Intravital Microscopy to Analyze Blood Cell-Endothelial Interactions in an Inflammation Mouse Model
Trascrizione
Begin with an anesthetized mouse that has been injected intraperitoneally with lipopolysaccharide to induce inflammation.
Incise the abdomen to expose the abdominal cavity. Maintain organ hydration with saline. Inject rhodamine 6G retro-orbitally. The fluorescent dye molecules enter the circulation and label leukocytes and platelets for their visualization.
Now, exteriorize a loop of ileum with its mesentery vessels. Keep the tissue hydrated with pre-warmed saline for optimal imaging. Under a fluorescence microscope, focus on a mesentery vein for intravital microscopy.
The lipopolysaccharide injection causes endothelial cell activation with the upregulation of adhesion molecules, including selectins. Circulating platelets are recruited to the endothelium, where they bind via specific adhesion molecule-ligand interactions, forming adhesion complexes.
Owing to these interactions, platelets get activated and release granules containing molecules, including serotonin, promoting leukocyte recruitment.
Leukocytes move to the endothelial surface and bind to activated platelets. Further, leukocytes roll along the endothelium and bind to the endothelial cells through weak adhesive interactions. Rolling motion activates leukocyte integrins for firm adhesion to endothelial cells, crucial for leukocyte-endothelial transmigration.
Using intravital microscopy, visualize the fluorescent leukocytes and platelets to determine their real-time interactions with endothelial cells.
