Neomycin-induced hair cell death and rapid regeneration in the lateral line of zebrafish (Danio rerio)
Neomycin-induced hair cell death and rapid regeneration in the lateral line of zebrafish (Danio rerio)
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DOI：
10.1007/s10162-002-3022-x
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发表时间：
2003-06-01
影响因子：
2.4
通讯作者：
Rubel, EW
中科院分区：
文献类型：
--
作者：
Harris, JA;Cheng, AG;Rubel, EW
机械感受性毛细胞对包括新霉素在内的氨基糖苷类抗生素极其敏感。在幼体野生型斑马鱼侧线神经丘最初暴露于一系列新霉素浓度1小时后，于4小时评估毛细胞的存活情况。使用荧光活性染料DASPEI选择性标记毛细胞，在活鱼中对每个侧线神经丘的毛细胞存在与否进行评分。在暴露于500μM新霉素4小时后，所有神经丘都没有DASPEI标记的毛细胞。活性DASPEI染色与固定幼体中通过乙酰化微管蛋白免疫细胞化学和鬼笔环肽标记所确定的每个神经丘的毛细胞数量成比例。还分析了新霉素诱导损伤后侧线神经丘毛细胞再生的时间进程。在新霉素处理后12小时和24小时，首先使用活的DASPEI染色观察到再生的毛细胞。分析了增殖在毛细胞再生中的潜在作用。使用溴脱氧尿苷（BrdU）掺入的1小时脉冲固定方案来识别神经丘中的S期细胞。在药物暴露4小时后，新霉素损伤的神经丘中的BrdU掺入与对照神经丘没有差异，但在12小时后显著上调。在新霉素处理后12小时的1小时期间识别的增殖细胞能够在药物处理后24 - 48小时产生新的毛细胞。此处呈现的结果为研究和识别影响脊椎动物毛细胞在耳毒性损伤后的死亡、存活和再生的基因提供了一种标准化的制备方法。
Mechanoreceptive hair cells are extremely sensitive to aminoglycoside antibiotics, including neomycin. Hair cell survival was assessed in larval wild-type zebrafish lateral line neuromasts 4 h after initial exposure to a range of neomycin concentrations for 1 h. Each of the lateral line neuromasts was scored in live fish for the presence or absence of hair cells using the fluorescent vital dye DASPEI to selectively label hair cells. All neuromasts were devoid of DASPEI-labeled hair cells 4 h after 500 muM neomycin exposure. Vital DASPEI staining was proportional to the number of hair cells per neuromast identified in fixed larvae rising immunocytochemistry for acetylated tubulin and phalloidin labeling. The time course of hair cell regeneration in the lateral line neuromasts was also analyzed following neomycin-induced damage. Regenerated hair cells were first observed using live DASPEI staining 12 and 24 h following neomycin treatment. The potential role of proliferation in regenerating hair cells was analyzed. A 1 h pulse-fix protocol using bromodeoxyuridine (BrdU) incorporation was used to identify S-phase cells in neuromasts. BrdU incorporation in neomycin-damaged neuromasts did not differ from control neuromasts 4 h after drug exposure but was dramatically upregulated after 12 h. The proliferative cells identified during a 1 h period at 12 h after neomycin treatment were able to give rise to new hair cells by 24-48 h after drug treatment. The results presented here provide a standardized preparation for studying and identifying genes that influence vertebrate hair cell death, survival, and regeneration following ototoxic insults.