Brain-localized and Intravenous Microinjections in the Larval Zebrafish to Assess Innate Immune Response.

Creating a robust and controlled infection model is imperative for studying the innate immune response. Leveraging the particular strengths of the zebrafish model system, such as optical transparency, ex utero development, and large clutch size, allows for the development of methods that yield consistent and reproducible results. We created a robust model for activation of innate immunity by microinjecting bacterial particles or live bacteria into larval zebrafish, unlike previous studies which largely restricted such manipulations to embryonic stages of zebrafish. The ability to introduce stimuli locally or systemically at larval stages provides significant advantages to examine host response in more mature tissues as well as the possibility to interrogate adaptive immunity at older larval stages. This protocol describes two distinct modes of microinjection to introduce lipopolysaccharide (LPS) or bacteria into the living larval zebrafish: one localized to the brain, and another into the bloodstream via the caudal vein plexus. Graphic abstract: Schematic shows the two distinct modes of larval zebrafish microinjection, either in the brain parenchyma or in the blood stream intravenously. Reagents introduced into the zebrafish to assess immune response are depicted in the "injection components" as described in the protocol.

[Background] The complex interactions during an infection require the use of in vivo animal models to fully understand the dynamic interplay between the pathogen and its host. Studying this phenomenon requires a controlled and reliable method of pathogen delivery. Zebrafish has been used as a model for studying the immune response to a variety of pathogens (Menudier et al., 1996 (Yang et al., 2014). Extending from previously published methods, we developed a protocol for microinjection of LPS or bacteria in the larval zebrafish, either directly into the brain parenchyma or into blood circulation to cause a robust innate immune response starting in the 4 dpf larvae. Since adaptive immunity does not begin until about 4 weeks after fertilization in zebrafish (Davis et al., 2002), we can leverage the early larval stages of 3 www.bio-protocol.org/e3978  zebrafish to study specific innate immune functions independent of adaptive immunity. Co-injection of immune activators with a fluorescently labeled dextran, or direct injection of fluorescently tagged immune activators allows for a quick visual verification of a successful injection as well as subsequent labeling of the macrophage response based on phagocytosis of the reporter. This protocol describes two different modes of microinjection with distinct target sites: first, the caudal vein plexus for systemic distribution throughout blood flow, and second, the brain tectum that briefly localizes the injected substance in the brain but is subsequently drained out into circulation (Yang et al., 2020). Although we describe our protocol for 3-5 dpf larvae, these methods are applicable to later larval stages at least up to 10 dpf (Yang et al., 2020).

Video 2. Stepwise demonstration of mounting larvae for microinjection
1. Use a plastic transfer pipette to transport the larvae to the center of a clean Petri dish lid and remove as much water as possible.
Note: Petri dish lids are used because the sides of the lid have a low profile which allows more latitude to position the needle to the desired target. Several larvae can be mounted at once; for brain injections we can mount upwards of 20-30 zebrafish larvae at once.

Heat 1.5% low-melt agarose (solid form) in microwave to melt it to a liquid form.
Note: Approximately 20 s to less than 1 min is needed to melt 100 ml of agarose. Low melt agarose heats up very fast so you will want to stand nearby to monitor the heating to prevent boil over.
3. Use pipette to collect a small amount of low-melt agarose.
a. Immediately after microwaving, the agarose will be very hot. If you see steam/condensation within the pipette then the agarose is too hot and will burn the larvae. Periodically monitor the temperature of the agarose by carefully touching the outside of the pipette where agarose is located. The agarose should be warm to touch, not hot, and remain fluid. make orienting the needle more difficult and cause unwanted bending of your glass needle.

Use forceps to orient the larvae.
a. This is the most time sensitive step because larvae must be correctly positioned before the agarose re-solidifies (1-2 min). Use fine forceps to quickly orient each larva, depending on desired injection site, but without concern for the exact orientation or body alignment. Be cautious to not injure the larvae by poking them, instead use the agarose around them to nudge and move them into position (see Video 2). b. For brain and intravenous injections, position the larvae on their dorsal or ventral-lateral sides, respectively. 6. Wait for agarose to cool down and solidify before starting injections.   F. Brain microinjection 1. Conduct the microinjections under a fluorescent stereomicroscope in order to monitor and validate each successful injection by fluorescence. The mounted larvae in low-melt agarose will need to be oriented with brain side up for needle access (see Figure 2).
2. Place needle directly above the brain tectum (see Figure 2).
3. Use the micromanipulator to slowly puncture the skin with the needle.
Note: Force of the puncture can drive the needle further into the brain than desired. Pull the needle backwards until the needle is only superficially piercing the brain. 4. Press the PicoPump foot pedal twice to eject 1 nl. 5. Screen for successful injection by visualizing fluorescence in the brain tectum at the site of injection (see Figure 2B).
Note: It is normal for the injection liquid to spread into the hindbrain ventricle. Remove any larvae that are not injected correctly by using forceps to grab the larvae out of the agarose.

Figure 2. Brain and intravenous microinjection sites.
A. Red arrow points to brain injection site in the right tectum and white box outlines the right tectum region of the larval brain at 3 dpf.
B. 30 s after fluorescent dextran injection into the brain tectum (red arrow), the fluorescent tracer can be readily observed to disperse into the brain ventricles and into the spinal canal (red arrowheads). C. Red arrow points to the intravenous injection site at the caudal vein plexus located beneath the dorsal aorta (top red arrowhead) and near the urogenital opening (bottom red arrowhead). D. Full body image 30 s after intravenous injection shows fluorescent dextran