耳蜗血管纹组织块的缘细胞培养

目的 :建立豚鼠耳蜗血管纹 (SV)组织块缘细胞 (MCs)的培养方法 ,为进一步研究药物耳毒性及其作用机制奠定基础。方法 :2 6只豚鼠按SV培养时间随机分成 4组 :2 4h组 (n =8) ;72h组 (n =8) ;>72h组 (n =8) ;对照组 (新鲜SV固定组 ,n =2 )。显微解剖数段连同螺旋韧带的SV组织块 ,置于 5 %CO2 / 95 %空气的二氧化碳恒温 (37℃ )培养箱中进行培养 ,分别进行形态学和组织学观察。结果 :培养 2 4hSV组织块保持良好活性 ,其组织学结构与新鲜固定的SV结构无明显差异 ;培养 72hSV组织块与新鲜固定的SV在组织学结构方面有显著性差异 ,不能观察到正常的SV结构 ,组织结构松散 ,缘细胞从组织块离心性生长出来 ;从SV组织块培养出的缘细胞能在培养皿内存活 13d。结论 :采用组织块培养技术 ,成功地建立了豚鼠耳蜗SV组织块的缘细胞培养方法 ;培养 2 4h的SV组织块光镜下保持了良好活性和正常组织学结构 ,可用来进一步研究药物耳毒性及其作用机制。     

成年大鼠耳蜗血管纹缘细胞的体外培养

目的:建立成年大鼠耳蜗血管纹缘细胞(MC)的体外培养体系。方法:采用活体组织分离及培养技术,获取培养的大鼠耳蜗MC,透射电镜观察细胞超微结构,免疫组织化学法检测上皮细胞标志性中间丝角蛋白的表达及纯度,RT-PCR检测MC特征性的中间丝角蛋白mRNA的表达。结果:体外培养的MC呈不规则多角形,细胞增殖相互连接,紧密排列呈现培养上皮细胞典型的"铺路石"样外观,并可见由数百个MC密集生长组成的折光性强的球形结构(dome);透射电镜观察可见细胞周围有微绒毛样结构;免疫细胞化学检查,纯化后95%以上的MC细胞质呈桔黄色,而对照组无此着色。RT-PCR扩增出标志中间丝角蛋白基因表达的PCR产物,条带位于300~400 bp,与目的条带(368 bp)相符。结论:采用组织块培养技术成功建立成年大鼠耳蜗血管纹MC的体外培养体系,为进一步研究成熟期MC的功能提供合适的细胞模型。     

Primary culture of vital marginal cells from cochlear explants of the stria vascularis

Summary Explants of the stria vascularis and spiral ligament were dissected from guinea pig cochleae and were successfully cultivated for several weeks. After 2 days, fibroblast-like cells of the spiral ligament covered the bottom of the cell culture dish around the explant. Marginal cells of the stria vascularis proliferated and grew on the luminal surface towards the border of the explant at a rate of 15 m/day. At day 6 in culture the proliferating marginal cells reached the border of the explant and then advanced to the bottom of the cell-culture dish. There the marginal cells replaced fibroblast-like cells and built an epithelial hexagonal-shaped monolayer. Light microscopic and transmission electron microscopic investigations revealed that the cultured cells were viable and that typical morphological characteristics of marginal cells were preserved. Cultivation of these cells provides a unique model for studies of physiological properties of marginal cells of the stria vascularis.     

Quinacrine staining of marginal cells in the stria vascularis of the guinea-pig cochlea: a possible source of extracellular ATP?

There is accumulating evidence for a purinergic humoral system involved in the control of cochlear function. Evidence of specific P₂ purinoceptors on cochlear tissues implies a role for extracellular adenosine triphosphate (ATP) in the cochlea. To further this hypothesis a study was undertaken to determine if there was any specific source of purine compounds in cochlear tissues. Cochlear tissues (the sensory epithelium and lateral wall) from the guinea pig were incubated with the acridine derivative quinacrine dihydrochloride (5×10⁻⁶ M in phosphate-buffered saline for 30 min at room temperature) which fluoresces on binding to high concentrations of ATP. Most cochlear tissues showed a diffuse green fluorescence slightly above the background level. However, a region of the marginal cells of the stria vascularis showed a specific punctate fluorescence. Optical sectioning of these cells by confocal microscopy revealed that the fluorescent structures in these marginal cells was confined to a region up to 10 μm from their endolymphatic surface. Similar cells studied by transmission electron microscopy showed membrane-bound vesicles located in the same region of the cell. These data imply that purine compounds are localized in discrete structures, perhaps vesicles, within the marginal cells which could serve as a source of extracellular ATP in the cochlea.     

Endocytosis of CF in marginal cells of stria vascularis regulated by ROCK and MLCK signaling cascade,but not G-proteins

Objective The endocytosis of cationized feritin (CF) via a clathrin-mediated pathway is regulated by a signaling network. Marginal cells showed the active endocytosis of CF via a clathrin-mediated pathway. The internalization of receptors through this clathrin-mediated pathway is an important regulatory event in signal transduction. Numerous kinases are involved in endocytosis, and each endocytic route is subjected to high-order regulation by cellular signaling mechanisms. In this study, we investigated whether ROCK and MLCK signaling cascades and G-proteins regulate the endocytosis of CF in marginal cells of the stria vascularis.Methods CF was infused into the cochlear duct with pertussis toxin (PTX),Clostridium botulinum C3 toxin (BTX), guanosine(g-thio)-triphosphate (GTP-γS), ML-7, Y-27632. Endocytic activity was measured at 30 min after the start of infusion under an electron microscope.Results In marginal cells, CF was internalized via a clathrin-mediated pathway that depends on F-actin and microtubules (MT). Its processes were controlled by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK), but not affected by G-protein-coupled receptor (GPCR) or the RhoA signaling cascade.Conclusion Our previous study showed that the main endocytotic pathway of microperoxidase (MPO) did not depend on the Rho/ROCK molecular switch or actin/myosin motor system, but was mainly regulated by the RhoA signaling cascade. The present study results indicate that these signaling cascades regulating CF internalization completely differ from the cascades for MPO internalization.     

新生大鼠耳蜗血管纹缘细胞释放ATP的机制

目的 证实体外培养的新生大鼠耳蜗血管纹缘细胞能够释放ATP,并进一步探讨缘细胞释放ATP的机制.方法 分离、培养新生大鼠耳蜗血管纹缘细胞,采用生物发光法分别检测巴佛洛霉素A1、己二酸二癸酯( didecyl adipate,DDA)、细胞外K+、毒胡萝卜素、细胞外Ca2、U73122及马兜铃酸钠对细胞外液中缘细胞ATP释放的影响.结果 随着巴佛洛霉素A1浓度的增加,细胞外液中ATP的浓度明显下降;当DDA浓度增加时,细胞外液中ATP的浓度几乎呈线性增加.随着细胞外液中的K+浓度的增高,缘细胞释放的ATP浓度呈现上升趋势,当细胞外液中的K+浓度为9.15 mmol/L时,ATP的释放量达到峰值,之后随着K+浓度的继续升高ATP的释放量呈下降趋势.随着毒胡萝卜素浓度的增加,缘细胞释放的ATP浓度呈现明显下降的趋势.当细胞外Ca2+浓度为0 mmol/L时,缘细胞仍然释放ATP;Ca2+浓度增加与ATP的释放呈负相关,但当细胞外的Ca2+浓度达到1.25 mmol/L以上时,ATP的释放量维持在一个较稳定的水平.U73122的浓度在0.25～1.25 μmol/L时,其与缘细胞释放的ATP浓度呈负相关.当马兜铃酸钠的浓度为12.5 ～ 100.0 μmol/L,缘细胞ATP释放呈明显下降的趋势;当其浓度＞100.0 μmol/L时,ATP释放浓度趋于平稳.结论 体外培养的新生大鼠耳蜗血管纹缘细胞能够释放ATP,其释放量与钙泵、K+通道状态以及细胞内信号传导通路相关酶的活性有关.     

Kinocilia in the developing stria vascularis of the rat pup

The mammalian stria vascularis undergoes certain developmental changes in the postnatal rat. The present study was designed to examine the ultrastructure of the stria vascularis in rat pups from immediately after birth to 20 days postpartum. The cochlea were removed with the animals under xylazine (Rompun) anesthesia and were prepared for transmission electron microscopy. Each of the three cell types in the stria were found to contain kinocilia up until 12–17 days of age. The presence of kinocilia in the intermediate and basal cells has not been previously described. Findings suggest that these organelles may serve a motile and/or sensory function to assist in the maturation of cell functions, particularly ion transport, during early stages of development.     

大鼠耳蜗血管纹缘细胞氧化性损伤体外模型的建立

目的 建立大鼠耳蜗血管纹缘细胞氧化性损伤的体外模型.方法 在体外培养的大鼠耳蜗血管纹缘细胞中加入过氧化氢(H2O2),观察细胞形态结构变化;采用CCK-8(cell counting kit-8)法检测200、300、400、600、800μmoL/LH2O2作用0.5、1、2、4、16、24 h对血管纹缘细胞活性的影响;检测不同浓度H2O2作用2 h后血管纹缘细胞脂质过氧化产物丙二醛含量的变化;利用碘化丙锭染色流式细胞仪测定细胞的凋亡率;通过免疫印迹法(Western blot)检测凋亡因子半胱氨酸天冬氨酸蛋白酶3(cmpase-3)活化片段cleaved-caspase-3的表达.结果 H2O2作用后血管纹缘细胞出现核固缩、边缘化,胞质浓缩,被膜包裹、隆起,产生凋亡;随着H2O2浓度的增加、作用时间的延长,缘细胞活性降低;200 μmol/L的H2O2作用2 h,即可诱导缘细胞凋亡率升高,差异具有统计学意义(P<0.05);cleaved-caspase-3在正常缘细胞呈微弱表达,H2O2作用后cleaved-caspase-3表达增强,并随H2O2浓度的增高而增强,但当H2O2达到600 μmol/L时,表达开始减弱,800 μmol/L时仅见微弱表达.结论 利用H2O2可成功建立耳蜗血管纹缘细胞氧化性损伤的体外模型,caspase-3的激活参与了缘细胞的氧化损伤过程.     

Morphological observation of the stria vascularis in midkine and pleiotrophin knockout mice

Objectives

Midkine and Pleiotrophin are low molecular weight basic proteins with closely related structures and serve as growth/differentiation factors. They have been reported to be expressed in the cochlea during the embryonic and perinatal periods. In the present study, we focused on the roles of midkine and pleiotrophin in the stria vascularis and investigated morphological changes using mice deficient in these genes.

Methods

Midkine knockout, pleiotrophin knockout, and double knockout mice were used and compared to wild-type mice. Auditory brain stem responses (ABRs) and cochlear blood flows were measured in each type of mice. Pathological changes in the stria vascularis were examined by light microscopy, including immunohistochemical staining with anti-Kir4.1 antibody, and electron microscopy.

Results

Hearing thresholds examined by ABRs were significantly higher in midkine knockout and pleiotrophin knockout mice than in wild-type mice. Double knockout mice showed higher thresholds compared to midkine knockout and pleiotrophin knockout mice. Blood flow in the lateral walls did not significantly differ and light microscopy examination showed an almost normal appearance of the stria vascularis in these knockout mice. However, the expression of Kir4.1 was weak in the knockout mice and severe vacuolar degeneration was observed by electron microscopy in the intermediate cells of the double knockout mice.

Conclusion

The present study demonstrates that midkine and pleiotrophin play some roles for the morphological maintenance of intermediate cell in the stria vascularis.     

Scanning electron microscopy of the human cochlea — the stria vascularis

Summary The endolymphatic surface of the stria vascularis from normally hearing human ears has been studied with the scanning electron microscope. The cells in contact with the endolymph usually have a hexagonal-shaped surface and possess many microvilli, whilst depressions or pits are rarely seen. There is marked heterogeneity of these cells with variations in the size of the cells and the number of microvilli present on each cell.A transitional zone occurs between the stria vascularis and the spiral prominence. The surface of the cells of the transitional zone are larger and elongated whilst the cells of the spiral prominence have a smaller and more regular appearance.This work has been supported by grants from the Medical Research Council of Great Britain and from the Merseyside Regional Health Authority     

内皮舒张因子对豚鼠耳蜗血管纹血管的保护作用

本实验利用活体显微镜摄像技术、透射电镜技术,观察了内皮舒张因子（EDRF）对豚鼠耳蜗微循环的保护作用。结果提示：①速尿组（F）动物,经静脉注射药10min后,耳蜗微动脉缺血,血管内皮细胞损伤;②对速尿／L-精氨酸组（F／L－Arginine）动物,EDRF能使速尿引起的微动脉缺血明显改善,血管纹血管超微结构的损伤程度较单纯F组减轻;③速尿／L-硝基-精氨酸组（F／L－NNA）动物,耳蜗的缺血程度较F组加重。结论提示;EDRF能通过增加局部血流的灌注而改善和保护耳蜗微循环。本研究提供的实验结果,于临床开展微循环致聋疾病的治疗有所启迪。     

胎儿耳蜗血管纹的扫描电镜观察

目的：借助扫描电镜技术观察血管纹的表面结构,以便对血管纹的结构和功能提供新的信息。方法：标本取自尸检3个足月胎儿的6个颢骨,在死后尽可能快速取出耳蜗,经处理后,借助扫描电镜技术观察人胎儿耳蜗血管纹的超微结构。结果：借助扫描电镜技术可从耳蜗基底圈到顶圈,上起前庭膜嵴,下到基底膜的基底嵴全面观察血管纹的各个部分,血管纹边缘细胞表面结构呈圆球形．细胞表面有许多微绒毛。螺旋凸部位有一个细胞移行区,这个区域的边缘细胞表面形态明显不同,细胞细K或呈不规则的多角形细胞,细胞边界微绒毛丰富,显出明显的细胞界限,细胞表面分布均匀的微绒毛。血管纹断面的观察还可获得中间细胞、基底细胞和毛细血管结构。结论：扫描电镜观察胎儿耳蜗血管纹,可获得整个血管纹全貌的表面精细结构,尤其是边缘细胞的表面形态,通过对血管纹断面的观察还可获得中间细胞、基底细胞的精细结构特征,为认识血管纹的结构和功能提供新的信息。     

Respiratory quotient of stria vascularis of guinea pig in vitro

Summary The respiratory quotient of the stria vascularis was measured in vitro by means of Cartesian diver microgasometry. A value of about 1.2 was found when the incubation medium was phosphate-buffered serum substitute with glucose as the sole substrate. This value suggests that endogenous lipids and amino acids do not contribute significantly to strial respiratory metabolism and that carbohydrate is the primary fuel in vitro. A high activity of the hexose monophosphate pathway may be responsible for raising the respiratory quotient above unity.Supported by the grant NS 06575 from the National Institutes of Health and the grant 77-16842 from the National Science Foundation     

Cell membrane alterations in the stria vascularis of the guinea pig after ethacrynic acid treatment studied by freeze-fracture

Summary A freeze-fracture examination of the stria vascularis during the first 2 h after injection of ethacrynic acid was performed. This showed a re-distribution of the particles on the membrane fracture faces of both marginal and intermediate cells. As oedematous spaces developed, particle-poor, vesicle-like structures were found associated with both cell types. The tight junctions at the apices of the marginal cells and around basal cells were unaffected.This work was supported by the Wellcome Trust in the form of a Research Fellowship     

Simultaneous investigations of elemental changes in individual cells of the stria vascularis and in endolymph

Summary Using the microprobe for energy dispersive X-ray microanalysis, the elemental compositions of both the individual cells of the stria vascularis and of the endolymph were followed simultaneously under normal conditions and after the administration of 120 mg/kg ethacrynic acid (EA). Marginal cells and intermediate cells showed reversible increases in potassium and decreases in sodium concentrations. Shifts in the ionic composition of endolymph occurred later than after elemental changes in the strial cells. The present results indicate that the marginal and the intermediate cells are the primary target for EA-induced ototoxicity. However, generalized toxic effects of EA are also indicated, with a general leakage of different elements occurring during the 30–60 min period after EA administration.Supported by grants from the Swedish Medical Research Council (12X-7305 and 12X-720), the Ragnar and Torsten Söderberg Foundation, the Foundation Tysta Skolan, the University of Umeå and Karolinska Institute     

庆大霉素对豚鼠血管纹黑色素的影响及其机制

目的了解庆大霉素(gentamicin,GM)对豚鼠血管纹黑色素的影响及其作用机制.方法豚鼠杂色40只和白色20只每日肌内注射庆大霉素150 mg/kg体重7 d后用脑干诱发电位仪检测两种豚鼠的听阈变化,以透射电镜观察杂色豚鼠用药前后血管纹内黑色素含量及酪氨酸酶活性的变化,并用免疫组化法观察血管纹增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)表达的变化.结果庆大霉素作用后,杂色和白色豚鼠的听阈与用药前比较,差异有非常显著性意义(P<0.001),且两组豚鼠间的阈移差异也有非常显著性意义(P<0.001).杂色豚鼠对照组和实验组(各10只)血管纹中间细胞内平均(±s,以下同)每个观测区域(300 μm2)的黑素体含量分别为(19.83±2.74)个和(58.33±16.22)个,差异有非常显著性意义(P<0.001);酪氨酸酶活性反应产物面积与测定区域面积之比从1.65%±0.40%增加为3.45%±0.41%,差异有非常显著性意义(P<0.001);但对照组与实验组PCNA阳性中间细胞分别为(14.08±2.76)个和(13.58±2.09)个,差异无显著性意义(P>0.05).结论庆大霉素作用后血管纹内黑色素含量的增加,可能是由于药物促进了酪氨酸酶活性的增强,而不是促进黑素细胞的增殖能力增强.黑色素可保护豚鼠内耳免受庆大霉素的耳毒性作用.     

The resting potential of marginal cells in stria vascularis explants

Summary In order to examine the intracellular potential of stria vascularis marginal cells (MCs) under direct visual control, a short-term organ culture of guinea pig stria vascularis (SV) was developed. The experimental conditions allowed exposure of the luminal surface of the SV to artificial endolymph or perilymph. Using conventional microelectrodes and inverted bright-field microscopy, impalement of MCs from the endolymphatic side through the luminal cell membrane was achieved. With artificial perilymph at the luminal side a small positivity at the cell surface and low negative intracellular potentials were recorded at room temperature (23°C). The initial recording was –6.5 ± 5.0 mV while the stable recording was –4.0 ± 3.6 mV. Similar results were obtained at a bath temperature of 37°C. Furthermore, subtotal exposure of the luminal cell surface to artificial endolymph did not result in a significant potential change.     

Changes in the stria vascularis of the guinea pig due to cis-platinum

Summary The microscopical and ultramicroscopical changes in the stria vascularis due to cis-diamminedichloroplatinum II (DDP) were studied. Sixteen healthy adult guinea pigs were used for the experiment. A standard dosage DDP (1.5 mg/kg/d) was administered over a period of 5–20 days. A clear degeneration pattern was found (ranging from no changes to cystic degeneration with protrusion of marginal cells followed by loss of marginal cells). DDP seems to be especially toxic for marginal cells in stria vascularis in the guinea pig.Supported by grants from the Heinsius Houbolt Foundation     

Ultrastructure of guinea pig stria vascularis processed by rapid freezing and freeze substitution

The rapid-freezing and freeze-substitution method fixes a specimen as if it were prepared before excision. We used this method to study the stria vascularis of guinea pigs using electron microscopy. Findings were essentially the same as those obtained with conventional chemical fixation, although freeze substitution made it possible to observe the membrane structures in a smoother and more linear manner. This method seems to be the procedure of choice for studying the instantaneous movement and behavior of cells.