Microinjection of Interleukin-6 into the Bloodstream of TSC Zebrafish Larvae to Study Neuropsychiatric Disorder-Like Behaviors

My research investigates how IL-6 induced inflammation interacts with genetic risk mutations for autism to influence brain development and function. Unlike broad LPS-induced inflammation, this protocol selectively elevates IL-6, enabling precise investigation of cytokine specific neurodevelopmental effects. To begin, gather the microinjection needles, required reagents, and two-day post-fertilization zebrafish embryos.

Prepare a 1%weight per volume agarose solution in E3 medium, and heat it until the agarose is completely dissolved and the solution becomes clear. Allow the agarose solution to cool briefly. Pour approximately 30 milliliters into a 100 millimeter Petri dish to ensure an even layer across the surface.

While the agarose is still liquid, but beginning to solidify, gently place a silicone mold onto the surface to create uniform grooves. Transfer approximately 100 chorionated embryos at two-day post-fertilization into a small 50 millimeter agarose-coated Petri dish. Add four milliliters of freshly prepared pronase solution to the dish, ensuring the embryos are fully submerged.

Incubate at room temperature for three to five minutes. Under a stereo microscope, monitor the release of embryos from the chorions. Gently rotate or swirl the dish intermittently to facilitate enzymatic removal of the chorions.

Continue until the majority of embryos are released from their chorions. Wash the embryos thoroughly by transferring them through five to six changes of fresh E3 embryo medium. Ensure that all chorions and pronase residues are removed to prevent damage to the larvae and to maintain optimal culture conditions.

Using a glass pasture pipette, transfer approximately 50 dechorionated larvae into a 100 millimeter Petri dish containing 40 milliliters of E3 medium. Maintain the larvae in E3 medium until microinjection. Under a stereo microscope, use fine forceps to carefully break the tip of the previously prepared glass capillary to generate a micropipette with an opening of approximately three micrometers in diameter.

Prepare a 0.9%weight per volume sodium chloride solution for control injections. And a two picogram per nanoliter Interleukin 6 or IL-6 solution in 0.9%sodium chloride for the IL-6 injections. Add one microliter of 0.05%Trypan blue for every 10 microliters of injection solution to facilitate visualization during injection.

With a pipette, load the solution into a microloader tip. Then use the microloader tip to backfill the prepared micropipette injection needle with approximately three microliters of injection solution, ensuring no air bubbles are introduced. Secure the loaded injection needle into the capillary holder to be connected to the microinjection system.

Then place a small drop of mineral oil onto a calibration slide. Calibrate the injection volume by expelling droplets into the mineral oil. Measure the droplet diameter using the calibration slide with a 0.01 millimeter scale.

Adjust the injection pressure to achieve a droplet volume of approximately one nanoliter. Anesthetize the larvae in small batches by adding tricaine to the E3 medium at a final 1X concentration to minimize stress and ensure consistent sedation. Transfer individual two-day post-fertilization larvae onto a solidified agarose injection plate.

Place the dish under the stereo microscope. Using a glass pipette, gently orient each larvae so that the duct of Cuvier is clearly visible and accessible. Position the micropipette tip at the entry point of the duct of Cuvier by tracking blood cells moving toward the heart under the stereo microscope.

Inject the first droplet of the prepared solution into the duct of Cuvier. After approximately 20 seconds, inject a second droplet into the duct of Cuvier. Following injection, gently transfer the larvae into a fresh 100 millimeter Petri dish containing 40 milliliters of E3 medium.

Return the larvae to an incubator at 28.5 degrees Celsius for recovery. On the following day, repeat the microinjection procedure as previously demonstrated. At five-day post-fertilization, use the larvae for downstream applications, including behavioral assays, brain imaging, or immunofluorescence analysis.

IL-6 microinjection did not affect larval viability compared to sodium chloride injections. Survival rates at five-day post-fertilization were indistinguishable between IL-6 injected and NACL-injected groups, indicating that the dosing regimen is well tolerated and does not introduce non-specific toxicity. Microinjections slightly reduced viability compared to uninjected siblings.

The Homozygous Tuberous Sclerosis Complex 2, or TSC2 mutants, displayed significantly higher baseline prostaglandin E2 or PGE2 levels than wild-type siblings. Heterozygous TSC2 mutants showed a modest, non-significant increase in baseline PGE2 compared with wild-type siblings. PGE2 levels rose sharply in heterozygous TSC2 mutants following IL-6 microinjection.

The open field test showed that TSC2 mutants displayed increased anxiety-like behavior compared with wild-type siblings in uninjected controls, and IL-6 microinjections did not affect this behavior. The heterozygous TSC2 and homozygous TSC2 mutants spent significantly more time in the outer zone of the arena and exhibited reduced center exploration than wild-type siblings in uninjected controls, as well as IL-6 injected TSC2 larvae. IL-6 injections increased repetitive, stereotype swimming patterns, particularly in heterozygous TSC2 mutants.

The proportion of IL-6 injected heterozygous TSC2 mutants exhibiting repetitive looping was significantly increased compared with sodium chloride injected controls. This protocol enables controlled IL-6 inflammation studies to assess gene environment effects on neurodevelopment and behavior in zebrafish. Key considerations when performing this protocol include consistent needle size, precise two nanoliter injection volume, and minimizing larval time outside E3.Future studies can investigate how IL-6 inflammation and genetic risk together affect brain development and behavior.