Name: accumulation and distribution of fluorescent microplastics in the early life stages of zebrafish
Uploaded: 2021-07-04T13:00:00
Description: Watch this Scientific Journal Video about Accumulation and Distribution of Fluorescent Microplastics in the Early Life Stages of Zebrafish at JoVE.com

本著作由中国国家自然科学基金委员会（21777145，22076170）和长江学者与创新研究团队项目（IRT_17R97）资助。

成年斑马鱼起源于中国斑马鱼资源中心（中国武汉）。这些实验是按照国家指南"动物福利伦理审查实验室动物指南（GB/T35892-2018）进行的。
1. 胚胎收集
<ol><li>在20升玻璃罐中保持鱼，在14：10 h光照下，在恒温下（28±0.5°C）重新循环木炭过滤的自来水系统（pH 7.0±0.2）： 黑暗。</li>
	<li>每天用 阿泰米亚·瑙普利喂鱼两次。建议每天以最多3%的鱼体重食用，每次<sup class="xr...

<ol>
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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+microplastics+impairs+digestive+performance,+stimulates+immune+response+and+induces+microbiota+dysbiosis+in+the+gut+of+juvenile+guppy+(Poecilia+reticulata).">Exposure to microplastics impairs digestive performance, stimulates immune response and induces microbiota dysbiosis in the gut of juvenile guppy (Poecilia reticulata).</a> Science of the Total Environment. 733, 138929 (2020).</li><li>Pr&#252;st, M., Meijer, J., Westerink, R. H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plastic+brain:+neurotoxicity+of+micro-+and+nanoplastics.">The plastic brain: neurotoxicity of micro- and nanoplastics.</a> Particle and Fibre Toxicology. 17, 24 (2020).</li><li>Jakubowska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chronic+exposure+to+microplastics+of+different+polymer+types+on+early+life+stages+of+sea+trout+Salmo+trutta.">Effects of chronic exposure to microplastics of different polymer types on early life stages of sea trout Salmo trutta.</a> Science of the Total Environment. 740, 139922 (2020).</li><li>Qiang, L., Cheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+polystyrene+microplastics+impairs+gonads+of+zebrafish+(Danio+rerio).">Exposure to polystyrene microplastics impairs gonads of zebrafish (Danio rerio).</a> Chemosphere. 263, 128161 (2021).</li><li>Hamed, M., Soliman, H. A. M., Osman, A. G. M., Sayed, A. E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidants+and+molecular+damage+in+Nile+Tilapia+(Oreochromis+niloticus)+after+exposure+to+microplastics.">Antioxidants and molecular damage in Nile Tilapia (Oreochromis niloticus) after exposure to microplastics.</a> Environmental Science and Pollution Research. 27, 14581-14588 (2020).</li><li>Burns, E. E., Boxall, A. B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplastics+in+the+aquatic+environment:+Evidence+for+or+against+adverse+impacts+and+major+knowledge+gaps.">Microplastics in the aquatic environment: Evidence for or against adverse impacts and major knowledge gaps.</a> Environmental Toxicology and Chemistry. 37, 2776-2796 (2018).</li><li>Ma, H., Pu, S., Liu, S., Bai, Y., Mandal, S., Xing, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplastics+in+aquatic+environments:+Toxicity+to+trigger+ecological+consequences.">Microplastics in aquatic environments: Toxicity to trigger ecological consequences.</a> Environmental Pollution. 261, 114089 (2020).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio reio). 4th ed. , (2000).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Developmental Dynamics. 203, 253-310 (1995).</li><li>Pikuda, O., Xu, E. G., Berk, D., Tufenkji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+assessments+of+micro-+and+nanoplastics+can+be+confounded+by+preservatives+in+commercial+formulations.">Toxicity assessments of micro- and nanoplastics can be confounded by preservatives in commercial formulations.</a> Environmental Science &amp; Technology Letters. 6, 21-25 (2019).</li><li>Lidster, K., Readman, G. D., Prescott, M. J., Owen, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+survey+on+the+use+and+welfare+of+zebrafish+Danio+rerio+in+research.">International survey on the use and welfare of zebrafish Danio rerio in research.</a> Journal of Fish Biology. 90, 1891-1905 (2017).</li><li>Pitt, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake,+tissue+distribution,+and+toxicity+of+polystyrene+nanoparticles+in+developing+zebrafish+(Danio+rerio).">Uptake, tissue distribution, and toxicity of polystyrene nanoparticles in developing zebrafish (Danio rerio).</a> Aquatic Toxicology. 194, 185-194 (2018).</li><li>Lin, S. J., Zhao, Y., Nel, A. E., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+An+in+vivo+model+for+nano+EHS+studies.">Zebrafish: An in vivo model for nano EHS studies.</a> Small. 9, 1608-1618 (2013).</li></ol>

根据《保护用于科学目的的动物的准则》，如欧盟2010/63/欧盟指令，在能够独立喂养（受精后5天）17岁之前，对斑马鱼早期生命阶段的实验，动物伦理许可不是强制性的。然而，最佳福利做法对于优化斑马鱼的使用非常重要，例如，麻醉和安乐死的人道方法应引起关注。乙基3-氨基苯甲酸酯（MS-222，或三甲酸酯），在大多数实验室中经常使用的制剂，在这里用于麻醉和安乐死。</p...

自20世纪首次合成以来，塑料在各个领域得到广泛应用，导致全球产量快速增长。2018年，全球塑料产量约为3.6亿吨。自然环境中的塑料会因化学、物理或生物过程而降解为细颗粒。一般来说，大小 <为5毫米的精细塑料颗粒定义为微塑料4。微塑料也设计用于特定的应用，如来自化妆品的微珠5。微塑料作为近乎永久性的污染物，在环境中积累，越来越受到科学家、决策者和公众的关注。先前的研究表明，微塑料可对鱼类造成不良影响，如胃肠道损伤7、神经毒性8、内分泌中断9、氧化应激10和DNA损伤11。然而，微塑料的毒性至今尚未完全暴露出来。
斑马鱼胚胎具有小尺寸、外部受精、光学透明度和大离合器等多种实验优势，被认为是研究早期污染物对鱼类影响的理想模型生物体。此外，评估生物反应只需要有限的测试物质。在这里，斑马鱼胚胎暴露在不同浓度的微塑料（0.1，1，10毫克/升）5天，并评估斑马鱼胚胎/幼虫微塑料的生物积累和分布。这一结果将促进我们对微塑料对鱼类毒性的理解，此处描述的方法可能被推广，以确定斑马鱼早期生命阶段其他类型的荧光材料的积累和分布。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Nikon, Japan</td>
			<td>Eclipse Ti-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Green fluorescently labeled polystyrene beads</td>
			<td>Phosphorex, USA</td>
			<td>2103A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma-Aldrich, USA</td>
			<td>A5040</td>
			<td></td>
		</tr>
	</tbody>
</table>

accumulation and distribution of fluorescent microplastics in the early life stages of zebrafish

微塑料作为一种新型的环境污染物，在水生环境中得到广泛应用，对水生生物构成极大威胁。微塑料的生物积累在其毒性作用中起着关键作用：然而，作为颗粒物，它们的生物累积与许多其他污染物不同。本文描述的是一种可行的方法，可以直观地确定使用荧光微塑料在斑马鱼胚胎或幼虫中的微塑料的积累和分布。胚胎暴露在直径为500纳米、时速为120小时的荧光微塑料的不同浓度（0.1、1和10毫克/升）中。结果表明，微塑料可以以依赖浓度的方式在斑马鱼胚胎/幼虫中生物积累。孵化前，在胚胎周围发现强烈的荧光：而在斑马鱼幼虫中，蛋黄囊、腹腔和胃肠道是微塑料的主要积累部位。结果表明，斑马鱼在早期生活阶段吸收和内化微塑料，这将为更好地了解微塑料对水生动物的影响提供依据。

荧光微塑料的分布和积累表现在图1和表1中。未曝光组（控制）中未观察到可见荧光。然而，在接触不同浓度的微塑料（24 hpf）后，在胆汁周围发现荧光积累。在幼虫中也检测到绿色荧光，荧光水平似乎以浓度和时间依赖的方式增加。蛋黄囊、腹腔和胃肠道是微塑料的主要积累部位（图2）。
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Watch this Scientific Journal Video about Accumulation and Distribution of Fluorescent Microplastics in the Early Life Stages of Zebrafish at JoVE.com

Accumulation and Distribution of Fluorescent Microplastics in the Early Life Stages of Zebrafish

斑马鱼胚胎/幼虫在外部发育，具有光学透明度。在鱼类早期生命阶段，微塑料的生物积累很容易用荧光标记的微珠进行评估。

斑马鱼早期荧光微塑料的积累和分布

As a new type of environmental pollutant, microplastic has been widely found in the aquatic environment and poses a high threat to aquatic organisms. The ...

斑马鱼早期荧光微塑料的积累和分布。介绍。微塑料的生物积累在它们的毒性作用中起着关键作用。然而，作为颗粒物，它们的生物累积与许多其他污染物不同。本文描述的是一种可行的方法，可以直观地确定使用荧光微塑料在斑马鱼胚胎或幼虫中的微塑料的积累和分布。这些实验是按照国家动物伦理审查指导实验室动物指南进行的。一是胚胎采集。成年斑马鱼起源于中国斑马鱼资源中心。鱼被保存在20升玻璃罐中，在恒温下循环木炭过滤的自来水系统，即28摄氏度，在14和10小时的光圈中，光：暗。鱼每天用阿特米亚纳普利喂两次。建议每天最多给予3%的鱼体重，每次应在五分钟内食用。发育良好的成年斑马鱼在繁殖前一天晚上以一雄二雌的比例被转移到产卵池中。第二天早上，鱼在光循环开始后开始产卵。鸡蛋使用巴斯德移液器收集，用10%Hank溶液冲洗数次，然后用显微镜检查受精情况。受精卵经过大约两个小时的受精后经过期，可以清楚地识别出来。受精胚胎在含有200毫升10%汉克溶液的500毫升烧瓶中孵育，1%甲基蓝在28摄氏度下消毒。每个 2 毫升溶液中的加载速率不超过一个胚胎。二、准备微塑料悬架。标称直径为500纳米的绿色荧光标签聚苯乙烯珠的库存溶液被声波化10分钟。用 10% Hank 的溶液稀释库存溶液，以产生所需的暴露解决方案，即每升 0.1、1 和 10 毫克。微塑料的暴露溶液在暴露前总是新鲜准备的。三、微塑料暴露。随机挑选了六个新受精胚胎，然后转移到每口井的六个井板中，其中含有五毫升不同浓度的微塑料溶液。包含包含 10% Hank 解决方案的对照组。三脚架井，共18个胚胎，用于每次治疗。胚胎在与成人相同的光暗周期和温度下孵育，每12小时观察一次。死者立即被移走。微塑料解决方案每 24 小时更新 90%。在暴露期间，鱼不喂食。一般来说，胚胎的孵化从受精后48小时开始，到受精后72小时左右完成。四、微塑料分布评估。在24-48-72-96和120小时后受精，胚胎和幼虫，从三个复制品各一个，随机选择和冲洗与10%汉克的解决方案。胚胎和幼虫被安排并准备观察。在此之前，幼虫被转移到培养皿中，并暴露在0.016%的三角肌麻醉中。用荧光显微镜观察鱼类，并使用成像软件进行成像。鱼的荧光强度通过图像J NIH进一步量化。代表性结果。结果表明，微塑料可以以依赖浓度的方式在斑马鱼胚胎和幼虫中生物积累。孵化前，在胚胎周围发现强烈的荧光：在斑马鱼幼虫中，蛋黄囊、腹膜和胃肠道是微塑料的主要积累部位。讨论。这一结果将促进我们对微塑料对鱼类毒性的理解，此处描述的方法可能被推广，以确定斑马鱼早期生命阶段其他类型的荧光材料的积累和分布。由于胆汁将作为对抗大尺寸粒子的有效屏障，因此可能需要在暴露前进行减压过程。然而，在考虑现实世界中暴露的状况时，更建议具有胆汁的胚胎评估污染物的生态毒性。微塑料在溶液中的行为对生物利用性也至关重要。应在暴露期间跟踪微塑料的物理化学特性。

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