The overall goal of the following experiment is to introduce two valid animal models leading to narrowing of the aorta via different mechanisms. The first model is balloon catheterization of the aorta to create the development of myo intimal hyperplasia. First, the infrarenal abdominal aorta is exposed and clamped.
Then the aorta is opened with a transverse incision and the balloon catheter is introduced. The endothelium of the aorta is then denuded with the inflated catheter and the surgery is completed. The second model is a dietary manipulation of transgenic APOE null mice to create atherosclerotic plaques.
These mice are housed under standard conditions and fed a high lipid western diet for four to six months. Ultimately, histological analysis of the surgically induced myo intimal hyperplasia and the dietary restriction model shows that they result in different luminal narrowing. Today we'll show you two different models to induce luminal narrowing the mouse model using a balloon catheter to induce vessel injury and the arterial sclerosis model.
Using transgenic mice in the Western diet, The balloon catheter model can help answer key questions in the stenosis field. The Transgenic homozygous upper E mutant mouse model is used to study the pathophysiology of atherosclerosis. For the intimal hyperplasia model, obtain C 57 black six J mice that weigh about 25 grams.
Maintain them under normal conditions with a normal diet. After anesthetizing the mouse with 2%isof fluorine, trim off the abdominal hair, provide carprofen by subcutaneous injection for analgesia. Next, alternatively, disinfect the skin with Provo iodine and 80%ethanol in two cycles.
Now check for an absence of reflexes using a toe pinch. Start the surgery by separating the skin and muscle along the linear alba. Next, wrap the intestines in a glove moistened with saline, then attentively, clear away the fatty tissue, sheeting the abdominal aorta.
Now expose the infrarenal aorta down to its bifurcation. Taking care not to injure any branch vessels. Now clamp the aorta.
First place a microsurgical clamp on the proximal infrarenal aorta to shut down the blood flow. Second, apply a second distal clamp next to the aortic bifurcation. When the blood flow is interrupted, make a small incision into the aorta close to the proximal clamp.
Insert the catheter towards the distal clamp of the aorta to make an endothelial injury. Denude the aorta by inflating the catheter balloon carefully and slowly pulling the catheter into the direction of the proximal clamp. Repeat this, maneuver twice for a total of three aorta inflations.
Now remove the catheter and close the aortic incision using 10 au proline running sutures. With sutures in place, carefully open the distal clamp if there is bleeding at the AOR autotomy site. Close the clamp again, locate the bleeding and stop it with additional interrupted stitches.
When there is no bleeding, slowly open the proximal clamp. An aortic pulse should be visible distal to the incision. Now arrange the intestines in C two.
Next, close the abdominal wall beginning with the muscle layer using six OTT proline running sutures. Then close the skin with five OTT sutures and transfer the mouse back to its cage. Over the first three postoperative days, provide metrazole to the drinking water for pain control.
Monitor the animal throughout its recovery, which takes about 28 days. For the atherosclerosis model, obtain four week old A POE deficient mice. House these mice under standard conditions with a high lipid western diet for four to six months.
Maintaining this diet throughout the experiment. Six hours before harvesting the aorta, take away access to food. Then prepare the mouse for surgery as described in the previous section.
Start the surgery by opening the chest. Next, cut off the apex of the heart and perfuse it under gentle pressure with three milliliters of 4%Paraform aldehyde through the aortic root to fix the aorta. Then free the aorta from the root to its bifurcation.
Try to remove as much of the fatty tissue as possible. Next, cut off the aortic arch and store it in 4%para formaldehyde. Now transfer the heart and aortic tree to a silicone line dish with PBS in the dish.
Cut one end of the aortic tree and fix it onto the silicone layer with fine pins. Open the aorta longitudinally and allow the aortic tissue to lie in PBS at room temperature for a few minutes. Then rinse the tissue once with 50%ethanol and immerse it into a bath of Sudan.
Three, staining solution for about 15 minutes. After staining, quickly rinse the tissue with 50%ethanol for a few seconds to remove just the excess Sudan. Three over rinsing will remove too much stain, immediately rinse the tissue with distilled water and proceed with imaging For the myo intimal hyperplasia model, denuded mouse aortas were harvested, paraffin embedded, and stained with chrome Im ratios were calculated from the tri chrome stain sections by measuring intima and media thickness.
Using computer analysis followed by calculation of the ratio, maximum plaque thickness was measured as the maximum intimal thickness in the lumen by computer morpho tree. The percentage of luminal obliteration was calculated as the ratio of the new inner lumen from the former lumen. The plaque area was calculated by subtracting the new inner lumen from the former lumen.
The same analyses were performed on tri chrome stained cross sections of the atherosclerosis model at the aortic valve, the aortic arch, and the descending aorta for just the atherosclerosis model. The descending aorta was opened longitudinally and stained with Sudan red. The plaque area and the total aortic area were measured with the aid of a computer For analysis of both models.
Other methods such as histopathology and the confocal immunofluorescence microscopy can be performed in order to answer questions like luminal oblation, morphology, and the cellularity. Thank you for watching and good luck with your experiments.
