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 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 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.
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 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.
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
2. Grouping the fish according to their body weight
3. Preparation of the needle and station for injection
4. IP Dox injection procedure
5. Post-injection fish management
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 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° (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°, a 45° 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 µg/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).
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. Please click here to view a larger version of this figure.
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 ± standard error. Please click here to view a larger version of this figure.
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° 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° needle penetration angle, used 3 batches (9, 12, 14) fish for Dox injection. For 0° 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 ± standard error. Please click here to view a larger version of this figure.
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 µg/g Dox (5 µL) was injected in four consecutive weeks. (B) The chronic DIC model manifests reduced EF% at 56 dpi. Values are shown as the mean ± standard error. Please click here to view a larger version of this figure.
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'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: >80%, preferably 100%, fish should be able to survive after the injection. All fish will survive until 56 dpi, when the majority of fish (>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° to 45°, 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.
The authors have nothing to disclose.
This study was supported by the NIH (HL107304 and HL081753) and the Mayo Foundation (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.
10 μL NanoFil-syringe | World Precision Instruments, Inc | NANOFIL | injection tool |
34 G needle | World Precision Instruments, Inc | NI34BV-2 | injcetion tool |
60 mm Petri dish | fisher scientific/fisherbrand | FB0875713A | placing the sponge |
Dissecting microscope | Nikon | SMZ800 | Injceting the Dox |
Doxorubicin hydrochloride | Sigma | D1515-10MG | drug for creating DIC model |
Echocardiography | VISUAL SONICS | Vevo 3100 | measuring cardiac function |
Foam Sponge | Jaece Industries | L800-D | placing the fish |
Hank's balanced salt solution (HBBS) | Thermo Fisher | 14025076 | Vehicle for Dox |
Microcentrifuge | southernlabware | MyFuge/C1012 | collect the Dox solution |
Precision Balance Scale | Torbal | AD60 | Digital scales |
Tricaine | Argent | MS-222 | Anesthetizing fish |
Tube | Eppendorf | 1.5 mL | storage |
vevo LAB software | FUJIFILM VISUAL SONICS | 5.6.0 | quantification of the heart |