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
<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 &#176;C for short-term storage (&#60;1 month), or -20 &#176;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 &#181;g/g.</li>
	<li>Use the following formula to calculate the injection volume: 
	<img alt="Equation 1" src="/files/ftp_upload/66500/66500eq01.jpg" style="margin: auto;" /></li>
	<li>Add 1x Hank&#39;s Balanced Salt Solution (HBSS) to dilute the Dox solution calculated in step 1 for injection, reaching a total volume of 5 &#181;L. 
	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 &#181;L 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 &#181;L 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&#176;, 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 &#181;L 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 effectively reduce organ injury, which results from minimized leakage of Dox during the penetration process. The needle&#39;s path is just beneath the silver skin, which can be continuously monitored by the investigator. Consequently, direct contact between the tip of the needle and the internal organs can be effectively avoided. After injection, all injected Dox stays in the abdominal cavity, and the only exit (puncture point) could be closed when the fish is awake and starts to swim forward.
Compared with the two existing IP injection methods4,10, the new IP injection method consistently results in minimized body damage and enhanced survival rates.This statement was further underscored by the feasibility of multiple injections in the same fish for generating a chronic zebrafish DIC model. Importantly, compared to the previous two IP injection methods4,10, the new method can be quickly mastered by researchers who have no previous experience in handling adult zebrafish. Reliable DIC models seem successfully obtained upon their first or second try in the hands of at least 4 different investigators. The success of the injection can be easily concluded within the first-week post-injection: &#62;80%, preferably 100%, fish should be able to survive after the injection. All fish will survive until 56 dpi, when the majority of fish (&#62;80%) should be free from obvious body damage. This is a significant improvement, because reliable DIC model using the first two IP methods can only be stabilized in the hand of a researcher after more than 3 rounds of practice with at least 10 fish in each round. This process could be time-consuming, because each practice required about 2 months to conclude on the success of his/her injection. Reducing tissue trauma not only improves the well-being of the fish but also minimizes confounding factors that could affect the outcomes of the study.
One critical step to ensure the success of the new method is the angle of penetration. As shown in Figure 2, when the angle of the penetration changed from 0&#176; to 45&#176;, significant fish death was noted, presumably owing to increased risk of contact between the tip of the needle and internal organs.
The primary limitation of the method is that the method cannot be used to deliver drugs to fish younger than 3 months. Because of their small body size, young fish cannot survive after the physical damage associated with the penetration of a needle. However, the method can be used in adult fish when their BW is larger than 0.15 g, including those aged from 3 months to 4 years old.
This new method would be widely used for drug delivery in adult zebrafish, especially when dealing with potentially toxic compounds such as Dox15,16. When it comes to non-toxic compounds, all three methods should be applicable.

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.

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 needle was inserted through the dorsal side of the fish (Figure 2A(i,ii)). By contrast, our method III has two major changes. First, the position of the injection is altered, with the abdomen facing upward and the needle directed from the midpoint of the pectoral fin toward the abdomen. The penetration site is guided by a natural hole in this area. Second, the angle of the needle is reduced, which should be close to 0&#176; (Figure 2A(iii), Figure 1).
Comparison among three intraperitoneal injection methods 
To compare the three IP injection methods, data obtained from Researcher I is presented, who did not have previous experience with zebrafish. When methods I and II were used for IP injection, significant fish mortality in the initial two weeks following IP injection was noted. In method I, death was observed three days post-injection, while in method II, it occurred after two days post-injection. 70% of the injected fish exhibited visible body damage, especially around the injection site, manifesting as drastically reduced body size and curved body shape one to two months post IP injection (Figure 2A (iv,v)). It was reasoned that the mortality and observed damage could be attributed to the toxicity of doxorubicin12, which might leak from the opening end of the injection needle during the injection process, damaging the internal organ along the route of the injection. In method I, the tip of the injection needle could touch the gut (Figure 2A(i)), while in method II, it could touch the swim bladder (Figure 2A(ii)). It was postulated that one could prevent the tip of the needle from touching any internal organ by using method III (Figure 2A(iii)). Indeed, a significantly improved survival rate was noted, with 80% of the fish surviving at 1 month post-injection (Figure 2B). This signifies a remarkable improvement compared to the survival rates for methods I and II, which were 53.33% and 33.33%, respectively. Importantly, 80% of the survived fish exhibited a normal body shape, in contrast to those generated by methods I and II (Figure 2A(vi)). To validate the successful generation of the DIC model, cardiac function was evaluated by measuring ejection fraction percentage (EF%). Indeed, a significant reduction in EF% was observed at 56 days post-injection in the group using method III (Figure 2C).
Reproducibility of method III by different researchers 
To prove that method III can be easily mastered by different researchers, 3 researchers were recruited to the study who had never worked with zebrafish before. Upon their first several tries with method III, all researchers were able to achieve 85.71%, 95%, and 83.33% survival rates 56 days after injection and a markedly decreased EF (Figure 3A,B). Of note, three researchers repeated their experiment 3 times, 3 times, and 2 times, respectively, and consistently obtained successful results. These data confirmed the repeatability of method III, allowing researchers without prior experience working with fish to generate reliable DIC models.
In one of the initial attempts using method III, researcher II tested a deviation of method III: instead of 0&#176;, a 45&#176; angle was used while penetrating the needle through the skin (Figure 2A(iii), dashed arrow). Only 15% of the fish survived two months after injection (Figure 3A), with 80% of the survived fish displaying considerable body damage. The data indicated that the angle of the penetration is a critical step for the success of method III.
Method III enables the establishment of a chronic DIC model 
It was postulated that significantly reduced body damage might enable multiple IP injections in the same fish, mimicking the chronic DIC model in mice13,14. Hence, method III was used to do a series of injections for 4 consecutive weeks, with 5 &#181;g/g Dox per week (Figure 4A). In this experiment, 100% of injected fish survived, and 80% of them exhibited no signs of body damage. Importantly, a significantly reduced ejection fraction was noted at 56 dpi, indicating the successful generation of a chronic DIC model in adult zebrafish (Figure 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66500/66500fig01.jpg" /> 
Figure 1: Location and angle of needle insertion for the new IP injection method. (A) The location of the needle insertion. The fish is upside down, exposing the abdominal side. The head is to the right. A natural hole is located between pectoral fins that makes penetration of the skin simpler. (B) The angle of needle insertion. It is near to zero degrees to the surface of the fish. (C) The track of the needle under the skin. The tip of the needle is continuously monitored once inside the abdominal cavity. The entry point appears different from Figure 1A because of the skin distortion during the penetration process. (D) A fish after Dox is released. The abdominal cavity becomes red after releasing the Dox. (E) A fish after IP injection. There is no sign of leakage after withdrawing the needle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66500/66500fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66500/66500fig02.jpg" /> 
Figure 2: Comparison of three IP injection methods in adult zebrafish. (A) Schematics of three IP injection methods. (i-iii) Shown are schematics of the classic IP injection (method I), alternative IP injection (method II), and the new method (method III). Arrows indicate the sites of penetration and angle of the needle for three injection methods. Dashed arrow, an unsuccessful angle of needle penetration. Scale bar: 5 mm. (iv-vi) Reduced body size and curved body shape in DIC models. Shown are representative fish from DIC fish at 56 dpi. (B) Comparison of survival curves among three IP injection methods. The number of survived fish was recorded weekly. (The experiment began with a total of n = 15). (C) Cardiac function assessment of fish at 56 dpi following Dox stress. n = 6 fish in the 1x HBSS control group, and the experiment began with a total of n = 15 fish in each 3 batches injected with Dox. Values are shown as the mean &#177; standard error. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66500/66500fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66500/66500fig03.jpg" /> 
Figure 3: Method III enables the zebrafish DIC model to be established consistently in the hands of 3 different investigators. (A) Survival curves of DIC fish using Method III by three different researchers. A high survival rate was noted in all three investigators. The numbers of live fish are recorded weekly. A low survival rate was noted for researcher II when an unsuccessful method III was used (45&#176; needle penetration angle, as shown by a dashed arrow in Figure 2A). (B) Cardiac function assessment of DIC fish using Method III by three different researchers. In total, Researcher II, for 45&#176; needle penetration angle, used 3 batches (9, 12, 14) fish for Dox injection. For 0&#176; needle penetration angle n = 6 fish were employed in the 1x HBSS as shared control group. The experiment began with a total of n = 85 fish with RII (9, 12, 14), RIII (8, 6, 6), and RIV (12, 18) employed in each of the 8 batches injected with Dox by three researchers (II, III, IV), respectively. Values are shown as the mean &#177; standard error. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66500/66500fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66500/66500fig04.jpg" /> 
Figure 4: Method III enables multiple injections of Dox in an adult zebrafish, recapitulating chronic DIC models in rodents. (A) Schematics of multiple injection model. 5 &#181;g/g Dox (5 &#181;L) was injected in four consecutive weeks. (B) The chronic DIC model manifests reduced EF% at 56 dpi. Values are shown as the mean &#177; standard error. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66500/66500fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

1.3K 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

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 ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

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

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

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

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

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

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

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

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

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

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

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

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

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 &#181;M after 30 min of incubation and the orange nucleic acid probe at 1 &#181;M after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 &#181;M concentrations and higher in both probes exhibited a type of non-specific staining, whereby &#39;live&#39; cells produced a target fluorescence, leading to an over representation of &#39;non-viable&#39; cell numbers. The positive controls (heat-killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively.

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

Microbial populations contain substantial cell heterogeneity, which can dictate overall behavior. Molecular probe analysis through flow cytometry can determine physiological states of cells, however its application varies between species. This study provides a protocol to accurately determine cell mortality within a cyanobacterium population, without underestimating or recording false positive results.

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120&#176;/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as cardiomyopathy. Because of its convenience and amenability to high throughput genetic manipulations, the generation of acquired cardiomyopathy models, such as the doxorubicin-induced cardiomyopathy (DIC) model in adult zebrafish, is opening the doors to new research avenues, including discovering cardiomyopathy modifiers via forward genetic screening. Different from the embryonic zebrafish DIC model, both initial acute and later chronic phases of cardiomyopathy can be determined in the adult zebrafish DIC model, enabling the study of stage-dependent signaling mechanisms and therapeutic strategies. However, variable results can be obtained with the current model, even in the hands of experienced investigators. To facilitate future implementation of the DIC model, we present a detailed protocol on how to generate this DIC model in adult zebrafish and describe two alternative ways of intraperitoneal (IP) injection. We further discuss options on how to reduce variations to obtain reliable results and provide suggestions on how to appropriately interpret the results.

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.