Avaliando interação leucócito-endotélio em condições de fluxo em um<em> Ex Vivo</em> Autoperfused Microflow Câmara Assay

The overall goal of this procedure is to create an ex vivo arteriovenous closed circuit to assess leukocyte interactions with endothelial adhesion molecules under physiologic flow conditions. This is accomplished by first coating a thin glass chamber with the endothelial adhesion molecule of interest. In the second step, polyethylene and silicone tubing are connected to the chamber and then surgically to the mouse.

Next blood from the carotid artery as allowed to flow through the tubing. And the subsequent interactions of the native leukocytes with the coated glass chamber are recorded in real time. Ultimately, the effects of various treatments on the displacement and rolling velocity of the leukocytes as the interact with the immobilized adhesion molecules can be calculated over time.

The main advantage of this technique over existing technique like a pro cytometry analysis, is that with this technique, leukocyte interaction with endothelial molecules is assessed under physiological flow conditions. The day before the experiment connect one centimeter of PE 10 polyethylene tubing to a six centimeter piece of silicone tubing, followed by five centimeters of polyethylene tubing. To prepare the tubing setup for the jugular side of the animal for the carotid side, connect the tubing as just demonstrated with the addition of a TTU within the six centimeter silicone tubing, about 1.5 centimeters away from the one centimeter polyethylene tubing.

Next, connect one end of a 10 centimeter piece of silicone tubing to the pressure transducer and insert the T end of the carotid side T tube into the other end. Ensure that the connection sites are tight and leak free. Then use a one milliliter syringe connected to a 25 gauge needle to infuse each microflow chamber with the freshly prepared endothelial surface coating protein of interest, and store the coated chambers in a 15 milliliter tube at four degrees Celsius overnight the next day.

To connect the jugular and carotid tubing to the microflow chamber, connect one centimeter silicone tubing to each side of the chamber, and then seal the connections with a small rectangular piece of param stretched over with the tubing meets the chamber. Use a syringe to fill the chamber and tubing with 0.1%bovine serum albumin. And then after checking for leakage, incubate to the chamber for 45 minutes.

At room temperature near the end of the incubation, stabilize the microflow chamber on a notch to Petri dish with a small drop of silicone at each notch. Then connect the jugular and carotid tubings to each side of the chamber. Then while the silicone is drying, connect a 60 mil locking syringe filled with heparin to the pressure transducer and run 100 USP grade heparin through the tubing and chamber until there are no air bubbles.

Before connecting the mouse to the microflow chamber, turn on the computer, the microscope, and the appropriate pressure recording device. Once the software is set up, secure a 10 to 12 week old mouse. In the ventral position, make an incision in the neck area with scissors and then dissect the thyroid gland to expose the trachea and vessels.

Clean the carotid artery until it is fully exposed. Taking care not to damage the vagus nerve. Then pass the bend of a folded piece of suture underneath the carotid artery and cut the suture at the bend.

So there are two pieces of suture underneath the artery. Loop each suture into knots without tightening, and then place the sutures for the left jugular vein in the same way. When the jugular sutures are in place, inject one milliliter of heparin into the mouse ip.

After the injection, tighten the upper suture on the carotid artery and place a vessel clamp as low as it can go on the exposed section of the artery. Use forceps to hold the artery by the upper suture and then use fine micro scissors to make a small incision about one eighth of the circumference of the artery close to the upper suture. Now insert the carotid artery tubing using a tube holder through the incision made in the artery and pass it down by the lower suture.

Then tie off the lower suture followed by the upper one. So the artery is sealed around the tubing, making several knots to ensure a tight seal. After repeating the same procedure for the jugular tubing, minus the vessel clamp, temporarily open the vessel clamp on the carotid artery tubing to test the flow.

If there are no leaks, move the mouse to the microscope and connect the carotid and jugular vein tubing to the microflow chamber tubing to record the rolling velocity of the leukocytes through the microflow chamber. Fully release the carotid clamp to fill the chamber. Allow the blood to circulate for three to five minutes so the flow conditions stabilize.

In the meantime, adjust the microscope stage so the chamber is level with the microscope objective and its edge is parallel with the video recording window on the screen. Next, build a Petri dish with room temperature water, and then lower the 10 x water immersion objective into the dish so the cells flowing through the chamber are visible, adjusting the light intensity as appropriate to simulate a medium physiological flow pressure, adjust the clamp on the tubing so the transducer reads a steady 30 millimeters of mercury. Then press start to initiate the recording of both the leukocyte trafficking and the pressure with the first time lapse close to the jugular end of the chamber and moving against the flow toward the carotid end until completed.

When the recording is finished, close the valve at the input site to stop the blood flow and replace the chamber with a new chamber coated with a different adhesion molecule if desired. During each recording, only interacting leukocytes are evident within the chamber. For example, in each of these images, the white arrowheads indicate individual leukocytes that were captured by the adhesion molecule of interest and that are rolling along the chamber among the other free flowing cells.

Within the blood flow, a common artifact outside the chamber can be seen with the large dark spheres in all of the images, which can help the viewer appreciate the leukocyte movement in relation to a fixed point. The graph illustrates a final plot of the data. The first experimental treatment was observed to decrease the rolling velocity evident as a shift to the left, and the second experimental treatment was observed to increase the velocity appearing as a shift to the right, both as compared to the control rolling velocity In combination with this procedure.

Other method like labeling the leukocyte and vivo x Viva can be utilized to further answer other questions like the contribution of certain leukocyte subtype disease progression.