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.     

川芎嗪对庆大霉素耳中毒耳蜗外毛细胞和血管纹的保护作用

目的：探讨在庆大霉素（gentamycin，GM）耳中毒情况下，川芎嗪（tetramethylpyrazine，TMP）对豚鼠耳蜗外毛细胞和血管纹边缘细胞的保护作用。方法：12只豚鼠随机分为GM组、联合用药组、TMP组及对照组，用药十天后处死，采用透射电镜观察耳蜗外毛细胞及血管纹边缘细胞超微结构，扫描电镜观察血管纹边缘细胞表面形态。结果：透射和扫描电镜显示，联合用药组外毛细胞及血管纹边缘细胞超微及表面结构破坏不均明显轻于庆大霉素组，特别是其中的线粒体结构破坏与数目减少更显著轻于庆大霉素组。结论：川芎嗪具有保护庆大霉素耳中毒耳蜗外毛细胞和血管纹结构的作用，从而拮抗庆大霉素耳毒性。     

Time sequence of degeneration pattern in the guinea pig cochlea during cisplatin administration. A quantitative histological study

We investigated the key tissues that are implicated in cisplatin ototoxicity within the time window during which degeneration starts. Guinea pigs were treated with cisplatin at a dose of 2 mg/kg/day for either 4, 6, or 8 consecutive days. Histological changes in the organ of Corti, the stria vascularis and the spiral ganglion were quantified at the light microscopical level. Outer hair cell (OHC) loss started between 4 and 6 days of cisplatin administration, but is only significantly different from the non-treated group after 8 days of treatment. Midmodiolar OHC counts were comparable to the cytocochleogram data. The cross-sectional area of the stria vascularis did not differ from the non-treated group, nor did an endolymphatic hydrops develop during the course of treatment. Spiral ganglion cell (SGC) densities did not decrease. After 6 days, however, detachment of the myelin sheath of the type-I SGCs was seen in the lower basal turn, whereas after 8 days it was also present in the more apically located turns. Myelin sheath detachment is the result of perikaryal shrinkage and swelling of the myelin sheath. The present study confirms that cisplatin at a daily dose of 2 mg/kg has a detrimental effect on the OHCs as well as on the type-I SGCs. These intracochlear effects occur simultaneously; OHC loss and SGC shrinkage start between the fourth and sixth day of cisplatin administration and appear to develop in parallel. At this dose, no histological effect on the stria vascularis could be observed, although previous electrophysiological experiments demonstrated a clear effect on the endocochlear potential     

Cisplatin-induced apoptotic cell death in Mongolian gerbil cochlea

Cisplatin is well known to cause cochleotoxicity. In order to determine the underlying mechanisms of cisplatin-induced cell death in the cochlea, we investigated the apoptotic changes and the expression of bcl-2 family proteins controlling apoptosis. Mongolian gerbils were administered 4 mg/kg/day cisplatin consecutively for 5 days. The cisplatin-treated animals showed a significant deterioration in the responses of both distortion product otoacoustic emissions and the endocochlear potential as compared with those of the age-matched controls, suggesting outer hair cell and stria vascularis dysfunction. The presence of DNA fragmentation revealed by a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling method was recognized in the organ of Corti, the spiral ganglion, and the stria vascularis in the cisplatin-treated animals whereas almost negative results were obtained in the control animals. The nuclear morphology obtained by Hoechst 33342 staining revealed pyknotic and condensed nuclei, confirming the presence of the characteristic features of apoptosis. A significant increase and reduction in the number of bax- and bcl-2-positive cells, respectively, following cisplatin treatment was observed in the cells of the organ of Corti, the spiral ganglion, and the lateral wall. These findings suggest a critical role for bcl-2 family proteins in the regulation of apoptotic cell death induced by cisplatin. The underlying mechanisms of the cisplatin-induced cell death are discussed.     

Immunohistochemical detection of platinated DNA in the cochlea of cisplatin-treated guinea pigs

Cisplatin-induced ototoxicity is correlated with functional and morphological changes in the organ of Corti, the stria vascularis and the spiral ganglion. However, the cochlear sites of cisplatin uptake and accumulation have not been properly identified. Therefore, we have developed an immunohistochemical method to, indirectly, detect cisplatin in semithin cryosections of the guinea pig cochlea (basal turn) using an antiserum containing antibodies against cisplatin-DNA adducts. Platinated DNA was present in the nuclei of most cells in the organ of Corti and the lateral wall after cisplatin administration. Nuclear immunostaining was most pronounced in the outer hair cells, the marginal cells and the spiral ligament fibrocytes. This study is the first to demonstrate the presence of cisplatin in histological sections of the cochlea.     

Expression of caspase-activated deoxyribonuclease (CAD) and caspase 3 (CPP32) in the cochlea of cisplatin (CDDP)-treated guinea pigs

Cisplatin, an anti-cancer drug, is known to induce apoptosis. During apoptosis, double-stranded DNA is broken into single-stranded DNA by the action of caspases and caspase activated deoxyribonuclease (CAD). We immunohistochemically examined the cochlea of guinea pigs for signs of the apoptosis after the administration of cisplatin. Cisplatin (10 mg/kg b.w.) was intraperitoneally injected to guinea pigs and 3 days later, the animals were sacrificed by intracardiac perfusion of 4% paraformaldehyde. The temporal bones were then removed and immunohistochemically stained for CAD and caspase 3, using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling method. CAD was observed in the stria vascularis and the spiral ligament. Caspase 3 was also detected in the stria vascularis, the spiral ligament and the supporting cells of the organ of Corti. These findings suggest that apoptosis is involved in the cochlear damage observed in cancer patients treated with cisplatin.     

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。     

Reduction of acute cisplatin ototoxicity and nephrotoxicity in rats by oral administration of allopurinol and ebselen

Cisplatin ototoxicity has been associated with the generation of toxic levels of reactive oxygen species (ROS) which can lead to injury or loss of outer hair cells in the organ of Corti, damage to the stria vascularis, and loss of spiral ganglion cells, resulting in permanent hearing loss. In an attempt to reduce the formation of ROS and to bolster the innate oxidative stress defenses of the cochlea, we tested individual and combined formulations of allopurinol, a xanthine oxidase inhibitor, and ebselen, a glutathione peroxidase mimic. We used an acute cisplatin toxicity rat model (16 mg/kg i.p.) to analyze allopurinol and ebselen alone and in combination for their ability to reduce cisplatin associated hearing loss and nephrotoxicity. The results from our studies indicate that a combined formulation of ebselen and allopurinol affords significant protection to the cochlea and kidney from cisplatin toxicity. In the cochlea, protection is dependent on the preservation of outer hair cell number, while in the kidney, protection is associated with the preservation of proximal tubular epithelia. Further evaluation of the chemoprotective effects of ebselen and allopurinol on cisplatin side effects in the presence of tumor appears warranted.     

Effects on cochlear morphology of repeated insults to the stria vascularis

The interrelationship of stria vascularis and organ of Corti integrity was investigated. Strial morphology was altered by repeated injections of ethacrynic acid in the chinchilla. Although prolonged temporary strial damage was created, neither strial atrophy nor organ of Corti damage resulted.     

The cochlea of the spontaneously diabetic mouse

Summary We used transmission electron microscopy to examine the cochleae of non-obese diabetic mice as animal models for human type I or non-insulin-dependent diabetes mellitus. Pathological changes were observed in the organ of Corti of the basal turn and in the stria vascularis of each turn. Major findings in the stria vascularis were protrusion or condensation of marginal cells, swelling of intermediate cells, and widening of the intercellular spaces. Principal findings in the organ of Corti involved degenerative changes of the outer and inner hair cells and replacement of hair cells by supporting cells. No prominent pathological changes were observed in the capillaries. The possible mechanism of diabetic involvement in cochlear pathology is discussed.     

The cochlea of the spontaneously diabetic mouse. II. Electron microscopic observations of non-obese diabetic mice

We used transmission electron microscopy to examine the cochlea of non-obese diabetic mice as animal models for human type I or non-insulin-dependent diabetes mellitus. Pathological changes were observed in the organ of Corti of the basal turn and in the stria vascularis of each turn. Major findings in the stria vascularis were protrusion or condensation of marginal cells, swelling of intermediate cells, and widening of the intercellular spaces. Principal findings in the organ of Corti involved degenerative changes of the outer and inner hair cells and replacement of hair cells by supporting cells. No prominent pathological changes were observed in the capillaries. The possible mechanism of diabetic involvement in cochlear pathology is discussed.     

Viral labyrinthitis: early pathology in the human

The histologic findings in the temporal bones of three patients who died from viral encephalopathy are presented. Pathology was restricted to the scala media, vestibular labyrinth, and internal auditory canal and was considered to be expressions of viral labyrinthitis. The changes were different degrees of degeneration of the organ of Corti, early encapsulation of the tectorial membrane, degeneration of the stria vascularis, and round cell infiltration of the modiolus and contents of the internal auditory canal. A new finding in the organ of Corti and early stages of cystic degeneration of the stria vascularis are documented. In all cases, the saccule was degenerated with sloughing of the otolithic membrane and vestibular labyrinth was involved in varying degrees.     

The influence of ischemia upon the energy reserves of inner ear tissues

Ischemic changes in the levels of glucose, glycogen, ATP and P-creatine are determined under “closed system” conditions in the organ of Corti, stria vascularis, ganglion spirale, cochlear nerve and vestibular sensory epithelia. From the resting levels of these compounds the total energy reserve in terms of equivalents of high energy phosphate, both preformed and potentially available from anaerobic glycolysis, is computed. The energy reserves are highest in the organ of Corti, intermediate in stria vascularis, spiral ganglion and vestibular structures, and lowest in the cochlear nerve. The rate of depletion of these energy reserves in ischemia is used as an indicator of the energy requirements of the respective tissues. The metabolic rate is by far the highest in the stria vascularis, intermediate in ganglion spirale and cochlear nerve and lowest in the organ of Corti and vestibular structures. There is no correlation between the total energy reserve and the initial energy use rates. The obtained data are compared with the dynamic patterns of the corresponding biopotentials and with pertinent results of enzymatic and respirometric studies; in addition, ischemic changes in glucose and lactate levels of perilymph are described.     

Ototoxicity of Vasocidin drops applied to the chinchilla middle ear

Some widely used ototopical preparations are potentially toxic to the middle and inner ear. Vasocidin Ophthalmic Solution (sulfacetamide sodium and prednisolone sodium phosphate) has been advocated as an alternative agent that may have fewer toxic side effects in the treatment of otorrhea. Vasocidin was introduced into the bullae of nine chinchillas to investigate the effects on the middle and inner ear. The organ of Corti and stria vascularis were found to be entirely normal in 17 of the 18 temporal bones studied. Changes observed in the middle ears at one week included inflammation, hemorrhage, and effusion. Examination of specimens at four weeks revealed resolution of most of the inflammatory changes. The results of this experimental study indicate that Vasocidin causes reversible middle ear inflammation with little or no toxic effect on inner ear structures.     

DPOAE监测一次性大剂量顺铂致豚鼠耳毒性的实验研究

目的观察一次性大剂量顺铂注射后豚鼠畸变产物耳声发射(DPOAE)和内耳形态学的依时性改变,探讨应用耳声发射监测顺铂耳毒性的最佳时机。方法将豚鼠随机分为对照组(A组)及实验组(B、C、D组),每组6只,A组皮下注射生理盐水,B、C、D各组一次性皮下注射顺铂(10mg/kg),分别于给药前及给药后第3(B组)、5(C组)、10(D组)天检测DPOAE,比较给药前、后DPOAE的幅值的变化,并行耳蜗铺片,观察毛细胞缺失率及耳蜗形态学的改变。结果给药后第3天,DPOAE的幅值仅8kHz处有明显改变(P<0.05);给药后第5、10天DPOAE幅值明显降低(P<0.01)。给药后第3、5、10天,耳蜗外毛细胞缺失率分别为4.34%±3.20%、16.14%±9.15%、18.25%±9.04%。在第5、10天后尚可见血管纹充血以及螺旋神经节细胞肿胀缺失。结论顺铂的耳毒性主要作用于耳蜗底回、中回的外毛细胞,用药后第3~5天应用畸变产物耳声发射检查能早期发现其耳毒性损伤。     

Toxic effects of cisplatin alone and in combination with gentamicin in stria vascularis of guinea pigs

The toxic effects on the stria vascularis of treatment with cisplatin alone and combined with the aminoglycoside antibiotic, gentamicin, were studied in guinea pigs. The toxicity induced in albino and pigmented guinea pigs was investigated morphologically with light and transmission electron microscopy, and functionally by brainstem-evoked response audiometry. The results of hearing thresholds were variable, ranging from no change in one ear in some of the animals to a hearing loss of 20 dB in one or both ears when treated with low-dose cisplatin alone or in combination with gentamicin. Bilateral deafness resulted from high-dose cisplatin combined with gentamicin. The combined treatment produced prominent structural damage in the stria vascularis. The results should be considered when aminoglycoside therapy is required in conjunction with cisplatin.     

Acoustic stimulation alters deoxyglucose uptake in the mouse cochlea and inferior colliculus

Deoxyglucose uptake and activities of hexokinase and glucose-6-phosphatase in auditory structures (organ of Corti, stria vascularis and spiral ligament, modiolar section of VIIIth nerve, inferior colliculus) and non-auditory tissues (heart, kidney, liver) of the mouse were analyzed. [³H]Deoxyglucose was given as a pulse into the tail vein and uptake was quantitated by microdissection of tissues and scintillation counting. Radioactivity in cochlear tissues was maximal after 45–60 min and declined with a half-life of 30–60 min. Deoxyglucose 6-phosphate represented ca. 60% of total radioactivity (heart, inferior colliculus. > 80%). The ratio of hexokinase to glucose-6-phosphatase activity was considerably lower in the auditory periphery than in brain. The rank order was inferior colliculus > VIIIth nerve ≈ heart > stria vascularis and spiral ligament > kidney > organ of Corti ≈ liver.Exposure to broadband noise increased glucose utilization in all auditory structures. Uptake was maximally (2- to 3-fold) stimulated at moderate noise intensity (55–85 dBA). In addition, the auditory system showed two salient features: at high intensities (100 and 115 dBA) deoxyglucose uptake decreased from the maximum; and the non-sensory tissues of the cochlea (stria vascularis and spiral ligament) responded to sound parallel to the sensory structures at all levels of stimulus intensity. There were no effects of acoustic stimulation on serum glucose levels, serum kinetics of deoxyglucose. or deoxyglucose uptake into other body tissues.     

Quantitative distributions of aspartate aminotransferase and glutaminase activities in the rat cochlea

The intra-cochlear distributions of aspartate aminotransferase and glutaminase, prominent enzymes of aspartate and glutamate metabolism, have been studied by quantitative microchemical techniques. Also measured was choline acetyltransferase, the enzyme synthesizing acetylcholine, and a marker for the olivocochlear bundle. Aspartate aminotransferase activity was highest in the stria vascularis, about half this high in the organ of Corti synaptic (hair cell) zones, somewhat lower in the organ of Corti non-synaptic (Hensen's cell) zones, lower yet in Reissner's and lowest in the tectorial membrane. Glutaminase, on the other hand, had its highest activity in synaptic zones, about a third of that activity in the organ of Corti non-synaptic zones, and a barely detectable activity in Reissner's and tectorial membranes, and stria vascularis. Seven days after transection of the olivocochlear bundle, no significant difference was found between lesion- and control-side aspartate aminotransferase or glutaminase activities, even though no choline acetyltransferase activity remained in the lesion-side of the organ of Corti. Both the distribution of aspartate aminotransferase activity and the lesion results would seem to implicate it in energy more so than neurotransmitter metabolism. The distribution of glutaminase activity could be consistent with a role in neurotransmission; however, the lesion data were unable to demonstrate a specific association with the olivocochlear bundle.