The overall goal of this procedure is to ligate the left pulmonary artery. This is accomplished by first intubating and ventilating an anesthetized rat. Next, a thoracotomy is performed and the left pulmonary artery is ligated.
After the ligation, the chest is closed and the rat is allowed to recover. Ultimately, the extent of neovascularization in the left lung can be assessed by measuring the level of fluorescence in the tissue or by other histological methods. The main advantage of this technique is that it provides an opportunity to study the in vivo development and neovascularization of the bronchial circulation in the lung.
This provides a a, an opportunity to study the complex 3D structure of this new vasculature in a ventilating lung. Now, this is very much different from what is currently done by most investigators who form who, who perform in vitro studies studying endothelial cell proliferation and chemotaxis or tube formation. Generally, individuals new to this procedure will struggle with trying to do the dissection between the left pulmonary artery and the left bronchus.
The only advice I can offer is to practice, to be patient and to view this video frequently to see if there are minor adjustments in the surgical approach. Demonstrating this procedure is John Jenkins, a technician from my laboratory. To begin place an anesthetized rat on a surgery board and attach a nose cone for the continuation of anesthesia, intubate the animal with a plastic catheter and connect the rat to a ventilator.
After applying ointment to the eyes, place the rat on its right side and tape down the appendages. Remove the hair over the surgical area and prepare the skin with repeated scrubs of Betadine and alcohol. When ready, make a transverse incision through the skin.
Next blunt, dissect through the layers of fat and tissue to reveal the rib cage. Once exposed, identify the third intercostal space and use forceps to make a blunt incision between the third and fourth rib. Insert rib separators into the opening and pull the ribs open gently until the lung is visible.
Secure the opening. First, move the left lung back with the right hand. Then with straight forceps, grab the left pulmonary artery and airway with the left.
The left pulmonary artery should lie on top of the airway and the forceps should be perpendicular to the table. Once aligned, properly use carved forceps to separate the artery from the left main stem bronchus at their natural borders. This separation line appears thin and white between the two individual structures.
Taking care not to go through the vessel. Slide the tips of the forceps along the separation. Continue in this manner until the left pulmonary artery lays on the curve of the forceps.
With the left hand gently release the straight forceps and grab a piece of precut suture. Open the curved forceps, cradling the artery enough to grab the suture. Gently pull the suture through in an upward motion relative to the curve of the forceps.
Once through, tighten the suture to occlude the left pulmonary artery and snip away any remaining suture while holding them together with forceps. Suture the ribs. Taking care not to suture the skin loosely.
Tie a square knot that will later close the ribs. Hyperinflate the lung by briefly occluding the ventilator expiratory line, and then tighten the suture. Secure the suture by making another full knot before snipping away the ends.
Remove the end expirator pressure and watch for 30 seconds to ensure that the lungs do not collapse. Lastly, apply five drops. Bupivacaine, close the skin with tissue glue.
Turn off the isof fluorine gas, but continue to ventilate the rat for one to two minutes on room air until spontaneous respiration returns to a steady state. Next, disconnect the tracheal tube from the ventilator and ensure that the rat is breathing spontaneously before removing it. Administer a local analgesic periodically over the next 24 hours.
Lastly, wipe the ointment from the eyes and monitor the animal until it is recovered at desired time. Points after ischemia labeled microspheres can be injected. To assess the magnitude of bronchial perfusion, a small volume infusion pump is used to deliver labeled microspheres into the left carotid artery at a constant rate.
Connect the catheter to a blunt 25 gauge needle and a four-way stopcock. To begin follow the surgical preparations demonstrated earlier. When ready, make an incision over the midline along the neck blunt.
Dissect the tissue to reveal the trachea and left carotid artery. Once isolated past two sutures under the carotid artery, tie the proximal suture firmly. Next, make a small cut in the artery and insert a saline filled catheter.
Secure this in place with the upstream suture. This vascular cast provides a remarkable visualization of bronchial angiogenesis in the lung. 28 days after left pary artery ligation here, an airway section from the left lung three days after ation is shown.
Note the two prominent bronchial vessels filled with red blood cells located within the airway wall. The inset is stained to show proliferating endothelial cells of the bronchial artery. This example shows a left lung section taken 14 days after ligation.
Note the increased size of the bronchial vessels at this time point and the withered pulmonary artery fluorescence used to assess the magnitude of systemic perfusion was virtually undetected in the left lung of naive rats. However, there was a substantial, significant and consistent level measured in the left lung from rats. Studied 14 days after ligation Once mastered.
This technique can be done in less than an hour if it is performed properly. After watching this video, you should have a good understanding of how to intubate the rat, perform the thoracotomy, and successfully ligate the pulmonary artery. Depending on your outcome variable, you should be able to cannulate the left carotid artery and infuse labeled microspheres for perfusion analysis.
