This protocol is capable of producing robust thoracic aortic aneurysms of consistent size and location in mice and serves as a guide for safe access to the murine chest. This protocol is a straightforward, consistent method for producing thoracic aortic aneurysms in mice over a reasonable amount of time compared to other models. After confirming an appropriate level of sedation in an eight to 10-week old male c57 black six mouse, place the mouse in the right lateral decubitus position and tape the right arm rostrally so the front paw is in line with the nose.
Pull the tail caudally to create a line of tension between the right arm and tail, producing extension of the spine, and tape the left leg in its natural position. Then, tape the left arm ventrally over rolled gauze. Before beginning the procedure, use clippers to shave the left flank from the left shoulder to the left abdomen.
Use a cotton-tipped applicator to brush first betadyne solution, then 70%ethanol over the surgical site. When the skin is dry, place a sterile drape over the animal. Make a two-centimeter lateral incision at the midpoint of the hemothorax and use hand-held electrocautery to divide the muscle layers until the ribs are visible.
Under 10 to 15x magnification, directly cauterize a two-millimeter portion between the ribs and use a saline-wetted, fine cotton-tipped applicator in the rib space in order to dissect into the pleural space through the cauterized portion. Place a saline-wetted, three by two-millimeter sponge into the thorax to help collapse the lung. Then, use scissors to widen the thoracotomy until the diaphragm is visible ventrally and the incision is several millimeters away from the paraspinus muscles dorsally.
To expose the aorta, place rib retractors into the thoracotomy incision and carefully remove the sponge from the surface of the lung. Cover the lung with a saline-wetted, six by four-millimeter sponge with the ends pointing rostrally and caudally and place a wide, flat lung retractor on the sponge. Then, gently slide the retractor ventrally until the descending thoracic aorta is exposed and use number seven forceps to dissect an approximately five-millimeter section of connective tissue and fat from the aorta.
To apply the elastase, first, use a micropipette to saturate a 0.5 by one-millimeter sponge with 12 microliters of porcine pancreatic elastase and place the sponge upon the exposed surface of the aorta. After three to five minutes, use number seven forceps to remove the elastase sponge and remove the lung retractor. Irrigate the chest cavity with approximately one half of a milliliter of sterile saline.
Remove the lung sponge and use a rolled two-by-two-inch piece of gauze to absorb the remaining saline irrigation. Remove the rib retractors and depose the ribs with three interrupted six-oh non-absorbable sutures, tying a loose knot in each suture without tying the sutures down. After reinflating the lung, tie the sutures and reapproximate the muscle layers with a running five-oh absorbable braided suture.
Close the skin with seven to 10 interrupted five-oh non-absorbable sutures. Then, monitor the animal until full recumbency. At the appropriate experimental end point, use scissors to incise the skin medially from the left flank to the central abdomen, taking care not to enter the peritoneum.
Incise the skin from the dorsal left flank rostrally to the left shoulder and make an incision at a 90-degree angle through the axilla to the sternum. Using cautery, dissect the skin flap toward the ventral aspect of the mouse to expose the left hemothorax and use scissors to enter the abdomen along the left costal margin from the ventral to dorsal direction to expose the underside of the left diaphragm. If the lateral-most edge of the diaphragm does not open, incise the diaphragm at its most lateral edge.
Placing the tip of the cautery in this hole, cauterize the diaphragm from the costal margin to the xiphoid process and use a damp, fine-tipped cotton-tipped applicator to gently free the lung from any adhesions to the chest wall. While ensuring the lung is out of the way, use electrocautery to cut through the ribs and chest wall from the costal margin to the first rib, dorsal to the mid-axillary line, but at least two millimeters from the aorta. Cut along the superior margin of the first rib and reflect the rib cage ventrally to expose the hemothorax.
Place retractors on the lung and pull medially. Place a retractor or saline-wetted gauze on the diaphragm and draw caudally to expose as much of the aorta as possible. Use a dry, cotton-tipped applicator to remove any adhesions from the aortic aneurysm and an unaffected distal segment and use video micrometry to measure the diameter of the unaffected control segment and the widest portion of the elastase-treated aneurysm.
To harvest the aorta, grasp the vessel with harms forceps just distal to the treated segment and use scissors to cut distal to the forceps. Dissect the aorta off of the spinal column and cut the vessel proximal to the treated segment to remove the aneurysmal tissue. Then, using a tuberculin syringe and needle, wash the aortic lumen with saline and process the sample according to the planned downstream experimental analysis.
The developed thoracic aneurysms as fusiform in shape and occur only in the treated portion on the aorta. In this representative video micrometry measurement, the aortic dilation was determined to be 130%at tissue harvest. Indeed, while type elastase treated mice exhibit a significant increase in aortic dilation at days three, seven, 14, and 21 post-treatment with the maximal dilation observed at day 14 compared to control wild type mice.
Histological analysis reveals that wild type aneurysms have elastin fibers that are thin and fragmented. There is also less smooth muscle cell staining with an increase in macrophage and interlukin one-beta expression. The murine lung is extremely delicate, and injury is usually fatal.
Take the utmost care with the initial entry into the chest and only manipulate the lung with saline-wetted sponges. This model has helped to elucidate the role of interlukin one-beta and interlukin six in the pathogenesis of descending thoracic aortic aneurysms and how their blockade could facilitate non-surgical management of these aneurysms.
