Neutrophils are important inflammatory cells that infiltrate wounds in response to locally-produced chemical ligands. This protocol allows visualization of receptor dynamics in response to such ligands in vivo. The use of chemokine receptor reporters could be expanded to other physiological settings, such as infection and tumor models and to other immune cells.
The method makes it possible to track when and where in the tissue leukocytes sense chem-attractant molecules. This can enable a better understanding of how immune responses unfold and resolve. Performing a ventral fin wounding in zebrafish larvae requires fine larvae manipulation, so some practice is required on larvae not intended for experimental purposes.
On day 2.5 to 3.5 post-fertilization, place the anesthetized zebrafish larvae under a fluorescent dissecting scope and select larvae with transgenic receptor expression. Transfer the selected larvae to a 120 millimeter Petri dish containing E3 medium supplemented Tricaine and use a sterile scalpel to cut the ventral fin of each larva deep enough cut to cause substantial neutrophil recruitment without cutting the caudal hematopoietic tissue vessels. Add 500 microliters of 2X Tricaine to a glass bottom dish, then add 500 microliters of agarose solution and mix by stirring gently with the pipette tip.
Use a pipette to transfer the wounded anesthetized zebrafish larvae into the dish and orient the embryo laterally while gently pushing down so that the caudal part of the fish is as close to the glass bottom as possible. When the embryo is in position, allow the agarose to cool. After five to 10 minutes, gently touch the gel with a small paintbrush to confirm solidification and add two milliliters of E3 medium supplemented with 0.2 milligrams per milliliter of Tricaine to the plate.
Image the wound site using a spinning disk confocal microscope. As soon as possible, after the agarose sets, transfer the embryo to the confocal imaging spinning disk platform and use the stage joystick and microscope objective to locate the fish. Using the 30 to 40X objective, focus on the wound area and select the field to image around the wound.
Select the laser and adjust the exposure time, then set up a timelapse every 30 seconds for the desired duration and obtain a Brightfield image to document the field of view. To quantify neutrophil receptor internalization at the wound site, open the image datasets in Fiji and use the time slider to select a representative timeframe of interest for each dataset. In MATLAB, create a new script and include functions for image reading, opening, and manual selection of the points of interest.
Run the script to open the frame of interest and click the neutrophils for analysis. Record an estimation of their centroids, both in the ventral fin wound and in the caudal hematopoietic tissue. Segment the neutrophils in each frame using the active contours technique and create the script to add the calculation of the gray level co-occurrence matrix for each neutrophil including the calculation of contrast of the neutrophils based on the gray level co-occurrence matrix.
Add commands to save the values separately for individual neutrophils in the ventral fin including the calculation of the mean neutrophil contrast value from all of the caudal hematopoietic tissue neutrophils. Include the normalization of the contrast value of individual neutrophils at the wound site to the calculated mean contrast of the caudal hematopoietic tissue neutrophils to obtain a normalized contrast that reflects how dotty the appearance of a receptor is within individual responding cells relative to the control non-responding cells. Then click run to run the script.
Ventral fin wounding is followed by rapid neutrophil mobilization from the caudal hematopoietic tissue into the ventral fin and clustering at the wound margin within 30 to 60 minutes of injury induction. In this analysis, the chemokine receptors CXCR1 and CXCR2 expressed by zebrafish neutrophils were imaged using spinning disk confocal microscopy. The pattern of receptor distribution was quantified using the contrast metric, which reports differences in intensity between neighboring pixels.
An alternative method is to quantify the ratio of receptor levels over the levels of a controlled membrane marker. Using the contrast metric to detect receptor internalization in neutrophil clusters at the wound, visible differences between CXCR1 and CXCR2 trafficking in neutrophils at the wound site can be quantified. For example, in this analysis, fluorescently-tagged CXCR1 internalization in cells located at the wound site increased over time, while fluorescently-tagged CXCR2 remained on the membranes of neutrophils at the wound.
Suppression of CXCR1 and CXCR2 ligands through morpholino treatment results in differential fluorescently-tagged CXCR1 internalization at the wound site. In addition, in early embryos, fluorescently-tagged CXCR1 is markedly internalized in embryos in which the receptor ligand is co-expressed. When attempting this protocol, keep in mind that the imaging should be performed as quickly as possible after wounding to capture receptor dynamics during neutrophil recruitment to wounds.
Following the imaging of chemokine receptors in normal larvae, researchers can perform comparative imaging in mutant larvae with dysregulated inflammation.
