Changes in permeability of strial vessels following vibration given to auditory ossicle by drill

We produced drill-induced damage of the auditory ossicles of guinea pigs to study changes over time in the permeability of the blood vessels of the stria vascularis to horseradish peroxidase (HRP). In group A, the stimulus was applied for 10 seconds after intravenous injection of HRP. In group B, it was applied for 30 seconds, and in group C, for 60 seconds. The cochlea was fixed with 2% glutaraldehyde perfused through the round window, and the guinea pigs were then decapitated. The stria vascularis of the basal and third turns was examined. The leakage of HRP from the blood vessels of the stria vascularis significantly increased in relation to the duration of the stimulus in both the basal and third turns. The damage to intermediate cells also tended to be in relation to the duration of the stimulus. Extravascular permeation of HRP took place through the tubules in the endothelial cytoplasm. The vibratory stimulation presumably opened channels that are not normally open.     

Morphological changes in the stria vascularis and hair cells after mastoid-vibration using a cutting bur

Severe sesorineural deafness develops occasionally after tympanoplasty. Since a vibration induced by the drill during surgery is thought to be one of the cause, we examined its effect on the inner ear in this study. Five guinea pigs aged about 1 month were used. The right ear of each animal was used as the control, and the left ear underwent stimulation with the drill induced vibration. The vibration was applied to the temporal bone for 60 seconds using a cutting bur. After the vibration, the stria vascularis and hair cells in the cochlea were examined. For the investigation of the permeability of the stria vascularis, horseradish peroxidase (HRP) was injected intravenously as a tracer, and the tissue was observed with a light and an electron microscopes. Three sections of the stria vascularis from each of the superior turn (the third turn) and the inferior turn (the basal turn) of bilateral cochleae were randomly prepared, and they were classified into Types I to IV according to the degree of HRP leakage. When a hair cell had even one vacuole inside, it was classified as a vacuole-positive cell. After stimulation with the drill-induced vibration, about 10% of the blood vessels of the stria vascularis showed severe leakage of HRP (Types III, IV). In the intermediate cells, partial degeneration of mitochondria was found. No significant difference in the permeability of the blood vessels was found between the turns. The route of leakage of HRP from blood vessels of the stria vascularis was neither the transport via pinocytotic vesicle nor passage through the intercellular space of endothelial cells. The leakage through a tubular-like structure in the endothelial cells was observed. Since no HRP was found in this tubular-like structure ordinarily, a channel might have been opened by injury due to the vibration. The leakage from the blood vessels of the stria vascularis in the control side could scarcely be observed. Many hair cells in the drill-stimulation side in both the first and the third turns showed vacuoles with loss of intracellular structure. Slight loss of auditory hairs was found in this study, suggesting that the development of a vacuole was the first reaction to the drill-stimulation. Since the sensitivity toward stimulation of the drill-induced vibration was different among individual animals, it appears possible that the inner ear is injured in some cases without direct touch to the auditory ossicles owing to technical failure.     

Quantitative evaluation of pinocytosis of capillaries of the stria vascularis under normal and experimental conditions

In order to examine evidence of high activity of pinocytosis in capillaries of the stria vascularis, quantitative morphometry of pinocytotic vesicles was carried out ultrastructurally by the horseradish peroxidase (HRP) tracer method. In guinea pigs, there was a significant difference between the stria vascularis and spiral ligament in the number of vesicles per micron 2 (p less than 0.05) and labelled vesicles per micron 2 (p less than 0.001). In normotensive control rats, the number of labelled vesicles per micron 2 in capillaries of the stria vascularis was significantly greater (p less than 0.001) than that of the spiral ligament. Results of acute hypertensive and acute hypotensive experiments both indicated that enhanced permeable capillaries of the stria vascularis showed no significant increase in the number of pinocytotic vesicles per micron 2, but that they showed a more significant increase in the number of labelled vesicles per micron 2 than non-enhanced permeable capillaries of the stria vascularis (p less than 0.01) and capillaries of the spiral ligament (p less than 0.001). These findings provide ultrastructural confirmation of our previous studies (4, 8, 9) that pinocytosis contributes to the high permeability of capillaries of the stria vascularis under normal and experimental acute hypertensive and acute hypotensive conditions.     

Quantitative evaluation of pinocytosis of capillaries of the stria vascularis under normal and experimental conditions

In order to examine evidence of high activity of pinocytosis in capillaries of the stria vascularis, quantitative morphometry of pinocytotic vesicles was carried out ultrastructur-ally by the horseradish peroxidase (HRP) tracer method. In guinea pigs, there was a significant difference between the stria vascularis and spiral ligament in the number of vesicles per urn(2) (p<0.05) and labelled vesicles per urn(2) (p<0.001). In normotensive control rats, the number of labelled vesicles per u,m(2) in capillaries of the stria vascularis was significantly greater (p<0.001) than that of the spiral ligament. Results of acute hypertensive and acute hypotensive experiments both indicated that enhanced permeable capillaries of the stria vascularis showed no significant increase in the number of pinocytotic vesicles per urn(2), but that they showed a more significant increase in the number of labelled vesicles per um(2) than non-enhanced permeable capillaries of the stria vascularis (p<0.01) and capillaries of the spiral ligament (p<0.001). These findings provide ultrastruc-tural confirmation of our previous studies (4, 8, 9) that pinocytosis contributes to the high permeability of capillaries of the stria vascularis under normal and experimental acute hypertensive and acute hypotensive conditions.     

颈上神经节切除对豚鼠耳蜗血流及听性脑干反应阈的影响

目的研究豚鼠颈交感神经节对耳蜗血流及听觉生理功能的调节作用。方法在正常豚鼠单侧耳蜗螺旋蜗轴动脉局部滴加逆行追踪剂辣根过氧化物酶(horseradishperoxidase,HRP),观察双侧交感颈上神经节、星状神经节内的阳性神经元分布;建立单侧颈上神经节切除模型,观察术后不同时间点双侧耳蜗血流、听性脑干反应的情况;建立单侧颈上神经节切除后噪声性听损伤模型,观察颈上神经节切除对噪声损伤导致的听觉阈移是否有保护作用。结果耳蜗螺旋蜗轴动脉局部给予逆行追踪剂后同侧颈上神经节内可见阳性神经元;单侧颈上神经节切除1周后,术侧耳蜗底回血管纹处血流较对侧升高,听性脑干反应阈值无明显变化;颈上神经节切除对噪声导致的听损伤有一定的保护作用。结论交感颈上神经节切除对豚鼠耳蜗血流及听觉生理功能有一定的影响。     

杂色和白色豚鼠听反应阈及耳蜗血管纹细胞增殖活性的差异

目的研究杂色和白色成年豚鼠正常听反应阈及耳蜗血管纹细胞增殖活性的差异。方法用听觉诱发电位仪分别测定杂色和白色豚鼠40Hz听觉相关电位(40HzAERP)及听性脑干反应(ABR)阈值;用免疫组化法检测两种豚鼠耳蜗血管纹增殖细胞核抗原(PCNA)表达的差异。结果杂色和白色豚鼠耳蜗血管纹中间细胞PCNA染色均呈阳性,但前者PCNA的表达显著强于后者;两种豚鼠的听反应阈值无显著性差异。结论正常杂色和白色成年豚鼠血管纹中间细胞均具有增殖能力,但前者的增殖能力显著高于后者,不过这种差异并不造成两种豚鼠的正常听反应阈有明显差异。     

Changes in the capillaries of the stria vascularis after hemorrhagic shock

As a result of severe hemorrhagic shock in guinea pigs, the blood flow in the capillaries of the stria vascularis was blocked, but that of the spiral ligament was unaffected. It is therefore postulated that the thick layer of basal cells and fibrocytes around the stria vascularis and the capillaries leaving the stria vascularis plays an important role in the formation of blood sludging.     

庆大霉素对豚鼠血管纹黑色素的影响及其机制

OBJECTIVE: To investigate the effect and its mechanism of gentamicin(GM) on melanin in stria vascularis of guinea pig. METHODS: The differences of auditory thresholds between pigmented and albino guinea pigs, given GM of 150 mg/kg for 7 days, were studied. Moreover, the content of melanosomes, activity of tyrosinase and expression of proliferating cell nuclear antigen(PCNA) in intermediate cells of stria vascularis in gentamicin-treated pigmented guinea pigs were compared with those in control animals by electron microscope and immunohistochemistry, respectively. RESULTS: After gentamicin exposure, the auditory thresholds of all animals increased significantly (P < 0.001), whereas threshold shifts averaged across all frequencies of pigmented animals were much less than those of the albinos(P < 0.001). The number of melanosomes of each examined area (300 microns 2) in intermediate cells was obviously increased from 19.83 +/- 2.74 to 58.33 +/- 16.22. The ratio of tyrosinase reaction products area to the total measured area was significantly increased from 1.65% +/- 0.40% to 3.45% +/- 0.41% after gentamicin exposure. However, the numbers of positive intermediate cells expressing PCNA were 14.08 +/- 2.76 and 13.58 +/- 2.09 before and after gentamicin treatment, respectively. But there was no statistically significant difference between them (P > 0.05). CONCLUSION: The increase of content of melanin in stria vascularis after GM exposure does not result from the change of proliferating activity of melanocytes, but from the enhanced tyrosinase activity. Melanins in stria vascularis may possess the ability to protect the inner ear from ototoxicity of gentamicin.     

Ultrastructural study of the effect of acute hypertension on the stria vascularis and spiral ligament

The effect of acute hypertension, induced in rats by intravenous injection of methoxamine chloride (Mexan), on the stria vascularis and spiral ligament was studied electronmicroscopically with the tracer method of horseradish peroxidase (HRP). Considerable extravasation of HRP occurred in the stria vascularis, due to the increased vesicular transport. The leaked HRP spread into intercellular spaces, but was prevented from spreading towards the endolymph by zonulae occludentes between marginal cells and towards the perilymph by zonulae occludentes between basal cells. The reaction product was occasionally found between basal cells. No leakage of HRP from capillaries was observed in the spiral ligament, although some labelled micropinocytotic vesicles were present in the endothelium. It is suggested that, under acute hypertensive conditions, areas of zonulae occludentes bordering the stria vascularis play an important role as a barrier to HRP, whereas capillaries in the spiral ligament themselves act as a barrier to it.     

噪声对豚鼠耳蜗血管纹紧密连接蛋白claudin-5表达及血迷路屏障通透性的影响

目的研究噪声对豚鼠耳蜗血管纹紧密连接蛋白claudin-5表达及血迷路屏障功能的影响。方法40只豚鼠随机分为正常对照组(20只)和噪声暴露组(20只),噪声暴露组给予声强为115dB SPL白噪声暴露,每天6小时,连续两天,对照组不予噪声暴露。于实验前及噪声暴露后次日对两组豚鼠行ABR检测,第二次ABR检测后对两组豚鼠耳蜗基底膜行鬼笔环肽-异硫氰酸荧光素(Phalloidin-FITC)染色;用硝酸镧透射电镜和常规透射电镜观察两组豚鼠内耳血迷路屏障通透性和紧密连接的变化;Western blot和免疫组织化学染色观察两组豚鼠耳蜗血管纹和螺旋韧带处claudin-5的表达。结果噪声暴露组豚鼠ABR波Ⅲ反应阈较正常对照组阈移40dB以上,差异有统计学意义(P<0.05);基底膜铺片FITC染色示噪声暴露组底回基底膜毛细胞大量缺失,其余部分毛细胞纤毛排列紊乱并出现融合,而对照组毛细胞排列整齐,其纤毛呈V或W型,两组间外毛细胞计数比较差异有统计学意义(P<0.05);透射电镜下,可见噪声暴露组血管内皮细胞间和边缘细胞间的紧密连接均遭到破坏,细胞间隙增大;硝酸镧透射电镜下,大量镧颗粒从血管腔内渗漏到管腔外的组织间隙,而对照组镧颗粒仅局限于血管腔内;Western blot结果示,两组耳蜗外侧壁血管纹均有claudin-5表达,但噪声暴露组的表达量明显低于正常组;免疫组织化学染色也表明,两组豚鼠耳蜗外侧壁毛细血管内皮细胞、边缘细胞等处均有claudin-5阳性表达,但噪声组表达量显著低于正常组(P<0.05)。结论噪声通过下调紧密连接蛋白claudin-5的表达,破坏细胞间紧密连接,增加豚鼠血迷路屏障的通透性,从而导致内耳微环境的破坏,是噪声性聋可能的发病机制之一。     

Effect of sympathectomy on capillaries of the cochlear lateral wall

The right superior cervical ganglia of 11 cats were removed. After intravenous injection of horseradish peroxidase, both sides of the cochleas were infused with 2% glutaraldehyde and the lateral wall of the cochlea was then microdissected. As control group, both sides of the cochleas of 4 cats were extracted while the superior cervical ganglia remained intact. The samples were incubated after a modified Graham-Karnovsky method and observed by light microscopy. The diameters of the capillaries of the stria vascularis and spiral ligaments were measured. In 8 cats (73%), the diameters of the capillaries of the stria vascularis on the side on which sympathectomy was performed were significantly greater than those on the opposite side. There was no statistical difference between the diameters of the capillaries of the spiral ligament on either the sympathectomized side or the intact side, except in 2 cats. In the control group (in which the superior cervical ganglia were left intact), no statistical difference was found as regards the diameters of the stria vascularis capillaries on either the left or right side. In summary, sympathectomy affected only the capillaries of the stria vascularis and not those of the spiral ligament.     

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.     

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。     

Electron microscopy of the stria vascularis and its response to etacrynic acid. A study using electron-dense tracers and extracellular surface markers

Lanthanum nitrate (La3+), ruthenium red (RR) and tannic acid (TA) have been used to examine the cell coat and permeability pathways in thin sections of the stria vascularis of normal and etacrynic-acid (EA)-treated guinea pigs. The tight junctions around the stria, at both endolymphatic and spiral ligament sides, excluded tracers even when EA-associated oedema was well advanced. La3+ was also unable to enter oedematous stria from the capillaries. A dense fibrillar coat was revealed on the endolymphatic marginal cell surface. This coat was disorganised during EA intoxication. Both RR and TA crossed the apical membrane and produced enhanced contrast in some, but not all, marginal cells in EA-treated animals. The results suggest that EA may affect the structure of the apical membrane of the marginal cells and that there is heterogeneity amongst the marginal cell population. These possibilities are discussed.     

An experimental study of auditory dysfunction associated with hyperlipoproteinemia

We sought to clarify whether or not hyperlipoproteinemia induces auditory dysfunction. In so doing, we studied the general states and cochlear pathologies of guinea pigs after the administration of a hyperlipid diet for 3 months. Serum biochemistries indicated marked elevations of cholesterol, high density lipoprotein (HDL)-cholesterol and low density lipoprotein (LDL) levels. An increased auditory threshold varying from 10 to 20 dB was observed in 40% of the guinea pigs using auditory brainstem responses. Histochemical study of the inner ear revealed variations in lipid metabolism and partial disorders of the outer hair cells. Electron microscopic observations showed vacuolar and parenchymal protrusions on the surfaces of the stria vascularis and Corti's organ, and vacuolar degeneration was seen around the capillary vessels of the vascular stria. Our data has shown that the auditory dysfunction present in the inner ear was less marked than were the morphological changes seen. Our findings suggest that other factors besides hyperlipoproteinemia are involved in the development of severe auditory damage.     

速尿对离体培养豚鼠血管纹毒性作用的观察

目的：观察速尿对离体培养的豚鼠血管纹组织的影响，探讨速尿耳毒性的作用机制。方法：20只花色豚鼠随机分成二组：速尿组（n=16），正常对照组（n=4）。应用组织块培养的方法，将血管纹组织培养24小时，随即对不同的实验组分别应用不同终浓度的的速尿（60、300、600、1250、2500μg/ml），分别继续培养30分钟和90分钟，观察培养的血管纹组织学结构。结果：速尿组在组织学方面均未出现血管纹水肿、缘细胞胞浆肿胀、细胞间隙扩大和中间细胞皱缩等病理改变，与对照组相比较血管纹结构无显著性差异。结论：速尿对体外培养的豚鼠血管纹组织无明显诱导水肿的作用，提示速尿耳毒性的产生，可能是间接的作用机制。     

An experimental study of auditory dysfunction associated with hyperlipoproteinemia

Summary We sought to clarify whether or not hyperlipoproteinemia induces auditory dysfunction. In so doing, we studied the general states and cochlear pathologies of guinea pigs after the administration of a hyperlipid diet for 3 months. Serum biochemistries indicated marked elevations of cholesterol, high density lipoprotein (HDL)-cholesterol and low density lipoprotein (LDL) levels. An increased auditory threshold varying from 10 to 20 dB was observed in 40% of the guinea pigs using auditory brainstem responses. Histochemical study of the inner ear revealed variations in lipid metabolism and partial disorders of the outer hair cells. Electron microscopic observations showed vacuolar and parenchymal protrusions on the surfaces of the stria vascularis and Corti's organ, and vacuolar degeneration was seen around the capillary vessels of the vascular stria. Our data has shown that the auditory dysfunction present in the inner ear was less marked than were the morphological changes seen. Our findings suggest that other factors besides hyperlipoproteinemia are involved in the development of severe auditory damage.     

The volume density of cells and capillaries of the normal stria vascularis

The purpose of this study was to provide morphometric (i.e. quantitative anatomical) data on the normal chinchilla stria vascularis. Five normal chinchillas were used in the present investigation and four regions of the cochlea were examined in each animal. The width, radial area and number of marginal cells across the stria's width increased from the cochlear apex toward the base. The increase in strial width and area appeared to be due to hyperplasia of the marginal cells. The mean total endolymphatic surface area of the stria vascularis was estimated to be 7.4 mm2 (S.E. = 1.23). The mean total volume of the stria vascularis was estimated to be 0.15 microliter (S.E. = 0.01). In addition, using a stereological method we found that the volume density of the cells and capillaries of the stria vascularis was constant along the length of the scala media. The mean (+/- S.E.) volume density of the stria cells and capillaries was estimated to be: marginal cells = 0.528 (0.013), intermediate cells = 0.212 (0.026), basal cells = 0.163 (0.009) and capillaries = 0.097 (0.009).     

豚鼠耳蜗微血管内皮细胞通透性分子调控机制的初步研究

目的：探讨豚鼠耳蜗微血管内皮细胞通透性的分子调控机制。方法：使用体外培养的豚鼠耳蜗血管纹微血管内皮细胞,建立耳蜗微血管内皮细胞通透性调控的体外模型;用霍乱毒素（CT）和速尿作为处理因素,检测其对豚鼠耳蜗微血管内皮细胞环磷酸腺苷（cAMP）和F-肌动蛋白（F-actin）含量的影响,以及对牛血清白蛋白和伊文氏兰通透率的影响。结果：CT能使豚鼠耳蜗微血管内皮细胞cAMP的含量显著增加（P＜0．01）,F-actin的含量显著减少（P＜0．05）,且核周F-actin减少尤为明显,使牛血清白蛋白和伊文氏兰的通透率显著降低（均P＜0．01）;相反,速尿能使耳蜗微血管内皮细胞cAMP的含量显著减少（P＜0．01）,F-actin的含量显著增加（P＜0．01）,且核周F-actin增多尤为明显,使牛血清白蛋白和伊文氏兰的通透率显著增高（P〈0．01和P〈0．05）。结论：cAMP是体外耳蜗血管纹微血管内皮通透性调控的主要分子机制之一。耳蜗血管纹微血管内皮细胞通透性的改变与内皮细胞形状的改变及肌动蛋白丝在内皮细胞中的分布和含量的变化有关。     

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。