組織と血液サンプリング手順の標準化による閉胸ブタ心筋梗塞モデルの改善

The overall goal of this porcine closed chest myocardial infarction model is to investigate myocardial ischemia reperfusion injury. This method can help to answer key questions in cardiovascular research such as investigating the pathophysiology of ischemia reperfusion injury in the context of acute myocardial infarction. The main advantage of this technique is that it allows an accurate determination of the infarct size and precise sampling technique for further analysis.

Prepare the pig for the surgery and administer anesthetics as described in the text protocol. Start the dissection by freeing the carotid arteries on both sides. Cannulate them with 7-French sheaths.

Next, cannulate the left jugular vein with a 7-French sheath for venous blood sampling. Then use standard blood collection tubes to withdraw baseline blood samples from the venous line. Collect five and 2.9 milliliters of blood into citrated and EDTA plasma collecting tubes respectively and immediately centrifuge at 2, 000 g for 15 minutes at four degrees Celsius.

Next, collect 2.9 milliliters of blood into a serum tube and allow it to coagulate for 30 minutes at room temperature, then centrifuge it at 2, 000 g for 15 minutes at four degrees Celsius. Next, use fluoroscopic guidance to insert a pressure catheter via the previously placed sheath into the left carotid artery. Advance it into the left ventricle and then connect it to an acquisition system to record left ventricular pressure.

Set up the acquisition system to continuously calculate and record the heart rate, the developed pressure, the contractility of the left ventricle, and the relaxation of the left ventricle during the entire experiment. Next, insert a guiding catheter via the previously placed sheath into the right carotid artery. Using x-ray guidance, advance the catheter into the left coronary artery and then to the LAD or Left Anterior Descending coronary artery.

Once in position, inject contrast medium into the guiding catheter from a 20 milliliter syringe to perform a baseline coronary angiography. Next, assembly the PCI catheter and the appropriately sized coronary guide wire, then connect the PCI catheter to the inflation device prefilled with contrast medium. Insert the assembled system into the lumen of the guiding catheter and advance the guide wire into the LAD until it reaches beyond the first diagonal branch of the LAD.

Next, use fluoroscopic guidance to advance the PCI catheter until it reaches about the middle of the LAD. Choose the LAD blocking site as described in the text protocol. Now remove the guide wire and increase the pressure in the inflation device to seven to 10 bar to inflate the balloon of the PCI catheter and induce myocardial ischemia.

Keep the balloon inflated for one hour. Just after starting the ischemia, record five to 10 seconds of video while injecting contrast medium to have an angiogram of the balloon of the PCI catheter. Repeat this after 10 minutes to verify the occlusion of the LAD.

From 15 to 40 minutes into the ischemia, gradually increase the fraction of inspired oxygen from 50 to 60%Always be quick to treat cardiac arrhythmias which typically occur between 20 and 40 minutes after coronary occlusion. An effective treatment to reestablish a regular heartbeat is to gently massage the neck on both sides just below the cheek. Start this treatment when extra systoles occur more than three times per minute, then inject the test substance intravenously through the central venous line and flush the line with 20 milliliters of saline.

Next, perform another angiogram to confirm that the LAD is still occluded, then deflate the balloon of the PCI catheter and remove it from the guiding catheter. Immediately after removing the catheter, confirm the perfusion of the LAD distal to the occlusion using another angiogram. Take another angiogram after 10 minutes and whenever signs of myocardial ischemia are visible by ECG for more than five minutes.

The ischemic myocardium is now allowed to reperfuse for two hours. Take plasma and serum samples at 10, 30, 60, and 115 minutes into the reperfusion. Also monitor blood gas analysis at 60 and 115 minutes into the reperfusion.

After two hours of reperfusion, perform another angiogram, then reocclude the LAD. Reinsert the PCI catheter together with the guide wire to exactly the same position as used for the ischemia. Inflate the PCI catheter and confirm LAD occlusion by angiogram.

Now remove the pressure catheter from the left ventricle and stop the recording. Finally, inject 100 milliliters of 2%Evans Blue in PBS into the central venous line and proceed with euthanizing the animal. After excising the heart, rinse it out with room temperature saline and weigh it.

Within 30 to 40 minutes, cut the heart into slices starting about three to five millimeters from the apex and moving toward the chordae tendineae of the mitral valve. Cut perpendicular to the long axis using a sharp knife. Next, cut away the right ventricle and weigh the left ventricle slices to obtain its total weight.

Then use a scalpel to dissect the slices to separate the Evans Blue negative tissue, which is ischemic, from the non-ischemic tissue which is Evans Blue positive. First, analyze the Evans Blue negative sections. They are the areas at risk.

Weigh them all and put them all into a plastic container, then submerge them in 100 to 150 milliliters of freshly made TTC solution and incubate them for 20 minutes. During the incubation time, take samples from the most distal part of the injury. Embed them in OCT compound and store them at minus 80 degrees Celsius for further analysis.

Transfer the remaining tissue into a 4%formaldehyde solution and store it at room temperature for future analysis. Now remove the pieces of the areas at risk from the TTC solution. The red-stained tissue is viable ischemic tissue and the non-stained tissue is necrotic ischemic tissue.

Cut two small blocks of similar mass from both tissue types and embed them into OCT compound, then store at minus 80 degrees Celsius for further analysis. Store the remaining tissue in 4%formaldehyde at room temperature for 24 hours. The tissue is then stained and the Areas AT Risk or AAR were compared with the areas Not At Risk or ANR.

The AAR expressed as a percentage of the LV showed no statistically significant differences between the FXIIa treated group and the control group. The infarct size also showed no differences between the groups. These data suggest that FXIIa inhibitor alone at the concentration used and duration of application could not protect the heart from myocardial IR injury.

The cardiac muscle damage marker troponin-I was monitored in the blood. There was almost no change after one hour of ischemia, but after reperfusion, troponin-I levels steadily increased. After watching this video, you should have a good understanding of how to calculate accurately the infarct size and perform correct sampling.

Once mastered, this technique can be done in six hours if it is performed properly. While attempting this procedure, it's important to remember to have a similar ischemic area at risk, inject Evans Blue intravenously, use freshly prepared TTC, and accurately calculate the NIT. Following this procedure, other methods like changing the ischemia and the reperfusion time can be performed in order to answer additional questions like the long-term effect of the treatment used to reduce reperfusion injury.