斑马鱼模型评价抗真菌药物的耳毒性

目的利用斑马鱼模型研究三种抗真菌药物-氟康唑、联苯苄唑、制霉菌素-的耳毒性。方法1.分别探索三种药物的最大非致死浓度(MNLC)和10%的致死浓度(LC10);2.每种药物分别选取LC10、MNLC、1/3 MNLC和1/10 MNLC四个浓度进行研究。利用荧光立体显微镜和解剖显微镜观察并分析斑马鱼平衡囊、躯体形态及毛细胞数量的改变。5μg/mL的庆大霉素为阳性对照组,0.1%DMSO为阴性对照组,养鱼用水为空白对照组。结果1.氟康唑、联苯苄唑、制霉菌素三种药物的MNLC分别是653.8μg/mL、0.37μg/mL和10.5μg/mL。LC10分别是705.4μg/mL、0.52μg/mL和11.8μg/mL。2.三种药物均能导致斑马鱼平衡囊形态改变及身体屈曲。在LC10、MNLC、1/3 MNLC和1/10 MNLC四种浓度下,氟康唑组斑马鱼毛细胞损伤率分别为99%、95%、30%和28%;联苯苄唑组分别为94%、87%,、63%和49%;制霉菌素组分别为99%、89%、83%和25%。庆大霉素组斑马鱼毛细胞损伤率为55%。当与空白对照组比较,差异均具有统计学意义(P<0.001)。结论三种抗真菌药物均具有斑马鱼耳毒性,导致斑马鱼平衡囊形态发生改变并导致斑马鱼身体屈曲。三种药物均导致斑马鱼毛细胞数量的减少,并存在剂量依赖关系。     

庆大霉素对豚鼠耳肾毒性的相关性实验研究

目的 探讨豚鼠庆大霉素耳性与肾毒性的关系。方法 通过ＡＢＲ测试,耳蜗铺片毛细胞片数,血液庆大霉素药代动力学分析,血ＢＵＮ、Ｃｒ值测定,肾标光镜下观察等方法,观察肌注庆大霉互后豚鼠的耳蜗功能及肾功能变化。结果 肌注庆大霉素二周组ＡＢＲ的ＩＶ波反应阈阈移明显高于肌注庆大霉素一周组及其生理盐水对照组。光镜下耳蜗铺片毛细胞计数二周组毛细胞缺失数明显多于一周组对于对照组。肌注纱二周组的血清庆大霉素清除率明显     

水杨酸盐预防庆大霉素耳毒性的试验研究

目的 探讨大剂量应用庆大霉素时,观察水杨酸钠是否仍有预防耳毒性的作用。方法 选33只健康雄性豚鼠。随机分为A(11只)、B(11只)、C(11只)三组。A组腹腔注射药物庆大霉素+水杨酸钠;B组腹腔注射庆大霉素;C组腹腔注射生理盐水。每组动物用药前、用药后第2、4、6、8、10天分别行双耳ABR的阈值测试。耳蜗铺片光镜下毛细胞记数,分别用SPSS软件进行统计学处理。结果 体重:A组体重平均增加10.2克,B组体重平均减少5.2克,对照组体重平均增加16.5克,A组与B组比较差异有显著性(P<0.01)。ABR检查:第10天A组平均阈值为43.15±6.96,较B组平均83.93±19.33有显著改善(P<0.01),A组与对照组比较无显著性差异(P>0.05)。毛细胞计数:A组比B组损伤毛细胞数减少有显著性差异(P<0.01),B组毛细胞损伤明显增加,与对照组相比有显著性差异,A组与对照组无显著性差异。结论 提示在临床上为了在短期内控制细菌感染采用大剂量的庆大霉素时,仍可用水杨酸来预防其耳毒性。     

还原型谷胱甘肽拮抗庆大霉素耳毒性作用观察

目的观察还原型谷胱甘肽对庆大霉素耳蜗毒性的拮抗效果,并且比较了两种给药方法对其效果的影响.方法健康黑目豚鼠41只,随机分为五组,各组动物在观察期结束后,检测由8kHz、4kHz、2kHz频率短音诱发的脑干听觉诱发电位Ⅲ波反应阈;扫描电镜观察外毛细胞形态变化;采用耳蜗基底膜铺片的方法,对深染的外毛细胞表皮板进行计数.结果谷胱甘肽组耳蜗三排外毛细胞呈V字型排列有序.庆大霉素组外毛细胞纤毛散乱、倒伏,深染的表皮板数明显增加,Ⅲ波反应阈明显提高.合并给药组深染的表皮板数较庆大霉素组明显减少,Ⅲ波反应阈的提高幅度明显减小(P＜0.05).后继给药组深染的表皮板数和8kHz、4kHz的Ⅲ波反应阈与庆大霉素组比较均无统计学差异(P＞0.05),然而2kHz的Ⅲ波反应阈与庆大霉素组比较差异有显著性(P＜0.05),谷胱甘肽组与生理盐水组差异无显著性(P＞0.05).结论还原型谷胱甘肽与庆大霉素合并应用可以显著地减轻庆大霉素的耳毒性;庆大霉素停药后再给予还原型谷胱甘肽,对耳蜗高频段的毛细胞所受庆大霉素毒性可能难以产生拮抗作用,但是对耳蜗中、低频段的毛细胞可能提供一定程度的保护作用.     

丹参对庆大霉素耳毒性的减毒作用

目的　为探讨丹参对庆大霉素耳毒性的减毒作用。方法　将 2 4只沙鼠分为 4组 ,每组 6只 ,①生理盐水对照组。②丹参组 (SA)。③庆大霉素加丹参组 (GM SA)。④庆大霉素组 (GM)。所有药物均经肌肉注射 ,GM组和GM SA组每天注射庆大霉素 15 0mg/kg ,共 10d ;GM SA组和SA组同时注射丹参 2 g/kg ,共 10d ;生理盐水组每天注射生理盐水 2ml/kg ,共 10d。在注射前和注射后 4周和 8周进行CAP检测 ,第 8周听力测定后处死动物行扫描电镜检查。结果　 4周和 8周GM组高频CAP阈明显上升 ,而GM SA组听阈明显低 ,统计学处理有显著差异 (P <0 .0 1,t检验 ) ;GM组毛细胞损伤率底圈和第二圈分别为 35 %和 30 % ,GM SA组分别为 9%和 7% ,差异非常显著 (P <0 .0 1,χ2 检验 )。结论　丹参能明显降低庆大霉素对沙鼠的耳毒性作用 ,且不降低血液中庆大霉素的浓度。电生理检测结果与形态学变化一致。     

经圆窗应用地塞米松对豚鼠庆大霉素耳毒性的拮抗作用北大核心CSCD

目的探讨经圆窗局部应用地塞米松和全身应用地塞米松对庆大霉素致聋豚鼠耳蜗损害的拮抗作用。方法选用听力正常豚鼠60只,随机分为4组:①庆大霉素组,肌肉注射庆大霉素120mg·kg-1·d-1,1次/天,共10天;②全身用药组,肌肉注射庆大霉素(用法同庆大霉素组),同时腹腔注射地塞米松2.5mg·kg-1·d-1,1次/天,共10天;③局部用药组,肌肉注射庆大霉素(用法同庆大霉素组),右耳经圆窗局部用明胶海绵浸地塞米松(5mg/ml)约0.1ml,共1次。④对照组:肌肉注射与庆大霉素等体积的生理盐水,1次/天,共10天,右耳经圆窗局部用地塞米松1次。于实验前、停药后第1天和第10天分别检测各组动物的听性脑干反应(ABR),显微镜下观察中耳粘膜反应,扫描电镜观察耳蜗毛细胞的形态学变化。结果停药第10天,除对照组外,庆大霉素组、全身用药组、局部用药组动物ABR反应阈较实验前均有不同程度的升高,其中庆大霉素组的反应阈明显高于局部用药组(P<0.05)。扫描电镜下可见庆大霉素组动物耳蜗毛细胞损害严重,而全身和局部用药组动物耳蜗毛细胞损伤程度明显减轻;各组动物中耳粘膜正常。结论地塞米松可以在一定程度上减轻庆大霉素所致的耳毒性,经圆窗局部给药的效果优于全身应用。     

α—硫辛酸对庆大霉素所致大鼠耳毒性损害的保护作用

目的 探讨高效抗氧剂α-硫辛酸是否对庆大霉素所致的大鼠耳蜗毒性有防护作用.方法 将30只Wistar大鼠随机分成庆大霉素(GM)组、α-硫辛酸(α-LA)+庆大霉素(GM)组及正常对照组,GM组每日腹腔注射GM100mg/kg;GM+α-LA组每日同时腹腔注射GM100mg/kg和α-LA注射液10mg/kg;正常对照组每日腹腔注射生理盐水100mg/kg.3组皆连续用药10d,用药前后测试DPOAE.最后将动物处死,取听泡,观察毛细胞形态.实验数据进行统计学分析.结果 DPOAE停药后,GM+α-LA组DPOAE幅值有轻度降低;GM组DPOAE幅值明显降低; 2组用药前后比较有显著性差异(P<0.01).GM+α-LA组大鼠听功能受损程度明显轻于GM组.正常对照组大鼠用药前后DPOAE幅值及形态学无明显变化.形态学观察:给药后GM组耳蜗底回外毛细胞均有较重损伤,前庭毛细胞空泡样改变,纤毛倒伏缺失明显.GM+α-LA组耳蜗底回外毛细胞损伤明显减轻,前庭毛细胞散在缺失.正常对照组耳蜗底回外毛细胞和前庭毛细胞基本正常.结论 在使用GM的同时联合应用α-LA可有效减少GM对大鼠的内耳毒性.     

灯盏花对庆大霉素内耳毒性的防护作用

目的　探讨灯盏花对庆大霉素耳毒性的防护作用。方法　选用听力正常豚鼠 4 0只 ,随机分为 2组 :非治疗组 (庆大霉素组 ) ;治疗组 (庆大毒素 +灯盏花组 )。两组皆肌肉注射庆大霉素注射液 (12 0mg·kg-1·d-1) ,治疗组同时腹腔注射灯盏细辛注射液 (4 5mg·kg-1·d-1)连续 10天。分别于停药后第 1、7、14、2 1天随机抽取一定数量的豚鼠处死行毛细胞及血管纹的电镜及光镜观察 ,于处死前行畸变产物耳声发射 (DPOAE)、听性脑干反应 (ABR)检测。结果　非治疗组耳蜗功能和结构损害严重 ,外毛细胞的外形及核已固缩 ,血管纹毛细血管数量随用药后时间的延长逐渐稀少、管径狭窄。治疗组耳蜗功能和结构损伤较轻 ,除可见轻度胞质水肿及线粒体固缩外 ,外毛细胞基本正常 ,血管纹毛细血管管径有所增宽 ,DPOAE振幅和ABR波潜伏期、阈值两组比较有显著性差异 (P <0 .0 1)。结论　灯盏花对庆大霉素耳毒性有一定的防护作用     

一氧化氮对庆大霉素耳毒性的影响

目的研究耳蜗内不同水平的一氧化氮(NO)含量对庆大霉素(GM)耳毒性的影响.方法将实验动物豚鼠随机分为GM组;庆大霉素加左旋精氨酸(GM加L-Arg)组;庆大霉素加NG-单甲基-L-精氨酸(GM加L-NMMA)组和正常对照组.实验过程中观察动物体重变化,检测ABR反应阈;取标本检测血清和耳蜗组织NO含量,耳蜗铺片观察毛细胞损失程度.结果对照组动物体重增加明显,各实验组体重增加缓慢,其中GM加L-Arg组体重略有下降.实验第4周,GM加L-Arg组ABR反应阈升高明显,与GM组比较有统计学意义(P<0.05),GM加L-NMMA组ABR反应阈升高较少,与GM组比较有统计学意义(P<0.05).GM加L-Arg组血清和耳蜗NO含量明显升高,GM加L-NMM A组NO含量升高不明显,与GM组比较差异具有显著性(P<0.05).耳蜗铺片各组毛细胞损失程度与ABR变化相对应.结论增加耳蜗NO含量可增加GM耳毒性,减少耳蜗NO含量可减轻GM耳毒性.     

FK506拮抗庆大霉素耳毒性的实验观察

目的 初步探讨FK506拮抗庆大霉素耳毒性的效果。方法 通过分别检测庆大霉素损伤组与庆大霉素加不同剂量FK506（1mg、2mg）保护组实验动物听性脑干反应(ABR)阈值的变化,并结合耳蜗铺片琥珀酸脱氢酶（SDH)染色方法观察动物耳蜗形态学变化,观察FK506拮抗庆大霉素耳毒性的作用。结果 实验动物的听觉生理检查显示FK506能够拮抗庆大霉素耳毒性,FK506保护组动物听阈明显低于庆大霉素损伤组(P<0.01),随剂量增加,保护作用越明显;同时形态结果显示FK506保护组外毛细胞缺失明显少于庆大霉素损伤组(P<0.01)。结论 FK506对庆大霉素引起的耳毒性有拮抗作用。     

Comparative ototoxicity of gentamicin in the guinea pig and two strains of rats

Gentamicin ototoxicity and nephrotoxicity were compared in two strains of rats, Sprague-Dawley and Fisher-344, and in the Hartley albino guinea pig. Treatment groups consisting of 8 male rats of each strain and four male guinea pigs were dosed subcutaneously for 14 days with either 80 or 100 mg/kg of gentamicin sulfate in saline. Brainstem auditory evoked response (BAER) thresholds were recorded from each animal in each group on day 11 post-administration. Blood urea-nitrogen and serum creatinine were measured in blood obtained on day 11 post-administration as measures of nephrotoxicity. Kidney weight/body weight ratios were also determined. Loss of sensory hair cells was observed in the basal region of the organ of Corti from all animals treated with 100 mg/kg of gentamicin. The hair cell loss and BAER threshold elevations were greatest in the guinea pigs. Fisher-344 rats showed more extensive hair loss and greater BAER threshold elevations than Sprague-Dawley rats. The Fisher-344 rats exhibited increased blood urea-nitrogen and kidney weight/body weight ratios. Sprague-Dawleys did not suffer any nephrotoxic effects. These data indicate that the Fisher-344 rat is useful animal in which to study aminoglycoside ototoxicity as it exhibits both functional and morphological changes after gentamicin administration.     

The use of zebrafish for assessing ototoxic and otoprotective agents

Zebrafish and other fish exhibit hair cells in the lateral-line neuromasts which are structurally and functionally similar to mammalian inner ear hair cells. To facilitate drug screening for ototoxic or otoprotective agents, we report a straightforward, quantitative in vivo assay to determine potential ototoxicity of drug candidates and to screen otoprotective agents in zebrafish larva. In this study, a fluorescent vital dye, DASPEI (2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide), was used to stain zebrafish hair cells in vivo and morphometric analysis was performed to quantify fluorescence intensity and convert images to numerical endpoints. Various therapeutics, including gentamicin, cisplatin, vinblastine sulfate, quinine, and neomycin, which cause ototoxicity in humans, also resulted in hair cell loss in zebrafish. In addition, protection against cisplatin-induced ototoxicity was observed in zebrafish larva co-treated with cisplatin and different antioxidants including, glutathione (GSH), allopurinol (ALO), N-acetyl l-cysteine (l-NAC), 2-oxothiazolidine-4-carboxylate (OTC) and d-methionine (d-MET). Our data indicate that results of ototoxicity and otoprotection in zebrafish correlated with results in humans, supporting use of zebrafish for preliminary drug screening.     

Amelioration of Cisplatin-Induced ototoxicity by fosfomycin

The continued chemotherapeutic application of cisplatin (cis-diamminedichloroplatinum [II]) necessitates reduction of its doselimiting toxicity without decreasing its tumoricidal effect. This research project evaluated the efficacy of fosfomycin, a phosphonic acid antibiotic, in decreasing or ameliorating the ototoxicity (high frequency sensorineural hearing loss) and nephrotoxicity (renal tubular necrosis and interstitial nephritis) of cisplatin. Experimentally, fosfomycin effectively inhibits aminoglycoside-induced ototoxicity and nephrotoxicity in animals and humans. The efficacy of fosfomycin in blocking platinum-induced toxicity in the guinea pig was evaluated histologically and functionally using cytocochleography and light microscopy of the organ of Corti and the auditory brain stem evoked response (ABR), and light microscopy of renal corticomedullary tissues, small bowel, liver, lung, and peripheral nerve. The results demonstrate that fosfomycin ameliorates the acute renal tubular necrosis and interstitial nephritis and markedly inhibits the elevation of ABR thresholds and simultaneous outer hair cell loss that can result from cisplatinum administration. Fosfomycin should be considered apotential antidote for the dose-limiting ototoxicity and nephrotoxicity of cisplatin chemotherapy.     

Synaptophysin immunohistochemistry during vestibular hair cell recovery after gentamicin treatment

In the present study, morphometric and immunohistochemical techniques were used to evaluate the degree of synaptic recovery in the chinchilla crista sensory epithelia during various post-gentamicin-treatment periods of hair cell loss and recovery. For this purpose, two groups of animals were treated with Gelfoam pellets impregnated with 50 micro g of gentamicin implanted in the perilymphatic space within the otic capsule of the superior semicircular canal. Animals were sacrificed 1, 2 and 4 weeks after treatment. The degree of synaptic reinnervation was evaluated in the horizontal crista of the first group of animals using immunohistochemical techniques and antibodies against synaptophysin, a marker for synaptic reinnervation and synaptogenesis. Quantification of immunoreactivity in this group was made in the mid-region of the crista using the NIH 'Image' program. The second group of animals was used for quantification of the number of hair cells and supporting cells in the horizontal crista. In the normal sensory epithelium, synaptophysin immunoreactivity was found in the areas corresponding to the known distribution of afferent and efferent nerve terminals. Immunoreactivity was predominantly located within the afferent calyces of type I hair cells. No immunoreactivity was found in the supporting cells. Seven days after treatment there was a significant loss of hair cells and synaptophysin-stained area (SSA). In the mid-region of the crista the loss of synaptophysin immunoreactivity was quantitatively the greatest within the central zone of this region (93%) while the loss of hair cells was the smallest. These results suggest that afferent and efferent nerve terminals were also severely affected by the ototoxic treatment. Four weeks after treatment corresponding to the end of the recovery phase of gentamicin ototoxicity, there was a proportional increase in the number of hair cells and of the degree of SSA in the mid-region of the crista. The number of hair cells recovered to 58% with a recovery of SSA to 54% of normal. These results suggest that a greater fraction of synaptophysin expression within the sensory epithelium depends on the presence of afferent calyceal endings, which are greatly affected by gentamicin. Also, these results demonstrate a significant level of reinnervation of the newly regenerated hair cells, forecasting the potential for functionality of the regenerated hair cells.     

地塞米松对顺铂致斑马鱼毛细胞损伤的保护作用研究

目的 探讨地塞米松对顺铂致斑马鱼侧线神经丘毛细胞损伤的保护作用.方法 ① 将受精后5天(5 dpf)斑马鱼幼鱼分为对照组、顺铂组、顺铂+地塞米松组,每组10条.对照组置于系统水中;顺铂组斑马鱼幼鱼中加入50μmol/L顺铂孵育24小时;顺铂+地塞米松组预先给予25μmol/L地塞米松作用24小时,再给予50μmol/L...     

Rescue of auditory hair cells from ototoxicity by CEP-11 004, an inhibitor of the JNK signaling pathway

The hair cells (HCs) are the most vulnerable elements in the cochlea and damage to them is the most common cause of sensorineural hearing loss (SNHL). Understanding the intracellular events that lead to the death of HCs is a key to developing protective strategies. Recently, it has been shown that the c-Jun-N-terminal kinase (JNK) pathway is activated in HCs in response to aminoglycosides and CEP-1347, an inhibitor of the JNK signaling pathway protected HCs from ototoxicity. We have studied another inhibitor (CEP-11 004) of this signaling pathway in its ability to protect HCs from aminoglycoside ototoxicity in vitro. Organ of Corti explants from p5 rat basal turns were maintained in tissue culture and treated with CEP-11 004 for 12 hours. They were then treated with CEP-11 004 plus gentamicin for 72 hours. Significantly less HC death was observed compared to gentamicin alone. CEP-11 004 alone had no effect on HCs. We conclude that the JNK signaling pathway plays a role in aminoglycoside ototoxicity signaling.     

Effects of alpha-tocopherol on cisplatin-induced ototoxicity in guinea pigs

Cisplatin (CDDP), an antitumor agent widely used in the treatment of head and neck cancers, has dose-limiting side effects such as ototoxicity and nephrotoxicity. Recently, evidence has been accumulated to demonstrate that these side effects are closely related to oxidative stress. In the present study, we attempted to suppress CDDP-induced ototoxicity and nephrotoxicity in guinea pigs by administering alpha-tocopherol, a naturally occurring antioxidant. Hartley albino guinea pigs (250 approximately 300 g) were treated with CDDP (4 mg/kg intraperitoneally (I.P.)) for 3 days in the presence and absence of alpha-tocopherol (50 mg/kg I.P.) injection for 6 days. The combined treatment of animals with alpha-tocopherol distinctly improved the CDDP-induced side effects. These were: loss of Preyer's reflex at high frequencies; distinct elevation of auditory brain stem response threshold at 16 kHz; increased lipid peroxidation in the cochlea determined by the malondialdehyde-thiobarbituric acid method; substantial losses of outer hair cells in the basal and second turns of the cochlea; fragmentation of nuclear DNA detected by the TUNEL method in cochlear hair cells and cells in the stria vascularis; and increases in serum BUN and Cr. These results strongly suggest that alpha-tocopherol suppresses CDDP-induced ototoxicity and nephrotoxicity via the suppression of the increased production of reactive oxygen species.