这项研究得到了 NIH（HL107304 和 HL081753）和梅奥基金会（生物医学发现和心血管研究中心）对 X.X. J.L. 的支持。JL 由中南大学中央大学基本研究基金第 56021702 号资助。特别感谢 Beninio Gore 和 Quentin Stevens 管理斑马鱼设施。

所进行的所有程序均已获得妙佑医疗国际机构动物护理和使用委员会的批准，并遵守"实验动物护理和使用指南"（美国国家科学院出版社，2011 年）中概述的标准。研究中的所有斑马鱼都属于野生印度核型 （WIK） 菌株。用于研究的试剂和设备的详细信息列在 材料表中。
1. 阿霉素原液的制备和储存
<ol><li>从商业来源获得阿霉素原液。 注意:阿霉素对光敏感，因此请以粉末形式获取并储存在不透明的容器中，以保护其免受光照。在化学罩内执行 Dox 粉末制备的所有步骤。</li>
	<li>将 Dox 粉末完全溶解在蒸馏水中，并制备终浓度为 5 mg/mL 的储备液。</li>
	<li>将原液分成 1.5 mL 试管，将其分装。</li>
	<li>用铝箔包裹管子以保护它们免受光线照射。</li>
	<li>将等分试样的 Dox 溶液储存在 4 °C 下进行短期储存（<1 个月），或 -20 °C 进行长期储存10。</li>
</ol>2. 根据鱼的体重对鱼进行分组
<ol><li>将 BW 差异小于 10% 的鱼分组在一起进行后续注射。 注意:为了在这个阶段省力，BW 差异小于 10% 的鱼被归类为相同大小。</li>
	<li>注射前让鱼禁食 24 小时。</li>
	<li>使用含有 0.16 mg/mL 三卡因的胚胎水麻醉鱼 1 分钟。</li>
	<li>用三卡因将鱼从水中取出，然后用干净的滤纸轻拍鱼体的两侧，以去除多余的水。</li>
	<li>测量并记录每条鱼的 BW，然后立即将鱼放回装满新鲜系统水的回收箱中。 注意:对 3 个月大的鱼进行 Dox 注射。在这项研究中，研究人员利用了 3 个月到 10 个月大的鱼。成熟 WIK 品系斑马鱼的 BW 可能在 0.2 g 到 0.5 g 之间变化。持续 5 分钟以上的长时间麻醉，然后进行 Dox 注射，导致鱼类死亡率高。</li>
</ol>3. 准备注射针头和注射站
<ol><li>根据平均体重确定每条鱼所需的 Dox 储备溶液（例如，5 mg/mL）的注射量，以达到 20 μg/g 的目标剂量。</li>
	<li>使用以下公式计算进样体积: <img alt="Equation 1" src="/files/ftp_upload/66500/66500eq01.jpg" style="margin: auto;"></li>
	<li>加入 1x Hank's 平衡盐溶液 （HBSS） 以稀释在步骤 1 中计算的 Dox 溶液以进行注射，达到 5 μL 的总体积。 注意:根据 BW 对每组鱼使用散装溶液，并在每组中额外包括 3 条鱼，以确保在实验过程中不缺乏注射溶液。</li>
	<li>轻轻敲击试管，然后以最高速度短暂微量离心，以在室温下收集溶液 10 秒。</li>
	<li>将准备好的溶液放在冰上，并使其避光。</li>
	<li>将带有海绵的干净 100 毫米培养皿放在解剖显微镜下方，然后调整焦距。 注意:海绵包含一个 4 厘米长的凹槽。海绵的弹性回缩将为鱼提供支撑并保持鱼体就位。海绵可以重复使用。</li>
	<li>为 10 μL 微量注射器配备 34 G 斜面针头。</li>
	<li>用 1x HBSS 缓冲液冲洗针头，以消除任何气泡并清除注射器中的潜在堵塞物。</li>
	<li>测量 5 μL 在步骤 4 中制备的用于注射的溶液。</li>
</ol>4. IP Dox 注射程序
<ol><li>将成鱼放入含 0.16 mg/mL 三卡因的水中 1 分钟，以诱导昏迷状态。</li>
	<li>将鱼放在嵌入海绵的凹槽中，腹部朝上（图 1A）。</li>
	<li>以接近 0° 的角度插入针头，从胸鳍的中点开始向心腔的后侧插入（图 1B）。 注意:手术过程中避免与心脏接触。</li>
	<li>将针头对准尾巴，进入银色皮肤下方。 注意: 将针头放在靠近银色皮肤的位置，注意避免任何刮擦或刺穿。</li>
	<li>在整个手术过程中监测腹腔内的针尖（图 1C）。 注意: 避免损伤肝脏、肠道、鱼鳔和其他器官。确保针头到达肠末端，靠近泄殖腔孔。</li>
	<li>逐渐均匀地分配 5 μL Dox 溶液，然后沿着原始路径缓慢抽出针头以防止任何泄漏（图 1D）。</li>
	<li>通过观察 Dox 溶液的红色来监测腹腔是否存在 Dox（图 1E）。</li>
	<li>迅速将注入的鱼移动到装满淡水的干净过境水箱中，以帮助鱼恢复。 注意:在两次注射之间，用 1x HBSS 缓冲液冲洗针头一次。</li>
</ol>5. 注射后鱼类管理
<ol><li>注射后将鱼放回系统并循环。</li>
	<li>将所有注射的鱼再禁食 24 小时以促进其恢复。</li>
	<li>在第一周，密切关注鱼。尽快将死鱼移走，以免感染其他鱼。 注意:最初 24 小时内的鱼类死亡可能是由于注射或长时间麻醉造成的身体伤害。记录鱼的数量以生成生存曲线。</li>
	<li>在注射后 56 天对注射 Dox 的鱼进行超声心动图表型11。 注:确保注射 HBSS 溶液的相应对照组的条件和程序的一致性。</li>
</ol>

优化腹腔注射用于成年斑马鱼化合物递送

<ol><li>Tavares, B., Lopes, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+importance+of+Zebrafish+in+biomedical+research%5BTitle%5D)+AND+%22Acta+Medica+Portuguesa%22%5BJournal%5D)">The importance of Zebrafish in biomedical research.</a> Acta Medica Portuguesa. 26 (5), 583-592 (2013).</li>
<li>Dang, M., Henderson, R. E., Garraway, L. A., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+drug+administration+in+the+adult+zebrafish+using+oral+gavage+for+cancer+preclinical+studies%5BTitle%5D)+AND+%22Disease+Model+Mech%22%5BJournal%5D)">Long-term drug administration in the adult zebrafish using oral gavage for cancer preclinical studies.</a> Disease Model Mech. 9 (7), 811-820 (2016).</li>
<li>Sciarra, J. B., Tyler, A., Kolb, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+gelatin-based+diet+for+oral+dosing+juvenile+to+adult+zebrafish+%28Danio+rerio%29%5BTitle%5D)+AND+%22Lab+Animal+Sci+Prof%22%5BJournal%5D)">A gelatin-based diet for oral dosing juvenile to adult zebrafish (Danio rerio).</a> Lab Animal Sci Prof. , https://api.semanticscholar.org/CorpusID:85611302 32-35 (2014).</li>
<li>Kinkel, M. D., Eames, S. C., Philipson, L. H., Prince, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intraperitoneal+injection+into+adult+zebrafish%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Intraperitoneal injection into adult zebrafish.</a> J Vis Exp. (42), e2126(2010).</li>
<li>Pugach, E. K., Li, P., White, R., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retro-orbital+injection+in+adult+zebrafish%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Retro-orbital injection in adult zebrafish.</a> J Vis Exp. (34), e1645(2009).</li>
<li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intraperitoneally+delivered+mesenchymal+stem+cells+alleviate+experimental+colitis+through+THBS1-mediated+induction+of+IL-10-competent+regulatory+B+cells%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Intraperitoneally delivered mesenchymal stem cells alleviate experimental colitis through THBS1-mediated induction of IL-10-competent regulatory B cells.</a> Front Immunol. 13, 853894(2022).</li>
<li>De Smet, L., Ceelen, W., Remon, J. P., Vervaet, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+drug+delivery+systems+for+intraperitoneal+therapy+to+extend+the+residence+time+of+the+chemotherapeutic+agent%5BTitle%5D)+AND+%22The+Scientific+World+Journal%22%5BJournal%5D)">Optimization of drug delivery systems for intraperitoneal therapy to extend the residence time of the chemotherapeutic agent.</a> The Scientific World Journal. 2013, 720858(2013).</li>
<li>Dakwar, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanomedicine-based+intraperitoneal+therapy+for+the+treatment+of+peritoneal+carcinomatosis-Mission+possible%5BTitle%5D)+AND+%22Adv+Drug+Deliv+Rev%22%5BJournal%5D)">Nanomedicine-based intraperitoneal therapy for the treatment of peritoneal carcinomatosis-Mission possible.</a> Adv Drug Deliv Rev. 108, 13-24 (2017).</li>
<li>Al Shoyaib, A., Archie, S. R., Karamyan, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intraperitoneal+route+of+drug+administration%3A+should+it+be+used+in+experimental+animal+studies%5BTitle%5D)+AND+%22Pharmaceutical+Res%22%5BJournal%5D)">Intraperitoneal route of drug administration: should it be used in experimental animal studies.</a> Pharmaceutical Res. 37, 1-17 (2020).</li>
<li>Ma, X., Ding, Y., Wang, Y., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+doxorubicin-induced+cardiomyopathy+model+in+adult+zebrafish%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">A doxorubicin-induced cardiomyopathy model in adult zebrafish.</a> J Vis Exp. (136), e57567(2018).</li>
<li>Wang, L. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standardized+echocardiographic+assessment+of+cardiac+function+in+normal+adult+zebrafish+and+heart+disease+models%5BTitle%5D)+AND+%22Disease+Model+Mech%22%5BJournal%5D)">Standardized echocardiographic assessment of cardiac function in normal adult zebrafish and heart disease models.</a> Disease Model Mech. 10 (1), 63-76 (2017).</li>
<li>Christidi, E., Brunham, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulated+cell+death+pathways+in+doxorubicin-induced+cardiotoxicity%5BTitle%5D)+AND+%22Cell+Death+Dis%22%5BJournal%5D)">Regulated cell death pathways in doxorubicin-induced cardiotoxicity.</a> Cell Death Dis. 12 (4), 339(2021).</li>
<li>Zhu, W., Shou, W., Payne, R. M., Caldwell, R., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mouse+model+for+juvenile+doxorubicin-induced+cardiac+dysfunction%5BTitle%5D)+AND+%22Pediatric+Res%22%5BJournal%5D)">A mouse model for juvenile doxorubicin-induced cardiac dysfunction.</a> Pediatric Res. 64 (5), 488-494 (2008).</li>
<li>Podyacheva, E. Y., Kushnareva, E. A., Karpov, A. A., Toropova, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+models+of+doxorubicin-induced+cardiomyopathy+in+rats+and+mice.+A+modern+view+from+the+perspective+of+the+pathophysiologist+and+the+clinician%5BTitle%5D)+AND+%22Frontiers+Pharmacol%22%5BJournal%5D)">Analysis of models of doxorubicin-induced cardiomyopathy in rats and mice. A modern view from the perspective of the pathophysiologist and the clinician.</a> Frontiers Pharmacol. 12, 670479(2021).</li>
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</ol>

与现有的两种 IP 注入方法 4,10 不同，新的 IP 注入方法具有以下鲜明的特点。首先，使用独特的针刺角（接近零）;其次，针头通过一个独特的位置穿透鱼，即鱼腹面上的天然孔，这将有利于注射;最后，针头的运动是从前到后。这些调整有效地减少了器官损伤，这是由于在穿透过程中最大限度地减少了 Dox 的泄漏。针头的路径就在银色皮肤下方，?...

斑马鱼 （Danio rerio） 作为研究人类疾病的实验模型而受到关注，因为这种动物与人类具有高度的基因和器官同源性、外部受精、易于遗传操作以及早熟时的身体透明度，这促进了无数的成像应用1。与将斑马鱼胚胎和幼虫的药物直接输送到水中的简单过程不同，对成年斑马鱼给药是一项更加复杂和具有挑战性的工作2。
在成年鱼中，化合物可以通过被动药物输送技术（例如直接给药到水中）或通过口服药物输送方法（如管饲法）输送 2。其他方法包括用化合物包衣鱼食，然后喂鱼3，以及直接施用预定浓度的水不溶性药物，包括眼眶后或腹膜内注射 4,5。腹膜内给药是疾病模型体内研究的首选，因为它具有独特的药代动力学优势6。该方法提供高药物浓度和延长腹膜腔内的半衰期，为药物输送提供了有效的途径 7,8。该方法通常用于研究环境，以确保最佳的药物吸收和分布 9。虽然基于注射的方法被证明对单次注射有效，但长时间和重复注射通常会导致身体损伤和慢性感染2。
目前，成年 zerbafish 有两种 IP 注射方法 4,10。然而，这两种方法在输送阿霉素等有毒化合物时都有局限性，导致体型严重受损和鱼类死亡。副作用会使数据解释变得非常复杂。尽管这些挑战可以通过广泛的培训来解决10，但显然需要一种新的 IP 注射方法，以最大限度地减少副作用。
在这里，我们的目标是开发一种新的 IP 注射方法，该方法针对将阿霉素有效输送到成年斑马鱼中进行了优化，促进生成可靠的阿霉素诱导的心肌病 （DIC） 模型，将身体损伤和相关死亡率降至最低。

<table><tbody><tr><td>10 &amp;mu;L NanoFil-syringe</td><td>World Precision Instruments, Inc</td><td>NANOFIL</td><td>injection tool</td></tr><tr><td>34 G needle</td><td>World Precision Instruments, Inc</td><td>NI34BV-2</td><td>injcetion tool</td></tr><tr><td>60 mm Petri dish</td><td>fisher scientific/fisherbrand</td><td>FB0875713A</td><td>placing the sponge&amp;nbsp;</td></tr><tr><td>Dissecting microscope</td><td>Nikon</td><td>SMZ800</td><td>Injceting the Dox</td></tr><tr><td>Doxorubicin hydrochloride</td><td>Sigma</td><td>D1515-10MG</td><td>drug for creating DIC model&amp;nbsp;</td></tr><tr><td>Echocardiography</td><td>VISUAL SONICS</td><td>Vevo 3100</td><td>measuring cardiac function</td></tr><tr><td>Foam Sponge</td><td>Jaece Industries</td><td>L800-D</td><td>placing the fish</td></tr><tr><td>Hank&#039;s balanced salt solution (HBBS)</td><td>Thermo Fisher</td><td>14025076</td><td>Vehicle for Dox</td></tr><tr><td>Microcentrifuge&amp;nbsp;</td><td>southernlabware</td><td>MyFuge/C1012</td><td>collect the Dox solution&amp;nbsp;</td></tr><tr><td>Precision Balance Scale</td><td>Torbal</td><td>AD60</td><td>Digital scales</td></tr><tr><td>Tricaine</td><td>Argent</td><td>MS-222</td><td>Anesthetizing fish</td></tr><tr><td>Tube</td><td>Eppendorf</td><td>1.5 mL</td><td>storage&amp;nbsp;</td></tr><tr><td>vevo LAB&amp;nbsp; software</td><td>FUJIFILM VISUAL SONICS&amp;nbsp;</td><td>5.6.0</td><td>quantification of the heart</td></tr></tbody></table>

an intraperitoneal injection technique in adult zebrafish that minimizes body damage and associated mortality

成年斑马鱼 （Danio rerio） 在基因上是可及的，正被用作研究心肌病等人类疾病的有价值的脊椎动物模型。腹膜内 （IP） 注射是一种重要的方法，可将化合物输送到体内，以测试治疗效果或生成疾病模型，例如阿霉素诱导的心肌病 （DIC）。目前，有两种 IP 注入方法。在处理阿霉素等有毒化合物时，这两种方法都有局限性，这会导致副作用表现为严重损害体型和鱼类死亡。虽然这些缺点可以通过广泛的研究者培训来克服，但需要一种副作用最小的新 IP 注射方法。本文报道了一种能够处理有毒化合物的独特 IP 注射方法。心脏功能持续下降可能会导致鱼类严重死亡。对成年斑马鱼经验最少的研究人员可以很容易地掌握这项技术。

以前，已采用两种腹膜内 （IP） 方法对成年斑马鱼施用阿霉素 4,10。在方法 I，也称为 Kinkel 等人4 描述的经典 IP 注射方法中，将针头与腹腔鳍之间的中线成 45° 角插入，腹部朝上。在方法 II 或 马 et al.10 描述的替代 IP 注射方法中，针头通过鱼的背侧插入（图 2A（i，ii））。相比之?...

Read this Scientific Article Protocol about Author Spotlight: Novel Intraperitoneal Injection Method for Zebrafish Cardiotoxicity Models at JoVE.com

作者聚焦: 斑马鱼心脏毒性模型的新型腹腔内注射方法

描述了成年斑马鱼中的一种新的腹膜内 （IP） 注射方法。在处理阿霉素等有毒化合物时，该程序比以前报道的两种 IP 方法更有效。该技术旨在让对斑马鱼模型经验有限的研究人员轻松采用。

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 ...

我们的目标是在成年斑马鱼中生成蒽环类药物诱导的心脏毒性模型，以便我们能够发现新的治疗基因和化合物。可靠的 IP 注入方法对于确保此项目的成功至关重要。因此，以前开发的 IP 注入方法是经典和替代技术。经典方法通过腹鳍之间的中线从腹部穿透鱼，而替代方法从位于骨盆和臀鳍之间的背侧穿透鱼。由于阿霉素的毒性，使用目前的两种 IP 方法已经注意到鱼的严重身体损伤和死亡率。新的研究人员需要接受广泛的培训，然后才能可靠地建立疾病模型。我们通过调整针头穿透位置和针头穿透角度，设计了一种新的 IP 注射方法。我们通过位于胸鳍之间腹侧区域的天然孔注射，并通过独特的前后方向渗透。

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

1.5K 次观看.  Mayo Clinic. 我们的目标是在成年斑马鱼中生成蒽环类药物诱导的心脏毒性模型，以便我们能够发现新的治疗基因和化合物。可靠的 IP 注入方法对于确保此项目的成功至关重要。因此，以前开发的 IP 注入方法是经典和替代技术。经典方法通过腹鳍之间的中线从腹部穿透鱼，而替代方法从位于骨盆和臀鳍之间的背侧穿透鱼。由于阿霉素的毒性，使用目前的两种 IP 方法已经注意到鱼的?...

视频: 作者聚焦: 斑马鱼心脏毒性模型的新型腹腔内注射方法

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

肾再生的成年斑马鱼庆大霉素性损伤

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

科研

发育生物学

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 ...

医学

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

在成年斑马鱼的眼窝射出

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

生物学

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

腹腔注射到成年斑马鱼

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Optimized Intravenous Injection in Adult Zebrafish

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in mammalian research models. However, its application in adult zebrafish for drug delivery, stem cell transplantation, and regenerative and cancer studies has been limited due to the challenges posed by their small body size and intricate blood vessels. To overcome these limitations, alternative injection techniques such as intracardiac and retro-orbital (RO) injection have been explored in the past for stem cell transplantation in adult zebrafish. However, these techniques have their drawbacks, including the need for meticulous injection techniques or increased risk of mortality.
In this study, we have developed a refined and optimized IV injection procedure specifically tailored to adult zebrafish, addressing the challenges associated with their unique anatomy. To demonstrate the effectiveness of this technique, we performed successful IV injections of whole kidney marrow cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish. The subsequent visualization of injected cells and dyes using a fluorescence microscope confirmed their successful delivery and engraftment within the zebrafish. Furthermore, we demonstrated that compared with the intracardiac and RO injections, the IV injection resulted in improved survival rates and engraftment efficiency in treated zebrafish. This approach enables precise delivery and localization of substances and holds great potential for large-scale drug and chemical screening using adult zebrafish. Additionally, the ability to visually track the injected cells and dyes provides invaluable insights into their engraftment, migration, and interactions with host tissues, enabling a more comprehensive evaluation of therapeutic effects and biological processes in zebrafish models.

Here, we present an optimized and effective protocol for the intravenous injection of cells or pharmacological agents into adult zebrafish, resulting in enhanced cell engraftment and increased survival rates of the treated zebrafish.

成年斑马鱼的优化静脉注射

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in ...

癌症研究

Intrathoracic Injection for the Study of Adult Zebrafish Heart

The adult zebrafish heart provides a powerful model in cardiac regeneration research. Although the strength of this system is based on transgenic approaches, a rapid delivery of exogenous factors provides a complementary technique in functional studies. Here, we present a method that relies on administration of a few microliters of solution into the pericardial cavity without causing myocardial damage. Intrathoracic (IT) injections can efficiently deliver proteins and chemical compounds directly onto the heart surface. The injected substances diffuse through the epicardium into the underlying cardiac tissues. Compared to intraperitoneal (IP) injections, the main advantage of intrathoracic injections is the focal administration of the tested factors on the target organ. The delivery of molecules directly into the pericardium is a suitable strategy for studies of cardiac preconditioning and regeneration in adult zebrafish.

This method relies on the injection of 0.5&#8722;3 &#956;L of solution into the thorax of adult zebrafish. The procedure efficiently delivers proteins and chemical compounds into the proximity of the zebrafish heart without damaging the organ. The approach is suitable for testing effects of exogenous factors on various tissues of the heart.

胸腔内注射治疗成年斑马鱼心脏的研究

The adult zebrafish heart provides a powerful model in cardiac regeneration research. Although the strength of this system is based on transgenic ...

Intramuscular (IM) Injection: Compound Delivery into the Dorsal Muscle of an Adult Zebrafish

Source: Myllym&#228;ki, H., et al. <a href="https://www.jove.com/v/58453/" target="_blank">Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines</a>. J. Vis. Exp. (2018).
This video describes the technique of intramuscular injection for delivering compounds into the dorsal muscles of an adult zebrafish.

肌内 （IM） 注射:化合物输送到成年斑马鱼的背肌中

Source: Myllym&#228;ki, H., et al. Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines. J. Vis. Exp. (2018).
This video ...

JoVE Encyclopedia of Experiments

蓝斑马（斑马鱼）

成年

Intraperitoneal Injection: A Method of Solution Delivery into the Abdominal Cavity of an Adult Zebrafish

Source: Luukinen, H., et al. <a href="https://www.jove.com/v/58299/" target="_blank">Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish</a>. J. Vis. Exp. (2018).
This video describes a technique of intraperitoneal injection, a method for delivering solutions into the abdominal cavity of Adult Zebrafish.

腹腔注射:一种将溶液输送到成年斑马鱼腹腔的方法

Source: Luukinen, H., et al. Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish.  J. Vis. Exp. (2018).
This video describes a ...

Intrathoracic Injection: Compound Delivery to the Adult Zebrafish Heart

Source: Bise, T., et al. <a href="https://www.jove.com/v/59724/" target="_blank">Intrathoracic Injection for the Study of Adult Zebrafish Heart</a>. J. Vis. Exp. (2019).
This video describes the technique of delivering compounds into the heart of adult zebrafish by using intrathoracic injection.

胸腔内注射:化合物输送到成年斑马鱼心脏

Source: Bise, T., et al. Intrathoracic Injection for the Study of Adult Zebrafish Heart.  J. Vis. Exp. (2019).
This video describes the technique of ...

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

副本

摘要

探索更多视频

肾再生的成年斑马鱼庆大霉素性损伤

在成年斑马鱼心肌梗死的感应使用Cryoinjury

在成年斑马鱼的眼窝射出

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

腹腔注射到成年斑马鱼

成年斑马鱼的优化静脉注射

胸腔内注射治疗成年斑马鱼心脏的研究

肌内（IM）注射:化合物输送到成年斑马鱼的背肌中

腹腔注射:一种将溶液输送到成年斑马鱼腹腔的方法

胸腔内注射:化合物输送到成年斑马鱼心脏

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

副本

摘要

探索更多视频

肾再生的成年斑马鱼庆大霉素性损伤

在成年斑马鱼心肌梗死的感应使用Cryoinjury

在成年斑马鱼的眼窝射出

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

腹腔注射到成年斑马鱼

成年斑马鱼的优化静脉注射

胸腔内注射治疗成年斑马鱼心脏的研究

肌内 （IM） 注射:化合物输送到成年斑马鱼的背肌中

腹腔注射:一种将溶液输送到成年斑马鱼腹腔的方法

胸腔内注射:化合物输送到成年斑马鱼心脏

肌内（IM）注射:化合物输送到成年斑马鱼的背肌中