Name: a doxorubicin-induced cardiomyopathy model in adult zebrafish
Uploaded: 2018-06-07T13:30:00.000+00:00
Duration: 8 min 9 s
Description: Watch this Scientific Journal Video about A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish at JoVE.com

这项工作得到部分支持, 由美国心脏协会 (14SDG18160021) 的科学家发展补助金, 美国 NIH R01 赠款 hl 81753 和 hl 107304 到 xx, 和梅奥基金会 xx。

此处描述的所有程序都是按照《实验室动物护理和使用指南》 (国家科学院出版社 2011) 进行的, 并获得梅奥诊所机构动物护理和使用委员会的批准。
1. 成年斑马鱼的准备
<ol><li>在交叉槽中设置足够的繁殖对, 至少要获得 DOX 注射液所需的总鱼的两倍。如果比较不同遗传背景的鱼类, 在同一周内养殖所有鱼类, 以确保与年龄相符的控制。</li>
	<li>第二天早上收集鱼胚, 将它们转移到100毫米培养皿中, 并将它们保存在28.5 摄氏度的孵化器中。保持低密度的胚胎 (< 100 胚胎/培养皿)。</li>
	<li>每天刷新胚水以避免性别失衡, 并使用转移吸管手动清除死卵。</li>
	<li>把相同数量的胚胎在每个坦克 (例如, 60 embryos/3 L 中型坦克最初), 以确保密度匹配的控制。</li>
	<li>开始草履虫喂养4天后受精 (dpf)。</li>
	<li>在青少年阶段每天检查鱼。根据需要调整鱼号以确保鱼的密度相似。</li>
	<li>当鱼达到4周的年龄, 转移多达20条鱼到每一个新的3升中型坦克进一步增长。开始用活孵化的盐水虾喂鱼。</li>
</ol>2. DOX 库存解决方案的制备和贮存
注: DOX 可以从各种生物公司购买。这种化合物通常是在深褐色的容器中获得的粉末。
<ol><li>彻底溶解 DOX 粉在脱电离水, 以确保没有团块是可见的, 最终浓度为5毫克/毫升作为库存解决方案。整除1毫升的 DOX 储存到每1.5 毫升安全锁管。用铝箔纸包裹1.5 毫升的管子, 以保护 DOX 不受光照射。 注: 在化学罩中执行此步骤。</li>
	<li>保持 DOX 库存解决方案在4摄氏度的存储。对于 DOX 库存解决方案的长期存储 (> 4 周), 请执行下面描述的可选节3。</li>
</ol>3. 使用斑马鱼胚胎 DOX 的质量控制 (可选)
注: DOX 是水分和光敏感, 因此它可以失去它的药物功效的建模后, 扩大存储。对于从不同公司购买的 DOX, 甚至来自同一公司的不同批次, 在对成年鱼进行实验之前, 使用野生型斑马鱼胚胎对其药效进行校准是有用的。这种方法是从报告的斑马鱼胚胎 DIC 模型21。
<ol><li>从至少2对鱼中收集斑马鱼的胚胎。Dechorionate 胚胎在24小时后受精 (hpf) 手动使用注射器与微针。或者, 在30摄氏度孵化器中, 用蛋白酶 K 在最终浓度为10µg/毫升的10-15 分钟治疗胚胎。在 dechorionation 后刷新胚水。去除死胚, 并保持每批的至少36个胚胎。</li>
	<li>将新鲜胚水中的 DOX 溶液稀释至最终浓度为100µM。每3个胚胎的溶液体积为100µL。用涡流混合稀释的 DOX 溶液。最后稀释的溶液应该是浅色的, 红色的。</li>
	<li>添加100µL/井稀释 DOX 溶液的清洁96井透明板。</li>
	<li>用一个塑料转移吸管带 3 dechorionated 胚胎, 并保持胚胎靠近吸管尖端的末端。用 DOX 溶液将吸管尖端放入每个井中。让尖端的末端接触到溶液, 让胚胎游入井中。 注意: 避免人工推胚胎, 这将增加更多的水入井和稀释 DOX 溶液。</li>
	<li>在 48 hpf 刷新 DOX 解决方案。到这个时候, 观察井下显微镜下的10X 放大, 以确定死亡胚胎 (心跳停止) 或胚胎水肿。及时计数和清除任何死胚, 否则, 与死胚接触的剩余胚胎也会很快死亡。</li>
	<li>检查 72 hpf 的胚胎并计算它们。DOX 治疗被认为是 "良好的药物功效", 如果 > 25% 死亡 (心跳停止) 可以观察到两批胚胎。</li>
</ol>4. 预注射制剂
注: 8 周至6月的鱼用于 DOX 注射液。被注射的成熟的野生印度核型 (WIK) 鱼的体重 (BWs) 可以从 0.2-0.5 克不等。
<ol><li>在注射前把鱼快24小时。</li>
	<li>麻醉含有0.16 毫克/毫升 tricaine 的胚水。用干净的滤纸把身体两侧的水晾干。测量每条鱼在秤上的体重。组鱼在10% 的差异, 在生物武器一起为以后注射。 注: 为了尽量减少这一步骤的工作量, 在10% 的体重差异的鱼被认为是相同的大小;因此, 根据他们的平均体重, 准备一个 DOX 的工作解决方案。</li>
	<li>计划为每条成年鱼注射5µL 的溶液。根据鱼数和 BWs 计算 DOX 的工作浓度。 注: 研究慢性心脏长达6月, 使用 DOX 剂量为20毫克/千克。为了研究 DOX 的急性心脏, DOX 剂量可以增加到50毫克/千克的体重。</li>
	<li>稀释1x 汉克平衡盐溶液 (HBSS) 中的 DOX 库存, 以达到相应的工作浓度。涡流混合溶液。简要地向下旋转以收集解决方案。</li>
</ol>5. DOX 注射成人鱼
<ol><li>放置一个干净的100毫米培养皿与海绵内, 解剖显微镜下, 然后调整焦点。切海绵, 使一个大约4厘米长的洞, 以保持鱼。为更大的鱼制造一个更长的洞。</li>
	<li>用10µL 微型注射器准备一根34克的针。用 1x HBSS 缓冲器冲洗针头, 从注射器和油管中取出任何气泡和块。</li>
	<li>麻醉的胚水中含有0.16 毫克/毫升 tricaine 2 分钟的成年鱼。 注: 长麻醉超过5分钟后 DOX 注射液可轻易导致鱼死亡。</li>
	<li>用 tricaine 将海绵浸泡在胚水中, 将鱼转移到海绵中注射。</li>
	<li>
通过下面描述的两种方法之一执行 IP DOX 注入。
	<ol>
<li>经典 IP 注入29<ol>
<li>把鱼和腹部放在海绵的洞里。快速插入针, 与45°角度的鱼体进入中线之间的骨盆鳍, 并穿透大约1-2 毫米. 慢慢释放所有 DOX 溶液。在拔针前等5秒。在鱼腹中用可见的红色来检查 DOX 的递送。</li>
		</ol>
</li>
		<li>替代 IP 注入<ol>
<li>将鱼横向放在海绵上, 右前方。用左手用钝端钳轻轻地稳定鱼, 用右手握住微注射器。</li>
			<li>将针放在骨盆鳍上方的侧线下方, 倾斜朝上。指向7点在45°角度的位置, 插入针3-4 毫米到位于骨盆和肛门鳍之间的鱼腔, 然后慢慢地压低柱塞。在鱼腹中用可见的红色来检查 DOX 的递送。</li>
		</ol>
</li>
	</ol>
</li>
	<li>快速将注入的鱼转移到充满新鲜系统水的干净的交叉水箱中, 让鱼恢复。一次性用 1x HBSS 缓冲针冲洗针头。</li>
</ol>6. 注射后鱼类管理
<ol><li>注射后, 将鱼返回系统, 循环运行。如有可能, 应将 DOX 处理的鱼与主系统分开维护, 以避免在共享循环的不同水箱间产生交叉污染。</li>
	<li>快速注入的鱼为另一个24小时的恢复。每天在第一周观察鱼。及时清除死鱼, 避免感染其他鱼类。 注: 头24小时内的鱼死亡很可能是由于注射引起的物理损伤。</li>
	<li>进一步保持 DOX 压力鱼的纵向观察。及时清除死鱼, 以避免在水箱中感染其他鱼类。 注意: 记录鱼数以生成生存曲线。</li>
	<li>用不同的实验测定表型 DOX 压力鱼, 如超声心动图30, 心脏功能报告转基因线23, 游泳挑战26, 并量化其他病理重塑标记23。</li>
</ol>

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作者声明没有利益冲突。

为了模拟渐进性 DIC, 20 毫克/千克 DOX 的剂量以实验的方式确定为最高剂量, 在1的过程中不会导致大量的鱼死亡, 但仍然导致鱼死亡和4度后心脏功能的减少 (图 3和图 4C)。这种剂量可与啮齿动物 DIC 模型中常用的 (15-25 毫克/千克) 和极限累计剂量 (550 毫克/米2, 相当于15毫克/千克)4, 7, 31,32,33. 更高剂量的 DOX, 如50毫克/千克, 在1的化学治疗中显示出明显的鱼类死亡, 因此它们可用于研究对 DOX23、28的急性心脏毒性反应。
在这个 DIC 模型的实施过程中, 我们注意到新的调查人员最初很难重现同样的结果。即使有经验的调查人员, 在不同的注射 (图 3) 中, 慢性期死亡率的大约20% 的变化仍然可以发现, 这表明 uncharacterized 生物混杂因素在当前模型。尽管一个不太完美的模型, 我们仍然认为, 目前的 DIC 模型足以使声音发现, 因为以下证据。首先, 经过几次实践, DIC 模型的结果可以在大多数 (即使不是全部) 调查人员中注册。其次, 基于这种 DIC 模型, 我们确定了4种有意义的基因修饰符。现有证据从文献报告支持3他们作为心肌病基因34,35,36,37。4是 DnaJ 同源亚科 B 成员6 (DNAJB6), 这已被证明是一个新的心肌病基因支持的人类遗传学研究28。因此, 我们得出结论, 基于实验组的结果, 使用严格的年龄匹配的控制仍然是高度可重复性的, 尽管 DIC 模型目前的形式是不可靠的相互实验比较。
造成我们目前 dic 模型不一致的混淆因素可能包括: (1) 衰老和性别差异被认为是心血管疾病的关键危险因素, 包括 dic38, 39,40。虽然这两个假设创始人仍然是在我们的模型测试, 特别是, 我们确实注意到, 老鱼往往更敏感的 DOX 毒性 (数据没有显示)。使用两种 IP 方法观察到的批次间变化 (图 3) 也可能有助于这两个创始人。(2) 与大动物模型不同的是, 成年斑马鱼的体型较小。因此, DOX 注射液引起的局部损伤会更严重, 而且变化更大。(3) 注射鱼的死亡可能是其他器官 (如肾脏毒性) 的 DOX 毒性所致, 除了心脏。虽然需要更仔细设计的实验来解决上述每一个混淆因素, 以前的研究表明, 以下注意事项将有助于减少表型变异: 首先, 保证 DOX 的疗效至关重要。DOX 粉应始终保持在干燥和黑暗的区域, 解决方案应小心处理, 以减少暴露于光。建议 DOX 的工作解决方案, 每次在鱼注射前都要新鲜地准备。我们通常不使用库存 DOX 解决方案后4周的存储。当有疑问时, 请使用快速胚胎 DIC 模型在本协议中执行可选的3节, 以校准每个 DOX 批次的药物功效。第二, 成年鱼的同步是关键。同样的鱼株需要在相同的密度下提高, 以确保类似的体型。然后, 我们预先选择鱼与类似的 BWs 剂量计算。建议在这一预选过程中至少维持两次鱼的数量。所有鱼类在预选前的过程中禁食24小时。第三, 建议始终使用类似年龄的鱼类, 并在 DOX 注射液前对鱼进行性反应, 因为它们的生长速率不同, 对 DOX 有潜在的不同的心脏应答。
我们注意到, 大多数不一致的结论可以归咎于一个可疑的对照组。因此, 我们建议研究者新的 DIC 模型进行 DOX 注射之前进行真正的实验。当20毫克/千克 DOX 注射使用替代的 IP 注射方法, 一个良好的注射技术可以表明, 近零鱼死亡期间, 1 的 DOX 和一个相对一致的死亡率在2-3 月后 DOX 注射。成功建模的最终证据是心脏功能指数的降低, 这可以通过超声心动图25,30, 使用一个精灵来量化;Tg (cmlc2: nusDsRed)转基因线, 或使用新开发的以体内为基础的心脏功能检测 (未显示数据)。
除了 IP 注射, 其他药物传递路线, 如复古轨道注射41, 口服喂养42, 和水孵化也经常使用的成年斑马鱼。我们没有采取复古轨道注射方法, 尽管它的直接药物释放到循环系统, 由于缺乏一种方法来验证成功的药物传递, 因为 DOX 的红肿可以很容易掩盖的潜在出血。我们尝试通过嵌入 DOX 与可吸收面筋42, 这是混合与膳食喂养成年鱼的口头传递协议。不幸的是, 随着150毫克/千克的累计剂量在4周的喂养期内使用, 我们没有观察到任何严重的心脏, 表明口服 DOX 分娩无效。另外, 还可以进一步探讨口服饲技术43。此外, 在含有 DOX 的溶液中浸泡鱼的孵化协议也是一个潜在的交付路线, 可以在未来进行测试。
我们承认, 目前 DIC 模型的主要局限性之一是单丸注射法, 这可能会对内脏产生高毒性和局部损伤。这一方法的目的是减少工作量和增加吞吐量, 以便基因筛选可以在高吞吐量的成年鱼28进行。今后, 应进行多项注射 DOX 的模型, 以更好地复述在癌症患者化疗治疗中所观察到的 DIC。

阿霉素 (DOX), 也命名为阿凡霉, 已发展为抗肿瘤药物自二十世纪六十年代1,2。它现在仍然积极地作为一种重要的化疗药物的广泛的肿瘤。然而, DOX 的临床应用因其剂量依赖性毒性而受到阻碍, 特别是心脏症状, 从无症状心电图改变到心包炎和失代偿型心肌病等。1,2. 迄今为止, 至少有三个主要假说被提出来解释 DIC, 包括活性活性氧 (ROS)1、3、4、5、抑制拓扑异构酶 II β (TOP2β)6,7, 和调制细胞内钙释放1,8,9。积累的证据还表明, 遗传易感性是 DIC10,11,12,13的关键危险因素。然而, 与这些 DIC 倾向相关的基因标识仍然很不清楚。右雷佐生是美国食品药品监督管理局 (FDA) 批准的唯一的佐剂, 用于治疗 DIC, 但实施14、15、16的限制, 强调需要确定其他治疗策略。因此, 为这些目的探索了 DIC 的动物模型。由于它们的易接近性和简单, 对 DIC 模型的机械研究可能会对其他类型的心肌产生更广泛的影响: 常见的发病机制可能在不同病因的心肌中共享, 特别是在以后病理阶段17,18,19,20。
除了 DIC 的啮齿动物模型外, 还开发了具有较高吞吐量的斑马鱼 DIC 模型, 以促进发现新的遗传因素和治疗方法。在透明斑马鱼胚胎中建立了一个胚胎 DIC 模型, 用于筛选治疗化合物21。鉴于心肌是成人发病疾病的渐进性发病机制, 成年斑马鱼心肌病模型已发展22,23,24,25,26。我们产生了第一个获得的模型, 由慢性贫血引起的心肌病24, 其次是 DIC 作为第二获得性心肌病模型在成年斑马鱼23。我们发现, 注射一丸的 DOX 到成年斑马鱼诱导心脏, 包括一个急性期大致在1周后注射 (), 其次是慢性心肌病期长达6月后注射。haploinsufficiency 雷帕霉素(mtor) 变好心肌病在慢性期的机械靶向性, 在急性期夸大鱼类死亡率, 强调成人 DIC 模型对分期依赖性的鉴别价值机制23。我们进一步证明, 成人 DIC 模型可以用来强调收集的斑马鱼插入心脏 (ZIC) 突变体正在通过座子的插入突变方法产生27。一个先导屏幕确定了3已知的心肌病基因以及 DnaJ (Hsp40) 同源、亚乙型和成员 6b (dnajb6b) 作为新的 DIC 易感性基因28。因此, 在斑马鱼的成人 DIC 模型的产生导致了一个新的方法, 系统地使鉴定的 DIC 基因修饰语, 这补充了现有的全基因组的关联研究 (GWAS) 和数量性状的轨迹 (QTL分析.
在成年斑马鱼 DIC 模型的生成和实施过程中, 我们注意到不同研究者和/或甚至在同一调查者的不同注射中有显著的差异。模型的纵向性质对注册不同调查人员的结果和顺序的故障排除过程提出了挑战。为了便于研究界使用这种简单的心肌病诱发应力方法, 我们详细描述了我们的协议, 提出了两种类型的 IP 注入, 并讨论了减少不同研究者之间差异的考虑因素。

<table><tbody><tr><td>Crossing tank</td><td>Aquaneering</td><td>ZHCT100</td><td>Fish breeding</td></tr><tr><td>Incubator</td><td>ThermoFisher</td><td></td><td>Maintaining embryo</td></tr><tr><td>3 L medium tank</td><td>Aquaneering</td><td>ZT280</td><td>Maintaining fish</td></tr><tr><td>Paramecia</td><td>Carolina</td><td>131560</td><td>Food for juvenile fish</td></tr><tr><td>Live hatched brine shrimp</td><td>in house</td><td></td><td>Food for adult fish</td></tr><tr><td>Doxorubicin hydrochloride</td><td>Sigma</td><td>D1515-10MG</td><td></td></tr><tr><td>1.5 ml safe-lock tube</td><td>Eppendorf</td><td>No. 022363204</td><td>For drug storage</td></tr><tr><td>Aluminum foil paper</td><td>Fisher</td><td>1213104</td><td>For preventing light exposure</td></tr><tr><td>Proteinase K</td><td>Roche</td><td>No. 03115887001</td><td>For dechorionating embryo</td></tr><tr><td>Hank&#039;s balanced salt solution (HBBS)</td><td>ThermoFisher</td><td>14025076</td><td>Vehicle for DOX</td></tr><tr><td>100 mm petri dish</td><td>Falcon</td><td>431741</td><td></td></tr><tr><td>10 &amp;mu;L NanoFil micro-syringe</td><td>WPI</td><td>NANOFIL</td><td>For injection</td></tr><tr><td>34 gauge needle</td><td>WPI</td><td>NF34BV-2</td><td>For injection</td></tr><tr><td>Tricaine</td><td>Argent</td><td>MS-222</td><td>Anesthetizing fish</td></tr><tr><td>96 well plate</td><td>Costar</td><td>3539</td><td>For embryo drug treatment</td></tr><tr><td>Transfer pipette</td><td>Bel-art product</td><td>F37898</td><td>For transfering embryo</td></tr></tbody></table>

a doxorubicin-induced cardiomyopathy model in adult zebrafish

基因可接触的成年斑马鱼 (斑马斑马) 已越来越多地被用作一种脊椎动物模型, 用于了解诸如心肌病等人类疾病。由于它的方便和顺从的高通量遗传操作, 生成的获得性心肌病模型, 如阿霉素诱发心肌病 (DIC) 模型的成年斑马鱼, 正在打开新的研究途径的大门,包括通过前基因筛选发现心肌病修饰语。不同于早期的斑马鱼 dic 模型, 早期急性和晚期心肌病的慢性阶段可以确定在成年斑马鱼 dic 模型, 使研究阶段依赖的信号机制和治疗策略。但是, 即使在经验丰富的调查人员手中, 也可以通过当前模型获得可变结果。为了促进 dic 模型的未来实施, 我们提出了如何在成年斑马鱼中生成这种 dic 模型的详细协议, 并描述了两种不同的腹腔 (IP) 注射方法。我们进一步讨论如何减少变化, 以获得可靠的结果, 并提出如何正确解释结果的建议。

本文给出了在成年斑马鱼中对 DIC 模型进行 IP 注射的两种方法。在使用经典的, 建立的 IP 注入方法29, 它指出, 注入 DOX 溶液 (红色) 有时可能会渗出的地方, 针穿透。替代的 IP 注入使用不同的位置, 针穿透, 是3-4 毫米远离腹膜的 DOX 被释放 (图 1A), 这有效地防止泄漏 (图 1B, 1C)。成功地交付 DOX 入腹膜为两种方法是由红色颜色的快速的分布证明在整个鱼腹部, 在注射的轨迹的相反边是可看见的。
注射 DOX 剂量为50毫克/千克使用替代 IP 方法导致严重的毒性, 其中大多数鱼死在一个星期内 (图 2)。相比之下, 使用替代 IP 方法注射 DOX 剂量为20毫克/千克, 导致在头2周内几乎没有鱼死亡, 10% 条鱼死亡时间为 4 (图 3A)。使用经典的 IP 方法注射 DOX 剂量为20毫克/千克, 30% 条鱼死于 4 (图 3C)。用这两种方法注射的鱼都有类似的20% 条鱼死亡, 从4到 10 (图 3B, D)。
我们已经利用了精灵;Tg (cmlc2: nusDsRed)鱼来评估 DIC 模型中心脏功能障碍的进展 (图 4A)23。透明的身体可以在荧光灯显微镜下, 在收缩 (图 4B) 和舒张 (图 4C) 阶段的红色心脏的文献。采用替代 IP 方法注射20毫克/千克 DOX 后, 可从 4 (图 4D) 开始检测心室功能下降。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57567/57567fig1.jpg"> 图 1: 注塑路线.(1A) 经典 ip 注入 (b) 和替代 ip 注入 (a) 方法的示意图。红色圆圈表示两种注入方法的常用 DOX 释放点。绿色圆圈表示针穿透点。距离估计的基础上的成年 WIK 鱼体重约0.3 克. (1B, 1C) 代表结果表明, 成功的 DOX 交付使用两种腹腔注射方法。在成年鱼腹内发红可以在注射后立即注意到。注入的鱼在5分钟恢复以后再被检查在一个淡水系统水。HBSS: 1x. 汉克的平衡盐溶液。WIK 斑马鱼被雇用。刻度条: 5 毫米.<a href="/files/ftp_upload/57567/57567fig1large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57567/57567fig2.jpg"> 图 2: 以50毫克/千克 DOX 注射液为 DOX 应力的成年斑马鱼的典型生存曲线.显示3套 DOX 注射在不同批次的野生成年鱼在3-6 月的年龄。在 1x HBSS 控制组中, 共使用 n = 24 条鱼, 在每批 DOX 中使用 n = 8 条鱼。两种注射方法之间没有发现鱼类存活的差异。WIK 斑马鱼被雇用。<a href="/files/ftp_upload/57567/57567fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57567/57567fig3.jpg"> 图 3: 以20毫克/千克 DOX 注射液为 DOX 的成年斑马鱼的代表性生存曲线和两种腹腔注射方法的比较.(3A) 经典型腹腔注射 DOX 分娩后0-4 周内鱼类存活。0周的鱼数百分比被认为是100%。(3B) 4 周后通过经典腹腔注射 DOX 分娩后的鱼存活。4周的鱼数百分比被认为是100%。(3C) 0-4 周内通过选择性腹腔注射 DOX 分娩后的鱼类存活。0周的鱼数百分比被认为是100%。(3D) 4 周后通过选择性腹腔注射 DOX 分娩后的鱼存活。4周的鱼数百分比被认为是100%。在 (3A) 和 (3B) 中显示的数据是9种不同批次的 DOX 注射液, 从223条注射鱼到3-6 月的年龄。在 (3C) 和 (3D) 中显示的数据是14种不同批次的 DOX 注射液, 从335条注射鱼到2-6 月的年龄。活鱼的数量是每周记录的。误差线表示每周不同批次中的生存百分比的标准偏差。WIK 斑马鱼被雇用。<a href="/files/ftp_upload/57567/57567fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57567/57567fig4.jpg"> 图 4: 有代表性的心功能评估后 DOX 应激使用马来;Tg (cmlc2: nusDsRed)鱼。 (4A) 一张成人精灵的照片;Tg (cmlc2: nusDsRed)鱼。刻度条 = 1 厘米 (4B) 在成熟的小精灵末端收缩期的红色心室的代表形象;Tg (cmlc2: nusDsRed)鱼。破折号线代表末端收缩直径 (ESD)。(4C) 在成熟的精灵的舒张末期, 一个红色心室的代表形象;Tg (cmlc2: nusDsRed)鱼。在 (4B) 和 (4C) 中的图像从使用6.3x 放大器的荧光解剖显微镜拍摄的跳动心脏的电影中提取。破折号线代表末端舒张直径 (EDD)。刻度条 = 1 毫米 (4D) 有代表性的心功能, 使用马来测量;Tg (cmlc2: nuDsRed)成年斑马鱼经过20毫克/千克 DOX 注射液。EDD 和 ESD 的测量显示在 (4B) 和 (4C) 的个别鱼类, 和心室分数缩短是计算公式 (EDD − ESD)/EDD。4周后发现心室分数缩短明显减少。采用选择性腹腔内法。值显示为 "平均" 标准错误。每组中的 n ≥3。学生t检验用于两组的比较。*p < 0.05。这些数字已经从丁等地改了。23<a href="/files/ftp_upload/57567/57567fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish at JoVE.com

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

阿霉素诱发成年斑马鱼的心肌病模型

本文介绍了一种在成年斑马鱼 (斑马斑马) 中产生阿霉素诱发的心肌病模型的方法。本文介绍了两种不同的腹腔注射方法, 并讨论了减少各实验组差异的条件。

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as ...

a-doxorubicin-induced-cardiomyopathy-model-in-adult-zebrafish

Nanyang Technological University

Research

JoVE Journal

Medicine

9.7K 次观看.  Mayo Clinic Graduate School of Biomedical Sciences. 本文介绍了一种在成年斑马鱼 (斑马斑马) 中产生阿霉素诱发的心肌病模型的方法。本文介绍了两种不同的腹腔注射方法, 并讨论了减少各实验组差异的条件。

视频: A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

在成年斑马鱼心肌梗死的感应使用Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

科研

医学

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.

心肌机械变形（DIAMOND）的位移分析揭示了胚胎斑马鱼心脏功能的分段异质性

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

发育生物学

High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its embryonic transparency, genetic tractability, and amenity to rapid, high-throughput studies. However, the loss of transparency limits heart function analysis at the adult stage, which complicates modeling of age-related heart conditions. To overcome such limitations, high-frequency ultrasound echocardiography in zebrafish is emerging as a viable option. Here, we present a detailed protocol to assess cardiac function in adult zebrafish by non-invasive echocardiography using high-frequency ultrasound. The method allows visualization and analysis of zebrafish heart dimension and quantification of important functional parameters, including heart rate, stroke volume, cardiac output, and ejection fraction. In this method, the fish are anesthetized and kept underwater and can be recovered after the procedure. Although high-frequency ultrasound is an expensive technology, the same imaging platform can be used for different species (e.g., murine and zebrafish) by adapting different transducers. Zebrafish echocardiography is a robust method for cardiac phenotyping, useful in the validation and characterization of disease models, particularly late-onset diseases; drug screens; and studies of heart injury, recovery, and regenerative capacity.

We describe a protocol to assess heart morphology and function in adult zebrafish using high-frequency echocardiography. The method allows visualization of the heart and subsequent quantification of functional parameters, such as heart rate (HR), cardiac output (CO), fractional area change (FAC), ejection fraction (EF), and blood inflow and outflow velocities.

高频超声超声心动图评估斑马鱼心脏功能

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

生物学

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

免疫染色的解剖斑马鱼胚胎心脏

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

在 Vivo 表面心电图中成人斑马鱼

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...

优化腹腔注射用于成年斑马鱼化合物递送

An Intraperitoneal Injection Technique in Adult Zebrafish that Minimizes Body Damage and Associated Mortality

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as cardiomyopathy. Intraperitoneal (IP) injection is an important method that delivers compounds to the body for either testing therapeutic effects or generating disease models such as doxorubicin-induced cardiomyopathy (DIC). Currently, there are two methods of IP injection. Both&#160;methods have limitations when handling toxic compounds such as doxorubicin, which result in side effects manifesting as severe damage to the body shape and fish death. While these shortcomings could be overcome by extensive investigator training, a new IP injection method that has minimal side effects is desirable. Here, a unique IP injection method that is able to handle toxic compounds is reported. Consistently reduced cardiac function can result without incurring significant fish death. The technique can be easily mastered by researchers who have minimal experience with adult zebrafish.

作者聚焦: 斑马鱼心脏毒性模型的新型腹腔内注射方法

A new intraperitoneal (IP) injection method in adult zebrafish is described. When handling toxic compounds such as doxorubicin, this procedure is more effective than the two previously reported IP methods. The technique is designed to be easily adopted by researchers with limited experience in the zebrafish model.

一种成年斑马鱼腹腔注射技术，可最大限度地减少身体损伤和相关死亡率

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as ...

阿霉素诱发成年斑马鱼的心肌病模型

摘要

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此视频中的章节

在成年斑马鱼心肌梗死的感应使用Cryoinjury

心肌机械变形（DIAMOND）的位移分析揭示了胚胎斑马鱼心脏功能的分段异质性

高频超声超声心动图评估斑马鱼心脏功能

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼

免疫染色的解剖斑马鱼胚胎心脏

恢复成年斑马鱼的心脏的高通量应用

在 Vivo 表面心电图中成人斑马鱼

一种成年斑马鱼腹腔注射技术，可最大限度地减少身体损伤和相关死亡率

一种成年斑马鱼腹腔注射技术，可最大限度地减少身体损伤和相关死亡率

一种成年斑马鱼腹腔注射技术，可最大限度地减少身体损伤和相关死亡率