的定位、分布及内耳组织形态学特征,确定OME大鼠耳蜗中的酪氨酸硝基化情况,探寻OME导致内耳损伤的酪氨酸硝基化机制。方法将40只健康成年雄性SD大鼠一侧耳通过阻塞咽鼓管的方法构建分泌性中耳炎模型。造模成功后分组行耳蜗石蜡切片HE染色和免疫组织化学,Tunel凋亡检测和神经节细胞的透射电镜观察。采用SPSS16.0软件包进行统计学分析,以p<0.05作为有显著性差异。结果造模后大鼠的耳蜗毛细胞没有明显的缺失,HE染色发现中耳及内耳慢性炎症改变,免疫组化检测3-NT在耳蜗毛细胞、血管纹、神经纤维细胞、神经节细胞均有阳性表达,而对照组在相应部位均阴性表达。Tunel凋亡检测见耳蜗的神经纤维细胞、神经节细胞出现凋亡。透射电镜观察示神经节细胞线粒体肿胀、出现空泡化,并有神经节细胞间质的淋巴细胞浸润。结论通过构建OME模型初步发现OME能够导致耳蜗损伤,OME可能通过酪氨酸硝基化作用导致耳蜗毛细胞的凋亡、螺旋神经节细胞及神经纤维的变性,即酪氨酸硝基化是OME导致耳蜗损伤的可能机制之一。     

Involvement of nitric oxide synthase in the physiology and pathophysiology of facial nerve function and dysfunction

To date few reports have discussed the presence and function of nitric oxide (NO) in structures of the facial nerve. We performed nicotinamide adenine dinucleotide phosphate (NADPH-d)-diaphorase-histochemistry and immunohistochemistry on the intratemporal portion of the facial nerve, including the geniculate ganglion, of guinea pigs using specific antibodies to the three known isoforms of NO synthase and soluble guanylyl-cyclase (sGC). Normal facial nerves were compared to those treated intratympanically with bacterial lipopolysaccharides (LPS) and tumor necrosis factor-α (TNF-α). Both constitutive NOS isoforms and sGC could be detected in the bipolar ganglion cells of normal animals, while the inducible isoform (iNOS or NOS II) was not found. Endothelial NOS (NOS III) and sGC were present in blood vessels and were predominantly found in the perineurial sheath and less in the endoneurium. sGC could be detected in all fibers in a cross section of the facial nerve. LPS and TNF treatment led to the detection of iNOS in the perikaryia of the geniculate ganglion and the perineural sheath. These findings imply that NO may be involved in neurotransmission at least in the visceroafferent system. NO regulates vascular tone of nutrient blood vessels in the perineural sheath and endoneurium. The presence of sGC indicates that NO acts via its second messenger cGMP. NOS II expression may be a contributing factor to facial nerve palsy via two different mechanisms: NOS II-generated NO may lead to an overstimulation of the visceroefferent nerve fibers and motor fibers of the facial nerve. Dysregulation in facial nerve blood vessels could lead to edema and elevated pressure on the nerve within its osseous canal. Received: 13 April 1999 / Accepted: 12 August 1999     

Localization of the NO/cGMP-pathway in the cochlea of guinea pigs.

The presence of nitric oxide synthase (NOS) in substructures of the cochlea of guinea pigs is an issue of current focus. Moreover, information concerning the localization of cells effected by the NO/cGMP-pathway are rare. Paraffin sections of guinea pig cochlea were incubated with specific antibodies to the three known NOS isoforms, soluble guanylyl cyclase (sGC) and cyclic guanosine-monophosphate (cGMP), the second messenger system of NO. While detection of inducible iNOS failed in all cochlear structures, expression of endothelial eNOS was found in the spiral ligament, in the stria vascularis, in cells of the organ of Corti, in nerve fibers and in some perikaryia of the spiral ganglion. The cochlear nerve showed an accentuated affinity for immunostaining in distal, basal segments of the cochlea. Neuronal bNOS was found predominantly in the endosteum of the modiolus and cochlea and was less intensively present in all perikaryia of the spiral ganglion and in the spiral ligament. Supporting cells of the organ of Corti and cells in the limbus spiralis displayed only modest immunostaining, while bNOS was not found in outer and inner hair cells. NOS detection was accompanied by immunoreactivity to sGC and to cGMP. The presence of NOS and its second messenger system gives evidence for a possible involvement in neurotransmission, regulation of the cochlear amplifier and in homeostasis.     

Localization of Nitric Oxide Synthase Isoforms in the Human Cochlea

The location of nitric oxide (NO) in the structures of the cochlea is a topical issue. Nitric oxide synthase (NOS) has been detected previously in mammalian cochleae, but information on its presence in the human cochlea is still sparse. The location of NOS isoforms I, II and III in substructures of the human cochlea was studied by immunohistochemistry (fluorescein isothiocyanate technique) using monoclonal antibodies to NOS I, II and III. NOS I was the predominant isoform and staining could be observed in cells of the spiral ganglion (SG), in nerve fibres and in the outer hair cells (OHC). Furthermore, the supporting cells of the organ of Corti and the stria vascularis showed a fluorescent reaction to NOS I. Staining for NOS III was less intense and was located in the OHC, supporting cells and SG cells, while the stria vascularis remained unstained. By contrast, NOS II showed weak staining in a few neuron fibres only. The results imply that NO in the human cochlea could act as a neurotransmitter/neuromodulator at the level of neural cells and may be involved in the physiology of the supporting cells and stria vascularis. Moreover, because NO is both a mediator of excitotoxicity and a non-specifically toxic radical, it may also play a role in neurotoxicity of the human cochlea.     

Localization of nitric oxide synthase isoforms in the human cochlea

The location of nitric oxide (NO) in the structures of the cochlea is a topical issue. Nitric oxide synthase (NOS) has been detected previously in mammalian cochleae, but information on its presence in the human cochlea is still sparse. The location of NOS isoforms I, II and III in substructures of the human cochlea was studied by immunohistochemistry (fluorescein isothiocyanate technique) using monoclonal antibodies to NOS I, II and III. NOS I was the predominant isoform and staining could be observed in cells of the spiral ganglion (SG), in nerve fibres and in the outer hair cells (OHC). Furthermore, the supporting cells of the organ of Corti and the stria vascularis showed a fluorescent reaction to NOS I. Staining for NOS III was less intense and was located in the OHC, supporting cells and SG cells, while the stria vascularis remained unstained. By contrast, NOS II showed weak staining in a few neuron fibres only. The results imply that NO in the human cochlea could act as a neurotransmitter/neuromodulator at the level of neural cells and may be involved in the physiology of the supporting cells and stria vascularis. Moreover, because NO is both a mediator of excitotoxicity and a non-specifically toxic radical, it may also play a role in neurotoxicity of the human cochlea.     

Histochemical and immunocytochemical study of nitrergic innervation in human nasal mucosa.

Nitric oxide (NO) is a free radical gas that has been found to be produced in neuronal cells by the action of the enzyme brain nitric oxide synthase (bNOS). The aim of this study was to identify NO-containing nerve structures in the human nasal mucosa by localizing bNOS and to find out whether NO production is attached to the parasympathetic system. For this purpose, immunocytochemistry with antibodies to bNOS and neurofilament was performed. Additionally, nicotinamide-adenine dinucleotide phosphate diaphorase (NADPH-d), an enzyme that correlates with the localization of NO synthase, and acetylcholinesterase were visualized in a histochemical double staining technique on frozen sections. The NADPH-d and bNOS reactions were found in axons of nerve bundles and in subepithelial, glandular, and vascular nerve fibers. Arteries showed a distinctly developed nitric innervation, whereas no activity was found in nerve fibers supplying veins. A high coexistence of NADPH-d in parasympathetic nerves could be detected. These findings suggest that NO takes part in the nerve control functions of the human nasal mucosa.     

化学修饰电极对雏鸡耳蜗神经元一氧化氮的定性检测

目的比较雏鸡和哺乳动物耳蜗生理功能的异同。过去已有哺乳动物耳蜗及前庭神经元中一氧化氮合酶（ＮＯＳ）活动证据的报道，其所释放的一氧化氮（ＮＯ）在听觉和平衡活动中可能发挥重要作用。方法用经化学修饰的碳纤维微电极，定性检测雏鸡离体耳蜗神经元在不同激动剂的诱发下所释放的ＮＯ。根据电极所检测的电流变化的幅度和持续时间判断ＮＯ的释放。结果由激动剂乙酰胆碱和Ｌ精氨酸所诱发的ＮＯ释放反应幅度相似，但乙酰胆碱的高电流持续时间较短，而ＡＴＰ所致者幅度大，持续时间长。预加ＮＧ硝基Ｌ精氨酸，再加上述激动剂，电流无变化。结论雏鸡耳蜗神经元细胞中存在ＮＯＳ。     

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。NOSⅢ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。     

化学修饰电极对雏鸡耳蜗神经元一氧化氮的定性检测

目的 比较雏鸡和哺乳动物耳蜗生理功能的异同。过去已有哺乳动物耳蜗及前庭神经元中一氧化氮合酶（ＮＯＳ）活动证据的报道，其所释放的一氧化氮（ＮＯ）在听觉和平衡活动中可能发挥重要作用。方法 用经修饰的碳和有、定性检测鸡离体耳神经元在不同激动剂的诱发下所释放的ＮＯ。根据电极所检测的电流变化的幅度和持续时间判断ＮＯ的释放。结果 由激动剂乙酰胆碱和Ｌ－精氨酸所诱发的ＮＯ释放反应幅度相似，但乙酰胆碱的高电流持续     

Detection of nitric oxide in the guinea pig inner ear, using a combination of aldehyde fixative and 4,5-diaminofluorescein diacetate

Localization of nitric oxide (NO) production sites in the inner ear of the guinea pig was investigated using a combination of glutaraldehyde fixative and a new fluorescence NO indicator. 4,5-diaminofluorescein diacetate (DAF-2DA). The cochlea and vestibular end organs were examined to locate NO production sites. The fluorescence persisted after glutaraldehyde fixation and embedding with water-soluble resin. NO production in the cochlea was observed in the outer and inner hair cells, nerve endings, nerve fibers and supporting cells of the organ of Corti, stria vascularis, spiral ligament, ganglion cells, etc. In the vestibular end organs, both type I and type II sensory cells, nerve fibers, blood vessels and dark cells displayed fluorescence. This localization was exactly identical to that of NO synthase. Thus, detection of intracellular NO production by using a combination of glutaraldehyde fixation and DAF-2DA is useful for examining the function of NO in cells, both in situ and in vivo.     

Expression of inducible nitric oxide synthase (iNOS/NOS II) in the hydropic cochlea of guinea pigs

Immunohistochemical investigations of the guinea pig cochlea, using a specific antibody to the inducible isoform of NO synthase (iNOS/NOS II), have been performed 3 weeks after closure of the right endolymphatic duct (n=7). Endolymphatic hydrops, the morphological substrate of Meniere's disease, became evident by distension of the Reissner's membrane. iNOS expression could be noted in endothelium, spiral ganglion cells, in nerve fibers, in supporting cells of the organ of Corti and cells of the spiral ligament. Temporal bones of non-operated controls (n=6) as well as of sham-operated animals (n=3) did not show structures positive to iNOS. These findings imply that iNOS-generated NO could be involved in the pathophysiology of cochlear dysfunction in Meniere's disease.