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.     

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.     

Profiles of resting potentials across the stria vascularis in kanamycin-deafened guinea pigs

The resting potentials of the marginal cells in the stria vascularis of the guinea pig were determined from changes in the combined electrode-tissue resistance of the electrode. The resistance of the electrode was 45.5±16.0 MΩ (n=20) before penetration of the stria vascularis and 46.7±17.3 MΩ (n=20) after penetration. The resistance drops across the luminal membrane of the marginal cells were 46.0±22.6 MΩ (n=12) in kanamycin-deafened guineal pigs and 54.5±33.1 MΩ (n=9) in normal guinea pigs. The endocochlear potential (EP) and resting potentials in the marginal cells were 90.1±6.0 mV (n=14) and 70.4±11.3 mV (n=14) in kanamycin-deafened guinea pigs and 84.8±5.1 mV (n=29) and 74.7±11.7 mV (n=29) in normal guinea pigs. The resting potentials in the marginal cells decreased gradually and were approximately 0 mV around 20 min after anoxia in both kanamycin-deafened and normal guinea pigs. These changes were comparable to those of EP in kanamycin-deafened guinea pigs during anoxia. The mechanism of the EP in kanamycin-deafened guinea pigs is discussed.     

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.     

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。     

Structural abnormalities in the stria vascularis following chronic gentamicin treatment

Freeze-fracture and thin-sectioning have been used to examine the stria vascularis of albino guinea pigs chronically treated with gentamicin. Immediately following the end of treatment, most marginal cells showed lipid bodies in the cell body region and freeze-fracture revealed alterations to the marginal cell plasma membrane. Intermediate cells also showed peculiarities including a dilation of the rough endoplasmic reticulum. A co-incidence was noted in the location in the cochlea in which effects in the stria and in outer hair cells occurred. At 4 weeks post-treatment, the stria was significantly thinner than normal and appeared less structurally complex. A minority of marginal cells degenerated. Some morphological features associated with degeneration resembled those of apoptosis, a process of controlled, cellular self-destruction. There were also indications of turnover of gap-junctions throughout the post-treatment period examined. The results indicate significant ototoxic effects of gentamicin occur in the stria and that changes to plasma membranes are one of the initial alterations.     

低能量超声照射对豚鼠耳蜗血管纹的影响

目的 探讨低能量超声照射豚鼠耳蜗后血管纹的酶组织化学变化.方法 以A型脉冲超声波诊断仪为超声发射器,分别以2.5 mHz、8.0 mHz超声照射豚鼠耳蜗6.0小时后间隔0.5小时、8.0小时,行豚鼠耳蜗血管纹铺片观察琥珀酸脱氢酶(succinate dehydrogenase,SDH)组织化学观察.以Kruskal and Wallis法对血管纹琥珀酸脱氢酶活性(吸光度A值)行统计学处理.结果 2.5 mHz、8.0 mHz超声照射豚鼠耳蜗6.0小时后间隔0.5小时、8.0小时,不同部位耳蜗血管纹SDH酶吸光度A值降低,且照射后8.0小时组较照射后0.5小时组SDH酶吸光度A值有明显升高,这种变化与耳蜗毛细胞SDH酶吸光度A值降低区域大体相对应.结论不同频率低能量超声照射豚鼠耳蜗达一定剂量可引起耳蜗血管纹不同部位SDH酶活性降低,这种变化在一定照射剂量下是可逆或部分可逆的.提示暴露低能量超声达一定剂量可引起耳蜗一定程度的病理改变.     

Results of an ultrastructural study comparing stria vascularis with organ of Corti in guinea pigs treated with kanamycin

Ultrastructural study of ototoxicity is well documented with two points of interest: organ of Corti for aminoglycosides and stria vascularis for loop diuretics. As a previous study suggested initial lesions of stria vascularis, an attempt of comparison and of chronological study was made between the organ of Corti and stria vascularis lesions by kanamycin intoxication. The method was devised by J. M. ARAN, with electrophysiological control. We failed to find in the stria vascularis a radial or longitudinal pattern of lesions. We could not discern a chronological injury between the organ of Corti and stria vascularis because both were damaged even in the less deafened animals. Nevertheless, two facts were clarified: hair cell lesions are lysosomial as for the kidney lesions, while stria vascularis lesions are mitochondrial, melanine granulations play a part in drug metabolism (increased number, secretory aspect) and deserve further study.     

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     

Turn-specific and pigment-dependent differences in the stria vascularis of normal and gentamicin-treated albino and pigmented guinea pigs.

The aims of the present study were to determine which structures in the stria vascularis (SV) may depend upon the presence of pigmented melanocytes both for normal morphology and for the expression of gentamicin ototoxicity in the inner ear. These pigment-dependent influences were inferred through comparisons of the SV in pigmented guinea pigs and in albinos containing nonpigmented melanocytes. Results were obtained from 6 albino and 8 pigmented guinea pigs given gentamicin, and from 3 albino and 3 pigmented control animals not receiving the drug. One-month old animals received gentamicin daily (100 mg/kg) for 14 days and recovered for an additional 14 days before being prepared for electron microscopy. The SV from each of the 4 cochlear turns was analyzed using stereological point counting procedures. In control animals, differences were found in the higher cochlear turns, where volume density for the marginal cells in albinos was abnormally large (turns 3 and 4), while the volume density for intermediate cells (melanocytes) was abnormally small (turn 3). Cell volume estimates for the intermediate cells were significantly smaller in the albino than pigmented control animals in the higher cochlear turns, indicating that functional abnormalities may be found in the albino cochlea. In animals exposed to gentamicin, marginal cell volume density was reduced significantly in turn 4 of albinos, but not in any region of the pigmented inner ears. Radial area of SV and estimates of the absolute volumes for marginal cells in albinos given gentamicin also were significantly reduced in turn 1 compared to their controls; such differences were not observed in the pigmented animals. The results indicate that marginal cell size is significantly reduced in albino but not pigmented animals 14 days after gentamicin exposure, and further suggest a role of pigmented melanocytes in ameliorating gentamicin-induced cochlear damage.     

Analysis of structural changes in the stria vascularis following chronic gentamicin treatment

Changes in the stria vascularis following chronic gentamicin treatment have been examined using quantitative methods. Albino guinea pigs were given gentamicin at 100 mg.kg-1.day-1 subcutaneously for 10 days. Comparisons were made of strial tissue from the treated animals sacrificed either 1 h or 4 weeks following the last injection with that from saline-injected controls. Strial width (spiral prominence to Reissner's membrane) and marginal cell (MC) number across the stria were determined from scanning electron micrographs. Strial thickness (endolymphatic surface to spiral ligament) and the volume fractions of the strial components (MCs, intermediate cells (ICs), basal cells (BCs) and capillaries) were derived from thin sections. Qualitative changes to both MCs and ICs were apparent 1 h after the last injection. At four weeks post-treatment, there was a small, but statistically significant, decrease in the number of marginal cells and a highly significant decrease in strial thickness. This was almost entirely due to a highly significant decrease in the volume fraction of MCs (i.e. shrinkage). The volume fraction of ICs was increased but this could be accounted for by MC shrinkage; after allowing for the reduction in strial thickness, the volume occupied by ICs was unchanged. Thus, following chronic gentamicin treatment, the stria is affected but significant progressive and permanent structural effects are confined to the marginal cells.     

Carbohydrates in the guinea pig stria vascularis demonstrated with lectin-gold techniques.

Soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), Ricinus communis agglutinin-II (RCA-II), Limax flavus agglutinin (LFA) and wheat germ agglutinin (WGA) were employed to determine the localization of specific carbohydrates on thin sections of lowicryl K4M embedded guinea pig striae vasculares using the lectin-gold and glycoprotein-gold techniques. SBA, HPA and RCA-II gold labeling was observed in many of the cytoplasmic vesicles, endosomes and apical tubules located in the supranuclear region as well as on the microvilli and micropinocytotic invaginations of the luminal plasma membrane of the marginal cells. LFA labeling was found on the basal plasma membrane of the marginal cells as well as in the basement membrane of the perivascular spaces. WGA binding sites were detected along the plasma membrane of all types of cells constituting the stria vascularis. Our present results revealed that the membranes of internalization and many of the cytoplasmic vesicles, endosomes and apical tubules in the supranuclear region of the marginal cells are associated together and it is suggested that these structures may be related to the regulation of endolymph.     

Alterations in the stria vascularis in relation to cisplatin ototoxicity and recovery

We have investigated whether or not cisplatin-induced depression of the endocochlear potential (EP), and its subsequent recovery, possesses a morphological correlate in the stria vascularis. Guinea pigs implanted with round window electrodes were treated daily with cisplatin (1.5 mg/kg/day) until the compound action potential showed a profound hearing loss (> or =40 dB at 8 kHz after 5-18 days). Animals were either sacrificed immediately after the shift in hearing threshold ('SHORT' group) or allowed to recover for > or =4 weeks and subsequently sacrificed ('LONG' group). Control animals ('CONTROL' group) were not treated with cisplatin. Using stereological methods we measured the total strial cross-sectional area together with the areas occupied by the different strial components: the marginal, intermediate and basal cells. The total strial cross-sectional area in the basal turn of the LONG group was found to be significantly smaller than that of the SHORT and the CONTROL groups, whereas the EP was normal in the LONG group (in comparison to the CONTROL group) and markedly decreased in the SHORT group. The smaller area in the LONG group was mainly due to a decrease in the area occupied by the intermediate cells and to a lesser extent to a decrease in the marginal cell area. The area occupied by the basal cells did not change. Thus, the marked decrease in EP after 5-18 days of cisplatin administration was not related to shrinkage of the stria vascularis. Moreover, 4 weeks later the EP showed full recovery, whereas the stria vascularis had shrunk markedly.     

Effects of a single high dose of cisplatin on the melanocytes of the stria vascularis in the guinea pig

The antineoplastic drug cisplatin is known to cause a reduction in endocochlear potential. The hypothesis to be tested was whether a single high dose of cisplatin affects the melanocytes by altering the expression of melanin. Pigmented guinea pigs received a bolus injection of cisplatin (8 mg/kg as a 15-second intravenous infusion). Auditory brainstem response (ABR) thresholds and morphological analysis of the hair cells and the stria vascularis were made 96 h after injection. ABR thresholds were elevated (15-40 dB) at 12-30 kHz and a significant loss of outer hair cells in the more basal regions was found. Cisplatin caused a significantly lower density of melanin in the intermediate cells in the basal region without any signs of apoptosis. Changes in melanin content were not noted in the middle or apical cochlear regions. Significant correlations were found between melanin density, ABR threshold shifts and outer hair cell loss in the region corresponding to 30 kHz. The findings reported here further support the multiple cytotoxic effect of cisplatin on the inner ear.     

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     

Respiratory rate and atp content of stria vascularis of guinea pig in vitro

Stria vascularis from guinea pig cochleae was incubated in vitro to determine its metabolic response to variations in substrate and ion composition of the incubation medium. The respiratory rate at 37° in a medium containing glucose and pyruvate as substrate was 17.3 ± 1.33 (SEM, n = 51) μI O₂/mg dry weight-hour. The stria could not maintain constant respiration by relying solely upon endogenous fuel stores. With substrate supplied, the ATP level could be maintained at about 73% of that existing in vivo. Glucose appears to be an adequate substrate for stria in vitro since glutamate, pyruvate, and fumarate did not increase the respiratory rate. Succinate increased respiration markedly but did not increase the ATP level. Ouabain (10^?4 M) caused a 48% decrease in the respiratory rate. Incubation in Na⁺-free and K⁺-free medium, each resulted in irreversible decrease of respiratory rate comparable to (or greater than) that caused by ouabain. These data are in accord with the high activity of Na⁺-K⁺-ATPase in the stria and the pronounced sensitivity of the endolymphatic potential to ouabain.     

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。