顺铂对耳蜗螺旋神经节细胞毒性作用及机制

目的通过透射电镜及免疫组化法观察顺铂对耳蜗螺旋神经节细胞的毒性作用。方法16只豚鼠随机均分为2组。顺铂组连续5天腹腔注射顺铂2mg/kg/d;对照组连续5天腹腔注射生理盐水2mg/kg/d;用药前后测试听力,处死动物后制作耳蜗标本,透射电镜观察及免疫组化法测定诱导型一氧化氮合酶(induciblenitricoxidesynthase,iNOS)的表达。结果ABR:顺铂组听力下降明显,阈值显著升高(P<0.01)。透射电镜观察:顺铂组螺旋神经节细胞的细胞器损伤严重,核变形,线粒体肿胀,大量空泡样变,粗面内质网增多,有髓神经纤维的髓鞘增厚;对照组螺旋神经节细胞核无变形,核仁基本居中,线粒体结构正常。免疫组织化学显示:顺铂组螺旋神经节有iNOS阳性反应显色,灰度值降低,iNOS活性升高,与对照组比较,有显著性差异(P<0.01)。结论顺铂可致耳蜗螺旋神经节细胞损伤并呈iNOS阳性表达,导致听力下降。     

顺铂对大鼠耳蜗螺旋神经节细胞外向延迟整流钾电流的影响

目的:观察记录顺铂作用下急性分离新生大鼠耳蜗螺旋神经节细胞(SGNs)延迟整流钾通道的电流曲线,分析顺铂对SGNs钾电流激活动力学的影响,并初步探讨其耳毒性机制。方法:采用全细胞膜片钳技术记录SGNs外向延迟整流钾电流及顺铂对此电流的影响。结果:钳制电压为-60mV,刺激电压从-60mV到 80mV逐渐去极化,阶跃电压为10mV,持续时间为500ms,可在SGNs上记录到外向钾电流,该电流对TEA-Cl(4-乙基胺)敏感,具有延迟整流特性;在细胞外液中加入10μmol/L顺铂,能明显抑制SGNs延迟整流钾电流;顺铂对此电流的抑制作用与细胞外液中顺铂的浓度呈剂量依赖性;外液洗脱后SGNs电流可基本恢复正常。结论:钾通道与SGNs动作电位的产生密切相关,顺铂可抑制SGNs钾通道电流,导致听觉功能障碍。     

顺铂致豚鼠螺旋神经节细胞凋亡及Caspase-3活化的研究

目的：探讨顺铂的耳毒性作用与螺旋神经节细胞（sprial glanglion cell,SGC)凋亡的关系，及SGC的凋亡是否与凋亡蛋白酶Caspase-3信号转导有关。方法：取40只听力正常的豚鼠，随机分为顺铂1、2、4天组和对照组，每天检测听性脑干反应（ABR）和40Hz相关电位以观察豚鼠高，低频的听阈变化，并用TNEL法检测凋亡细胞数量变化，免疫组化检测SGC中Caspase-3p20活性片段的表达。结果：随着注射顺铂时间的延长，豚鼠听力逐渐下降，同时SGC的TUNEL染色明显增强，Caspase-3p20活性片段的表达增高，与对照组比较均有显著性差异（P＜0．01），结论：顺铂的耳毒性损害机制与SGC的凋亡有关；顺铂所致的SGC凋亡过程中有Caspase-3p20的激活。     

顺铂对小鼠耳蜗螺旋神经节细胞延迟整流钾电流的影响

目的探讨耳毒性药物顺铂对耳蜗螺旋神经节细胞延迟整流钾电流的影响,了解顺铂耳毒性的离子机制。方法选择状态良好培养2—14d的耳蜗螺旋神经节细胞在电压钳制条件下记录的钾电流作为对照组,浴液中分别加入100,500,1000,5000μM不同浓度的顺铂溶液后记录的电流为实验组,观察其对钾通道电流电生理学特性的影响,并观察冲洗后电流的恢复情况。结果实验显示耳蜗螺旋神经节细胞钾通道电流的电生理学特性受不同浓度顺铂溶液的影响,并具有浓度依赖性,表现在其活化动力学和电流幅度的改变。本实验中顺铂的50％的电流被抑制时的浓度（IC50）为294μM,冲洗后钾通道电流逐渐恢复。结论顺铂可以改变耳蜗螺旋神经节细胞钾通道电流的电生理学特性,这种变化在短期内可逆,揭示其耳毒性的形成具有一定的离子机制。     

顺铂作用下沙鼠耳蜗螺旋神经节神经元和Corti器细胞的凋亡

目的　探讨顺铂是否可引起沙鼠耳蜗螺旋神经节 (spiralganglion ,SG)神经元和Corti器细胞的凋亡。方法　对沙鼠进行顺铂连续腹腔注射 ,每日 4mg kg体重 ,分别于健康对照组及给药 4、5、6、7d各处死沙鼠 12只 ,取左侧耳蜗 ,每组动物取 10只耳蜗做平行蜗轴的石蜡切片 ,另 2只耳蜗做底回蜗轴及基底膜超薄切片。通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记技术 (terminaldeoxynucleotidyltransferase mediateddUTPnickendlabelingmethod ,TUNEL)及透射电镜检测连续用药不同时间后底回SG及Corti器的细胞凋亡情况。结果　连续应用顺铂 5～ 7d ,在透射电镜下可以观察到底回SG神经元及外毛细胞中出现凋亡特征性病理改变 ,TUNEL标记的石蜡切片中可见给药 5d后SG和Corti器出现的阳性细胞明显高于健康对照组 ,给药 6～ 7d阳性细胞进一步增加。结论　细胞凋亡是顺铂损伤沙鼠SG神经元和Corti器细胞的重要方式     

顺铂作用下沙鼠耳蜗螺旋神经节神经元和Corti器细胞的凋亡

目的 探讨顺铂是否可引起沙鼠耳蜗螺旋神经节(spiral ganglion，SG)神经元和Corti器细胞的凋亡。方法 对沙鼠进行顺铂连续腹腔注射，每日4mg／kg体重，分别于健康对照组及给药4、5、6、7d各处死沙鼠12只，取左侧耳蜗，每组动物取10只耳蜗做平行蜗轴的石蜡切片，另2只耳蜗做底回蜗轴及基底膜超薄切片。通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记技术(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method，TUNEL)及透射电镜检测连续用药不同时间后底回SG及Corti器的细胞凋亡情况。结果 连续应用顺铂5—7d，在透射电镜下可以观察到底回SG神经元及外毛细胞中出现凋亡特征性病理改变，TUNEL标记的石蜡切片中可见给药5d后SG和Cord器出现的阳性细胞明显高于健康对照组，给药6～7d阳性细胞进一步增加。结论 细胞凋亡是顺铂损伤沙鼠SG神经元和Cord器细胞的重要方式。     

腺病毒介导bcl-2基因转染对顺铂所致大鼠螺旋神经节细胞损伤的拮抗作用

目的 通过腺病毒(adenovirus,Ad)载体介导bcl-2基因转染体外培养的新生大鼠耳蜗螺旋神经节细胞(spiral ganglion cells,SGC),探讨bel-2蛋白过度表达对顺铂所致SGC损伤的拮抗作用.方法 体外培养新生大鼠SGC,携带绿色荧光蛋白(green fluorescent protein,GFP)基因的腺病毒载体Ad-GFP转染SGC,并行神经丝蛋白(NF200)免疫细胞化学染色鉴定,激光共聚焦扫描荧光显微镜下观察.携带bel-2基因的腺病毒载体Ad-bel-2转染SGC,蛋白质印迹法(Western Blot)检测bcl-2蛋白表达.设立Ad-bcl-2转染加顺铂组(A组),Ad-GFP转染加顺铂组(B组),顺铂组(C组)和正常对照组(D组),顺铂作用浓度为2μg,/ml;顺铂作用48 h后,行各组SGC计数,并通过lmageJ软件测量各组SGC轴突长度.结果 成功分离并培养新生大鼠SGC.激光共聚焦扫描荧光显微镜下观察到腺病毒载体可安全高效转染体外培养的SGC.Ad-bcl-2转染3 d后,Western Blot检测有外源性人bcl-2基因的高效表达,而Ad-GFP转染组和顺铂组未检测到表达.顺铂作用后,A、B、C组部分SGC细胞突起变短、萎缩,胞体缩小、变圆,甚至浮起.A组SGC数目明显多于B组和C组(P值均＜0.01),但少于D组(P＜0.05);A组SGC轴突长度明显长于B组和C组,但短于D组(P值均＜0.01).结论 腺病毒能够安全高效地转染体外培养的新生大鼠SGC.bel-2蛋白过表达对顺铂所致SGC损伤有一定的拮抗作用.     

脑源性神经营养因子对耳蜗螺旋神经节的保护作用

目的 观察腺疾病携带的脑源性神经营养因（brain derived neurotrophic factor,BDNF）在豚鼠耳蜗中的表达，及噪声损伤后螺旋神经节的保护作用。方法 27只白色纯豚鼠，暴露于135dBSLP，4kHz的窄带噪声4h。7d后，12只经圆注入腺病毒携带BDNF（adenoviral-mediated BDNF,ad-BDNF），12只经圆窗注入ad-LacZ,3只注入人工外淋巴液。分别于1、4、8周后取材，石蜡包埋中轴切片后，用免疫组化方法（ABC法）检测BDNF的表达。于光镜下计数螺旋神经节细胞。结果 在耳蜗各回中BDNF均有表达，4、8周组动脉较1周组织动物表达弱。在8周，注入ad-BNDF组较adLacZ组和人工淋巴组螺旋神经发生退行性病变的数目少，螺旋神经节细胞计数结果经统计学t检验，P＜0．01，差异有显著性。结论 腺病毒携带的神经营养因子在耳蜗中能高效表达，在噪声损伤情况下腺病毒携带的脑源性神经营养因子对螺旋神经节有保护作用。该研究为基因治疗感音神经性聋提供了坚实的实验基础。     

耳蜗毛细胞和螺旋神经节及其神经纤维的联合定量观察

目的：将耳蜗基底膜取材技术与耳蜗切片技术相结合以观察同一耳蜗中几个主要结构在病理发展中的相互关系。方法：耳蜗铺片用于定量观察毛细胞，骨性螺旋板切片用于定量观察缰孔内的神经纤维，中轴切片用于定量观察螺旋神经节。结果：将卡铂耳中毒灰鼠的耳蜗标本与正常灰鼠进行比较，显示内毛细胞，神经纤维和螺旋神经节的损失百分比基本一致。结论：卡铂对内毛细胞，神经纤维和螺旋神经节都有破坏作用，至于在卡铂病变早期，究竟哪一     

顺铂致聋后大鼠耳蜗螺旋神经元NeuroD的表达

目的检测神经分化因子NeuroD在大鼠耳蜗螺旋神经元顺铂损伤后的表达变化。方法通过免疫组织化学及Real-TimePCR技术,观察耳蜗螺旋神经元损伤1d、3d、5d后NeuroD的表达变化。正常成年SD大鼠32只随机分为对照组(生理盐水5ml/kg,1次/d,腹腔注射,连续5d),用药1d组(顺铂5mg/kg,腹腔注射),用药3d组(顺铂5mg/kg,1次/d,腹腔注射,连续3d),用药5d组(顺铂5mg/kg,1次/d,腹腔注射,连续5d),每组8只,建立顺铂耳毒性模型。采用Real-TimePCR、免疫组织化学染色检测不同时间螺旋神经元NeuroD的mRNA及蛋白表达变化。结果成功建立顺铂耳毒性大鼠模型,随着用药时间的延长,神经分化因子NeuroD在耳蜗螺旋神经元中呈动态变化。NeuroD的mRNA和蛋白表达在用药1d及3d组分别为2.17±0.39、1.15±0.20及7.02±0.69、2.42±0.40,与对照组及用药5d组比较具有统计学意义(P值<0.01)。结论 NeuroD在用药1d后开始增加,3d后达到高峰,5d后下降;在用药早期有一过性表达增强,后期表达下降同时听力损失明显。表明NeuroD可能参与顺铂损伤螺旋神经元后的修复过程。     

碱性成纤维细胞生长因子对耳蜗螺旋神经节细胞谷氨酸钠毒性作用的影响

目的了解谷氨酸钠致单离培养的小鼠耳蜗螺旋神经节细胞损伤后，碱性成纤维细胞生长因子（ｂＦＧＦ）对其生存及生长的影响，以及二者对细胞内游离钙离子浓度的影响。方法在培养液中加入２×１０２ｍｏｌ／Ｌ的谷氨酸钠２小时后，实验组分别换成含ｂＦＧＦ２５、５０、１００μｇ／Ｌ的培养液，对照组加空白培养液。结果２４小时后可见部分细胞裂解、死亡，对照组明显多于实验组。培养１４天后甲苯胺蓝染色证实，实验组的细胞生存数及细胞突起长度明显高于对照组，差异有显著性（Ｆ检验，Ｐ＜０．０１），且随着ｂＦＧＦ浓度增加，细胞生存数及细胞突起长度均增加，不同浓度实验组间差异有显著性（Ｐ＜０．０１）。用Ｆｌｕｏ３染色，激光扫描共聚焦显微镜观察１０个螺旋神经节细胞，培养液中加入谷氨酸钠后细胞内钙离子浓度迅速上升，在１０～１５秒内达峰值，１００秒左右恢复到正常水平；加入ｂＦＧＦ后细胞内钙离子浓度无明显变化（ｔ检验，Ｐ＝０．２０４）。结论谷氨酸钠可致螺旋神经节细胞内钙离子浓度升高，ｂＦＧＦ可以降低谷氨酸钠对螺旋神经节细胞的细胞毒性。     

Branching of spiral ganglion neurites is induced by focal application of fibroblast growth factor-1

OBJECTIVES/HYPOTHESIS: During the terminal innervation of the developing organ of Corti, fibroblast growth factor-1 (FGF-1) messenger RNA has been shown to be transiently expressed in the sensory epithelium of the rat, suggesting that this growth factor may contribute to developmental processes such as innervation and synaptogenesis of the inner and outer hair cells. In a previous study it has been demonstrated that exogenous FGF-1 supports rat spiral ganglion neurite extension in vitro, whereas a secreted form of FGF-1 produced by transfected fibrocytes induces neurite branching and targeting. STUDY DESIGN: Response of spiral ganglion neurites to FGF-1-coupled beads was compared with the response to noncoupled control beads. METHODS: Effects of multiple focal sources of FGF-1 to outgrowing spiral ganglion neurites were investigated on explants from postnatal day 4 rat spiral ganglion samples that were cultured in the presence of FGF-1 covalently coupled to polybead microspheres. After fixation and immunocytochemical labeling of the explants the growth patterns of the extending neuronal processes were evaluated. RESULTS: When spiral ganglion neurites were observed near clusters of FGF-1 beads, they formed a plexus-like network characterized by significantly higher branching in the vicinity of the beads. However, fibers did not appear to terminate on the beads. Plexus-like formations were not seen at a distance from FGF-1 coupled beads or in the vicinity of control beads lacking FGF-1 on their surface. CONCLUSION: The results of the study indicate that spiral ganglion neurites branch in response to focal sources of FGF-1, suggesting an important role of this growth factor for hair cell innervation during the terminal development of the sensory epithelium.     

Quisqualate excites spiral ganglion neurons of the guinea pig

The effects of quisqualate on primary afferent auditory neurons were examined by comparing the unit activity of guinea pig spiral ganglion neurons before, during, and after the infusion of artificial perilymph containing 1 mM quisqualate into the basal turn scala tympani. In 10 of 13 preparations quisqualate infusion increased unit activity above baseline rates established prior to infusion while control infusions of quisqualate-free artificial perilymph had no appreciable influence on the unit activity of four preparations. Postexcitatory depression typically followed peak evoked excitation, but no purely inhibitory responses were observed. Postexcitatory depression developed into a temporary cessation of spike production in three cases, suggesting depolarization blockade had developed; however, all of these preparations gradually regained some degree of spontaneous unit activity following the termination of quisqualate infusion. Quisqualate-induced excitation may be attributable to the activation of receptors for the afferent neurotransmitter released by hair cells. This interpretation is consistent with our working hypothesis that the primary afferent neurotransmitter is L-glutamate or a structural analog of this excitatory amino acid.     

Qualitative and quantitative observations of spiral ganglion development in the rat

The postnatal development of the spiral ganglion cells in the rat was studied from birth until the adult stage. At birth, a single population of ganglion cells is present. Some of them are surrounded by one or two layers of satellite cell processes. With maturation, the satellite cell processes increase in number around the cell body and its processes. At the end of the first postnatal week, two important events occur. The first is the appearance of myelin lamellae between the 4th and the 6th postnatal day in both ganglion cell processes, and between the 6th and the 8th day in the cell body. The second event is the appearance of a new type of cell (the Type II spiral ganglion cell) on the 6th to the 8th day postpartum. At this stage, the Type II cell is mainly characterized by densely packed neurofilamentous structures in the cytoplasm. Comparison between the myelination of the cell body and its processes reveals three main differences! There is a time lag of approximately 2 days between the onset of myelination in the cell body and in its processes. The kinetics of myelination are different in the cell processes and in the cell body. The myelination of the cell body starts slowly, whereas it is very fast in the processes. Later, the kinetics of myelination decrease in the processes, and increase in the cell body. At all stages including the adult, the fibers have a myelin sheath composed of more lamellae than the cell body. These observations are discussed with respect to development in other species.     

The protective effect of olfactory ensheathing cells on post-injury spiral ganglion cells

Conclusions: Transplantation of OECs into the cochlea may protect and increase the survival of SGCs.

Objective: To investigate the protective effect of the transplantation of olfactory ensheathing cells (OECs) on injured spiral ganglion cells (SGCs) in rats.

Methods: OECs were transplanted into the cochlea in rats with SGCs that were injured by kanamycin sulfate (KM). An equal volume of D-Hanks was injected into the cochlea of control rats. Auditory brainstem responses (ABRs) were recorded from the rats in both groups to monitor changes in hearing thresholds. Immunofluorescence was employed to examine the density and morphology of SGCs to assess the ototoxic condition of the cochlea.

Results: There was no significant difference in the ABR threshold at each frequency between the control and experimental groups. Notably, in the experimental group, a number of Hoechst 3334-labeled nuclei were detected from the apex to the basal turn of the cochlea, demonstrating that the OECs were successfully transplanted and survived in the cochlea. In the experimental group, most of the SGCs were tightly arranged, and the nuclear membrane, chromatin, and nucleolus were all clear. The SGCs in the control group were loosely arranged, and only a few normal SGCs were observed in this group.     

小鼠耳蜗螺旋神经节细胞电生理学特性的研究

目的：应用膜片钳技术记录小鼠耳蜗螺旋神经节细胞的全细胞电流,了解电压依赖性离子通道的基本电生理学特性,并比较耳蜗顶、底转螺旋神经节细胞电生理学特性的差异。方法：应用全细胞构型电压钳制技术,采用不同的电极内液及阻断剂,在不同的刺激参数下记录耳蜗顶、底转螺旋神经节细胞的电压依赖性离子通道电流,并进行分析比较。结果：实验记录到了内向的钠电流、延迟整流钾电流、超极化激活内向阳离子通道电流及瞬时外向钾电流,并发现耳蜗顶、底转螺旋神经节细胞的延迟整流钾电流及瞬时外向钾电流的电生理学特性具有显著性差异（P〈0．05）。结论：实验记录到的各种离子电流数据表明耳蜗螺旋神经节细胞具有完成动作电位的形成、传导并对其功能进行调节的离子通道基础;耳蜗顶、底转螺旋神经节细胞电生理学特性的差异有助于听觉的形成过程。     

低剂量哇巴因对螺旋神经节细胞损伤的保护作用

目的:探讨低剂最哇巴因对B27剥夺诱发的螺旋神经节细胞损伤的保护作用,并初步探讨其保护机制.方法:螺旋神经节细胞培养第7天开始分组实验,设B27剥夺损伤组(损伤组)、B27剥夺加10 nmol/L哇巴因保护组(保护组)和正常培养对照组(对照组).各组继续培养48 h后用FITC标记的Annexin-V和碘化丙啶双染试剂盒染色,用流式细胞仪检测各组螺旋神经节细胞损伤情况.各组螺旋神经节细胞分别于加药后6、12 h两个时间点用免疫细胞化学方法检测细胞内Bel-2表达水平.另外,应用螺旋神经节组织块分组培养48h后光学显微镜下观察轴(树)突生长情况.结果:流式细胞仪检测结果显示,保护组螺旋神经节细胞损伤率为(9.0±1.3)%,显著低于损伤组(32.8±2.4)%,与对照组(6.3±0.3)%相比无明显差异;免疫细胞化学结果显示,保护组螺旋神经节细胞6h点Bcl-2水平较损伤组和对照组增加.螺旋神经节组织块培养显示,保护组轴(树)突生长明显优于损伤组.结论:低浓度哇巴因对B27剥夺诱发的螺旋神经节细胞损伤具有保护作用.此作用可能通过上调螺旋神经节细胞内Bcl-2表达水平来实现.     

The spiral ganglion and hair cells of Bronx waltzer mice

Inner hair cells and spiral ganglion cells were counted in a mutant mouse strain (Bronx waltzer) reported to have very few of these cells (6) in order to determine if the remaining ganglion cells would be predominantly type II cells. These cell counts indicate a 50% reduction of spiral ganglion cells in Bronx waltzer cochleas compared to normal mice. Averaged throughout the cochlea about 11% of the remaining cells are type II cells while in normal mice this percentage is 5%. In some regions however, as many as 20% of the remaining cells are type II cells. Counts of IHC in surface preparations reveal 37 normal looking IHC (about 5% of the normal population) in each of two Bronx waltzer mouse cochleas. There were also about 120 shrunken IHC in each cochlea, representing 17% of the normal cell population. While there appears to be an increased proportion of type II cells in the Bronx waltzer spiral ganglion there are also many more type I cells than might be expected from the small number of IHC.