A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

This protocol identifies four key areas to help improve the methodology and have reliable lung transplantation. First is a reproducible cuffing technique. Second is accurately and reproducibly identifying the fourth intercostal space, a thorough closure of the wound, and then how to recover and obtain samples for analysis.

These techniques help develop a reliable method of lung transplantation to work on molecular mechanisms and develop therapeutic interventions. Begin with the surgical device setup by turning on the heart rate/oxygen saturation monitoring equipment and warming up the board to 42 degrees Celsius. Also, turn on the ventilation and anesthesia machine to prewarm the isoflurane evaporator.

Fill an anesthesia syringe with 10 milliliters of liquid isoflurane and mount the syringe onto the ventilation and anesthesia machine. Next, turn on the surgical microscope with the height and focus adjusted to the preferences and start the electrocautery device. Likewise, prepare and lay out the surgical tools.

Determine the correct gauge size of the angio-catheter to be used to make the cuffs based on the body weight of the rat by referring to table one. After preparing the cuffs for the pulmonary artery, bronchus, and pulmonary vein, place the angio-catheter of the appropriate size on a sterile surface under the surgical microscope. Then, use a rib-back surgical blade number 11 to cut the angio-catheter to a 90 degree angle to form a two millimeter long cuff body with a one millimeter width and height at the top of the cuff body.

After preparing the wrap for the surgery, make a three to four centimeter-long midline skin incision, mid neck, with the scissors, and carefully dissect out the subcutaneous tissues and muscles using forceps. For the endotracheal intubation, thread a 4-0 silk suture around the trachea. Then, insert 16 gauge angio-catheter into the trachea and tie the suture around the trachea tightly with a double knot, followed with a single knot to hold the angio-catheter in place.

Connect the angio-catheter to the ventilator to maintain a surgical plane of anesthesia in the rat with 1 to 2%isoflurane. Use the scissors to perform a laparo-sternotomy, as a combined midline and transverse incision. Then, inject 1, 000 units per kilogram of heparin with an insulin syringe via the inferior vena cava, or IVC, and allow 10 minutes for systemic circulation.

While the lungs are are ventilating, cut the right and left auricles with micro dissecting spring scissors and then introduce an 18 gauge angio-catheter through the pulmonary artery to gravity flush the lungs with 20 milliliters of preservation solution in a syringe hanging at 28 centimeters of water, connected to the tubing and angio-catheter. Disconnect the ventilator from the endotracheal tube to connect to a five milliliter syringe filled with a proper volume of air based on body weight. After one hour of the warm ischemia, excise the heart-lung block with the micro dissecting spring scissors and forceps and place on a sterile gauze dampened with ice cold PBS on a sterile Petri dish on ice.

Carefully trim the left lung hilar area with Vannas-Tubingen spring scissor and procure the left pulmonary vein, pulmonary artery, and bronchus. Use a mosquito hemostat to grab the cuff tab. Then, take the distal end of the pulmonary vein, pulmonary artery, or bronchus through the appropriate cuff body and evert the extra tissue around the cuff before securing with 8-0 nylon suture.

Once done, use Vannas-Tubingen spring scissors to trim the extra tissue and the cuff around the cuff body. To prepare the recipient rat, draw a line on the chest of the rat from the suprasternal notch to the xiphoid process. At the middle of this line, draw a line along the left side of the chest that measures half of the measurement from the suprasternal notch to the xiphoid process.

Using the fiber optic cable connected to an LED light from the endotracheal intubation kit, visualize and intubate the recipient with a 16 gauge angio-catheter. Once the lungs are pulled out, place a sterile gauze around the left lung while holding it with Dieffenbach bulldog clamp and apply a Yasargil clamp on the left lung hilar area as proximally as possible. When the rat is prepared, proceed for the pulmonary vein anastomosis by placing 7-0 nylon suture around the recipient's pulmonary vein.

Incise the recipient's pulmonary vein using the Vannas-Tubingen spring scissors by transversely cutting the upper and lower segmental veins as distally as possible, and flush out blood with 0.2 milliliters of one international unit per milliliter heparinized saline using an insulin syringe. Place the donor lung wrapped with an ice-cold, wet, sterile gauze into the thoracic cavity, and insert the donor's cuffed pulmonary vein into the recipient's pulmonary vein before securing the anastomosis with the prepositioned 7-0 nylon suture. For the bronchial or bronchus anastomosis, place a 7-0 nylon suture around the recipient's bronchus before incising the recipient's bronchus by transversely cutting the upper and lower segmental airways as distally as possible.

After inserting the donor's cuffed bronchus into the recipient's bronchus, secure the anastomosis with the prepositioned 7-0 nylon suture. Next, perform the pulmonary artery anastomosis as described earlier. To allow the reprofusion and ventilation of the transplanted donor lung, remove the Yasargil clamp on the hilum, followed by closing the thoracotomy incision with a 6-0 nylon suture.

Collect the BAL fluid from the native or transplanted lungs as desired. The wet to dry weight ratio of the lobes was calculated to measure the pulmonary edema. The transplanted lobe showed a significant increase in the wet to dry weight ratio compared to the donor's or recipient's native lobe, indicating no pulmonary edema.

In our opinion, the two critical steps of this procedure are to one, accurately use anatomic landmarks to identify the fourth intercostal space, and then two, select and appropriately use the cuffs to have a reproducible cuffing technique. A rat lung transplantation model that is both reproducible and consistent is a basis for molecular investigations into ischemia reprofusion and transplantation in general.