Name: Author Spotlight: Novel Intraperitoneal Injection Method for Zebrafish Cardiotoxicity Models
Uploaded: 2024-03-29T14:20:00
Description: Read this Scientific Article Protocol about Author Spotlight: Novel Intraperitoneal Injection Method for Zebrafish Cardiotoxicity Models at JoVE.com

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

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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

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 ...

Мы стремимся создать модели кардиотоксичности, индуцированной антрациклинами, у взрослых рыбок данио, чтобы мы могли открыть новые терапевтические гены и соединения. Надежный метод внедрения IP имеет важное значение для обеспечения успеха этого проекта. Таким образом, ранее разработанные методы внедрения IP являются классическими и альтернативными методиками.В то время как классический метод проникает в рыбу от брюшной области через среднюю линию между брюшными плавниками, альтернативный метод проникает в рыбу из спинного участка, расположенного между брюшным и анальным плавниками. В связи с токсичностью доксорубицина при использовании двух современных методов ВП были отмечены значительные повреждения организма и смертность рыб. Новые исследователи должны пройти всестороннюю подготовку, прежде чем в их руках можно будет надежно установить модели заболеваний.Мы разработали новый метод инъекции IP, регулируя место проникновения иглы и угол проникновения иглы. Мы вводим инъекцию через естественное отверстие, расположенное в вентральной области между грудными плавниками, и проникаем через уникальное направление спереди назад.