The overall goal of the surgical procedure is to provide a simple platform for studying myocardial hypertrophy, remodeling, and dysfunction. This method can help answer key questions in the cardiovascular field such as the signaling involving cardio hypertrophy and remodeling. As well as the discovery of novel therapeutic targets in hypertensive heart disease.
The main advantage of this technique is that it is a minimally invasive method that requires very simple surgical techniques and yet the results are highly reproducible. The implications of these techniques extend to our therapy of hypertensive heart disease. Since constriction of the abdominal aorta induce pressure overload and eventually leads to cardio hypertrophy, remodeling, and heart failure.
To begin prepare a 22 gauge syringe needle by using a honing stone to blunt the tip. Then using pliers, plicate the needle at a right angle. Prepare the required surgical materials including the recovery cage and autoclave the surgical instruments.
Maintain the rats around 200 grams and keep them under 12 hour light-dark cycles at a controlled temperature with free access to food and water. Place the rat in a supine position on a surgery platform with a heating pad to maintain the body temperature. Completely shave the surgical area to avoid contamination of the wound through contact with fur.
Then, with betadine or another cleasing reagent, scrub the cleanly shaven abdomen. Next, using a scalpel, make a two centimeter incision along the midline of the abdomen. Then, using normal saline, moisten the abdominal organs and keep the organs moist during surgery.
Using cotton balls carefully displace the digestive organs to the side to expose the inferior vena cava and the posterior peritoneal region. Then identify the abdominal aorta which lies juxtaposed to and usually to the left of the inferior vena cava. Now, with forceps, pierce the peritoneum to uncover the vessels beneath.
Gently isolate the abdominal aorta adjacent to the renal arteries and pass an eight centimeter long 4/0 silk suture underneath the vessel between the origins of the right and left renal arteries. Make a loose double knot with the suture and leave a three millimeter diameter loop. Then place the blunted and bent 22 gauge needle inside the loop.
Tighten the knot around the aorta and the needle and then immediately remove the needle to achieve a 0.7 millimeter diameter constriction. With 6/0 sutures close the abdominal cavity. Then use simple interrupted sutures to close the muscle and skin incisions.
Use iodine tincture to scrub the surgical site to prevent infection. To prevent postsurgical pain treat the animal with acetaminophen and closely observe the animal until it regains sufficient consciousness as indicated by free movement and food intake and maintains sternal recumbency. At 10 weeks postsurgery weigh the rat.
After anesthetizing the animal and confirming the depth of anesthesia place the rat on a metal tray. Now make a five centimeter incision at the thoracic region around the midline of the xiphoid process. Using sharp forceps pierce the diaphragm.
Then, with scissors, cut and remove the rib cage along the midclavicular lines on both sides to expose the heart. Carefully excise the heart along the cardiac and vascular borders. Then gently remove the heart without grabbing the tissue.
Mount the heart on a modified Langendorff perfusion apparatus by tying the aortic trunk to the perfusion needle. Then use Krebs buffer to perfuse the heart and wash out the blood. Weigh the heart and calculate the heart weight to body weight ratio.
Then, while wearing a mask, place the organ in 4%paraformaldehyde and fix it on ice overnight. After fixing the heart tissue place it on a tissue sectioning device, cut it into two millimeter thick slices, and place the sections in an embedding cassette. Following paraffin embedding and additional sectioning according to the text protocol, deparaffinize the slides by placing them into a tank with xylene for 30 minutes.
Then rehydrate the tissue in a sequentially diluted alcohol series followed by distilled water. Next use adequate picrosirius red solution to completely cover the tissue sections and incubate for one hour. Then use a 0.5%acetic acid solution to rinse the slides two times.
And rinse the samples twice in absolute alcohol. Air-dry the samples and mount them in synthetic resin with a cover slip. Then use visible light under a microscope at 200X magnification to image the slides.
Finally, calculate the percentage of picrosirius red positive zone over the total area which indicates the extent of fibrosis. As shown here, cardiac volume increased after abdominal aortic constriction surgery. As demonstrated by a higher heart weight to body weight ratio which is an indicator of cardiac hypertrophy.
By using picrosirius red staining, fibrotic myocardium with increased collagen content can be distinguished from normal areas. As demonstrated in these stained samples, cardiac fibrosis was increased after abdominal aortic constriction surgery as compared to controls. Once mastered the surgery can be done in 30 minutes if it is performed properly.
Following this procedure, other methods like echo cardiography can be performed in order to answer additional questions about cardiac function. After it's development this technique paved the way for researchers in the field of hypertensive heart disease to explore the mechanisms involving cardiohypertrophy, remodeling, and failure. After watching this video you should have a good understanding of how to perform abdominal aortic constriction to induce cardiohypertrophy and myocardial remodeling in rats.
