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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G., 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=Development+of+doxorubicin-induced+chronic+cardiotoxicity+in+the+B6C3F1+mouse+model.">Development of doxorubicin-induced chronic cardiotoxicity in the B6C3F1 mouse model.</a> Toxicol Appl Pharmacol. 266 (1), 109-121 (2013).</li><li>Zhu, W., Shou, W., Payne, R. M., Caldwell, R., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+juvenile+doxorubicin-induced+cardiac+dysfunction.">A mouse model for juvenile doxorubicin-induced cardiac dysfunction.</a> Pediatr Res. 64 (5), 488-494 (2008).</li><li>Chatterjee, K., Zhang, J., Honbo, N., Karliner, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin+cardiomyopathy.">Doxorubicin cardiomyopathy.</a> Cardiology. 115 (2), 155-162 (2010).</li><li>Bang, C., 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=Cardiac+fibroblast-derived+microRNA+passenger+strand-enriched+exosomes+mediate+cardiomyocyte+hypertrophy.">Cardiac fibroblast-derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy.</a> J Clin Invest. 124 (5), 2136-2146 (2014).</li><li>Rassaf, T., Kelm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+from+diabetic+cardiomyopathy+-+putative+role+of+the+retinoid+receptor-mediated+signaling.">Protection from diabetic cardiomyopathy - putative role of the retinoid receptor-mediated signaling.</a> J Mol Cell Cardiol. 59, 179-180 (2013).</li><li>Wahbi, K., 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=Dilated+cardiomyopathy+in+patients+with+mutations+in+anoctamin+5.">Dilated cardiomyopathy in patients with mutations in anoctamin 5.</a> Int J Cardiol. 168 (1), 76-79 (2013).</li><li>Zhou, M. D., Sucov, H. M., Evans, R. M., Chien, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoid-dependent+pathways+suppress+myocardial+cell+hypertrophy.">Retinoid-dependent pathways suppress myocardial cell hypertrophy.</a> Proc Natl Acad Sci U S A. 92 (16), 7391-7395 (1995).</li><li>Hershman, D. L., 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=Doxorubicin,+cardiac+risk+factors,+and+cardiac+toxicity+in+elderly+patients+with+diffuse+B-cell+non-Hodgkin's+lymphoma.">Doxorubicin, cardiac risk factors, and cardiac toxicity in elderly patients with diffuse B-cell non-Hodgkin's lymphoma.</a> J Clin Oncol. 26 (19), 3159-3165 (2008).</li><li>Silber, J. H., Barber, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin-induced+cardiotoxicity.">Doxorubicin-induced cardiotoxicity.</a> N Engl J Med. 333 (20), 1359-1360 (1995).</li><li>Von Hoff, D. D., 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=Risk+factors+for+doxorubicin-induced+congestive+heart+failure.">Risk factors for doxorubicin-induced congestive heart failure.</a> Ann Intern Med. 91 (5), 710-717 (1979).</li><li>Pugach, E. K., Li, P., White, R., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injection+in+adult+zebrafish.">Retro-orbital injection in adult zebrafish.</a> J Vis Exp. (34), (2009).</li><li>Zang, L., Morikane, D., Shimada, Y., Tanaka, T., Nishimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+protocol+for+the+oral+administration+of+test+chemicals+to+adult+zebrafish.">A novel protocol for the oral administration of test chemicals to adult zebrafish.</a> Zebrafish. 8 (4), 203-210 (2011).</li><li>Collymore, C., Rasmussen, S., Tolwani, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gavaging+adult+zebrafish.">Gavaging adult zebrafish.</a> J Vis Exp. (78), (2013).</li></ol>

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)<sup class="...

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&#39;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 &#956;L NanoFil micro-syringe</td>
			<td>WPI</td>
			<td>NANOFIL</td>
			<td>For injection</td>
		</tr>
		<tr>
			<td>34 gauge needle&#160;</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?...

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

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

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

斑马鱼模型的糖尿病和代谢记忆

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

多囊肾一种可能的斑马鱼型号:击倒<em&gt; Wnt5a的</em&gt;原因在斑马鱼肾囊肿

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

心动过速诱发的心肌病作为猪慢性心力衰竭模型的研究

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

从成年小鼠分离心膜肌细胞

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

使用左前部降冠动脉的永久结扎的月心肌梗死模型

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac performance in the absence of hypertension, valvular, congenital, or ischemic heart diseases, and which may be related to infection, autoimmune or metabolic abnormalities, or family inheritance. It can progress into congestive heart failure with a poor prognosis. Doxorubicin (Dox) is widely employed as a chemotherapeutic drug, but its use is limited because it causes DCM-like changes of the myocardium. Its myocardial toxicity is attributed to oxidative stress, chronic inflammation, and cardiomyocyte apoptosis. A model of DCM exploiting these Dox-induced DCM symptoms has not been established.

Described is a protocol to establish a Doxorubicin-induced dilated cardiomyopathy (DCM) model in mice via long-term intraperitoneal injection of Doxorubicin.

阿霉素诱导的体内扩张型心肌病小鼠模型

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac ...