The overall goal of this procedure is to monitor the formation of an induced thrombosis in an exposed mouse vessel. This is accomplished by first exposing the vessel of interest. Next, a small piece of filter paper soaked with ferric chloride is deposited on the vessel to induce the thrombus on the mesenteric vessel.
The aggregation of the thrombus forming cells are monitored by in vial microscopy on the carotid artery. The changes in the blood flow induced by the thrombus is measured with a doppler flow meter. The use of chloride to induce a thrombosis in the CED artery of reds has been first established in 1990 by Professor Coz and colleagues.
Since then, it has been adapted to mice and has been used to study many different vessels. This method is now the most commonly used thrombosis model in our research field, and it has helped to answer key question such as the wall of vulnerable factor and the application of the P two Y 12 platelet receptor in thrombo disformation. Before starting the procedure, cut one by two millimeter strips of filter paper and a small three by five millimeter piece of white plastic from a syringe wrapper.
Next, after confirming a lack of response to tow pinch, apply vet ointment to each of the animals'eyes. Then to induce a mesenteric arterial thrombus, place the mouse in a small Petri dish on a 37 degree Celsius heating pad and open the abdomen with a three centimeter incision, followed by a careful incising of the peritoneum. Position the mouse on the side of the Petri dish and gently exteriorize the intestines.
Then using two cotton buds, carefully spread the mesentery to bring a suitable vessel to the surface of the dish and carefully dry the tissue with a delicate wiper. Next, dilate the tail vessels with a warm water soak, followed by a 30 microliter intravenous injection of freshly prepared Domine six G To label the circulating leukocytes and platelets. Then place the Petri dish under an inverted microscope and use the Brightfield channel to loosen the chosen arterial.
Now soak a strip of filter paper with freshly prepared 6%ferric chloride, and use one pair of forceps to pick up the paper and carefully press it onto only the arterial of interest. Observe the formation of the thrombus by intravital inverted fluorescence microscopy through the filter paper. Within the first 10 seconds of the deposition, the aggregation of the circulating domine six G labeled leukocytes and platelets will allow an easy identification of the thrombus.
After one minute of exposure, remove the filter paper. Then while monitoring the thrombus formation, wash the vessel with PBS and record the dynamic formation of the thrombus as highlighted by the labeled blood cells. Still images of the thrombus can also be captured to facilitate measurement of the vessel occlusion over time.
To assess carotid artery thrombus formation, fix the legs of an anesthetized mouse with sticky tape onto a 37 degree Celsius heating pad positioned under an operating microscope. Then tape a small pillow fashioned out of a wiper under the animal's head, and use a thread loop with tongs to pull the snout down by the upper teeth. When the carotid artery region is exposed, make a small incision in the skin directly below the jaw down to the sternum.
Then dissect the fascia and isolate a fragment of either the left or right common carotid artery above the bifurcation. Next, carefully introduce tweezers between the artery and the nerve to separate them taking care not to damage the nerve. Then use one pair of forceps to pick up the small piece of white plastic and another to slowly slide it under the isolated section of the vessel so that the iron chloride will not soak the surrounding tissue.
Carefully dry the artery with wipers. Now soak a band of the filter paper in four or 6%ferric chloride, and place the band around the artery. After three minutes, remove the filter paper, rinse the vessel with PBS and dry the tissue with wipers.
Then place a doppler flow, probe around the vessel at the injured area, and record the changes in the flow.Here. The progressive formation of a partial thrombus was monitored in a 200 micrometer mesentary vessel by intra vial fluorescence microscopy. In this experiment, the thrombus became clearly identifiable after the first minute of ferric chloride exposure.
40 seconds after the removal of the filter paper, the thrombosis rapidly progressed with the complete thrombus finally observed on the wall of the whole vessel section. As these data demonstrate, an overall constant blood flow of around 1.1 milliliters per minute is typical in a non-injured carotid artery. After three minutes of exposure to 4%ferric chloride.
In this experiment, an occlusive thrombus was obtained with an occlusion time of 13 minutes and 30 seconds. With a 6%ferric chloride exposure. The occlusion time decreased by a full four minutes.
The intravital microscopy observation provides a simple and accurate way of testing the lytic capacities of many novel drugs in vivo by visualization of the thrombus degradation. The flow monitoring on the cate artery has been a useful tool to study the efficiency of anti trombotic drugs and to evaluate if the drugs can prevent or delay occlusion.
