A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

This method can help answer key questions about how biomaterials influence susceptibility to infection, how they influence the progression of such infections, and how they affect immune cell behavior around such biomaterials in vivo. The main advantage of this technique is that it provides a novel animal model that allows in vivo visualization and intravital analysis of biomaterial-associated infections. Visual demonstration of this method is critical as the injection of biomaterial microspheres is a very delicate procedure.

To generate a bacteria-only suspension, first transfer four to five colonies of the fluorescent Staph aureus mCherry strain, cultured on tryptic soy agriculture plates supplemented with 10 micrograms per milliliter of chloramphenicol into 10 milliliters of tryptic soy broth supplemented with chloramphenicol, for growth in the mid-logarithmic growth phase at 37 degrees Celsius with shaking. When the culture reaches an optical density measurement at 620 nanometers of 0.4 to 0.8, collect the bacteria by centrifugation, and wash the cells two times with one milliliter of sterile PBS per wash. After the second wash, resuspend the pellet in 1.1 milliliters of PVP, and vortex the bacterial suspension before measuring the optical density again.

Then adjust the concentration of the bacterial suspension with PVP according to the experimental requirements to generate the bacteria-only suspension. To generate a bacteria microsphere suspension, centrifuge commercial polystyrene microspheres, PS-10, and resuspend the microsphere pellet in the bacteria-only suspension. Dilute the bacteria microsphere suspension with a 1/2 volume of PVP to reach an appropriate concentration of bacteria in the suspension that is approximately 2/3 of that in the bacteria-only suspension.

Mix the suspension by vortexing. To check the concentration of the bacteria-only and bacteria microsphere suspension, add 100 microliters of each suspension to individual wells of a 96-well plate. Serially dilute 10 microliter aliquots of the suspensions in 90 microliters of sterile PBS in subsequent wells, using fresh tips for each dilution step.

Apply duplicate 10-microliter aliquots of the undiluted and diluted bacterial suspensions onto agar plates, and incubate the plates overnight at 37 degrees Celsius. The next morning, count the colonies to calculate the number of bacteria within the prepared bacteria-only and bacteria microsphere suspensions. After collecting the zebrafish embryos, discard any non-transparent unviable embryos, and incubate approximately 60 embryos per 100-milliliter Petri dish at 28 degrees Celsius in fresh E3 medium.

After anesthesia, sequester the GFP-positive embryos for fluorescence microscopy. Transfer them to a 100-milliliter Petri dish of E3 medium. To create an injection mold, fill a 100-milliliter Petri dish with one to 1.5%liquid agarose.

Use a plastic mold template to create grooves in the agarose. When the agarose has solidified, remove the mold, and cover the agar with E3 medium supplemented with 0.02%tricaine. One day three post-fertilization, use forceps to break the tip of a pulled glass microcapillary needle to achieve tips with an approximately 20 micrometer outer diameter under a light microscope with a scale bar in the ocular.

The adjustment of the opening of the needle tip is practical for the injection of microspheres. The diameter of the opening needs to be adjusted for microspheres of different sizes. Then use a Microloader pipette tip to load the needle with approximately 20 microliters of the bacteria microsphere or bacteria-only suspension, and mount the needle onto a micromanipulator connected to a microinjector.

Now transfer the selected embryos to the grooved agarose plate. After waiting five minutes for the embryos to be anesthetized, align the embryos within the grooves in a single orientation for their injection. Set the microinjector to the appropriate injection settings, and insert the needle into the muscle tissue of the first embryo at a 45 to 60-degree angle under a stereo microscope.

Gently move the needle back and forth to adjust the position within the tissue as necessary, and use the microinjector foot pedal to inject the loaded suspension. When injecting a bacteria microsphere suspension into the tissue of zebrafish embryos, the needle should be inserted gently but with a steady hand. After a successful insertion, a space should be created for the materials before injection.

When all of the embryos have been injected, score the embryos for a successful injection under a stereo fluorescence microscope, and maintain the embryos in E3 medium without tricaine in individual wells of 48-well plates with daily medium changes. To monitor the infection progression by colony-forming unit quantification, randomly transfer five to six viable infected embryos into individual two-milliliter microtubes shortly after injection, and gently wash the embryos with sterile PBS. After discarding the washes, add 100 microliters of sterile PBS to each tube, followed by two to three sterile two-millimeter-diameter zirconia beads.

Then crush the embryos in a homogenizer at 3, 500 rpm for 30 seconds, and culture the homogenate as demonstrated. Place a 100-millimeter Petri dish containing E3 medium supplemented with 0.02%tricaine on a stereo fluorescence microscope stage, and add 500 microliters of 2%methylcellulose into the Petri dish. To monitor the infection progress by fluorescence microscopy, equip the appropriate bright-field and fluorescent filters, align the anesthetized infected embryos horizontally in a spot of methylcellulose in a Petri dish.

Use the bright-field filter to bring the damaged injected tissue into focus at a 160x magnification. Set the Z-stack depth at 10 micrometers and the step size at five micrometers to allow the recording of three consecutive images. Then image the individual embryos under identical optimized settings at a 160x magnification.

Use the ObjectJ plugin in ImageJ to analyze the images. Intramuscular injection of Staph aureus initiates a dose-dependent infection in embryos, with viable infection progression observed in embryos administered high-challenge doses at days one and two post-injection. As demonstrated, it is possible to inject similar numbers of bacteria in the presence or absence of microspheres.

Typically, all embryos with more than 20 colony-forming units of Staph aureus mCherry are positive for fluorescence under microscopic scoring, although the presence of microspheres seems to not significantly influence the infection progression in the low-dose challenged embryos. In high-dosed challenged embryos, microscopic scoring does not demonstrate any differences in infected embryo frequency with or without microspheres, but quantitative culture reveals higher colony-forming unit numbers retrieved from embryos in the Staph aureus plus microsphere group compared to those harvested from the Staph aureus-only group at two days post-injection. The presence of a biomaterial influences both the immune cell response and the initial susceptibility to infection.

For example, at five hours post-injection, the macrophage infiltration in response to a Staph aureus-only injection is significantly higher than in response to a Staph aureus plus microspheres injection, while at one day post-injection, the embryos with microspheres exhibit significantly higher levels of Staph aureus infection than the embryos without microspheres. It is important to remember to always adjust the opening of the needle tip to the size of the biomaterials and to adjust the ratio of the bacteria concentration in the bacteria-only suspension and the bacteria microsphere suspension. This zebrafish embryo model allows the in vivo visualization and the intravital analysis of the infection progression and the provoked immune cell responses in the presence and absence of biomaterials.

This technique provides a new whole-animal model for the development of antimicrobial biomaterials and treatment strategies for safe medical devices.