Optimized Intravenous Injection in Adult Zebrafish

Our research leverages the zebrafish model to study human blood disorders, focusing on identifying novel biomarkers, therapeutic targets, and treatments for conditions such as leukemia. By utilizing the unique advantages of zebrafish, we aim to explore the molecular mechanisms underlying hematological diseases and develop innovative therapeutic strategies to improve patient outcomes. Intravenous injection into adult zebrafish is challenging due to their small size and complex vasculature.

We have developed an optimized IV injection method for precise and efficient delivery of substances or cells into adult zebrafish. Compared to the intracardiac and retro-orbital injection, the IV method we present here resulted in better fish survival and better cell engraftment. This method makes adult zebrafish more usable for non-term studies.

Overcoming some limits of novel models, the improved IV technique increases the accuracy of disease models and drug screenings, helping to advance new therapies. To begin, place the euthanized mpo:EGFP transgenic zebra fish on a dissecting table. Dissect the zebra fish to isolate kidney marrow.

Then transfer the kidney marrow into a 1.5 milliliter micro centrifuge tube containing cell suspension medium. Pipette the suspension to disaggregate the kidney marrow cells. Filter the cell suspension through a 40 micrometer nylon cell strainer.

Then centrifuge the cells at 500G for eight minutes and discard the supernatant, before re-suspending the pellet in 500 microliters of cell suspension medium. Using an automated cell counter count the cells with a 10 microliter aliquot. Thoroughly wash the Hamilton syringe three to four times with 75%ethanol.

Then rinse the syringe three to four times with ultrapure water to ensure all ethanol residue is removed. After administering anesthesia to the zebrafish, confirm the depth of the anesthesia by diminished movement and loss of reflex responses. Position the fish dorsal side up with the head oriented to the left on clean damp tissue paper to stabilize for injection.

Firmly grasp the Hamilton syringe and angle the needle at approximately 45 degrees to the caudal vein. For Casper zebrafish, aim at a visible section of the vessel through the translucent body. Gently insert the needle into the vessel and administer a two microliter of whole kidney marrow cells isolated from mpo:EGFP transgenic zebrafish.

After injection, apply gentle pressure to the injection site with a cotton bud for 10 seconds to control bleeding. Observe the injected zebrafish under a microscope to confirm GFP signals within the vessel. Allow the injected zebrafish to recover in freshly sterilized fish water for 10 minutes.

Then transfer the fish to a static tank. The intravenous injection method yielded the highest success rate with GFP signals visible in 41 out of 43 fish. While the retro-orbital method had GFP detected in only three out of 37 fish and the intracardiac method showed success in 11 out of 40 fish.

The intravenous injection method resulted in better survival rates in injected fish, especially with higher cell doses compared to the retro-orbital and intracardiac methods. GFP signals were detected as early as day three post transplantation in intravenous injected fish while retro-orbital and intracardiac methods showed delayed GFP signal onset. By day 17, intravenous injected fish displayed the most prominent GFP signals in kidney marrow.

Flow cytometry of kidney marrow showed about 18%GFP positive cells in intravenous injected fish with three times 10 to the power of five cells aligning with GFP levels in donor kidney marrow. In contrast, intracardiac and retro-orbital methods showed 5%and 2%GFP positive cells respectively.