Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The overall goal of this procedure is to micro inject zebrafish larva with a bacterial pathogen streptococcus IEA for live non-invasive imaging of the host immune response. This is accomplished by first loading the microinjection needle with a bacterial suspension and lining up anesthetized zebrafish larvae on the agarose injection mold. Next, the bacterial suspension is micro injected into the otic vesicle of the larvae.

Then the injected larva are mounted in low melt agarose. Gus finally host immune cells are photo labeled for easy tracking and the infection is imaged using a confocal microscope. Ultimately micro injection of streptococcus IEA into the otic vesicle and the subsequent photo labeling and confocal microscopy are used to study the host immune response of zebrafish larvae.

The main advantage of this technique over existing methods like coddle vein injection, is that otic vesicle injection generates a localized bacterial infection to allow non-invasive imaging of host pathogen interactions in the transparent zebrafish larva. To begin prepare a 1.5 to 2%high melt aro solution in E three medium and microwave until the solution is clear. Once cooled, pour some of the aros into a Petri dish, adding just enough to cover the bottom of the dish and swirl.

Once the aros has solidified, place a rinsed and dried gel comb on top so that the solid end is just resting on the top of the Petri dish and the notched end is touching the aros. Ensure that the comb is as horizontal as possible, creating a 30 degree angle with respect to the bottom aros layer. Pour an additional small amount of aros at the interface between the comb and bottom aros layer so that the fresh aros layer covers the wells of the comb.

Allow it to cool completely before removing the comb. Then using a pipette tip, remove any overhanging pieces of aros from the wells. Pour E three medium on top of the injection mold and store at four degrees Celsius.

After preparing the SNEA inoculum and zebrafish larvae for injections according to the text protocol, turn on the micro injector and set the time range to milliseconds. Open the valve on the carbon dioxide tank to let gas into the line. The pressure of the micro injector should read approximately 20 PSI vortex the prepared SNEA culture in phenol red and PBS and use a micro loader tip to load two to three microliters of the culture into a pulled capillary injection needle mount the loaded needle on a micro manipulator connected to a magnetic stand and position it under a stereo microscope so that the needle is at approximately a 45 to 65 degree angle with respect to the base of the microscope.

First break off the tip of the needle using a pair of fine tweezers, press the foot pedal of the micro injector to dispense a drop of the inoculum onto the tip of the needle. Use the scale bar in the ocular lens of the microscope to measure the diameter of the drop. The diameter of the drop should be approximately 0.10 millimeter, which is about one nanoliter in volume.

To adjust the drop size, adjust the duration setting on the micro injector or use fine tweezers to clip off more of the needle tip. The injection time should be between 20 and 35 milliseconds to avoid too much tissue damage. Next, using a plastic transfer pipette transfer one fish per well into each of the 12 wells of the injection mold.

Then with a glass rod, hair loop, or plastic tip, gently position the larvae so that the heads are pointed towards the back of the microscope and the yolk sacks are against the left side of the well. Point the left ear of the larvae up towards the ceiling. When the larvae are ready for injection, use the knobs on the micro manipulator to line up the loaded needle with the otic vesicle so that both are in the same field of view.

With the needle tip pierce the outer epithelial layer of the otic vesicle so that the tip is just inside the vesicle. Now using a low enough pressure so as not to rupture the cavity, press the foot pedal to inject one nanoliter of the desired dose of SNEA. If the injection is successful, the otic vesicle, but not the surrounding tissue should fill with a phenol red inoculum.

Carefully retract the needle out of the larvae and under five x magnification, move the injection plate by hand so that the larvae is in view. If injecting fluorescently labeled bacteria under a fluorescent microscope, visually scan the injected larvae and keep just those where deposition of bacteria has occurred only inside the otic vesicle to ensure the injection volume remains the same over the course of the experiment. After injecting every 48th embryo, inject a drop of bacterial suspension into a 1.7 milliliter centrifuge tube containing 100 microliters of sterile PBS.

Then plate the 100 microliters on THY plus P auger plates, and incubate at 37 degrees Celsius overnight. To determine the CFU in the injection volume, when an entire group of 12 larvae on the injection ramp has been injected, carefully use a plastic transfer pipette to remove the larvae from the wells and place them into a new Petri dish. Remove the trica solution and replace with approximately two milliliters of fresh E three medium to allow the larvae to recover.

Incubate the embryos according to the text protocol. After preparing aros with trica, according to the text protocol on the stage of the stereo microscope, use a transfer pipette to place four to five anesthetized larvae in a glass bottom dish. Remove the trica and aro solution from the 55 degree Celsius water bath and let it cool at room temperature for one to two minutes.

In the meantime, remove as much liquid from the anesthetized larvae as possible. Next, pour the cooled agros into the dish until about half of the surface is covered. Then swirl the dish to spread the aros.

Pick up larvae that have floated to the sides of the dish and pipette them back into the center. Then under the stereo microscope, use a long pipette tip to gently position the larvae as desired for imaging. The otic vesicle position the larvae so that the right otic vesicle is facing up and the left otic vesicle is flat against the bottom of the dish.

Let the aros cool for about 10 minutes before moving the dish. Then gently pipette some trica and E three solution on top of the aros layer to keep it moist. To visualize the samples, use a Zack scan With the 488 nanometer and 543 nanometer lasers.

Use continuous line scanning to adjust the laser power and detector gain. Verify that there is no accidentally photo converted red fluorescence on the image acquisition control window. Under stimulus setting, select the use scanner tool and choose main, select the 405 nanometer laser and set it to 70%power.

Then using the circle option, define the region of interest in the odic vesicle under the stimulus start setting select activation in series with a pre activation of one frame and an activation time of 60, 000 milliseconds on the acquisition setting window. Under the time scan heading, choose two intervals of one minute each, one for pre and one for post photo conversion. Start the time-lapse series to initiate photo conversion of the defined region of interest.

Finally, scan the sample using a Zack and the 488 and 543 nanometer lasers. To visualize the photo converted red fluorescence, as well as any remaining green fluorescence microinjection of SNEA in phenol red into the otic vesicle results in an initially localized host response that when injected correctly should only be seen in the otic vesicle and not in the surrounding tissue or blood. Alternatively, if labeled bacteria are injected, a quick scan of infected larvae immediately post-injection can confirm the bacteria are only in the otic scle and not the surrounding tissue.

Microinjection sites of initially localized infection are useful for studying leukocyte chemotaxis as shown here. Leukocyte recruitment can be quantified by either Sudan, black staining of neutrophil granules or formaldehyde fixation of fluorescent transgenic lines using transgenic zebrafish lines expressing the green fluorescent protein. Dendra two, specifically in macrophages or neutrophils revealed that injection of NEA into the otic vesicle triggered recruitment of neutrophils and macrophages and phagocytosis of bacteria within 60 minutes post-infection.

In this figure, DRA two labeled macrophages that were recruited to the otic vesicle at five hours post-infection were photo converted and then tracked over the following 24 hours. Although some photo converted cells remain in the otic vesicle or head region, some were also found disseminated throughout the body of the larvae as seen here. After watching this video, you should have a good understanding of how to micro inject streptococcus n EEA into the otic vesicle of zebrafish aged three days post fertilization.

This includes one lining up anesthetized larvae on the injection mold and loading the needle with the bacterial inoculum. Two micro injecting bacteria into the otic vesicle. Three, mounting infected larvae in low melt aeros, and four, using a confocal microscope to photo label host leukocytes and image leukocyte pathogen interactions in vivo.