The overall goal of this procedure is to produce ferric chloride-induced thrombosis in a mouse carotid artery model to obtain highly reproducible results as analyzed by intravital microscopy. These mice can help answer key questions about various disease or genetic disorders associated with platelet activation and the aggregation of closed vascular system. The main advantage of this technique are that it is simple and sensitive to both anticoagulant and antiplatelet drugs, and that it can be combined with other techniques.
This method can be used to gain valuable insight into the pathophysiology of thrombosis, discover new mechanisms and therapies, and evaluate the effects of Novel drugs, including targeted nanomedicine on thrombosis. Visualization of this method is critical for learning the strategies for obtaining accurate blood flow cessation time. After confirming a lack of response to toe pinch, use small electric animal clippers to remove the fur from the neck and upper chest of an eight to 12-week-old mouse.
Apply neutral petroleum eye ointment to animal's eyes, and tape the mouse in the supine position on the lid of a 15-centimeter tissue culture plate. Wrap a 4-0 suture around the incisors, and tape the ends of the suture to the lid to keep the neck straight. Then, move the animal under a surgical light source, and use an alcohol pad to sterilize the surgical site.
Next, use Micro-Adson forceps to grasp the skin, and use surgical scissors to make a two to three-millimeter incision. Holding the incision with forceps, insert the scissors with the blades closed, and push the scissors subcutaneously toward the manubrium. Then, open the blades to free the skin from the subcutaneous layer.
Now, make a midline incision from the manubrium to the hyoid bone, and use a fine hemostat and Graefe forceps to bluntly dissect the thin fascia between the submaxillary gland. When the trachea is visible, use the forceps to hold the soft tissue to the right of the manubrium, and insert the hemostat under the fascia toward the two o'clock position. Open the jaws of the hemostat to free the jugular vein from the surrounding tissue.
Then, continue the incision through the fascia, soft tissue, and skin toward the two o'clock position until the right jugular vein is exposed. Now, attach a 30-gauge needle attached to a one-milliliter syringe containing 100 microliters of rhodamine 6G solution, and use a needle-holder to bend two to three millimeters of the tip 90 degrees. Then, holding the syringe with one hand and the soft tissue with the distal site of the jugular vein with the Graefe forceps in the other, insert the needle into the vessel.
Stabilize the needle with the forceps, and inject the rhodamine. Once all of the platelet labeling solution has been administered, clamp the injection site with the forceps to prevent bleeding by opening the hemostat and inserting the instrument under the forceps to clamp the vessel wall of the injection site. Then, use a 6-0 suture to ligate the injection site.
Bluntly dissect the soft tissue and fascia around the left submaxillary gland, and pull the gland toward the seven o'clock position to expose the left sternocleidomastoid muscle. Then, bluntly dissect the fascia between the left sternocleidomastoid muscle and the omohyoid and sternohyoid muscles to the left side of the trachea. When the fascia has been displaced, pass a needle with a 15-centimeter, 6-0 silk suture under the middle of the sternocleidomastoid muscle, and pull the two ends of the suture laterally toward the 10 o'clock position.
Bluntly separate the thing sternohyoid and omohyoid muscles to expose the carotid artery. Then, use the hemostat to separate the soft tissue from the carotid artery without touching the artery. Using a fine-tipped forceps, lift the lateral fascia around the carotid artery while avoiding the vagus nerve and the artery.
Then, use another fine-tipped forceps to puncture the fascia between the carotid artery and the vagus nerve. Now, pass a blunt hook through the hole to gently lift the carotid artery, and slide the fine-tipped forceps under the artery. Moving the hook and forceps in opposite directions along the artery, strip about five millimeters of advantigial soft tissue from the vessel from the surrounding tissue.
Then, rinse a piece of U-shaped plastic with saline, and use the hook to gently lift the artery to allow for the placement of the plastic under the artery. The plastic will block the background fluorescence, preventing diffusing of the ferric chloride solution to the surrounding tissue, and they ensure that only the carotid artery is visible under the microscope. Immediately hydrate the carotid artery with two to three drops of saline.
Then, transfer the mouse onto a fluorescent microsope stage under the 10X water lens, and fill the space between the lens and the artery with saline. Now, start the digital video recording software application, and obtain images of the normal vessel. If the vessel wall is integrated and uninjured, transfer the mouse to the Gibraltar platform, and use the corner of a paper towel to carefully blot the saline around the carotid artery in the surgical field without touching the artery.
Then, use fine-tipped forceps to transfer a piece of ferric chloride-saturated filter paper directly on top of the carotid artery close to the distal end of the exposed artery. Make sure to place the filter paper close the distal end of the exposed carotid artery, which we will leave a short segment at the proximal side for observing the blood flow during the later phase of the injury. After one minute, remove the filter paper, and rinse the artery with at least two milliliters of saline.
Return the mouse to the microscope stage, and obtain images of the vessel. The formation of the thrombus can be identified by the accumulation of the fluorescent platelets. Continue to image the vessel for 10 seconds every minute for the first 10 minutes, followed by 10 seconds every other minute until the end of the experiment.
Tissue-type plasminogen activator profusion five minutes after ferric chloride injury demonstrates the initiation of thrombolysis about four minutes after the treatment. With the formed thrombi observed to undergo repeated size variations during the 30 minute observation period, but without a complete ablation of the platelet-mediated thrombus. Treatment with a polyclonal PAR4 antibody that delays PAR4 cleavage significantly prolongs the time to occlusive thrombus formation in the ferric chloride-induced carotid artery thrombosis model.
Non-platelet red blood cell labeling experiments indicate that, while ferric chloride-induced thrombi are made up exclusively of vessel wall-adherent platelets, red blood cells do become trapped within the thrombus, contributing to the shape and occlusive property of the clot over time. Interestingly, while the initial thrombus is visualized within about 15 seconds of the injury to the mesenteric artery and vein, the formation of the thrombus within the mesenteric vessels is dramatically delayed to approximately 17 minutes in the mesenteric vein. And to nearly 24 minutes in the mesenteric artery, due at least in part to diffusion of the ferric chloride through the adipose tissue surrounding the mesenteric vessels.
Once mastered, this refund ferric chloride injury induced thrombosis model can be finished in about a 30 to 60 minutes depending on the mastery used, if it is performed properly. While attempting this procedure, it is important to remember that the measure it will permit us is the elapsed attempt from the injury to the complete ways of occlusion, and a precise observation of the blood flow is critical for obtaining accurate results. This model may be combined with a transfusion of ex-vivo treated wild-type platelets to mimic platelet-specific knockouts for defining the function of regulatory pathways and platelets.
This technique paved the way for researchers in the field of vascular biology to explore not only the role of platelets in thrombosis, but also under various other endothelium inflammatory conditions. After watching this video, you should have a good understanding of how to set up a ferric chloride-induced carotid artery thrombosis model in mice to achieve highly reproducible result using intravital microscope. Don't forget that ferric chloride can be extremely hazardous, and that precautions such as wearing the proper personal protective equipment should always be taken while performing this model.
