This study was supported by the NIH (HL107304 and HL081753) and the Mayo Foundation&#160;(Center for Biomedical Discovery and Cardiovascular Research Center) to X.X. J.L. is funded by the Fundamental Research Funds for the Central Universities of Central South University, No. 56021702. Special thanks to Beninio Gore and Quentin Stevens for managing the zebrafish facility.

All procedures conducted were approved by the Mayo Clinic Institutional Animal Care and Use Committee, adhering to the standards outlined in the &#39;Guide for the Care and Use of Laboratory Animals&#39; (National Academies Press, 2011). All zebrafish in the study belong to the Wild Indian Karyotype (WIK) strain. The details of the reagents and the equipment used for the study are listed in the Table of Materials.
1. Preparation and storage of doxorubicin stock solution<...

Optimized Intraperitoneal Injection for Compound Delivery in Adult Zebrafish

<ol>
	<li>Tavares, B., Lopes, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+Zebrafish+in+biomedical+research.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+drug+administration+in+the+adult+zebrafish+using+oral+gavage+for+cancer+preclinical+studies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gelatin-based+diet+for+oral+dosing+juvenile+to+adult+zebrafish+(Danio+rerio).">A gelatin-based diet for oral dosing juvenile to adult zebrafish (Danio rerio).</a> Lab Animal Sci Prof. , 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraperitoneal+injection+into+adult+zebrafish.">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/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), e1645 (2009).</li><li>Liu, J., 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=Intraperitoneally+delivered+mesenchymal+stem+cells+alleviate+experimental+colitis+through+THBS1-mediated+induction+of+IL-10-competent+regulatory+B+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+drug+delivery+systems+for+intraperitoneal+therapy+to+extend+the+residence+time+of+the+chemotherapeutic+agent.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomedicine-based+intraperitoneal+therapy+for+the+treatment+of+peritoneal+carcinomatosis-Mission+possible.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraperitoneal+route+of+drug+administration:+should+it+be+used+in+experimental+animal+studies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+doxorubicin-induced+cardiomyopathy+model+in+adult+zebrafish.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+echocardiographic+assessment+of+cardiac+function+in+normal+adult+zebrafish+and+heart+disease+models.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+cell+death+pathways+in+doxorubicin-induced+cardiotoxicity.">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/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> 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+models+of+doxorubicin-induced+cardiomyopathy+in+rats+and+mice.+A+modern+view+from+the+perspective+of+the+pathophysiologist+and+the+clinician.">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><li>Chaoul, V., 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=Assessing+drug+administration+techniques+in+zebrafish+models+of+neurological+disease.">Assessing drug administration techniques in zebrafish models of neurological disease.</a> Int J Mol Sci. 24 (19), 14898 (2023).</li><li>Lu, X., Lu, L., Gao, L., Wang, Y., Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calycosin+attenuates+doxorubicin-induced+cardiotoxicity+via+autophagy+regulation+in+zebrafish+models.">Calycosin attenuates doxorubicin-induced cardiotoxicity via autophagy regulation in zebrafish models.</a> Biomed Pharmacother. 137, 111375 (2021).</li></ol>

Different from the two existing IP injection methods4,10, the new IP injection method is characterized by the following distinct features.Firstly, a unique needle penetration angle is used (close to zero); secondly, the needle penetrates the fish via a unique location, i.e., a natural hole on the ventral surface of a fish, which would facilitate the injection; and finally, the movement of the needle is from anterior to posterior. These adjustments effect...

Zebrafish (Danio rerio) has gained attention as an experimental model for studying human diseases because this animal encompasses high gene and organ homology to humans, external&#160;fertilization, ease of genetic manipulation, and body transparency into early maturity, which facilitates a myriad of imaging applications1. Unlike the straightforward process of delivering drugs directly to the water for zebrafish embryos and larvae, administering drugs to adult zebrafish presents a more intricate and challenging endeavor2.
In adult fish, compounds can be delivered through passive drug delivery techniques, such as direct administration into the water, or through oral drug delivery methods like gavaging2. Other approaches include coating fish food with the compounds and subsequently feeding the fish3, and direct administration of water-insoluble medications at a predetermined concentration, including retro-orbital or intraperitoneal injections4,5. Intraperitoneal administration is preferred for in vivo studies of disease models due to its distinct pharmacokinetic advantages6. This method provides a high drug concentration and an extended half-life within the peritoneal cavity, offering an effective route for drug delivery7,8. The approach is commonly utilized in research settings to ensure optimal drug absorption and distribution 9. While injection-based methods prove efficient for single delivery, prolonged and repeated injections often lead to body damage and chronic infection2.
Currently, there are two methods of IP injection in adult zerbafish4,10. However, both methods have limitations when delivering toxic compounds like doxorubicin, leading to severe damage to the body shape and fish mortality. The side effects can significantly complicate data interpretation. Although these challenges may be addressed with extensive training10, there is a clear need for a new IP injection method that minimizes side effects.
Here, our goal is to develop a new method of IP injection optimized for the effective delivery of doxorubicin into adult zebrafish, facilitating the generation of reliable doxorubicin-induced cardiomyopathy (DIC) models with minimized body damage and associated mortality.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 &#956;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&#160;</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&#160;</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&#39;s balanced salt solution (HBBS)</td>
			<td>Thermo Fisher</td>
			<td>14025076</td>
			<td>Vehicle for Dox</td>
		</tr>
		<tr>
			<td>Microcentrifuge&#160;</td>
			<td>southernlabware</td>
			<td>MyFuge/C1012</td>
			<td>collect the Dox solution&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>vevo LAB&#160; software</td>
			<td>FUJIFILM VISUAL SONICS&#160;</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

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.

Author Spotlight: Novel Intraperitoneal Injection Method for Zebrafish Cardiotoxicity Models

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

We aim to generate anthracycline induced cardiotoxicity models in adult zebrafish so that we can discover new therapeutic genes and compounds. A reliable IP injection method is essential to ensure the success of this project. So previously developed IP injection methods are the classic and alternative techniques.While the classic method penetrates the fish from the abdominal site through midline between the pelvic fins, the alternative method penetrates the fish from the dorsal site located between the pelvic and the anal fins. Owing to the toxicity of doxorubicin, significant body damage and mortality of the fish have been noted using the current two IP methods. New investigators need to be extensively trained before disease models can be reliably established in their hands.We designed a new IP injection method by adjusting the needle penetration location and the needle penetration angle. We inject through a natural hole located in the ventral region between pectoral fins and penetrate through a unique anterior to posterior direction.

Previously, two intraperitoneal (IP) methods have been employed for the administration of doxorubicin in adult zebrafish4,10. In method I, also known as the Classic IP injection method as described by Kinkel et al.4, the needle was inserted at a 45&#176; angle to the midline between the pelvic fins with the abdomen facing upward. In method II, or the Alternative IP injection method as described by Ma et al.10, the n...

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

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.

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

an-intraperitoneal-injection-technique-adult-zebrafish-that-minimizes

Research

JoVE Journal

Medicine

762 ビュー.  Mayo Clinic. 私たちは、成体のゼブラフィッシュにおけるアントラサイクリン誘導性心毒性モデルを作製し、新たな治療遺伝子や化合物の発見を目指しています。このプロジェクトを成功させるには、信頼性の高いIPインジェクション方法が不可欠です。したがって、以前に開発されたIPインジェクション方法は、古典的および代替的な手法です。従来の方法は、腹部から骨盤ヒ...

ビデオ: 著者スポットライト:ゼブラフィッシュ心毒性モデルのための新しい腹腔内注射法

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...

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intraperitoneal injection into adult zebrafish

Intraperitoneal Injection into Adult Zebrafish

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...

modified intraperitoneal injection of doxorubicin into adult zebrafish

Modified Intraperitoneal Injection of Doxorubicin into Adult Zebrafish

intraperitoneal injection: a method of solution delivery into the abdominal cavity of an adult zebrafish

This video describes a technique of intraperitoneal injection, a method for delivering solutions into the abdominal cavity of Adult...

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

a novel cell injection method with minimum invasion

Author Spotlight: A Novel Cell Injection Method with Minimum Invasion  

This method eliminates any major invasion during cell injections caused by the cell suspension...

A Novel Minimally Invasive Slow Injection Method for Reducing Tissue Injury

zebrafish cardiomyopathy model: drug-induced cardiotoxicity

This video describes the technique of creating a zebrafish cardiomyopathy model with minimal variations among experimental groups and in the sample, we create zebrafish cardiomyopathy model using the drug...

Zebrafish Cardiomyopathy Model: Drug-Induced Cardiotoxicity

A Novel Cell Injection Method with Minimum Invasion

generation, maintenance, and identification of germ-free zebrafish models from larvae to juvenile stages

Author Spotlight: Establishing Germ-Free Zebrafish for Microbiota-Host Relationship Studies

This protocol outlines the primary steps for obtaining germ-free (GF) fish embryos and maintaining them from larvae until the juvenile stage, including sampling and detecting their sterile status. The use of GF models with infection is important for understanding the role of microbes in host...

Optimizing GF Zebrafish Models: Generation, Maintenance, and Microbial Studies

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intraperitoneal injection of cells: a method of delivering cancer cells into the peritoneal adipose tissue of murine model

In this video, we perform an intraperitoneal injection of ovarian cancer cell suspension in a mouse model to study the colonization of ovarian cancer cells in peritoneal adipose tissues and their subsequent...

Intraperitoneal Injection of Cells: A Method of Delivering Cancer Cells into the Peritoneal Adipose Tissue of Murine Model

efficient gene knockdown in the liver via intrasplenic injection of adeno-associated virus serotype 8 (aav8)-delivered small hairpin rna

Author Spotlight: A Novel Intrasplenic Injection Protocol for AAV8-Mediated Gene Knockdown Targeting Nucleostemin in Liver Regeneration and Cancer Models

This protocol describes how intrasplenic injection of AAV8-delivered small hairpin RNA achieves the same gene knockdown efficiency in the liver as portal vein injection, representing a simpler procedure with much lower perioperative and postoperative mortality and...

Intrasplenic AAV8-shRNA Injection for Efficient and Safe Liver Gene Knockdown

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facilitating repeat intracarotid injections in mouse models by a novel injection site repair technique

Author Spotlight: Advancing Glioblastoma Treatment Through Intraarterial Delivery Strategies for Oncolytic Viruses and MicroRNAs

Repair of the intracarotid artery in a mouse model after injection returns blood flow to the artery without negatively impacting the distribution of the injected material. Injection site repair facilitates subsequent injections through the same artery and prevents cerebral ischemia in mouse strains that lack a complete Circle of...

Facilitating Repeat Intracarotid Injections in Mouse Models by a Novel Injection Site Repair Technique

systemic treatment for postnatal, juvenile, and runted adult mice by retrobulbar sinus injection

Author Spotlight: Overcoming Challenges in Gene Therapy for Organic Acidemias Through Retrobulbar Injection in Murine Models

This article provides a protocol and an accompanying video for the retrobulbar sinus injection of up to a total volume of 150 &#181;L for postnatal, juvenile, and runted adult mice. This procedure is particularly well suited for the injection of small mice (15 g) when tail vein injection is not...

Effective Systemic Delivery in Mice Using Retrobulbar Injection Technique

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zebrafish brain ventricle injection

Zebrafish Brain Ventricle Injection

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles.  We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live...

rapid isolation of stage i oocytes in zebrafish devoid of granulosa cells

Author Spotlight: Enhanced Method for Isolating Pure Zebrafish Stage I Oocytes

This protocol describes a modified procedure for rapidly isolating clean stage I oocytes in zebrafish devoid of granulosa cells, thereby providing a convenient method for oocyte-specific...

Improved Procedure to Obtain Granulosa Cell-Free Stage I Zebrafish Oocytes

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intrathoracic injection for the study of adult zebrafish heart

Intrathoracic Injection for the Study of Adult Zebrafish Heart

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...

Imagine a line across the abdomen, and locate a point on the animal&#39;s right side and close to the midline. The point of entry is cranial to and slightly medial of the last nipple. Prepare to insert the needle on the mouse&#39;s right side to avoid the cecum and reduce the risk of puncturing the intestines. Insert the needle at the lower lateral region of the mouse&#39;s abdomen to a depth of approximately 0.5 centimeters. Pull back on the plunger to confirm that the needle has not...

optimized intravenous injection in adult zebrafish

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...

Optimized Intravenous Injection in Adult Zebrafish

retro-orbital injection in adult zebrafish

Retro-orbital Injection in Adult Zebrafish

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

novel apparatus and method for drug reinforcement

Novel Apparatus and Method for Drug Reinforcement

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans.  In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in...