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 'Guide for the Care and Use of Laboratory Animals' (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
<ol>
	<li>Obtain the doxorubicin stock from a commercial source. 
	NOTE: Doxorubicin is light-sensitive, so acquire it in powder form and store it in opaque containers to protect it from exposure to light. Perform all steps for the Dox powder preparation within a chemical hood.</li>
	<li>Completely dissolve the Dox powder in distilled water and prepare a stock solution with a final concentration of 5 mg/mL.</li>
	<li>Aliquot the stock by dividing it into 1.5 mL tubes.</li>
	<li>Wrap the tubes in aluminum foil to shield them from exposure to light.</li>
	<li>Store the aliquot Dox solution at 4 °C for short-term storage (&lt;1 month), or -20 °C for long-term storage10.</li>
</ol>
2. Grouping the fish according to their body weight
<ol>
	<li>Group the fish with a BW difference of less than 10% together for subsequent injection. 
	NOTE: To save effort at this phase, fish with less than a 10% BW variance are categorized as the same size.</li>
	<li>Allow the fish to fast for 24 h prior to injection.</li>
	<li>Anesthetize the fish using embryo water containing 0.16 mg/mL tricaine for 1 min.</li>
	<li>Take the fish out of the water with tricaine and dab both sides of the fish body with clean filter paper to remove excess water.</li>
	<li>Measure and record the BW of each fish, then promptly return the fish to a recovery tank filled with fresh system water. 
	NOTE: Dox injection was performed on fish after reaching 3 months of age. In this study, researchers utilized 3-month to 10-month-old fishes. The BWs of mature WIK strain zebrafish may vary from 0.2 g to 0.5 g. Prolonged anesthesia lasting for more than 5 min, followed by a Dox injection, led to high fish mortality.</li>
</ol>
3. Preparation of the needle and station for injection
<ol>
	<li>Determine the injection volume of Dox stock solution (e.g., 5 mg/mL) needed for each fish based on the average body weight to achieve the targeted dose of 20 µg/g.</li>
	<li>Use the following formula to calculate the injection volume: 
	<img alt="Calculating fish injection volume formula, ratio of dose to concentration, equation diagram." src="/files/ftp_upload/66500/66500eq01.jpg" style="margin: auto;" data-altupdatedat="1747911167000" data-alt="Equation 1"></li>
	<li>Add 1x Hank's Balanced Salt Solution (HBSS) to dilute the Dox solution calculated in step 1 for injection, reaching a total volume of 5 µL. 
	NOTE: Utilize bulk solution for each group of fish based on their BW and include an additional 3 fish in each group to ensure that there is no shortage of solution for injection during the course of the experiment.</li>
	<li>Gently tap the tube and then briefly microcentrifuge at top speed to collect the solution at room temperature for 10 s.</li>
	<li>Place the prepared solution on ice and shield it from exposure to light.</li>
	<li>Place a clean 100 mm Petri dish with a sponge underneath a dissecting microscope, then adjust the focus. 
	NOTE: The sponge contains a 4 cm long groove. The elastic retraction of the sponge will provide the fish with support and keep the fish body in position. The sponge can be reused.</li>
	<li>Equip a 10 µL micro-syringe with a 34 G beveled needle.</li>
	<li>Flush the needle with 1x HBSS buffer to eliminate any bubbles and clear potential blockages from the syringe.</li>
	<li>Measure 5 µL of the solution prepared in step 4 for the injection.</li>
</ol>
4. IP Dox injection procedure
<ol>
	<li>Put the adult fish in water with 0.16 mg/mL tricaine for 1 min to induce a state of unconsciousness.</li>
	<li>Position the fish in the groove of the embedded sponge with the abdomen facing upward (Figure 1A).</li>
	<li>Insert the needle with an angle near 0°, starting from the midpoint of the pectoral fin towards the posterior side of the cardiac cavity (Figure 1B). 
	NOTE: Avoid any contact with the heart during the procedure.</li>
	<li>Direct the needle towards the tail and go beneath the silver skin. 
	NOTE: Position the needle in close proximity to the silver skin, taking care to avoid any scratching or piercing.</li>
	<li>Monitor the needle tip within the abdominal cavity throughout the entire operation (Figure 1C). 
	NOTE: Avoid damaging the liver, intestines, swimming bladder, and other organs. Ensure that the needle reaches the end of the intestine, near the cloacal foramen.</li>
	<li>Gradually and evenly dispense the 5 µL Dox solution, then slowly withdraw the needle along the original path to prevent any leakage (Figure 1D).</li>
	<li>Monitor the abdominal cavity for the presence of Dox by observing a red coloration of Dox solution (Figure 1E).</li>
	<li>Swiftly move the injected fish to a clean crossing tank filled with fresh system water to help the fish recuperate. 
	NOTE: Between injections, flush the needle once with 1x HBSS buffer.</li>
</ol>
5. Post-injection fish management
<ol>
	<li>Return the fish back to the system with circulation after the injection.</li>
	<li>Fast all the injected fish for an additional 24 h to facilitate their recovery.</li>
	<li>During the first week, keep a close eye on the fish. Remove the deceased fish as soon as possible to avoid infecting the other fish. 
	NOTE: Fish fatalities within the initial 24 h are likely due to physical injuries from the injection or prolonged anesthesia. Record fish numbers to generate a survival curve.</li>
	<li>Perform echocardiography to phenotype the Dox-injected fish at 56 days post-injection11. 
	NOTE: Ensure uniformity in conditions and procedures for the corresponding control group injected with HBSS solution.</li>
</ol>

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><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

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.

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° 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 needl...

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

Author Spotlight: Novel Intraperitoneal Injection Method for Zebrafish Cardiotoxicity Models

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.

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 ...

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

Research

JoVE Journal

Medicine

1.5K Views.  Mayo Clinic. 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.

Video: Author Spotlight: Novel Intraperitoneal Injection Method for Zebrafish Cardiotoxicity Models

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.

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

Developmental Biology

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.

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

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.

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

Biology

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.

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.

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.

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

Cancer Research

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.

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.

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

Danio rerio (zebrafish)

Adult

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.

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.

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