卡那霉素耳中毒后豚鼠耳蜗细胞凋亡的研究

目的探讨卡那霉素耳中毒后耳蜗毛细胞有无凋亡.方法取听力正常豚鼠12只,实验组6只,对照组6只分别给予4000mg·kg-1·     

耳蜗组织低温包埋切片技术

目的探索一种新的耳蜗组织切片方法，以获得高质量的切片，满足其免疫组织化学染色的需要。方法取1天龄大鼠、成年豚鼠、成年小鼠耳蜗标本经蔗糖快速浸透，用离心管定向并用新型组织包埋剂Tissue—Tek（OCT）包埋，液氮中快速冷冻后在恒冷箱切片机中切片，然后分别做苏木素、伊红染色和免疫组化染色，观察组织切片的形态结构。结果经免疫荧光和组织化学染色后组织切片形态结构完整，耳蜗基底膜平直，盖膜及前庭膜完整，毛细胞与支持细胞间结构完整，细胞核清晰。结论耳蜗组织免疫组化染色应选择低温包埋切片技术制作的切片。     

耳蜗石蜡切片原位杂交定位检测技术的方法学探讨

目的 :探讨原位杂交 (ISH)技术检测大鼠耳蜗石蜡切片的最佳方法。方法 :取成年雄性Wister大鼠1 5只 ,以自身小脑半球作对照 ,与听泡同时进行脱钙 ,石蜡包埋 ;作 5 μm切片进行ISH ,观察不同的贴片剂、蛋白酶K浓度及免疫组织化学检测方法对ISH的影响 ;并以γ 氨基丁酸Aα2 cDNA为探针 ,对小脑和内耳石蜡切片进行ISH。结果 :Vectabond处理过的载玻片 ,1×蛋白酶K在室温下消化 1 5min ,用碱性磷酸酶作免疫组织化学检测可得出预期结果。内耳蜗轴内的螺旋神经节细胞表达γ 氨基丁酸Aα2 mRNA受体。结论 :大鼠耳蜗石蜡切片作ISH可成功获得满意结果     

氧自由基中毒与耳蜗毛细胞凋亡

氧自由基的产生及继发性细胞损伤过程在细胞凋亡过程中起重要作用。细胞凋亡是内耳感觉上皮毛细胞损伤的一种重要方式。但它们之间存在何种有机联系呢?本着重就耳蜗氧自由基中毒与毛细胞凋亡的关系作一综述。     

内质网应激在老年大鼠耳蜗细胞凋亡中的作用

目的探讨内质网应激在老年大鼠耳蜗细胞凋亡中的作用,从而进一步探索老年性聋的发病机制。方法Wistar大鼠24只,其中12只为对照组(3～4月龄),12只为自然衰老组(22～24月龄);比色法测定内耳组织中超氧化物歧化酶(superoxide dismutase,SOD),丙二醛(malondialchehyche,MDA)水平;ABR检测听力学改变;免疫组化及western blot检测GRP78/BiP和Caspase-12蛋白的表达。结果老年大鼠听力下降;内耳组织SOD活性降低,MDA升高,与对照组相比差异有统计学意义(P<0.05);耳蜗切片中及western blot结果可见GRP78的表达与对照组相比差异无统计学意义(P<0.05);caspase-12的表达明显高于对照组,差异有统计学意义(P<0.05)。结论内质网凋亡途径是老年大鼠老年性聋的发生机制之一。     

耳蜗火棉胶包埋冰冻切片技术的应用

目的通过改进耳蜗火棉胶切片技术,比较传统火棉胶切片和耳蜗火棉胶包埋冰冻切片技术,为耳科实验室制作高质量火棉胶切片,获得高质量耳蜗组织形态学图片提供方法。方法将豚鼠耳蜗组织进行常规的梯度酒精脱水、梯度火棉胶浸润、12%的火棉胶固定制作成火棉胶组织块,然后平行于蜗轴沿耳蜗侧面切取一个平面,将耳蜗其它组织修掉使得火棉胶包埋块成为一个长方体。用OCT将火棉胶组织块固定在冰冻切片机上,进行连续切片。结果用火棉胶将耳蜗包埋成块,OCT固定冰冻切片机的模具上进行切片。不用火棉胶切片机切片,同样可以获得传统火棉胶切片方法所获得的制片质量,并且具有切片效果良好、组织切片完整、厚薄均匀、不易形成皱折等优点。还可以减少火棉胶切片的费用开销,便于耳蜗火棉胶包埋制片技术的推广和应用。结论耳蜗火棉胶包埋冰冻切片技术是耳科实验室制作高质量耳蜗火棉胶切片,获得高质量耳蜗组织形态学图片简便有效的方法。     

硫辛酸不同方式给药对急性声损伤耳蜗细胞凋亡的抑制效应

目的 比较抗氧化剂硫辛酸(lipoic acid,LA)经静脉与鼓室两种不同给药途径对急性声损伤后动物耳蜗内细胞凋亡过程的影响.方法 48只豚鼠随机分为4组.A:鼓室注射硫辛酸组,不给于噪声暴露;B:单纯噪声暴露组(110 dB SPL稳态噪声暴露5 h);C:噪声暴露+静脉注射硫辛酸组;D:噪声暴露+鼓室注射硫辛酸组.各组动物于处理前和处理后1、3、5、7 d取出豚鼠耳蜗,石蜡包埋、切片.应用原位末端标记技术(TUNEL)标记凋亡细胞,观察耳蜗Corti器、血管纹、螺旋神经节等部位细胞的凋亡特点. 结果光镜下Corti器、血管纹、螺旋神经节TUNEL阳性反应细胞表现为细胞核染成棕黄色或棕褐色.硫辛酸经鼓室注射组(A组)未见明显凋亡细胞.噪声暴露后经鼓室注射硫辛酸组(D组)与经静脉注射组(C组)同单纯暴露组(B组)相比,差异有统计学意义(P<0.05).但C、D两种方式抑制凋亡无明显的差异(P>0.05).结论 110 dB SPL噪声暴露5 h可导致急性声损伤,引起豚鼠耳蜗组织内细胞的凋亡.抗氧化剂硫辛酸经鼓室与静脉注射对噪声暴露后细胞的凋亡皆有明显的抑制效应,但鼓室注射较静脉注射无明显的优势.     

强噪声暴露下豚鼠耳蜗螺旋神经节细胞凋亡及Caspase-3的表达

目的探讨强噪声能否诱导豚鼠耳蜗螺旋神经节细胞（sprial ganglion cell,SGC）的凋亡及SGC的凋亡是否与凋亡蛋白酶Caspase-3信号转导有关。方法选用健康雄性白色豚鼠20只,随机分为4组,每组5只,其中3组为实验组,1组为对照组。将实验组动物暴露于4kHz、120dB SPL窄带噪声中4h,分别测试噪声刺激停止后1、4、14d及对照组豚鼠听性脑干反应（auditory brainstem response,ABR）。通过透射电镜和脱氧核糖核甘酸末端转移酶介导的缺口末端标记法（ternial-deoxynucleotidyl transferase mediated nick end labeing,TUNEL）,检测SGC凋亡细胞,免疫组织化学方法检查Caspase-3蛋白的表达。结果透射电镜观察发现实验组SGC出现了凋亡细胞的特征性改变。实验组SGC的TUNEL染色明显增强,Caspase-3的表达增高,与对照组比较差异均有显著统计学意义（P〈0.01）。结论强噪声可以诱导SGC凋亡;SGC凋亡与caspase-3的激活有关。     

顺铂诱导氧化应激引起耳蜗血管纹周细胞线粒体途径的细胞凋亡北大核心CSCD

目的:探讨顺铂是否通过诱导氧化应激水平升高引起耳蜗血管纹毛细血管周细胞线粒体途径的细胞凋亡。方法:将20只6~8周的雄性C57BL/6J小鼠分为对照组和顺铂组;同时原代培养小鼠耳蜗血管纹周细胞并鉴定,分为对照组、顺铂组和N-乙酰半胱氨酸（N-acetylcysteine,NAC）+顺铂组。听性脑干反应（ABR）检测小鼠听力变化,苏木精-伊红（HE）染色观察小鼠耳蜗血管纹形态学变化,总超氧化物歧化酶（superoxide dismutase,SOD）活性检测试剂盒（WST-1法）和硫代巴比妥酸（thiobarbituric acid,TBA）法分别检测SOD活性和丙二醛（malondialdehyde,MDA）含量,DCFH-DA荧光探针检测周细胞上活性氧的含量,Hoechst 33342和流式细胞术检测周细胞的凋亡率,免疫荧光技术检测耳蜗血管纹上周细胞的凋亡相关蛋白分布表达,免疫组化和蛋白免疫印迹法（WB）检测凋亡相关蛋白、线粒体相关蛋白的表达,Mito SOX TM-red和JC-1检测周细胞线粒体功能,伊文思蓝染色观察血迷路屏障（blood labyrinth barrier,BLB）的通透性。采用SPSS 18.0软件进行统计分析。 结果:与对照组相比,顺铂组可升高小鼠听力阈值和Ⅰ波潜伏期（ t值为4.72和12.25, P值均<0.05）,升高耳蜗和周细胞内氧化应激水平（ t=38.34, P<0.01）,使耳蜗血管纹结构紊乱皱缩,BLB通透性增加［伊文思蓝渗漏（1.08±0.42）AU比（0.55±0.23）AU, t=4.64, P<0.05］,差异均有统计学意义。顺铂组小鼠耳蜗血管纹细胞凋亡蛋白c-Caspase-3和Bax的表达增加（ t值为5.01和6.33, P值均<0.01）,且顺铂可使原代培养的周细胞凋亡并上调c-Caspase-3、Bax的表达（ P值均<0.05）,NAC+顺铂组可逆转顺铂诱导的周细胞凋亡（ P<0.05）。顺铂使周细胞线粒体功能损伤,诱导线粒体向胞浆释放凋亡诱导因子（apoptosis-inducing factor,AIF）和细胞色素C（cytochrome-c,Cyt-c）,NAC+顺铂组可逆转顺铂诱导的线粒体损伤（ P值均<0.05）。 结论:顺铂可升高耳蜗内氧化应激水平引起C57BL/6J小鼠耳蜗血管纹周细胞线粒体途径凋亡,从而提高BLB的通透性,造成听力损失。     

耳蜗组织连续切片显微结构的三维重建研究

目的:探讨生物组织连续切片显微结构的三维重建使用方法.方法:应用火棉胶切片技术获得耳蜗组织连续切片,显微镜下放大40倍后对耳蜗组织摄像.使用Adobe photoshop 6.0图像处理软件和Amira3.0三维重建软件对内耳结构进行图像拼接、对位和三维重建.结果:三维重建立体真实再现了耳蜗组织的解剖结构,重建效果表明该研究显微结构的数据输入、图像拼接、内定位技术和三维重建方法是可行的.结论:采用内定位技术,借助重建软件,可以在个人计算机上实现生物组织连续切片显微结构的立体重建,是一种实用的重建方法.     

TUNEL与碘化丙啶双染检测耳蜗毛细胞的损伤

目的应用TUNEL与PI（碘化丙啶）双染法检测耳蜗毛细胞损伤。方法听力正常豚鼠经庆大霉素致聋后，做耳蜗基底膜铺片，用TUNEL与PI双染检测耳蜗毛细胞损伤的情况。结果在核固缩、核碎裂及部分核肿胀的细胞中TUNEL染色为阳性．细胞核正常及部分核肿胀的细胞中TUNEL染色为阴性。所有有核细胞均能被PI染色。结论通过TUNEL与PI双染可以确定TUNEL染色为阳性的细胞为凋亡的细胞，而核肿胀的细胞中TUNEL染色为阴性者可能为胀亡细胞。     

Immunofluorescence localization of proteins in semithin 0.2–1 μm frozen sections of the ear

Summary The present work describes a high resolution technique for locating proteins in frozen sections of the inner ear by immunofluorescence. Dissected organs are encapsulated in gelatin, and sections 0.1–1 m thick are cut at –100°C in a cryoultramicrotome. These are labelled with antibodies against two cytoskeletal proteins, actin and tubulin. Actin, which had previously only been described in the sensory cells, is found in the supporting cells as well. Tubulin is identified in the supporting cells and in outer spiral nerve fibres.Supported by grants from the Swedish Medical Research Council (no. 04x-02461), the Ragnar and Torsten Söderberg Foundation, and the Foundation Tysta Skolan     

Anti-clarin-1 AAV-delivered ribozyme induced apoptosis in the mouse cochlea

Usher syndrome type 3 is caused by mutations in the USH3A gene, which encodes the protein clarin-1. Clarin-1 is a member of the tetraspanin superfamily (TM4SF) of transmembrane proteins, expressed in the organ of Corti and spiral ganglion cells of the mouse ear. We have examined whether the AAV-mediated anti-clarin ribozyme delivery causes apoptotic cell death in vivo in the organ of Corti. We used an AAV-2 vector delivered hammerhead ribozyme, AAV–CBA–Rz, which specifically recognizes and cleaves wild type mouse clarin-1 mRNA. Cochleae of CD-1 mice were injected either with 1 μl of the AAV–CBA–Rz, or control AAV vectors containing the green fluorescent protein (GFP) marker gene (AAV–CBA–GFP). Additional controls were performed with saline only. At one-week and one-month post-injection, the animals were sacrificed and the cochleae were studied by histology and fluorescence imaging.

Mice injected with AAV–CBA–GFP displayed GFP reporter expression of varying fluorescence intensity throughout the length of the cochlea in the outer and inner hair cells and stria vascularis, and to a lesser extent, in vestibular epithelial cells. GFP expression was not detectable in the spiral ganglion. The pro-apoptotic effect of AAV–CBA-delivered anti-clarin-1 ribozymes was evaluated by TUNEL-staining. We observed in the AAV–CBA–Rz, AAV–CBA–GFP and saline control groups apoptotic nuclei in the outer and inner hair cells and in the stria vascularis one week after the microinjection. The vestibular epithelium was also observed to contain apoptotic cells. No TUNEL-positive spiral ganglion neurons were detected. After one-month post-injection, the AAV–CBA–Rz-injected group had significantly more apoptotic outer and inner hair cells and cells of the stria vascularis than the AAV–CBA–GFP group.

In this study, we demonstrate that AAV–CBA mediated clarin-1 ribozyme may induce apoptosis of the cochlear hair cells and cells of the stria vascularis. Surprisingly, we did not observe apoptosis in spiral ganglion cells, which should also be susceptible to clarin-1 mRNA cleavage. This result may be due to the injection technique, the promoter used, or tropism of the AAV serotype 2 viral vector. These results suggest the role of apoptosis in the progression of USH3A hearing loss warrants further evaluation.     

Computer-aided serial section reconstruction of nerve endings on the outer hair cells of the cochlea. Semithin sections vs. ultrathin sections

Afferent and efferent nerve endings on several outer hair cells in different turns and rows of the guinea pig cochlea were three-dimensionally reconstructed from serial semithin sections or ultrathin sections by means of a new computer system. In the basal turn, a single afferent ending was sometimes isolated from the others and surrounded by efferent endings, suggesting that this type of afferent ending may have properties different from the others. The advantages and disadvantages of semithin sections and ultrathin sections in computer-aided reconstruction were discussed and it was concluded that the semithin section technique will be useful in reconstructions of specimens from a wide range, whereas the ultrathin section technique will be better for reproducing fine structures such as the distribution of cell organelles as well as synaptic membrane specializations.     

An immunohistochemical method for the study of aminoglycoside ototoxicity in the guinea pig cochlea using decalcified frozen sections

Summary An immunohistochemical technique with decalcified frozen sections was used to study aminoglycoside ototoxicity. Decalcified guinea pig cochleas were cut with a fine blade parallel to the plane of the modiolus to facilitate the penetration of inclusion material and the manipulation of frozen sections. Light microscopy was carried out and additional frozen sections were employed for an immuno-electron microscopic study. Twenty-four hours after a single transtympanic injection of 10 mg gentamicin, there was a definite distribution of the drug in only type I hair cells of the ampullae as well as in both inner and outer hair cells along the length of the cochlea. In those animals treated intraperitoneally with 200 mg/ kg amikacin for 8 days, the drug was located in the outer hair cells of the cochlea, with a tendency to decrease from base to apex and in the inner hair cells towards the apex.Read at the XVIIIth International Congress of Audiology, Prague, Czechoslovakia, 26 August 1986     

Morphological differences among radial afferent fibers in the cat cochlea: An electron-microscopic study of serial sections

The entire unmyelinated portions of 104 radial afferent fibers were reconstructed from serial ultrathin sections through the organ of Corti. Each radial fiber was unbranched and formed a single synapse with one inner hair cell. Fibers innervating the side of the hair cell facing the pillar cells were, in general, larger in diameter and richer in mitochondria than fibers innervating the side of the hair cell facing the modiolus. Some of the thin, mitochondrion-poor fibers had a more complex synaptic junction with the inner hair cell. A fiber classification scheme is suggested which may correlate with the classification of single auditory nerve units according to threshold and rate of spontaneous discharge [5].