塩化第二鉄によるイヌ総頚動脈血栓症: 血管損傷の大動物モデル

This method can help answer key questions in the thrombosis field, allowing the translation of therapeutic treatments in a large animal model. The main advantage of this technique is that the large animal model allows for monitoring of physiological parameters relevant to humans, such as complete blood count and platelet function. This experimental technique allows us to study thrombosis and the disorders that result in thrombosis.

The advantages include that it is reproducible, it can be monitored by angiography, we can capture histology, and perform serial blood sampling throughout the procedure. Before inducing the injury to determine the extent of canalization of the carotid artery and the length of the occlusive thrombus, feel for a pulse in the right inguinal region of an anesthetized adult canine and make a three-to five-centimeter ventral sagittal incision. Use blunt dissection to expose and separate the femoral artery from the surrounding tissues, and use a right-angle hemostat to place zero-silk sutures around the proximal and distal ends of the exposed artery.

Tie the distal zero-silk suture and clamp the loose zero-silk suture ends to the skin with hemostats to apply slight tension to the artery. Use an 18-gauge introducer needle to puncture the artery midway between the proximal and distal zero-silk suture ties and allow the insertion of a guide wire into the artery. Put an assembled 6 French sheath and dilator onto the guide wire, grasping the wire as it exits from the assembly.

Advance the dilator and sheath over the guide wire, taking care to maintain a firm grip on the wire as it protrudes from the dilator. After full insertion, tie the proximal zero-silk suture around the sheath to maintain hemostasis at the arterial puncture site. Then simultaneously remove the dilator and the guide wire, and open the one-way valve to verify the presence of pulsatile blood flow.

Flush the valve with three to five milliliters of normal saline, and connect the access sheath one-way valve to a fluid-filled extension line attached to a pressure transducer to monitor and record invasive blood pressure measurements. Preload a 0.35-inch guide wire into a 4 French angiographic catheter, and connect the catheter to the contrast injection manifold set with a two-way Y adapter. Pull the tip of the guide wire just inside the distal tip of the catheter, and flush the entire assembly with saline.

Insert the angiographic catheter into the hemostatic valve of the sheath, and advance the guide wire about five centimeters beyond the distal catheter tip. Under fluoroscopic guidance and using a retrograde transaortic approach, slowly advance the catheter into the aortic arch. Inject two to three milliliters of contrast agent diluted one to one with normal saline to identify the takeoff of the brachiocephalic trunk.

Use the guide wire to direct the catheter into the brachiocephalic trunk and selectively into the right common carotid artery. Remove the guide wire and inject two to three milliliters of diluted contrast to verify that the catheter is placed within the carotid. Then, inject three to four milliliters of undiluted contrast agent while recording angiographic runs using both digital subtraction and standard angiographic techniques.

Make an eight-to 10-centimeter incision of the skin directly on top of the right common carotid artery region, and dissect the fascia to isolate the vessel from the surrounding tissue. Carefully use forceps to separate the carotid artery and the vagus nerve. Place the Doppler flow probe around the carotid, allowing ample space to wrap the umbilical tape around the carotid without touching the flow probe, and record the baseline blood velocity for about five minutes before using a hemostat to place a piece of prepared ferric chloride umbilical tape below the flow probe.

After 15 minutes, remove and discard the tape and stabilize the occlusive thrombus for 45 minutes, adding additional saline to the probe as necessary to maintain signal. To determine the stroke or hemorrhage volume, in addition to the downstream thromboembolic events that may result from pharmacological interventions, the canine can be transported to a magnetic resonance imaging machine. While still under a deep surgical plane of anesthesia, collect whole blood as appropriate for later analyses.

After exsanguination, rinse the heart with PBS and flash-freeze the samples in liquid nitrogen if desired. Before removing the carotids, measure and record the clot length, and place a tissue marker on the middle of the clot. Mark the contralateral carotid at the same length, and remove the entire length of both vessels for fixation in 10%formalin.

Flash-freeze half of the contralateral carotid artery in liquid nitrogen for later analysis. Rinse the brain in PBS, and use a sharp scalpel to laterally remove two four-millimeter sections from the middle of the brain. Then place one four-millimeter section into 2%TTC for ischemic demarcation, and place the other tissue sample in 10%formalin for seven days for subsequent hematoxylin and eosin staining.

Data from the baseline flow velocity recording can be used to determine the percent of re-perfusion with carotid artery injury and treatment in this canine model. Staining of the contralateral and injured canine carotid sections can be used to verify the recanalization status at the time of sacrifice. Carotid artery angiography as demonstrated can be used to determine if the thrombus is occlusive.

In addition, using the square flow probe as a marker, the length of the thrombus can also be determined at each time point that the angiogram is obtained. As demonstrated in these diffusion and T2-weighted magnetic resonance images, the size of both the hemorrhage and the stroke volume can be identified in different levels in areas of the brain for quantification at each time point of interest. Further, TTC staining can be used to delineate brain tissue in which the cells are still metabolically active, while hematoxylin and eosin staining can be used to identify areas where the hemorrhage has occurred.

This technique, once mastered, should take approximately eight to 10 hours when properly performed. If imaging is going to be included as part of this procedure, please make sure to consider anesthesia as part of the transport to MR or CT for analysis. Using this protocol, procedures like endovascular thrombectomies can be utilized to look at the effect of recanalization or even the effect of the vascular device on the endothelial surface.

After watching this video, you should have a good understanding of how to perform a ferric chloride acid-induced injury model in a large animal. Remember, that when performing the angiography portion of this protocol that one, it's iodine-based dyes that are used, and so if there's an allergy present, one should be very careful. Secondly, the angiography utilizes radiation, so everybody in the room should have lead on.