The overall goal of this epifluorescence intravital video microscopy, or IVM protocol of blood vessels is to visualize and quantify leukocyte rolling and adhesion in corroded arteries in Angiotensin II-induced hypertension in mice. This method can help answer key questions in the pathology of vascular dysfunction and arterial hypertension such as mechanism of interaction between leukocytes and in particular the lysosomes and positive cells, and the vessel work. The main advantage of this technique is that we can see the in vivo interactions between circulating blood cells and the vascular understanding.

Prepare a male mouse for surgery after seven days of angiotensin two treatment with an osmatic pump according to the text protol. Apply ophthalmic ointment over the eyes to protect them during the procedure. Place the anesthetized animal in dorsal recumbance on a 39 degreese Celsius surgical plate and insert a rectal probe to maintain the temperature.

Use a cotton swab to spread and rub hair removal cream into the neck until the hair starts to fall out. Then, after an additional 2 minutes, use a spatula to remove the hairs and cream. Then, disinfect the skin by using an alcoholic skin antiseptic.

Next, under a stereo-microscope, make a 1 to 1.5 cm longitudinal incision in the skin of the neck, 1 cm from the trachea. Then, make 2 perpendicular incisions on either end of the first incision. Carefully remove the tissue just under the skin and remove the piece of skin covering the left jugular vein and the trachea.

Next, use two curved forceps to carefully isolate the sublingual and submaxillary corroded glands from the zone of interest. While taking care not to cut or damage the glands, position them to allow the best access to the jugular vein and corrideds. Then, to isolate the jugular vein, insert thin forceps between the vessel and surrounding tissue, and slowly open and close the instrument to gently free the vein from the tissue.

Once the jugular vein is completely clean, position the forceps under the vessel and place two 10 cm long 7-0 sutures underneath it. Tie two knots to close the suture proximal to the head and with the second suture, prepare one knot, but do not close it. Now, with forceps in one hand, hold the catheter filled with 37 degrees Celsius sterile saline, then with the other hand, use scissors or a curved 26 gauge needle to make a small incision into the vessel.

Insert the catheter into the jugular vein, and close the knot two times. Then, close the suture proximal to the head over the catheter to ensure complete immobilization. To isolate and prepare the corroded arteries, use thin curved forceps to dissect the area covering the trachea.

Then, once the corrodeds are free from surrounding tissues, remove the vegas nerve from the vessel without cutting it, by slowly closing and opening the thin forceps, if necessary, to gently mobilize the nerve. Once both arteries are completely clean, position the forceps under the first corroded artery, and while taking care to not overstretch the vessel, place a small, black, 3 mm by 2.5 cm plastic vessel holder under the vessel. Once the vessel holder is in place, ensure that blood flow is present.

To avoid stretching the vessel, the operator's hand should move as a little as possible with a single movement of the first forceps to isolate the corroded artery and the help of second forceps to make a hole under the vessel. When the vessel holder is correctly positioned, place the second artery over the device. If the mouse is not placed directly under the microscope, place the glands back over the trachea, and apply 37 degrees Celsius sterile saline to avoid drying of the zone.

In a 1 mL syringe, prepare a 1-4 dilution in sterile saline, of a 2 mg per mL thawed stock solution of acridine orange. Protect the syringe from light. To carry out measurements, position the mouse under a high-speed, wide-field florescence microscope using a long distance condenser and a 10x water-immersion objective with a monochromater, a beam-splitter, and a CCD camera.

Acquire images in real-time by focusing the objective in the middle of the corroded artery at the endothelial surface. Slowly inject 50 mL of acridine orange. Then, set the software to record 100 images, with an exposure time of 120 milliseconds per picture.

Record four videos per corroded artery at different locations on the artery for each video. If the fluorescent signal decreases, inject another 50 mL of acridine orange. To test different conditions, inject the acridine orange directly into the lateral tail vein if a jugular catheter was not inserted, and carry out the same image acquisition.

As a control, record florescence measurements from un-injected lys-Mcre-positive, IRG-positive mice. Quantify the rolling and adherence cells in a 200 micron by 250 micron rectangle view placed in the middle of the vessel for each video, displayed at a speed of 10 images per second. Count the rolling and adherence cells.

Adherence cells are defined as cells that do not move or detach from the endothelium within the 10 second video. To simplify quantification, remove the channel with the red fluorescence, and use only the green fluorescence to count rolling and adhering cells. In this study, the corrodeds of lys-mCre positive, IRG-positive mice infused with angiotensin 2, were observed using IVM.

As seen here at baseline, the presence of a jugular catheter resulted in the adhesion of lys-M-positive fluorescent cells. After acridine orange injection, the endothelial cells were also fluorescent. Then, reduction of the background to limit endothelial related fluorescence, revealed that all nucleated adhering cells were lys-M positive.

In this experiment, after one week of angiotensin 2 infusion, leukocyte endothelium interactions in corroded arteries of lys-M Cre-positive IRG-positive mice, were imaged and visualized by IVM, with or without acridine orange injection, via a jugular catheter or through the tail vein. Without any catheter or injection, rolling of lys-M positive cells significantly increased after angiotensin 2 infusion, compared to untreated mice. And adhesion was also increased.

Injection of acridine orange with a jugular catheter increased adhesion and rolling in angiotensin 2 treated mice, to a greater extent compared to mice without a catheter. Finally, as seen in these panels, injection of acridine orange in the tail vein, leads to similar adhesion and rolling compared to the mice that did not receive injection of acridine orange. Once mastered, this technique can be done in thirty minutes if it's performed properly.

When attempting this procedure, it's important to remember not to damage the tissues surrounding the vessels. After its development, this technique paves the way for researchers in cardiovascular and hemostatic fields to explore leukocytes and platelet adhesion to the vascular wall in vivo. After watching this video, you should have a good understanding of how to limit the impact of dye injection and surgery in IVM.

Don't forget that working with intercalating fluorescent dyes can be extremely hazardous, and skin contact with the dye must be avoided with the proper material, such as gloves.

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