The overall goal of this experiment is to study the influence of potential vasoactive drugs on microvascular thrombus formation in vivo. This method can help answer key questions in thrombus formation such as identifying new antithrombotic substances that could help to prevent microvascular thrombus formation, which is one of the main complications in sepsis and during tissue transplantation. The main advantage of this technique is that the ear model of the hairless mouse enables intravital fluorescence microscopy and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed.
Begin by weighing and recording the weight of the mouse. Then load the appropriate volume of the test substance into an insulin syringe. Administer the drug 30 minutes prior to thrombus induction.
After anesthetizing the mouse 15 minutes prior to thrombus induction, place the mouse back into the cage until the onset of anesthesia. To verify sufficient anesthesia, pinch the tail with forceps and note the absence of a reaction. Next, 0.05 ml of defrosted 50 Dextran into an insulin syringe.
After filling the syringe, ensure that no air bubbles remain, because even small intravenously administered air bubbles can be lethal for the animal. Place the anesthetized mouse on an acroglass pad with an integrated heating plate in the face-down position. Adjust the heating plate to 37 degress Celsius.
Cover the mouse eye corneas with ophthalmic ointment. Then stitch two sutures of polypropylene 7.0 into the cranial and caudal edge of the right ear. Place the stitches as close to the edge and as proximal to the base as possible.
Shift the mouse to a dorsal position. Fix all legs to the platform using adhesive bandage. Hook a suture under the front teeth and position the head in dorsiflexion by affixing the suture to the platform with adhesive bandage.
Place the animal on the platform under the stereo microscope. Use 10X magnification. For microscopy of the right ear, prepare the left jugular vein by first creating a 5 mm incision in the skin of the neck in a craniocaudal direction.
Then use microforceps and microscissors to dissect the subcutaneous tissue. Then, free the vein from its adventia without touching the vessel. Now, use the previously prepared insulin syringe for the injection of the fluoroscent dye.
Carefully grab the vessel wall with the microforceps without perforating the vein. Penetrate the distended vessel wall with the syringe needle and inject 50 Dextran intravenously. Withdraw the needle and stop the bleeding using cotton swabs.
Avoid blood and dye contamination of the ear. Transfer the animal on the heating plate to an acro glass construction with a slat for the hearing plate and 0.5 cm high plane for positioning the ear. Fix the animal in a prone position on the heating plate using adhesive bandage.
Place the convex cartilage at the base of the ear beside the plane for the ear so that the apical part of the ear can be positioned flat on the plane. Add one drop of room temperature aqua to the acro glass plate. Using cotton swabs, absorb the drop of aqua, and let capillary forces attach the ear plane to the acro glass.
The auricle must be positioned as flat as possible, even though the cartilage gives the auricle a convex shape. So only the more flexible distal part of the auricle should be positioned on the acrylic glass plate. To avoid tissue damage and extravasation of fluids and dye, touch the ear as little as possible with the forceps.
Next, add drop of aqua to the convex dorsal side of the ear. Carefully put one cover slip on the ear without compressing the basal vessels entering the ear. Use cotton swabs to remove as much aqua as possible from under the cover slip To minimize the distance between the cover slip and the ear target vessels.
Begin by adjusting the intravital fluorescence microscope for 50 Dextran visualization. Tranfer the prepared animal to the deck of the intravital fluorescence microscope. Using 5X, 10X and 20X magnification, and 20%light intensity, search for a venous vessel of 50 to 60 microns in diameter and with an anterograde blood flow.
Add one drop of room-temperature water to the cover slip for water immersion of the 63X magnification objective. Immediately after the application of the water drop, begin recording the vessel for 20 seconds for the baseline assessment of the diameter and blood flow. Start thrombus induction five minutes after the injection of 50 Dextran.
For this purpose, raise the light intensity to 100%During phototoxic thrombus induction, close the aperture of the microscope for two seconds within a 30-second period to check for occlusion of blood flow. If blood flow persists, open the aperture again. The vessel is classified as occluded if the flow stands still for 30 seconds or more, or if the blood flow is retrograde.
Select and occlude five vessels per ear within a one hour period after injection of 50 Dextran. Here, animals were treated with vehicle or with a cannabinoids, anandamide, WIN 155, 212-2 or cannabidiol before injection of 50 Dextan prior to thrombus induction. Anandamide, but not CBD, and WIN 155, 212-2 significantly accelerated thrombus growth after one-time treatment as compared to vehicle control.
In another setting, anandamide was combined with indomethacin to assess the impact of cyclooxygenase-dependent products on thrombus formation via anandamide. Co-administration with indomethacin neutralized the pro-thrombotic effect of anandamide. Therefore, acute exposure to anandamide elicits a cyclooxygenase-dependent pro-thrombotic effect in vivo.
Once mastered, thrombus induction in tight vessels can be performed in 90 minutes, including preparation. While attempting this procedure, it's important to handle the auricle very carefully every time it is touched to avoid extravasation of the fluids and dye. Though this method can provide insight into thrombus formation, it can also be applied to other systems, such as wound healing, flap failure, and angiogenesis.
After watching this video, you should have a good understanding of intravenous fluorescence dye application, position of the auricle, and illumination of specific vessels in order to perform intravital phototoxic thrombus induction.
