Effects of combined administration of furosemide and kanamycin on rat auditory nerve

Objective To determine the effects of combined administration of furosemide and kanamycin on inner ear structures and the auditory nerve in rats.Methods The rats in the treatment group received intrave...     

卡那霉素和速尿联合用药后的毛细胞死亡*

目的观察卡那霉素和速尿联合用药后豚鼠耳蜗毛细胞的死亡时程和方式.方法选用健康成年白色红目豚鼠,雌雄不限,随机分成健康对照组和药物致聋后6 h、9 h组(实验组),每组5只.实验组在选取的时间点完成听性脑干反应(ABR)检测后行耳蜗基底膜铺片、PI荧光染色,共聚焦显微镜下观察毛细胞.对照组不做处理.结果实验组半数豚鼠致聋(ABR阈值>95 dB SPL),致聋豚鼠耳蜗可见外毛细胞核固缩和核肿胀,与给药6 h组相比,给药9 h组外毛细胞核肿胀数目增多.检测给药6 h组和9 h组豚鼠末端脱氧核苷酸转移酶介导的dUTP缺口末端标记物(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling,TUNEL),没有观察到阳性着色细胞.正常对照组所有豚鼠ABR正常,其耳蜗各回毛细胞没有出现毛细胞核固缩或者核肿胀.结论卡那霉素和速尿联合用药后数小时即可导致豚鼠耳蜗毛细胞死亡,毛细胞损害为两种类型,即坏死和凋亡.     

Comparative analysis of combination kanamycin-furosemide versus kanamycin alone in the mouse cochlea

Combinations of aminoglycosides and loop diuretics have been known to have a synergistic effect in ototoxic injury. Because murine hair cells are relatively resistant to ototoxicity compared to those of other mammals, investigators have turned to combination therapies to create ototoxic lesions in the mouse inner ear. In this paper, we perform a systematic comparison of hearing thresholds, hair cell damage and monocyte migration into the mouse cochlea after kanamycin versus combined kanamycin/furosemide and explore the pathophysiology of enhanced hair cell loss in aminoglycoside ototoxicity in the presence of loop diuretic. Combined kanamycin-furosemide resulted in elevation of threshold not only in the high frequencies, but across all frequencies with more extensive loss of outer hair cells when compared to kanamycin alone. The stria vascularis was severely atrophied and stellate cells in the spiral limbus were missing in kanamycin-furosemide exposed mice while these changes were not observed in mice receiving kanamycin alone. Monocytes and macrophages were recruited in large numbers to the spiral ligament and spiral ganglion in these mice. Combination therapy resulted in a greater number of macrophages in total, and many more macrophages were present further apically when compared to mice given kanamycin alone. Combined kanamycin-furosemide provides an effective method of addressing the relative resistance to ototoxicity that is observed in most mouse strains. As the mouse becomes increasingly more common in studies of hearing loss, and combination therapies gain popularity, recognition of the overall effects of combined aminoglycoside-loop diuretic therapy will be critical to interpretation of the interventions that follow.     

Potentiation and its mechanism of cochlear damage resulting from furosemide and aminoglycoside antibiotics

The severe ototoxic interaction of the combined administration of furosemide and aminoglycoside antibiotics (kanamycin, streptomycin and gentamicin) was studied in rabbits, and its mechanism clarified. Severe damage occurred not only in the inner ear but also in the kidney when both furosemide and aminoglycoside antibiotics were administered to rabbits. Kanamycin concentration after a single injection of kanamycin with furosemide was much higher not only in the perilymph but also in the cerebrospinal fluid and serum than that after a single injection of kanamycin alone. The ototoxic interaction following the combined use of furosemide and aminoglycoside antibiotics seems to be caused mainly by the inhibitory effect of furosemide on the excretion of aminoglycoside antibiotics from the kidneys.     

卡那霉素联合呋塞米快速诱导小鼠耳蜗损伤

目的 探讨卡那霉索和呋塞米联合应用对小鼠耳蜗的毒性作用,建立一种可靠的小鼠感音神经性聋模型.方法 选用3～4周龄的CBA/J小鼠为实验对象,按1 g/kg的剂量皮下注射卡那霉素,30～45 min后按0.4 g/kg的剂量腹腔注射呋塞米.在注射前、注射后12 h、24 h、48 h、7 d、2周、4周及12周分别应用听性脑干反应(ABR)检测小鼠听觉功能的改变;应用异硫氰酸荧光素标记的鬼笔环肽及碘化丙啶染色、半薄切片甲苯胺蓝染色、脱氧核苷酸末端转移酶介导的dUTP缺口末端标记技术、扫描电镜等观察小鼠毛细胞死亡的模式和程度;同时通过透射电镜观察记录耳蜗血管纹形态及厚度的变化.结果 序贯应用卡那霉素及呋塞米后12 h小鼠ABR阈值开始上升,随后的36 h内持续进行性上升,继而稳定在90 dB左右.应用激光共聚焦显微镜在药物注射后12 h观察到耳蜗底回外毛细胞开始出现死亡,24 h时底回外毛细胞基本全部消失,同时顶回外毛细胞开始出现死亡,至48 h时整个耳蜗外毛细胞绝大部分死亡;而内毛细胞的损伤至给药后7 d时才开始出现,随时间推移仍有部分内毛细胞完好无损.死亡的毛细胞均具有典型的凋亡细胞特征.扫描电镜观察发现在药物注射后受损毛细胞出现纤毛消失、表皮板塌陷,随后支持细胞增生并在该处形成瘢痕.血管纹表面边缘细胞在给药后出现部分胞体融合并且有坏死物自胞内排出,随后边缘细胞表面大部分微绒毛消失,胞体呈"石块"样改变.透射电镜结果 显示血管纹厚度在给药后进行性下降,主要为边缘细胞萎缩造成.结论 单次序贯应用卡那霉素及呋塞米能快速诱导小鼠耳蜗毛细胞大量死亡,适合应用于建立小鼠感音神经性聋模型.     

Combined administration of kanamycin and furosemide does not result in loss of vestibular function in Guinea pigs

Aminoglycoside antibiotics are known to damage the vestibular and auditory sensory epithelia. Although loop diuretics enhance the cochleotoxic effect of aminoglycosides, it is not known whether concomitant administration of an aminoglycoside and a loop diuretic affects the vestibular system. The aim of our study was to investigate the effect of co-administration of kanamycin and furosemide upon the otolith organs and to compare it to the known vestibulotoxic effect of gentamicin. Five guinea pigs were injected with a single dose of both kanamycin (400 mg/kg, s.c.) and furosemide (100 mg/kg, i.v.), 5 animals received gentamicin (100 mg/kg, i.p.) for 10 days, and 5 untreated animals served as controls. After 7 days, vestibular function was assessed by measuring vestibular short-latency evoked potentials (VsEPs) to linear acceleration stimuli and cochlear function by auditory brainstem responses (ABRs) to clicks. Hair cell densities were determined in phalloidin-stained whole mounts of the utricles and saccules, and in midmodiolar sections of resin-embedded cochleae. Co-administration of kanamycin and furosemide had no significant effect on VsEPs and hair cell densities in the utricles and saccules were not reduced. ABR thresholds were increased to a great extent (by ～60 dB), and histologically a severe loss of cochlear hair cells was observed. The effect of gentamicin, both on vestibular and cochlear function, was just the opposite. VsEP thresholds to horizontal stimulation were elevated and suprathreshold amplitudes showed a decrease, whereas cochlear function was not reduced. With this protocol, we have a tool to selectively induce cochlear or vestibular damage, which may be of interest to researchers and clinicians alike.     

The effect of combined administration of cadmium and furosemide on auditory function in the rat

A number of heavy metals have been associated with toxic effects to the peripheral or central auditory system. These include lead, arsenic, mercury, platinum and organic tin compounds. In addition, the ototoxic effects of some metals may be potentiated by other factors. However, the auditory effects of cadmium have not previously been reported. The purpose of the present study was to investigate the potential ototoxic effects of cadmium from an acute dosage, and its potentiation by furosemide. Auditory brainstem response (ABR) thresholds were measured in adult Sprague-Dawley rats. Rats received either cadmium chloride (5 mg/kg, i.p.) followed by saline (4 ml/kg, i.p.). cadmium chloride followed by furosemide (200 mg/kg, i.p.), or furosemide alone. Follow-up ABRs were carried out 7 days post-treatment and threshold changes were compared between each treatment group. No significant threshold change was seen for the cadmium chloride plus saline treated or the furosemide treated animals. However, significant threshold elevations were observed in animals receiving cadmium chloride plus furosemide. In addition, scanning electron microscopy revealed extensive hair cell loss in animals treated with cadmium chloride and furosemide. Although functional auditory changes were not seen after the administration of cadmium alone, the potentiation of threshold changes by furosemide suggests that cadmium may be ototoxic under certain conditions.     

Effects of N-acetylcysteine on kanamycin ototoxicity in the guinea pig

It has been suggested that aminoglycoside antibiotics are ototoxic because they create free radicals (reactive oxygen species), which in turn attack membranes. If this hypothesis were correct, then it would be expected that any agent which protects against oxygen-linked toxic effects would ameliorate the ototoxic effects of aminoglycosides. N-Acetylcysteine, a drug in common clinical use, was used to examine this possibility. Three groups of guinea pigs received daily injections for 21 days. One group received 200 mg/kg kanamycin subcutaneously, one group received N-acetylcysteine, while animals in the third group received N-acetylcysteine followed one hour later by kanamycin. After 7 days recovery, all animals, together with a group of control animals receiving no injections, were anaesthetised (neuroleptanalgesia) and thresholds for detection of the compound action potential were measured at the round window. N-Acetylcysteine alone had no detectable effect on thresholds. Kanamycin alone produced a moderate (10–20 dB) hearing loss below 10 kHz and a more severe loss above 10 kHz. Animals receiving both N-acetylcysteine and kanamycin had severe hearing losses (40–60 dB) at all frequencies between 3 and 30 kHz. N-Acetylcysteine had no acute effect on thresholds. The results fail to support the prediction that free radical scavenging protects against kanamycin ototoxicity. On the contrary, N-acetylcysteine exerts a strong synergistic effect on kanamycin, so producing a more severe hearing loss and more severe cochlear damage. The physiological and biochemical bases of this synergy remain to be determined.     

二甲基亚砜对在体耳蜗毛细胞的毒性作用

目的研究二甲基亚砜(DMSO)对在体耳蜗毛细胞的毒性作用。方法取清洁级健康、ABR阈值正常SD大鼠32只,雌雄不限,体重100-120g。随机分成4组,人工外淋巴液对照组(即0%组)8只;0.1%DMSO溶液组8只;1%DMSO溶液组8只;5%DMSO溶液组8只。所有动物均取右耳作为实验耳。通过耳蜗鼓阶打孔显微注射向每个实验耳注入不同浓度DMSO溶液4ul。术前1天和术后7天分别进行ABR(click和toneburst)检测。激光共聚焦显微镜观察DMSO溶液导入7天后的毛细胞变化。结果人工外淋巴液对照组click、4kHz、8kHz、16kHz处阈移平均值均<5dBSPL,仅于32kHz处有约13dBSPL的阈移,形态方面未见明显损伤;0.1%浓度组在click、4kHz、8kHz处阈移平均值均<5dBSPL,而32kHz处阈移约25dBSPL,与人工外淋巴液对照组比较提示有统计学意义,激光共聚焦显微镜观察底回时偶见少数内毛细胞胀大;1%浓度即可引起OHC大量丢失,造成相应纤毛表皮板缺如,且以底回最重,各频率ABR阈移均>15dBSPL,32kHz处阈移>30dBSPL;当浓度增加到5%时,不仅损伤耳蜗底回的外毛细胞,也导致内毛细胞的丢失,所造成的听力损失在32kHz处较1%组严重。结论 DMSO对毛细胞的损伤存在剂量依赖性,损伤程度自耳蜗底回向顶回逐渐减轻。     

Ototoxicity and the olivocochlear bundle

Cochlear hair cell damage due to kanamycin sulfate was studied in chinchillas receiving 200 mg/kg kanamycin for nine days. The drug was given to a group of animals with lesions of the crossed olivocochlear bundle, a group that had received a sham operative procedure, and a group of normal animals. The animals were sacrificed 14 days after the last drug injection and the cochleae removed for examination by surface preparation. The lesioned animals showed markedly less hair cell loss than the control groups, and this was attributed to the fact that the loss of the efferent endings rendered these animals less susceptible to the ototoxic effects of kanamycin.     

Hair Cell Toxicity in Anti-cancer Drugs: Evaluating an Anti-cancer Drug Library for Independent and Synergistic Toxic Effects on Hair Cells Using the Zebrafish Lateral Line

Inner ear hair cell loss is the most common pathology seen after ototoxic drug injury. While certain drugs such as aminoglycosides and cisplatin are well-known to have dramatic ototoxic effects, it is probable that there are other drugs that cause occult degrees of hair cell loss and lesser degrees of hearing loss. Anti-cancer drugs are particularly strong candidates due to their general cytotoxicity. We have screened a library of 88 anti-cancer drugs (National Cancer Institute Approved Oncology Drugs Set) for drugs that damage hair cells of the zebrafish lateral line. The screen identified four out of five known ototoxic drugs. The screen also identified four out of seven suspected ototoxic drugs (drugs that have isolated case reports of patients developing hearing loss after administration). Five additional drugs with no known ototoxicity were identified as potentially novel ototoxins. Additional dose–response curves were performed to evaluate relative toxicity. Since anti-cancer drugs are often used clinically in combination, we also performed dose–response curves for a variety of anti-cancer drug combinations and demonstrated synergistic toxicity in five out of ten drug combinations that we tested. These findings support the use of the zebrafish lateral line as a screening tool to detect ototoxic effects in drugs and also suggest that ototoxicity should be considered in terms of drug regimens rather than individual drugs.     

Ototoxic interaction of kanamycin and 3-nitropropionic acid

Conclusion. Mitochondrial dysfunction in the cochlea potentiates the ototoxicity of aminoglycosides. Objective. This study examined whether mitochondrial dysfunction in the cochlea affects the ototoxicity of aminoglycosides. Materials and methods. Nineteen guinea pigs were treated with the mitochondrial toxin 3-nitropropionic acid (3-NP), kanamycin, both agents, or normal saline as control. After 14 days, hair cell loss and auditory brainstem response (ABR) were assessed. Results. The administration of 400 mg/kg of kanamycin caused neither hair cell loss nor ABR threshold shift. Administration of 3-NP caused mild ABR threshold shift without significant hair cell loss. Administration of 3-NP and kanamycin caused ABR threshold shift and significant hair cell loss.     

The reinnervation of regenerated hair cells in the basilar papilla of chicks after kanamycin ototoxicity

When newly hatched chicks were injected with kanamycin on 8 successive days, the hair cells were destroyed completely in the area 0.4 to 0.8 mm from the proximal end of the basilar papilla. At this time, and 1 and 7 days following the completion of 10 days of injections, the nerve fibres in the basilar papilla of chicks show no sign of injury. On the first day following 10 days kanamycin administration the regenerated hair cells obtained not only afferent innervation, but also efferent innervation. At 15 days following drug cessation, afferent innervation of the regenerated hair cells was already similar to the controls, and the thresholds measured at this time were significantly better than those at 1 and 7 days. The chalice efferent terminals did not appear until the 60th day of drug cessation. Efferent innervation of the regenerated hair cells also approached maturity at this time. Compared with the half month after the completion of drug injection, the hearing of birds had no evident increase. It was obvious that afferent innervation of the regenerated hair cells was related more to the recovery of hearing than efferent innervation. The regenerated hair cells beginning reinnervation early and maturing were important factors influencing the recovery of the birds' hearing function after kanamycin ototoxicity.     

Effect of 4-methylthiobenzoic acid on cisplatin-induced ototoxicity in the rat.

The purpose of this study was to investigate the effectiveness of 4-methylthiobenzoic acid (MTBA) as a protection agent against cisplatin (CDDP)-induced changes in organ of Corti surface structure, compared to electrophysiological changes. Electrophysiological change was assessed using auditory brainstem response (ABR) and morphological changes were assessed using scanning electron microscopy (SEM). Male Wistar rats underwent pre-treatment ABRs in response to clicks, and tone bursts at 2, 4, 8, 16, and 32 kHz. The three groups of rats were injected as follows: (1) MTBA (250 mg/kg, i.p.), (2) CDDP (16 mg/kg, i.p.), (3) CDDP+MTBA (16 mg/kg, i.p. + 250 mg/kg, i.p.). Post-treatment ABRs were performed 3 days after drug administration and rats were sacrificed. Their cochleae were harvested and SEM was used to examine the surface of the organ of Corti, specifically the number of inner hair cells (IHCs) and outer hair cells (OHCs) in the apical, middle and basal turns of the cochlea. Animal weight was measured on the first and final days. There was a good correlation between ABR threshold changes and hair cell loss in the high frequency region of the cochlea (basal turn), while threshold changes in the lower test frequencies (middle turn) appeared to be the result of more subtle changes in the cochlea. MTBA provided effective protection against cisplatin-induced ABR threshold changes at all test frequencies as well as hair cell loss. MTBA also protected against body weight loss.     

The Reinnervation of Regenerated Hair Cells in the Basilar Papilla of Chicks after Kanamycin Ototoxicity

When newly hatched chicks were injected with kanamycin on 8 successive days, the hair cells were destroyed completely in the area 0.4 to 0.8 mm from the proximal end of the basilar papilla. At this time, and 1 and 7 days following the completion of 10 days of injections, the nerve fibres in the basilar papilla of chicks show no sign of injury. On the first day following 10 days kanamycin administration the regenerated hair cells obtained not only afferent innervation, but also efferent innervation. At 15 days following drug cessation, afferent innervation of the regenerated hair cells was already similar to the controls, and the thresholds measured at this time were significantly better than those at 1 and 7 days. The chalice efferent terminals did not appear until the 60th day of drug cessation. Efferent innervation of the regenerated hair cells also approached maturity at this time. Compared with the half month after the completion of drug injection, the hearing of birds had no evident increase. It was obvious that afferent innervation of the regenerated hair cells was related more to the recovery of hearing than efferent innervation. The regenerated hair cells beginning reinnervation early and maturing were important factors influencing the recovery of the birds' hearing function after kanamycin ototoxicity.     

阿米卡星对幼年大鼠内耳毒性实验观察

目的探讨阿米卡星对幼年大鼠内耳的毒性作用。方法实验组9天龄sD大鼠皮下注射500mg&#183;k^-1&#183;d^-1阿米卡星连续1周,对照组皮下注射0．9％生理盐水连续1周。分别于用药后l天、1周和3周行听觉脑干反应测听（ABR）仪测试实验组和对照组大鼠耳蜗听功能,并对耳蜗进行扫描电镜观察和常规切片观察。结果实验组大鼠ABR阈值,用药后1天与对照组比较、用药后1周与用药后1天和对照组比较,差异均有统计学意义（P〈0．01）．而用药后3周和用药后1周比较,差异尢统计学意义（P〉0．05）。形态学观察,随用药后时间延长,何替器细胞损伤从感觉毛细胞到非感觉支持细胞,用药后3周出现立方上皮样结构。实验组大鼠无前庭功能障碍。结论阿米卡星连续用药可导致幼年大鼠耳蜗毛细胞的严重损伤,而对前庭功能影响较小。     

Systemic Lipopolysaccharide Induces Cochlear Inflammation and Exacerbates the Synergistic Ototoxicity of Kanamycin and Furosemide

Aminoglycoside antibiotics are highly effective agents against gram-negative bacterial infections, but they cause adverse effects on hearing and balance dysfunction as a result of toxicity to hair cells of the cochlea and vestibular organs. While ototoxicity has been comprehensively studied, the contributions of the immune system, which controls the host response to infection, have not been studied in antibiotic ototoxicity. Recently, it has been shown that an inflammatory response is induced by hair cell injury. In this study, we found that lipopolysaccharide (LPS), an important component of bacterial endotoxin, when given in combination with kanamycin and furosemide, augmented the inflammatory response to hair cell injury and exacerbated hearing loss and hair cell injury. LPS injected into the peritoneum of experimental mice induced a brisk cochlear inflammatory response with recruitment of mononuclear phagocytes into the spiral ligament, even in the absence of ototoxic agents. While LPS alone did not affect hearing, animals that received LPS prior to ototoxic agents had worse hearing loss compared to those that did not receive LPS pretreatment. The poorer hearing outcome in LPS-treated mice did not correlate to changes in endocochlear potential. However, LPS-treated mice demonstrated an increased number of CCR2⁺ inflammatory monocytes in the inner ear when compared with mice treated with ototoxic agents alone. We conclude that LPS and its associated inflammatory response are harmful to the inner ear when coupled with ototoxic medications and that the immune system may contribute to the final hearing outcome in subjects treated with ototoxic agents.     

新霉素致发育中大鼠毛细胞损伤的电镜观察

目的应用扫描电镜研究氨基甙类抗生素导致出生后发育过程中大鼠耳蜗毛细胞的损伤病理变化。方法７天龄ＳＤ大鼠肌肉注射８０ｍｇｋｇ－１ｄ－１新霉素连续８天,停药后７天处死动物制备样本进行扫描电镜观察。结果新霉素可造成出生后发育过程中的耳蜗毛细胞严重损伤,表现为底回和钩回三排外毛细胞全部损伤缺失,损伤病变累及顶回的外毛细胞,底回和钩回的内毛细胞出现纤毛脱落和细胞损伤丢失;外毛细胞损伤缺失的部位由顶部具有微绒毛的多边形细胞取代,该细胞形态与胚胎发育早期的毛细胞相似;毛细胞损伤区域未发现再生的毛细胞。结论出生后发育过程中大鼠耳蜗毛细胞对氨基甙类抗生素的敏感性较高,表面有微绒毛的多边形细胞在毛细胞损伤区域出现提示听觉感觉上皮细胞有再生的倾向。     

Round window pH manipulation alters the ototoxicity of systemic cisplatin

The effect of manipulation of pH on the ototoxicity of systemic cisplatin was studied in Wistar rats. After control auditory brainstem responses (ABR) were performed, the auditory bullae were opened and acidic (pH 6.0), neutral (pH 7.4) or basic (pH 9.0) phosphate-buffered saline (PBS) was applied to fill the round window niche (RWN). After 30 min, 13 mg/kg cisplatin solution or saline was administered intraperitoneally. After 3 days, follow-up ABRs were performed and cochleae were processed for morphological analysis. Animals that received basic PBS on the RWN and cisplatin intraperitoneally had significantly smaller ABR threshold shifts compared to rats pretreated with neutral pH buffer (P<0.05). Animals that received acidic PBS on the RWN and systemic cisplatin showed significantly greater ABR threshold shifts compared to those pretreated with neutral pH buffer (P<0.05). No significant threshold changes were observed in animals that received buffer of any pH on the RWN, followed by saline intraperitoneally. Semiquantitative analysis of hair cell survival confirmed a protective effect by basic PBS against cisplatin and a synergistic effect by acidic PBS on cisplatin ototoxicity (P<0.05). It appears that changes in cochlear pH can modulate the ototoxic effects of systemically applied cisplatin.     

Aminoglycoside ototoxicity: a human temporal bone study.

OBJECTIVE: Hearing loss after aminoglycoside administration has been thought to result primarily from hair cell injury. The purpose of the study was to determine the potential for direct injury of spiral ganglion cells and hair cells in cases of documented human aminoglycoside ototoxicity. STUDY DESIGN: Retrospective case review. METHODS: The clinical course of two individuals with aminoglycoside ototoxicity are documented, including the details of administration of tobramycin and other ototoxic medication and serial audiograms. The temporal bones were processed, and the cochlear elements quantified. RESULTS: Histopathological study of the temporal bones from the individuals in the study demonstrated reduction of both ganglion cell and hair cell populations. Spiral ganglion cell loss was not necessarily subadjacent to areas of hair cell loss in cases of aminoglycoside ototoxicity. Instead, spiral ganglion cell reduction may be present in segments of the cochlea with normal-appearing hair cells. CONCLUSIONS: The study suggests that aminoglycoside antibiotics can injure spiral ganglion cells directly, as well as hair cells. Thus, the characteristic hearing loss of ototoxicity can result from degeneration of either cochlear element.