Flentke George R, Klingler Rebekah H, Tanguay Robert L, Carvan Michael J, Smith Susan M
Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, Wisconsin.
Alcohol Clin Exp Res. 2014 May;38(5):1255-65. doi: 10.1111/acer.12360. Epub 2014 Feb 11.
Fetal alcohol spectrum disorders (FASD) are a leading cause of neurodevelopmental disability. Nonhuman animal models offer novel insights into its underlying mechanisms. Although the developing zebrafish has great promise for FASD research, a significant challenge to its wider adoption is the paucity of clear, mechanistic parallels between its ethanol (EtOH) responses and those of nonpiscine, established models. Inconsistencies in the published pharmacodynamics for EtOH-exposed zebrafish, alongside the use of comparatively high EtOH doses, challenge the interpretation of this model's clinical relevance.
To address these limitations, we developed a binge, single-exposure model of EtOH exposure in the early zebrafish embryo.
Brief (3-hour) EtOH exposure is sufficient to cause significant neural crest losses and craniofacial alterations, with peak vulnerability during neurogenesis and early somitogenesis. These losses are apoptotic, documented using TUNEL assay and secA5-YFP-reporter fish. Apoptosis is dose dependent with an EC50 = 56.2 ± 14.3 mM EtOHint , a clinically relevant value within the range producing apoptosis in chick and mouse neural crest. This apoptosis requires the calcium-dependent activation of CaMKII and recapitulates the well-described EtOH signaling mechanism in avian neural crest. Importantly, we resolve the existing confusion regarding zebrafish EtOH kinetics. We show that steady-state EtOH concentrations within both chorion-intact and dechorionated embryos are maintained at 35.7 ± 2.8% of EtOHext levels across the range from 50 to 300 mM EtOHext , a value consistent with several published reports. Equilibrium is rapid and complete within 5 minutes of EtOH addition.
The calcium/CaMKII mechanism of EtOH's neurotoxicity is shared between an amniote (chick) and teleost fish, indicating that this mechanism is evolutionarily conserved. Our data suggest that EtOHext concentrations >2% (v/v) for chorion-intact embryos and 1.5% (v/v) for dechorionated embryos have limited clinical relevance. The strong parallels with established models endorse the zebrafish's relevance for mechanistic studies of EtOH's developmental neurotoxicity.
胎儿酒精谱系障碍（FASD）是神经发育残疾的主要原因。非人类动物模型为其潜在机制提供了新的见解。尽管发育中的斑马鱼在FASD研究中具有很大潜力，但其更广泛应用面临的一个重大挑战是，其乙醇（EtOH）反应与已确立的非鱼类模型之间缺乏清晰的、机制上的相似性。已发表的关于暴露于乙醇的斑马鱼的药效学存在不一致之处，再加上使用相对较高的乙醇剂量，这对该模型临床相关性的解释提出了挑战。
为解决这些局限性，我们开发了一种早期斑马鱼胚胎乙醇暴露的单次暴饮模型。
短暂（3小时）的乙醇暴露足以导致显著的神经嵴损失和颅面改变，在神经发生和早期体节发生期间易感性达到峰值。这些损失是凋亡性的，通过TUNEL检测和secA5-YFP报告鱼得以证实。凋亡呈剂量依赖性，半数有效浓度（EC50）= 56.2 ± 14.3 mM EtOHint，这是一个在鸡和小鼠神经嵴中产生凋亡的临床相关范围内的值。这种凋亡需要CaMKII的钙依赖性激活，并重现了禽类神经嵴中描述详尽的乙醇信号传导机制。重要的是，我们解决了现有的关于斑马鱼乙醇动力学的困惑。我们表明，在50至300 mM EtOHext范围内，完整绒毛膜胚胎和去绒毛膜胚胎内的乙醇稳态浓度均维持在EtOHext水平的35.7 ± 2.8%，这一数值与多篇已发表报告一致。在添加乙醇后5分钟内即可快速且完全达到平衡。
乙醇神经毒性的钙/CaMKII机制在羊膜动物（鸡）和硬骨鱼之间是共有的，表明该机制在进化上是保守的。我们的数据表明，完整绒毛膜胚胎的EtOHext浓度>2%（v/v）以及去绒毛膜胚胎的EtOHext浓度>1.5%（v/v）的临床相关性有限。与已确立模型的强烈相似性支持了斑马鱼在乙醇发育神经毒性机制研究中的相关性。
