使用头太空气色谱对斑马鱼胚胎中的乙醇水平进行定量
Summary
这项工作描述了一种使用头部空间气相色谱从适当的暴露方法到胚胎加工和乙醇分析来量化斑马鱼胚胎中乙醇水平的协议。
Abstract
胎儿酒精谱系障碍 （FASD） 描述了乙醇引起的发育缺陷的高度可变连续体，包括面部畸形和神经损伤。与复杂的病理学，胎儿外病影响大约每100个孩子在美国出生每年。由于FASD的高度可变性，动物模型在我们目前对乙醇引起的发育缺陷的机械理解中已被证明是至关重要的。越来越多的实验室专注于使用斑马鱼来检查乙醇引起的发育缺陷。斑马鱼产生大量外部受精、遗传可移植的半透明胚胎。这使得研究人员能够精确控制多种遗传环境中乙醇暴露的时间和剂量，并通过活成像技术量化胚胎乙醇暴露的影响。这结合人类对遗传学和发育的高度保护，已经证明斑马鱼是研究乙醇致畸性力学基础的有力模型。然而，不同斑马鱼研究之间的乙醇接触方案各不相同，这混淆了这些研究中对斑马鱼数据的解释。下面是使用头空间气相色谱对斑马鱼胚胎中的乙醇浓度进行量化的协议。
Introduction
胎儿酒精谱系障碍（FASD）描述了与胚胎乙醇暴露相关的多种神经损伤和颅面畸形。多种因素，包括乙醇暴露的时间和剂量以及遗传背景，都促成了FASD2，3的变化。在人类中，这些变量的复杂关系使得研究和理解FASD的病因变得具有挑战性。动物模型已经证明对于我们理解乙醇致畸性的机械基础至关重要。各种各样的动物模型系统已经被用来研究FASD的多个方面，结果已经非常一致，在人类接触发现4。啮齿动物模型系统用于检查FASD的许多方面，其中小鼠是最常见的5，6，7。这项工作的大部分重点是发育缺陷，以早期乙醇暴露8，虽然后来接触乙醇已被证明会导致发育异常以及9。此外，小鼠的遗传能力大大有助于我们探索FASD10、11的遗传基础的能力。这些小鼠研究强烈表明，有基因-乙醇相互作用与声波刺猪通路， 视黄酸信号，超氧化物歧化酶， 一氧化二氮合成酶 I， Aldh2和Fancd2 8，10，11，12，13，14，15，16，17，1819，20，21.这些研究表明，动物模型对于促进我们对FASD及其基本机制的理解至关重要。
斑马鱼已成为一个强大的模型系统，以检查乙醇畸变的许多方面22，23。由于其外受精、高肥力、遗传可遗传性和活成像能力，斑马鱼非常适合研究乙醇畸变的时序、剂量和遗传学等因素。乙醇可以施用精确分期胚胎，然后可以成像研究乙醇在发育过程中的直接影响。这项工作可以直接与人类有关，因为斑马鱼和人类之间的遗传开发程序高度保守，因此可以帮助指导FASD人类研究24。虽然斑马鱼已经被用来检查乙醇畸形的产生，但缺乏共识报告胚胎乙醇浓度，使得与人类相比困难25。在哺乳动物系统中，血液-酒精水平与组织乙醇水平直接相关26。许多斑马鱼研究在胚胎的循环系统完全形成之前治疗。由于没有母体样本进行检测，需要一个评估乙醇浓度的过程来量化胚胎内的乙醇水平。在这里，我们描述了一个过程，以量化乙醇浓度在正在发展的斑马鱼胚胎使用头空间气相色谱。
Protocol
在这个程序中使用的所有斑马鱼胚胎是在既定的IACUC协议27下培育和繁殖的。这些协议得到了德克萨斯大学奥斯汀分校和路易斯维尔大学的批准。 注：本研究28使用斑马鱼线Tg（fli1：EGFP）y1。 本程序中使用的所有水都是无菌反渗透水。所有统计分析均使用图形板棱镜 v8.2.1 执行。 1. 制造胚胎介质 ?…
Representative Results
血液乙醇水平不能确定在早期胚胎斑马鱼，因为它们缺乏一个完全形成的循环系统。为了确定斑马鱼胚胎中的乙醇浓度水平，乙醇水平直接从均质胚胎组织测量。为了正确测量胚胎乙醇浓度，必须考虑胚胎体积。胚胎（附着的蛋黄）位于被胚胎外流体包围的胆囊（蛋壳）内（图1）。胚胎的任何体积测量都必须在胚胎暴露时进行，因为胚胎体积会随着胚胎大小的增大而改变。…
Discussion
斑马鱼作为一种开发模型系统，非常适合研究环境因素对发展的影响。他们产生大量的外部受精胚胎，这允许在乙醇研究中精确计时和剂量范式。这与活成像能力以及人类的遗传和发育保护相结合，使斑马鱼成为teratology研究的强大模型系统。本文使用头空间气相色谱法测量斑马鱼胚胎的胚胎乙醇浓度。
确定胚胎乙醇浓度对于了解乙醇对发育中的胚胎的影响至关重要。在啮齿?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
本文中介绍的研究得到了美国国家卫生研究院/国家牙科和颅面研究所（NIH/NIDCR）R01DE020884对J.K.E的先前资助。 以及国家卫生研究院/国家酒精滥用和酒精中毒研究所（NIH/NIAAA）F32A021320至C.B.L.以及国家卫生研究院/国家酒精滥用研究所（NIH/NIAAA）R00A023560向C.B.L.提供的赠款。我们感谢鲁本·冈萨雷斯提供和协助进行气致度表分析。我们感谢蒂娜·奥尼特罗斯和吉娜·诺布尔斯博士的写作协助。
Materials
| Air | Provided by contract to the university | ||
| Analytical Balance | VWR | 10204-962 | |
| AutoSampler, CP-8400 | Varian | Gas Chromatograph Autosampler | |
| Calcium Chloride | VWR | 97062-590 | |
| Ethanol | Decon Labs | 2701 | |
| Gas chromatograph vial with polytetrafluoroethylene/silicone septum and plastic cap 2 mL | Agilent | 8010-0198 | Can reuse the vials after cleaning, but not the caps/septa |
| Gas Chromatograph, CP-3800 | Varian | ||
| Helium | Provided by contract to the university | ||
| HP Innowax capillary column | Agilent | 19095N-123I | 30 m x 0.53 mm x 1.0 μm film thick |
| Hyrdogen | Provided by contract to the university | ||
| Magnesium Sulfate (Heptahydrate) | Fisher Scientific | M63-500 | |
| Microcentrifuge tube 1.5 mL | Fisher Scientific | 2682002 | |
| Micropipette tips 10 μL | Fisher Scientific | 13611106 | |
| Micropipette tips 1000 μL | Fisher Scientific | 13611127 | |
| Micropipette tips 200 μL | Fisher Scientific | 13611112 | |
| Petri dishes 100 mm | Fisher Scientific | FB012924 | |
| Pipetman L p1000L Micropipette | Gilson | FA10006M | |
| Pipetman L p200L Micropipette | Gilson | FA10005M | |
| Pipetman L p2L Micropipette | Gilson | FA10001M | |
| Polytetrafluoroethylene/silicone septum and plastic cap | Agilent | 5190-7021 | Replacement caps/septa for gas chromatograph vials |
| Potassium Chloride | Fisher Scientific | P217-500 | |
| Potassium Phosphate (Dibasic) | VWR | BDH9266-500G | |
| Pronase | VWR | 97062-916 | |
| Silica Beads .5 mm | Biospec Products | 11079105z | |
| Silica Beads 1.0 mm | Biospec Products | 11079110z | |
| Sodium Bicarbonate | VWR | BDH9280-500G | |
| Sodium Chloride | Fisher Scientific | S271-500 | |
| Sodium Phosphate (Dibasic) | Fisher Scientific | S374-500 | |
| Solid-phase microextraction fiber assembly Carboxen/Polydimethylsiloxane | Millipore Sigma | 57343-U | Replacement fibers |
| Star Chromatography Workstation | Varian | Chromatography software | |
| Thermogreen Low Bleed (LB-2) Septa | Millipore Sigma | 23154 | Replacement inlet septa |
Referenzen
- Elliott, E. J., Payne, J., Morris, A., Haan, E., Bower, C. Fetal alcohol syndrome: a prospective national surveillance study. Archive of Diseases in Childhood. 93 (9), 732-737 (2008).
- Cudd, T. A. Animal model systems for the study of alcohol teratology. Experimental Biology and Medicine. 230 (6), 389-393 (2005).
- Williams, J. F., Smith, V. C. Committee on Substance Abuse. Fetal Alcohol Spectrum Disorders. Pediatrics. 136 (5), 1395-1406 (2015).
- Patten, A. R., Fontaine, C. J., Christie, B. R. A comparison of the different animal models of fetal alcohol spectrum disorders and their use in studying complex behaviors. Frontiers in Pediatrics. 2, 93 (2014).
- Petrelli, B., Weinberg, J., Hicks, G. G. Effects of prenatal alcohol exposure (PAE): insights into FASD using mouse models of PAE. Biochemistry and Cell Biology. 96 (2), 131-147 (2018).
- Mayfield, J., Arends, M. A., Harris, R. A., Blednov, Y. A. Genes and Alcohol Consumption: Studies with Mutant Mice. International Review Neurobiology. 126, 293-355 (2016).
- Marquardt, K., Brigman, J. L. The impact of prenatal alcohol exposure on social, cognitive and affective behavioral domains: Insights from rodent models. Alcohol. 51, 1-15 (2016).
- Sulik, K. K. Genesis of alcohol-induced craniofacial dysmorphism. Experimental Biology and Medicine. 230 (6), 366-375 (2005).
- Lipinski, R. J., et al. Ethanol-induced face-brain dysmorphology patterns are correlative and exposure-stage dependent. PLoS One. 7 (8), 43067 (2012).
- Eberhart, J. K., Parnell, S. The genetics of fetal alcohol spectrum disorders. Alcoholism: Clinical and Experimental Research. 40 (6), 1154-1165 (2016).
- Becker, H. C., Diaz-Granados, J. L., Randall, C. L. Teratogenic actions of ethanol in the mouse: a minireview. Pharmacology, Biochemistry and Behavior. 55 (4), 501-513 (1996).
- Ahlgren, S. C., Thakur, V., Bronner-Fraser, M. Sonic hedgehog rescues cranial neural crest from cell death induced by ethanol exposure. Proceedings of the National Academy of Sciences of the United States of America. 99 (16), 10476-10481 (2002).
- Loucks, E. J., Ahlgren, S. C. Deciphering the role of Shh signaling in axial defects produced by ethanol exposure. Birth Defects Research Part A: Clinical and Molecular Teratology. 85 (6), 556-567 (2009).
- Hong, M., Krauss, R. S. Cdon mutation and fetal ethanol exposure synergize to produce midline signaling defects and holoprosencephaly spectrum disorders in mice. PLoSGenetics. 8 (10), 1002999 (2012).
- Aoto, K., Shikata, Y., Higashiyama, D., Shiota, K., Motoyama, J. Fetal ethanol exposure activates protein kinase A and impairs Shh expression in prechordal mesendoderm cells in the pathogenesis of holoprosencephaly. Birth Defects Research Part A: Clinical and Molecular Teratology. 82 (4), 224-231 (2008).
- Deltour, L., Ang, H. L., Duester, G. Ethanol inhibition of retinoic acid synthesis as a potential mechanism for fetal alcohol syndrome. The FASEB Journal. 10 (9), 1050-1057 (1996).
- Wentzel, P., Eriksson, U. J. Ethanol-induced fetal dysmorphogenesis in the mouse is diminished by high antioxidative capacity of the mother. Toxicological Sciences. 92 (2), 416-422 (2006).
- Karacay, B., Mahoney, J., Plume, J., Bonthius, D. J. Genetic absence of nNOS worsens fetal alcohol effects in mice. II: microencephaly and neuronal losses. Alcoholism: Clinical and Experimental Research. 39 (2), 221-231 (2015).
- Bonthius, D. J., Winters, Z., Karacay, B., Bousquet, S. L., Bonthius, D. J. Importance of genetics in fetal alcohol effects: null mutation of the nNOS gene worsens alcohol-induced cerebellar neuronal losses and behavioral deficits. Neurotoxicology. 46, 60-72 (2015).
- Bonthius, D. J., et al. Deficiency of neuronal nitric oxide synthase (nNOS) worsens alcohol-induced microencephaly and neuronal loss in developing mice. Brain Research. Developmental Brain Research. 138 (1), 45-59 (2002).
- Langevin, F., Crossan, G. P., Rosado, I. V., Arends, M. J., Patel, K. J. Fancd2 counteracts the toxic effects of naturally produced aldehydes in mice. Nature. 475 (7354), 53-58 (2011).
- Lovely, C. B., Fernandes, Y., Eberhart, J. K. Fishing for Fetal Alcohol Spectrum Disorders: Zebrafish as a Model for Ethanol Teratogenesis. Zebrafish. 13 (5), 391-398 (2016).
- Fernandes, Y., Buckley, D. M., Eberhart, J. K. Diving into the world of alcohol teratogenesis: a review of zebrafish models of fetal alcohol spectrum disorder. Biochemistry and Cell Biology. 96 (2), 88-97 (2018).
- McCarthy, N., et al. Pdgfra protects against ethanol-induced craniofacial defects in a zebrafish model of FASD. Development. 140 (15), 3254-3265 (2013).
- Lovely, C. B., Nobles, R. D., Eberhart, J. K. Developmental age strengthens barriers to ethanol accumulation in zebrafish. Alcohol. 48 (6), 595-602 (2014).
- Harris, R. A., Trudell, J. R., Mihic, S. J. Ethanol’s molecular targets. Science Signaling. 1 (28), (2008).
- Westerfield, M. . The Zebrafish Book: A guide for the laboratory use of zebrafish Danio (Brachydanio) rerio. , (1993).
- Lawson, N. D., Weinstein, B. M. In vivo imaging of embryonic vascular development using transgenic zebrafish. Entwicklungsbiologie. 248 (2), 307-318 (2002).
- Hagedorn, M., Kleinhans, F. W., Artemov, D., Pilatus, U. Water Distribution and permeability of zebrafish embryos, Brachydanio rerio. Journal of Experimental Zoology. 278 (6), 356-371 (1997).
- Lippi, G., et al. The alcohol used for cleansing the venipuncture site does not jeopardize blood and plasma alcohol measurement with head-space gas chromatography and an enzymatic assay. Biochemia Medica. 27 (2), 398-403 (2017).
- Poklis, J. L., Wolf, C. E., Peace, M. R. Ethanol concentration in 56 refillable electronic cigarettes liquid formulations determined by headspace gas chromatography with flame ionization detector (HS-GC-FID). Drug Testing and Analysis. 9 (10), 1637-1640 (2017).
- Heit, C., et al. Quantification of Neural Ethanol and Acetaldehyde Using Headspace GC-MS. Alcoholism: Clinical and Experimental Research. 40 (9), 1825-1831 (2016).
- Chun, H. J., Poklis, J. L., Poklis, A., Wolf, C. E. Development and Validation of a Method for Alcohol Analysis in Brain Tissue by Headspace Gas Chromatography with Flame Ionization Detector. Journal of Analytical Toxicology. 40 (8), 653-658 (2016).
- Schlatter, J., Chiadmi, F., Gandon, V., Chariot, P. Simultaneous determination of methanol, acetaldehyde, acetone, and ethanol in human blood by gas chromatography with flame ionization detection. Human and Experimental Toxicology. 33 (1), 74-80 (2013).
- Schier, C. J., Mangieri, R. A., Dilly, G. A., Gonzales, R. A. Microdialysis of ethanol during operant ethanol self-administration and ethanol determination by gas chromatography. Journal of Visualized Experiments. (67), e4142 (2012).
- Adalsteinsson, E., Sullivan, E. V., Mayer, D., Pfefferbaum, A. In vivo quantification of ethanol kinetics in rat brain. Neuropsychopharmacology. 31 (12), 2683-2691 (2006).
- Quertemont, E., Green, H. L., Grant, K. A. Brain ethanol concentrations and ethanol discrimination in rats: effects of dose and time. Psychopharmacology. 168 (3), 262-270 (2003).
- Flentke, G. R., Klinger, R. H., Tanguay, R. L., Carvan, M. J., Smith, S. M. An evolutionarily-conserved mechanism of calcium-dependent neurotoxicity. Alcoholism: Clinical and Experimental Research. 38 (5), 1255-1265 (2014).
- Reimers, M. J., Flockton, A. R., Tanguay, R. L. Ethanol- and acetaldehyde-mediated developmental toxicity in zebrafish. Neurotoxicology and Teratology. 26 (6), 769-781 (2004).
- Zhang, C., Ojiaku, P., Cole, G. J. Forebrain and hindbrain development in zebrafish is sensitive to ethanol exposure involving agrin, Fgf, and sonic hedgehog function. Birth Defects Research Part A: Clinical and Molecular Teratology. 97 (1), 8-27 (2013).
Diesen Artikel zitieren
Lovely, C. B. Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography. J. Vis. Exp. (156), e60766, doi:10.3791/60766 (2020).