The overall goal of this procedure is to study the development of intimal hyperplasia in a humanized model. This is accomplished by first denuding, a section of human mammary artery or IMA, using a two French fogerty catheter. Next, a stent is inserted into the IMA.
Then a segment of rat abdominal aorta is replaced with a stented IMA. Finally, the wound is sutured and the animal is allowed to recover the endothelial damage due to the invasive procedure of balloon dilatation and stenting results in inflammation and the production of inflammatory cytokines. This leads to the proliferation of smooth muscle cells, which progressively occlude the vessel lumen resulting in stenosis.
Ultimately, results can be obtained that show the proliferation of human smooth muscle cells through immunofluorescence confocal microscopy. Today we will show you a model for the assessment of wrist stenosis in human arteries in a red host. The advantage of this model of existing techniques like the abdominal wet aortic standing, is that we can assess human stents in human tissue.
We first had the idea for this method when we were aiming to find a translational in vivo model for vascular remodeling. To prepare the IMA begin by using a two French Fogarty arterial embolectomy catheter to denude the arterial endothelium by moving the catheter into the vessel to ensure endothelial damage. Insert a human stent of eight millimeters in length and 2.5 to three millimeters in diameter into the vessel.
Using the appropriate balloon pressure, deploy the stent to achieve the desired diameter. Store the stented IMA on ice at four degrees Celsius in RPMI and Heparin until transplantation. After anesthetizing, a rat with isoflurane, shave the abdominal hair, place a face mask over its nose and mouth to maintain 2%anesthesia using provo iodine, disinfect the abdominal area widely and follow it with 80%ethanol.
Repeat these steps twice. Pinch the hind feet to be sure that the rat is sufficiently sedated. Next, perform an upper median laparotomy to expose the infrarenal abdominal aorta.
Then place the intestines in a saline moisturized glove and fold the glove around the intestines to prevent loss of moisture Under a surgical microscope, dissect the aorta from the infrarenal region to the bifurcation, taking care not to cause damage on the branches of the vessels. To stop aortic blood flow, use micro clamps to first clamp the proximal end, then the distal end. Remove an approximately 0.5 to 0.7 millimeter aortic segment and flush the remaining aorta with 200 units of heparin.
Next, shorten the stented IMA to an adequate length and position it into the aortic gap. Then using running sutures, connect the IMA to the recipient aorta. Carefully open first the coddle and afterwards the cranial clamp.
There should be a visible pulse in the transplanted IMA and at the distal end of the aorta. Place the intestines back into the abdomen and flush it with prewarm sterile saline. Use six aut proline running sutures to close the muscle layer of the abdominal wall, followed by the skin layer.
While the rat is still under anesthesia subcutaneously. Inject four to five milligrams per kilogram of carprofen. Monitor the animal daily until removal of the IMA to identify the origin of proliferation.
Human IMA was stably transduced to overexpress GFP in the vessel. Histological sections were stained with antibodies against GFP shown here in green and human smooth muscle actin or SMA seen in red. The cell nuclei are in blue.
Shown here is emerged confocal image showing the origin of proliferated cells. This 3D video shows the colocalization of SMA in red and GFP in green Following surgery. Other methods like histopathology and the confocal immunofluorescence micro can be performed in order to answer the questions like luminal obliteration, morphology, and the cellularity.
This method is a promising model for translational research. Thank you for watching and good luck with your experiments.I.
