Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

The overall goal of this procedure is to investigate leukocyte, endothelial cell and platelet leukocyte interactions during inflammation in vivo. This is accomplished by first pretreating, a mouse with the substances of interest and labeling the blood cells an ileum loop, and the accompanying mesentary vessels are then exteriorized and the STO cell interactions that take place within a selected mesentary vein are imaged by intra vital microscopy. Ultimately, the interactions between the leukocytes and the platelets with the endothelial or each other can be analyzed.

This method can help answer key questions in the field of inflammation, such as what kinds of leukocyte endothelial, or platelet leukocyte interactions occur in vivo during different models of inflammation or after different pre-treatments of interest. Four hours before the intra vital imaging inject 20 milligrams per kilogram of LPS IP into a 16 to 20 gram, four to six week old male mouse to induce the mock bacterial inflammatory response. Prewarm a 0.9%saline solution in a 37 degree Celsius water bath for humidifying the plastic chamber and mesentary tissue at this time as well.

Next, confirm the appropriate depth of anesthesia by a lack of response to toe pinch in the LPS treated mouse, and then apply ointment to the animal size, dete the abdomen with a shaver, removing any loose hair with 70%ethanol soaked gauze. Then use a small curved forceps and scissors to open the abdomen, identify the epigastric vessels and open the peritoneum in the linear alba region to protect the vessels. Apply a few drops of prewarm saline into the abdominal cavity to keep the tissue moist, followed by a retroorbital injection of 50 microliters of rod Domine six G to label the circulating blood cells.

Then place the mouse in a 10 centimeter Petri dish, exteriorize a loop of illium and administrate the prewarm saline every other minute to keep the tissue moist. To visualize the inflammatory response by intravital microscopy, place the mouse underneath the microscope and bring a mesentary vein with the diameter of 200 to 300 micrometers and as little as possible visible surrounding fat into the field of view. Using the appropriate microscope software.

Record the blood cell endothelial interactions for one minute in four different veins per mouse. Then to analyze the cell to cell responses to confirm the stable and enter individually blood flow conditions. To quantify the number of rolling leukocytes, draw a vertical line through the vein and manually count all of the leukocytes that cross this line in one minute.

To determine the rolling velocity, draw two vertical lines through the vein 50 micrometers apart, and measure the time it takes a single leukocyte to cross between the lines while stably rolling on the endothelium. To measure the leukocyte adhesion, draw a 200 by 200 micrometer square in the vein. Then manually count the firmly adherent leukocytes that exhibit, no visible movement within the square for 30 seconds.

Finally, to quantify the platelet leukocyte interactions manually count the number of platelets bound to one leukocyte. Although there is a certain degree of activation in untreated animals due to the procedure itself, there is almost no slow rolling or firm adhesion of untreated leukocytes. Indeed, higher numbers of rolling and adherent leukocytes are observed with a decrease in the rolling velocity of the circulating blood cells in LPS challenged mice.

In this experiment, intravital microscopy was performed without further LPS challenge immediately after acute intraperitoneal treatment with fluoxetine. Note, the higher numbers of firmly adherent leukocytes and the lower rolling velocities in the fluoxetine treated animals indicating an influence of the acute fluoxetine treatment on leukocyte endothelial interactions. Further, as observed in this representative image of platelet leukocyte interactions with an e mesenteric vein during LPS induced peritonitis, a ting of the platelets around the leukocytes can be observed as well as the interactions of these complexes with the vessel wall.

After watching this video, you should have a good understanding of how to perform inal microscopy to investigate leukocyte endothelial and platelet leukocyte interactions.