This protocol helps people analyze endothelial cell morphology, and the expression of molecules in regions, under different fluid shear stress. The main advantage of this technique is that it provides a direct assessment of vascular endothelial cells under different fluid shear stress. When performing this procedure, key steps are to use freshly prepared paraformaldehyde and to perform a good perfusion.
Begin this procedure with anesthetization of a 12-week-old C57BL/6 mouse, as described in the text protocol. Confirm proper anesthetization by gently pinching the tail. Tape the mouse's paws to a stack of paper towels, with adhesive tape.
Hold up the skin of the mouse with forceps, and cut the skin with a pair of scissors from the abdomen to the top of the thorax. Open the abdominal cavity, below the ribcage, with a sharp pair of scissors. Lift the sternum with forceps, and cut the diaphragm.
Then cut away the ribcage to expose the thoracic cavity. Cut off the vena cava just above the liver with scissors. Pressure perfuse the arterial tree for five minutes, with pre-chilled normal saline containing 40 units per milliliter heparin, through the left ventricle, until the lungs and liver become pale.
Then perfuse with pre-chilled 4%paraformaldehyde in PBS for four minutes. Remove all the muscles, organs, and fat, until the aorta is exposed. Place the mouse under a dissecting microscope.
Use delicate forceps and a pair of delicate scissors to expose the aorta clearly under a dissecting microscope, and remove the connective tissue along the aorta, as cleanly as possible. Dissect the thoracic aorta from the heart to the celiac trunk with a pair of delicate scissors. Place the aorta into a six centimeter cell culture dish, with PBS.
Cut open the aorta longitudinally, first along the lesser curve, and then use the microscissors to cut open the three branches of the aortic arch, including the innominate, left common carotid, and the left subclavian artery along the greater curve until the straight segment is met. Permeabilize the aorta with 1%polyoxyethylene octyl phenyl ether, in PBS for 10 minutes. Then block the aorta with 10%normal goat serum in tris-buffered saline containing 2.5%polysorbate 20 for one hour at room temperature, in a 12 well cell culture plate.
Next incubate the aorta in the blocking buffer containing five grams per milliliter rabbit anti-VCAM1, and five grams per milliliter rat anti-VE-Cadherin, overnight at four degrees Celsius. After rinsing the sample three times with washing solution, apply the fluorescence conjugated secondary antibodies for one hour at room temperature, keeping in a dark place. Rinse another three times in the washing solution.
Counterstain the aorta with DAPI for 10 minutes, keeping in a dark place. Then rinse the stained aorta three times in washing solution. Place the aorta on a cover slip with the lumenal surface downward, and move it slowly to antifade mounting solution that has been previously dropped on the cover slip.
Gently stretch the aorta to keep the specimen flat. Invert the cover slip and put it on the glass slide. Take care to avoid any air bubbles between the specimen and the glass.
Examine the aorta with a laser scanning confocal microscope. Analyze color intensities of different channels from the desired region in the en face images, with Image-Pro Plus software. En face immunofluorescence staining images of vascular endothelial cells from the mouse aorta are shown.
En face immunofluorescence of the vascular cell adhesion protein one, and expression with VE-Cadherin as an endothelial marker, are shown under varying flow patterns from different regions of the mouse aorta. DAPI was also counterstained to show the cell nuclei for better visualization. When looking through the Z stacks under the microscope, the endothelial and smooth muscle cells can be easily distinguished by their cell nuclei morphology.
The endothelial cell nuclei are oval shaped, and bigger than the spindle shaped, smooth muscle cell nuclei. Expression of VCAM-1 was more abundant in regions under disturbed flow, at lesser curvature of the aorta arch, compared with regions under steady flow, at greater curvature of the aorta arch, and at the thoracic aorta. Here areas of lesser curvature of a mouse aorta are indicated as d-flow areas, and the greater curvature and thoracic.
With the right modification, this technique can be also applied to analysis of vascular permeability, and the position of macromolecules in the endothelial cell intima.
