Effects of gadolinium injected into the middle ear on the stria vascularis

Conclusion.The concentration of gadolinium (Gd) used clinically showed no remarkable effects on the stria vascularis; however, a higher concentration had adverse effects. The concentration of Gd must be borne in mind when injecting Gd into the tympanic cavity. Objective. Endolymphatic hydrops has been visualized using high resolution MRI with the intratympanic administration of Gd in patients with Meniere's disease. We attempted to investigate the effects of Gd on the stria vascularis. Materials and methods. Gd hydrate diluted eightfold with saline or non-diluted Gd or saline was injected into the tympanic cavity of guinea pigs. To investigate the effects of Gd on the stria vascularis, we measured endocochlear DC potential (EP) and observed the stria vascularis using transmission electron microscopy. Results. Intratympanic injections of Gd hydrate diluted eightfold with saline (1/8 Gd) and saline did not cause apparent changes in the EP. Moreover, the amplitude of the EP decreased significantly 60 min after non-diluted Gd was injected. Transmission electron micrographs of the stria vascularis revealed no significant morphological difference between the ears injected with 1/8 Gd and those injected with saline. There was significant morphological change in the ear injected with non-diluted Gd. The intercellular spaces were markedly enlarged.     

Effects of organic acids on the edema of the stria vascularis induced by furosemide.

Furosemide is a loop diuretic which is ototoxic. Investigations have shown the stria vascularis to be the target tissue of this ototoxic drug. The purpose of the present study was to investigate the effects of furosemide on the stria vascularis in chinchillas, in controls and in animals pretreated with the above organic acids. Control animals were injected with 0.5 ml alkalinized saline followed by furosemide IV 30 min later. Experimental animals received probenecid, penicillin or sodium salicylate IV. Thirty minutes later, furosemide was injected in the same dose as in the controls. The basal turn of the stria vascularis was rapidly removed at various times from 10 to 30 min after furosemide administration and processed for transmission electron microscopy. Control animals were found to have reversible edema of the stria vascularis. Experimental animals had variable findings. Those animals pretreated with penicillin had virtually no edema of the stria vascularis at any time. Salicylate and probenecid pretreated animals had significantly less edema from one to 10 min after furosemide injection, but more edema than controls at later times. These findings suggest a discrepancy between ultrastructural pathology and functional status of the cochlea in experimental animals pretreated with probenecid or sodium salicylate followed by furosemide. On the other hand, good structure function correlations were seen in controls and in experimental animals pretreated with penicillin.     

Effects of organic acids on stria vascularis ultrastructure and function in the chinchilla

Summary The purpose of this study was to compare the effects of several organic acids (probenecid, sodium salicylate and penicillin G) on the endocochlear potential (EP) and the ultrastructure of the stria vascularis of the chinchilla with the effects of furosemide on these parameters. chinchillas received 50 mg/kg i.v. doses of probenecid, sodium salicylate or penicillin G, or 25 mg/kg i.v. furosemide. The EP was monitored continuously before and for 60 min afterwards. The stria vascularis was removed at 10-min intervals from animals and from 10 to 60 min after the injection of these agents. Specimens were then processed for transmission electron microscopy. Only furosemide had an effect on the EP, causing a reversible reduction. The reduction of the EP was accompanied by the appearance of edema in the intercellular spaces of the stria vascularis. No significant edema was found after probenecid, sodium salicylate or penicillin G. This was consistent with the finding that none of these latter three agents affected the endocochlear potential.Offprint requests to: L. P. Rybak     

Mechanisms of endocochlear potential generation by stria vascularis

It is commonly accepted that the endocochlear potential (EP) of the cochlea is generated by an electrogenic transport of potassium into scala media by the marginal cells of stria vascularis. We have studied the potential and potassium concentration gradients as stria vascularis was penetrated with double-barreled potassium selective electrodes in the guinea pig cochlea. Our data demonstrate that a region exists in stria which is positively polarized (higher than the EP), but which has a low (perilymph-like) potassium composition. It is concluded that EP cannot be generated by the marginal cells alone but may involve passive potassium movement across the apical membranes of the basal cells. A model is presented which is consistent with many anatomical and physiological features of stria vascularis.     

窒息对豚鼠耳蜗内直流电位的影响

目的:探讨缺氧引起听功能障碍的发病机制,了解窒息对耳蜗内直流电位(EP)的影响。方法:选择听耳廓反射良好豚鼠,采用血管纹法检测耳蜗内直流电位,观察呼吸暂停时及恢复人工呼吸后耳蜗内直流电位的变化。结果:①正常EP初始值实验组为(76.4±8.4)mV,对照组为(80.8±8.4)mV,组间差异无统计学意义;②呼吸暂停时全部豚鼠经过8~34 s潜伏期后EP迅速下降,EP下降速度与潜伏期呈正相关;③呼吸暂停3 min后EP最低值平均为(-17.5±4.4)mV,与潜伏期及下降速度呈正相关;④恢复人工呼吸后平均(85.0±16.0)s EP恢复至初始值,并有7例出现过冲现象。结论:EP下降预示呼吸暂停时毛细胞生存环境出现异常;呼吸停止3min全部豚鼠EP变为负值,提示呼吸停止3 min不会导致全部毛细胞功能丧失、血管纹也不会发生不可逆功能障碍。     

Bumetanide-induced enlargement of the intercellular space in the stria vascularis requires an active Na+-K+-ATPase

OBJECTIVE: Loop diuretics such as bumetanide and furosemide cause an acute enlargement of the intrastrial space of the stria vascularis, with an associated decline in the endocochlear DC potential (EP). The aim of this study was to determine the role played by the Na+-K+-ATPase in the bumetanide-induced enlargement of the intrastrial space, and to examine the importance of the balance between the activities of the Na+-K+-2Cl- cotransporter and the Na+-K+-ATPase to the physiological function of the stria vascularis. MATERIAL AND METHODS: Albino guinea pigs were used in experiments involving perilymphatic perfusion, EP measurement and electron microscopy. The effects of bumetanide on the stria vascularis were examined following inhibition of the Na+-K+-ATPase by ouabain. Ouabain was administered to the perfusate and, when the EP reached 0 mV, both ouabain and bumetanide were administered. RESULTS: Although there was no enlargement of the intrastrial space, vacuoles were apparent in marginal cells. The vacuolar change in marginal cells was similar to that caused by ouabain alone. CONCLUSION: This study indicates that the enlargement of the intrastrial space requires not only the blockade of the Na+-K+-2Cl- cotransporter but also normal activity of the Na+-K+-ATPase, and suggests that the bumetanide-induced enlargement of the intrastrial space resulted from the imbalance between the activities of the Na+-K+-2Cl- cotransporter and the Na+-K+-ATPase.     

Sources of endolymph and EP. Does the perilymph or Reissner's membrane influence the ionic content of the endolymph and EP?

The source of EP and the production of the endolymph are often considered to be at the stria vascularis. However, the significance of Reissner's membrane in maintaining the ionic composition of the endolymph cannot be ruled out. We perfused the entire perilymphatic space of guinea pigs with paraffin oil to determine the effects on the endolymph and perilymph. After 1 and 2 h the endolymph was removed from the second turn and the ionic concentration of these samples was measured by flamephotometry. The results revealed no significant changes in Na+ and K+ concentration as compared to normal endolymph. EP was measured from the second turn under an equivalent experimental condition. During 1 h of oil perfusion, EP remained fairly constant. From these experiments we surmise that perilymph and Reissner's membrane do not influence the ionic composition of the endolymph and EP during 1 or 2 h of our experiments.     

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。     

Effect of a beta-stimulant on the inner ear stria vascularis

In vivo and in vitro experiments were conducted to investigate the effect of alpha-isoproterenol on the inner ear stria vascularis with intracellular cytochrome oxidase activity used as an index. Intraperitoneal injection of alpha-isoproterenol (5 mg/kg) was performed in 10 rats, and that of physiological saline in 4 rats, for 21 consecutive days. After the 3-week treatment, bilateral cochleas were excised for frozen sections and stained for cytochrome oxidase. The staining density of the stria vascularis for the enzyme was analyzed with a computer. Electron microscopic observation was also performed for some specimens. As for the in vitro experiments, bilateral cochleas from 6 normal rats were excised for cell culture. Cochlear cells from the right ear were cultured with medium containing alpha-isoproterenol (10-micromol/L concentration), and those from the left ear with medium alone. After 3-day culture, the enzyme activity of cytochrome oxidase in the stria vascularis was quantified by the same method used for the in vivo experiments. Cytochrome oxidase activity was markedly elevated in the alpha-isoproterenol group. The activity tended to be higher in the lower turns of the cochlea. Electron microscopy revealed that numerous mitochondria were present in marginal cells that protruded into the endolymphatic space. The enzyme activity was also elevated in the stria vascularis from cochlear specimens in the alpha-isoproterenol group of the in vitro experiment. The above results suggest that alpha-isoproterenol accelerated the metabolic activity of the cells that constitute the stria vascularis. The increase in activity was probably attributable to direct pharmaceutical effects of the beta-stimulant, rather than an increase in blood flow. It is possible that the cells that constitute the stria vascularis may have beta-receptors.     

内皮素在噪声性内耳损伤过程中的作用

目的研究噪声性内耳损伤过程中耳蜗微循环的病理变化以及内皮素(endothelin，ET)在这一病理过程中的作用及意义。方法30只大鼠按电脑产生随机数字法分为5组：对照组、噪声暴露(115dB白噪声每天8h)1、4、8和15d组。分别用扫描电镜观察毛细胞的形态，血管纹铺片观察耳蜗微循环的状态，放射免疫方法检测血浆ET含量，免疫组化染色观察ET-1、ETA、ETB的分布。结果噪声4、8、15d组出现了血管纹明显缺血，外毛细胞形态异常。血浆ET于4d组出现暂时性升高。ET-1于耳蜗组织中有广泛的阳性表达，噪声暴露前后差异无统计学意义；ETA主要分布于血管纹中间细胞与微血管壁周围的细胞，对照组与1d组表达呈弱阳性( )，噪声4、8、15d组表达呈强阳性( )。ETB主要分布于血管纹毛细血管内皮细胞，其变化趋势与ETA相似。结论噪声性内耳损伤过程中出现了明显的耳蜗微循环障碍，同时大鼠耳蜗组织的ET系统活性增高，二者在时间上有明显的一致性。推测噪声所致的耳蜗微循环障碍过程中ET起了重要的介导作用。     

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。     

Reduction in the endocochlear potential caused by Cs(+) in the perilymph can be explained by the five-compartment model of the stria vascularis

In an earlier publication (Takeuchi et al., Biophys. J. 79 (2000) 2572-2582), we proposed that K(+) channels in intermediate cells within the stria vascularis may play an essential role in the generation of the endocochlear potential (EP), and we presented an extended version of the five-compartment model of the stria vascularis. In search of further evidence supporting the five-compartment model, we studied the effects of Cs(+) added to the perilymph on guinea pig EP. Cs(+) is known as a competitive K(+) channel blocker. Both the scala tympani and the scala vestibuli of four cochlear turns were perfused at a flow rate of 10 microl/min, and the EP was recorded from the second cochlear turn. Cs(+) at 30 mM caused a biphasic change in the EP; the EP increased transiently from a control level of 89.6 mV to 94.8 mV within 10 min, and then decreased to a steady level of 24.5 mV within the next 40 min. We propose that the initial transient increase in the EP results from Cs(+)-mediated blockade of K(+) conductance in the basolateral membrane of hair cells, and that the subsequent EP decrease is due to effects of Cs(+) on the stria vascularis. We believe that Cs(+) in the perilymph is able to access the stria vascularis by being taken up by fibrocytes in the spiral ligament and then being transported to intermediate cells because it is known that Cs(+) is taken up via Na(+),K(+)-ATPase and that gap junctions connect fibrocytes in the spiral ligament to basal cells and basal cells to intermediate cells. To clarify the effect of intracellular Cs(+) on the electrophysiological properties of intermediate cells, these cells were dissociated from guinea pigs and studied by the whole-cell patch-clamp method. Intracellular Cs(+) depolarized intermediate cells in a dose-dependent manner. In addition, efflux of Cs(+) from the intermediate cell was much less than the efflux of K(+). Thus, Cs(+) may accumulate in the intermediate cell, which depolarizes the cell, which in turn decreases the EP. We conclude that the five-compartment model of the stria vascularis can explain the EP decrease caused by Cs(+) in the perilymph.     

Cisplatin ototoxicity in guinea pigs with special reference to toxic effects in the stria vascularis

The toxic effects of cisplatin (cis-diamminedichloroplatinum [II]) on the organ of Corti are well established. Few and conflicting data on this drug's effects on the stria vascularis exist. The present study presents animal experiments on the toxic effects of cisplatin in the stria vascularis and in the organ of Corti. Cisplatin-induced toxicity in albino and pigmented guinea pigs was evaluated morphologically and functionally, using light and transmission electron microscopy as well as auditory brainstem-evoked potentials on the organ of Corti and the stria vascularis. The results showed variability in hearing thresholds, ranging from no change to hearing loss of 30 dB, and prominent damage in the organ of Corti and in the stria vascularis. The toxic effects to both the organ of Corti and the stria vascularis should be considered when cisplatin is used in chemotherapy.     

小鼠耳蜗血管纹Na-K-2Cl联合转运子1在顺铂耳毒性发生时的改变

目的研究顺铂(cis-dichlorodiammine platinum,Cisplatin)耳毒性发生后耳蜗血管纹Na-K-2Cl联合转运子1(NKCC1)的表达情况,并初步探讨其机制。方法选取健康CBA/CaJ小鼠20只,随机分为对照组和实验组各10只,实验组动物连续腹腔注射顺铂3.5mg.kg-1.d-1,建立顺铂耳毒性小鼠模型,对照组注射等量生理盐水。以听性脑干反应(ABR)阈值作为评价听功能的指标,检测给药前后小鼠听功能的改变,并采用免疫组织化学(SP法)结合免疫荧光实验技术,观察对照组和实验组小鼠腹腔注射顺铂前后耳蜗血管纹NKCC1表达的变化。结果NKCC1在小鼠耳蜗血管纹主要表达于边缘细胞,而顺铂作用后血管纹边缘细胞的NKCC1表达明显减弱,图像分析显示两组平均灰度值差异有显著统计学意义(P<0.01)。结论小鼠顺铂耳毒性作用后血管纹边缘细胞NKCC1的表达量明显减弱,这可能是顺铂耳毒性发生机制中的一个重要环节。     

Specificity of action of vanadate to the organ of corti

Although vanadate strongly inhibits Na/K-ATPase activity of the stria vascularis in vitro, it initially causes no depression of the ouabain-sensitive endocochlear potential (EP) when perfused perilymphatically or via the vasculature. However, when the perilymph of scala tympani is replaced with artificial media containing 0.1 to 1 mM vanadate, there is a large (about 17 mV) increase in the EP of the second cochlear turn. Further experiments showed that the cochlear microphonics declined during the time in which the EP increased, and that the response of these two potentials to vanadate is greater in the second turn than in the first. Injection of 50 n1 of 1 mM vanadate (in artificial endolymph) into the endolymphatic space of the second turn caused no increase in the EP. These results support the notion that the early effects of vanadate are on the contra-luminal membranes of cells of the organ of Corti rather than on the stria vascularis. By superimposing anoxia or furosemide (i.v.) upon vanadate intoxication, we determined that the initial increase of the compound EP due to vanadate alone was due to a reduction in magnitude of the negative component of the EP. It is argued that of the three prevalent theories concerning the generation of the negative EP, the data tend to support the hypothesis that the intracellular potential of the hair cells gives rise to the negative EP.     

An experimental study using sodium salicylate to reduce cochlear changes induced by furosemide

Furosemide is a loop diuretic which has been found to be ototoxic in humans and experimental animals. The ototoxic effects seem to be directed primarily towards the stria vascularis, since its shrinkage and extracellular edema have been observed in correlation with electrophysiologic changes. The present study was designed to examine the interaction of sodium salicylate and furosemide on the cochlear microstructures. Chinchillas weighing 400-600 g were used in all tests performed. The endocochlear potential (EP) was monitored continuously through a microelectrode inserted through the basilar membrane. A control group of animals was injected with 0.5 ml saline intravenously (IV) 30 min before 25 mg/kg furosemide was given. The experimental group of animals was injected with 50 mg/kg sodium salicylate IV 30 min before 25 mg/kg furosemide. The control animals were found to have a mean decrease in EP of 61.1 +/- 7.0 mV. In contrast, the experimental group had very little alteration of the EP following furosemide injection (18.7 +/- 3.9 mV). These findings suggest that sodium salicylate markedly reduces the ototoxic effect of furosemide. This effect may be mediated by an alteration of local or systemic prostaglandin metabolism, or may be due to inhibition of organic acid uptake in the cochlea.     

Effect of potassium canrenoate on the EP and Na+, K+ activities in endolymph

The perilymphatic space was perfused with artificial perilymph containing 5 x 10(-3)M potassium canrenoate. The EP, K+ and Na+ activities in the scala media were measured with double barrelled K+ or Na+ selective microelectrode. Following the onset of the perfusion, the EP gradually declined and was stable after about 20 minutes. K+ activity also declined gradually but Na+ activity was unchanged. When the EP became stable, the artificial ventilation was stopped. The EP dropped to a large negative potential and K+ activity decreased gradually, but Na+ activity increased by degrees. The same results were observed in the untreated animals when the ventilation was stopped. There are no pathological changes both in the TEM view of the stria vascularis and in the SEM view of the hair cell. These results suggest that this drug may affect K+ conductance of the stria vascularis specifically.     

Increased capillary permeability of the stria vascularis to HRP, induced by experimental acute hypotension in rats

Acute hypotension was produced in rats by using two experimental techniques: intravenous infusion of a ganglion-blocking agent (Arfonad), and venesection of a femoral vein. Horseradish peroxidase (HRP) was injected intravenously after each procedure, and subsequently observed in the inner ear by light and electron microscopy. In both experimental models, a large amount of tracer spread into the intercellular spaces, but it was halted by tight junctions bordering the stria vascularis. The endothelium exhibited a high distribution density of labelled vesicles, which suggested increased vesicular transport. There was no extravasation of HRP from capillaries in the spiral ligament in spite of the presence of some labelled pinocytotic vesicles. The present study was concerned with the discovery of enhanced capillary permeability of the stria vascularis under acute hypotension, as in the case of acute hypertension (Sakagami et al., 1984).     

Time course changes of vasopressin-induced enlargement of the rat intrastrial space and the effects of a vasopressin type 2 antagonist

Conclusion: The intrastrial space was enlarged remarkably at 20 min after vasopressin (VP) injection, and this enlargement of the intrastrial space was reduced by administration of OPC-31260 before VP injection. These results suggest that VP increases the influx of water from the perlymph to the basal cells via aquaporin (AQP) 2 and causes the formation of endolymphatic hydrops. Objectives: To investigate a time course of changes of the stria vascularis after VP injection and the influence of OPC-31260 on experimentally induced enlargement of the intrastrial space caused by VP injection. Materials and methods: In the time course study, Wistar rats were injected with 50 µg/kg of VP subcutaneously. The stria vascularis specimens were harvested at 10, 20, 30, and 60 min after VP injection. For OPC-31260 administration, animals were administered 100 mg/kg of OPC-31260 orally 1 h before receiving 50 µg/kg of VP subcutaneously. The specimens were harvested 20 min after VP injection. These specimens were observed using transmission electron microscopy. Results: In the time course study, the incidence of intrastrial space enlargement was 50%, 100%, 25%, and 0% for 10, 20, 30, and 60 min, respectively. In the study with OPC-31260 administration, the stria vascularis showed no morphological changes.     

Effect of impulse noise exposure on the endocochlear potentials]

Effects of impulse noise exposure (25 impulses, peak level 165 dB SPL) on endocochlear potentials (EP) and CAP threshold were investigated in guinea pigs. Eight hours after exposure, EP recorded from the second turn of the cochlea was very low (19.0 +/- 1.7 mV) and the negative EP resulted from prolonged anoxia was markedly diminished (-EPmax = -6.4 +/- 1.3 mV). The return rate of EP after reventilation was also reduced. The positive EP returned to normal values 7 days after exposure but the negative EP and the CAP threshold did not. The results suggest that the stria vascularis was also damaged together with the organ of Corti, the degree of damage seemed to be heavier in the latter if only complete recovery of positive EP is taken into consideration. The stria vascularis was also not completely restored when examining the return rate of EP after reventilation. Mechanisms underlying the changes in EP and hearing sensitivity after noise exposure are discussed.