Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

The overall goal of this procedure is to model mucosal candidiasis using the juvenile zebrafish. This is accomplished by first loading a solution containing live the albicans into a microneedle. In the second step, a defined volume of the yeast cells is injected into the swim bladder of a juvenile zebra fish.

Next, the fish are screened for a homogenous inoculum and incubated for the desired time. In the final step, confocal microscopy is used to follow the dynamic interaction of the pathogen with the zebrafish immune system. Additionally, the swim bladder can be micros dissected to obtain higher resolution confocal images.

The method that we are presenting here answers key questions of fungal immunology as well as microbiology, such as what is the precise sequence of events leading to a failed immune response to the fungal pathogen and candidal beacons at the mucosal surfaces. Before beginning the procedure warm an injection dish containing 2%aros in a 33 degrees Celsius incubator. After 30 minutes, use a 25 milliliter pipette to remove 100 milliliters of the E three media plus PTU from a plate of day four, post fertilization zebra fish, taking care not to aspirate any of the fish, and add two milliliters of four milligrams per milliliter.

Trica methane sulfonate stock to the media to anesthetize the animals. After 15 minutes, use a dissecting microscope to identify the fish with inflated swim bladders and use a plastic transfer pipette to transfer 30 zebra fish onto the prewarm injection dish. Remove the excess media, then hold the dish at an angle and use a 0.012 inch diameter fishing line super glued into a boro silicate capillary to make three lines of 10 vertically aligned Fish each next thoroughly vortex a tube of freshly prepared sea albicans at a concentration of 1.5 times 10 to the seventh CFUs per milliliter.

And then load three microliters of the yeast solution to EC clipped microneedle. Set the pulse duration to the appropriate setting. Then rotate the injection dish to reach an angle of 20 degrees between the microneedle and the head of the fish aiming toward the back of the swim bladder.

Now push the microneedle into the swim bladder, making sure the bore of the needle is in the lumen and press the puddle. Once verifying which fish have been injected is relatively easy, as the bolus displaces the air bubble and the back of the swim bladder is subsequently filled with media, co injection of phenol red with the bolus can further facilitate the visualization of a successful injection. After injecting all 30 fish, flood the dish with E three media and then empty the fish into the recovery dish.

Transfer 10 animals into a 1.7 milliliter centrifuge tube containing one milliliter of E three media and euthanize the fish with 200 microliters of trica. Next, for each animal, arrange four drops of vacuum grease in a square, one centimeter apart on a micro slide, and add two drops of freshly prepared, 2%low melting to the middle of the square. Next place one fish on a separate glass slide under a dissecting microscope and to verify that the animal's heart has stopped beating and remove the excess water.

Now use one pair of fine tweezers to hold the head in a fixed position and another to pull down the anterior gut. Pick up the swim bladder by the pneumatic duct, separating it from the digestive tract, and place it at the center of one of the prepared micro slides before the agro solidifies. Finally, place a cover slip over the tissue gently pushing down until it touches the vacuum grease and the agarose and image the swim bladder within 10 minutes of harvesting.

Injecting four nanoliters of sea albicans at one to 1.5 times 10 of the seventh. CFU per milliliter gives the most consistent data resulting in a range of 10 to 50 yeast cells per fish. Narrowing the inoculum to 15 to 25 yeast cells gives an even tighter phenotype and response.

Neutrophils play a key role in the resistance to candidiasis in mice and humans, and the infection of the zebra fish swim bladder with live albicans leads to a rapid and sustained recruitment of neutrophils. That does not occur when heat kill yeast cells are injected. Filaments develop rapidly after injection of the yeast cells with filamentous growth continuing for the first 24 hours post-injection, after which this number starts decreasing the ability to follow individual fish non-invasively enables these longitudinal studies and highlights the individual to individual differences in disease progression and host responses.

An experimental protocol that remains impractical in the mouse imaging, the interactions of immune or epithelial cells with c albicans is relatively easy in the model presented here. However, dissecting the swim bladder as just demonstrated significantly improves the quality of the imaging. This is particularly useful when visualizing transgenic fish lines where the fluorescence is ubiquitously expressed, and there is extraneous fluorescence in the surrounding tissue as observed in these images of a dissected swim bladder from the alpha Catine citrine fish line.

Following this procedure. Other methods said such as morph technology or chemical inhibition treatment can be used to answer question like, what is the role of a particular immune cell or signaling pathway in mucosal candidiasis.