Visualization of Neutrophil Extracellular Traps in Mesenteric Venules After Mesenteric Ischemia-Reperfusion Injury via Intravital Microscopy

The role of particular genes on the onset and the development of thrombosis, in this case, we investigate the endothelial PAR1 expression on ischemia-reperfusion injury. We try to pinpoint the effect of the endothelium on the response, both after ischemia and during reperfusion. A really important aspect that is understudied so far is to pinpoint the role of gut commensals in mesenteric ischemia-reperfusion injury.

To study this question, our group combined intravital microscopy with a mesenteric ischemia-reperfusion injury model, and we combined that with germ-free mouse models. The application of thrombosis models to transgenic mice in combination with intravital microscopy, such technologies enable the in vivo observation of cellular behaviors during the onset of the disease. Performing experimental models on a living organisms is always a challenge, as the experiment is influenced by many factors including sex, age, anesthesia treatment, changes animal welfare regulations, et cetera.

Also, research in thrombosis started in the last century. Many contributing factors remain still unresolved. My group studies the role of the gut microbiota on thrombosis, atherosclerosis, platelet function, and vascularization.

To begin, position the anesthetized, shaved mouse in a dorsal recumbency on a heating pad. Connect the mouse to the oxygen system via a nose cone. Secure the mouse's limbs to the heated pad with surgical tape.

For the implantation of the catheter in the jugular vein, make a transverse incision over the trachea. Remove the skin covering the neck and isolate the right jugular vein. Then, using a five-centimeter silk thread, close the distal end of the vein and fix it with surgical forceps.

Place three silk threads approximately two centimeters below the vein, one near the distal end, and two near the proximal end of the jugular vein. Tie a surgical knot, but do not close the vessel. Fill a polyethylene catheter with 0.9%sodium chloride.

Remove air bubbles and plug it in a one milliliter syringe. Use scissors to cut a hole between the second and third knots and implant the catheter into the jugular vein. Aspirate the syringe to ensure that blood is present in the catheter.

Tie the silk thread to secure the catheter in the vein. Then, use a medical tape to secure the syringe. Fill one milliliter syringes with acridine orange at 0.5 milligrams per milliliter final concentration and nucleic acid dye with five micromolar concentration.

Inject 50 microliters of SYTOX Orange for neutrophil extracellular traps, or NETs. Disinfect the mouse's abdominal skin with alternating rounds of iodine-based scrubs and alcohol. Then, using a scalpel, perform a three to five-centimeter laparotomy along the linear alba.

Gently position the intestine out of the abdominal cavity using two saline-moistened cotton tips and identify a location with minimal fat coverage. Position small branches of the intestine on a black dough to reduce background signal. Inject 50 microliters of acridine orange through the jugular vein catheter to visualize the stained leukocytes.

Acquire the video of the intestine before ischemia reperfusion. Prepare a compress moistened with sodium chloride and place the intestine in it. Occlude the superior mesenteric artery with a small vascular clamp.

Gently push the intestine back into the abdominal cavity using saline-moistened cotton swabs. Use a 7-0 suture to close the abdominal wall and prevent loss of moisture and temperature. After one hour, remove the suture.

Following the ischemic period, apply a few drops of saline to the clipped area. Then, gently remove the microvascular clips using a clip applier. Using saline-moistened cotton tips, gently position the intestine again out of the abdominal cavity and position the small branches of the vein on a black dough.

Inject 50 microliters of acridine orange and capture the post-ischemic video of the vein. Turn on the high-speed, wide-field fluorescence microscope equipped with a long-distance condenser, a monochromator, a 10 times water immersion objective and a charge-coupled device camera. Set the exposure time to 30 milliseconds and select the 5B channel with installed Alexa Fluor 488 and Rhodamine Red filters to visualize the leukocytes and NETs.

Quantify cell recruitment within a 0.06 square millimeter region of interest. Then, quantify adherent leukocytes by counting cells that remained stationary or attached to the endothelial lining during a 20-second observation period. Identify NETs labeled with both leukocyte and nucleic acid fluorescent dyes.

Leukocyte adhesion to the ischemic mesenteric endothelium increased in both wild-type and F2r delta EC groups following ischemia-reperfusion injury, with a notably lower adhesion observed in the F2r delta EC group, implying a role of endothelial PAR1 in leukocyte recruitment. After ischemia-reperfusion injury, NETs were observed only in the wild-type group expressing normal levels of PAR1 on the endothelial cells, indicating a regulatory role of PAR1 in NETosis.