We are interested in studying the effects of ischemia reperfusion injury on outcomes following lung transplantation with the goal of understanding underlying cellular and molecular mechanisms. Neutrophils that enter the lung contribute to ischemia reperfusion injury, or IRI. We've recently discovered that B cells proceed neutrophils in entering the lung, and they play a role in the recruitment of neutrophils following IRI.
Left lung transplantation in the mouse is technically challenging and requires full-time dedicated training over several months to achieve consistent results. Additionally, it introduces allergenicity and surgical anastomotic trauma. Thus, we need a more accessible technique that can isolate the study of ischemia reperfusion injury.
Using our hilar clamp model, we have found that B cells enter the lung early after ischemia reperfusion injury, and they recruit classical monocytes in a B cell receptor into TLR4 dependent fashion. This ultimately results in increased neutrophil extravasation into the lung. Our protocol involves reversible suture ligation of the left pulmonary hilum with a slip knot.
The pliable and unobtrusive nature of the suture allows for chest closure, which is useful for studying longer periods of warm ischemia, as chest closure minimizes heat and insensible fluid losses in the mouse. To begin, place an anesthetized and intubated mouse in a right lateral decubitus position. Take both front paws to the top left corner and the hind paw to the bottom corners.
Disinfect the left chest with 70%alcohol prior to incision. After incising and opening the skin over the fourth intercostal space, use a handheld cautery pen to coagulate the visible blood vessels within the subcutaneous and muscle layers. With the scissors, divide the latissimus dorsi and serratus anterior muscles along the length of the skin incision.
Carefully elevate the fourth rib with a pair of fine-curved forceps. Make a pinpoint snip just above the fifth rib to enter the fourth intercostal space. When the negative pleural cavity pressure is lost and the lung retracts from the chest wall, extend the thoracotomy anteriorly and posteriorly in the fourth intercostal space.
Apply two rib retractors to spread open the rib space, ensuring a working window of at least one square centimeter is available for visualization. Use two pointed cotton-tipped applicators to elevate the cephalad and mobilize the left lung. Then bluntly divide the translucent inferior pulmonary ligament.
Reflect the lung anteriorly to visualize the posterior left pulmonary hilum. Then cut approximately 10 centimeters of a 6-0 silk tie, and place the midpoint of the tie posterior to the hilum. Now, flip the left lung posteriorly over the tie and pull both tie ends anteriorly.
Hold a curved mosquito clamp in the dominant hand and a pair of forceps in the other hand. Hold one free end A of the tie with the forceps and loop its midpoint over the closed clamp once. Next, grasp the midpoint of the other free end B with the clamp and pull with both hands to cinch the slip knot.
After tying the knot B should have a loop from the knot, while A should be straight. To confirm adequate occlusion of the bronchus, manually occlude the outflow tubing from the endotracheal tube to the ventilator. This will not result in inflation of the left lung.
Next, close the skin incision with one or two simple interrupted stitches made with a 6-0 nylon suture. After the required period of ischemia, gently pull on the B end of the tie to release the slip knot. Immediately start the reperfusion and occlude the outflow tubing of the endotracheal tube to the ventilator to reinflate the lung.
During reperfusion, close the fourth rib space with a single stitch 6-0 nylon suture. Place the mouse in a supine position 15 to 20 minutes before the end of the reperfusion time. Secure all four limbs with tape.
After performing a midline laparotomy, extend the laparotomy left and right to the axillary line following the curve of the inferior most rib. From the abdomen, make an incision on the anterior diaphragm to enter the chest. Then extend the incision to the left and right of the anterior axillary line along the inferior most rib.
Divide the bilateral ribs along the anterior axillary line. Extend upwards to create a clamshell thoracotomy. Now, reflect the anterior chest wall upwards to expose the heart and bilateral lungs.
Then apply a clamp to the anterior chest wall to facilitate retraction. Use a curved mosquito clamp in the midline to retract the diaphragm downwards for better chest visualization. Next, return the accessory lobe that extends past the midline into the left chest and back into the right chest.
With the help of a cotton-tipped applicator and scissors, divide the thin ligament that connects the lobe to the left chest. Reflect the entire lung anteriorly. Then place another 6-0 silk tie posterior to the right hilum.
Tie a slip knot around the right hilum while ensuring that all four lobes of the right lung are encircled. Next, code a half inch 31 gauge needle on a one CC tuberculin syringe with 200 microliters of heparin. After four minutes of right hilar clamping, draw the arterial blood from the left ventricle into the syringe.
Angle the needle towards the left neck. Multiple puncture may be necessary to obtain sufficient blood for the ABG evaluation. Left hilar clamping resulted in significantly lowered partial pressure of oxygen in the left lung relative to the sham thoracotomy.
ABG evaluation showed that most of the neutrophils were intravascular after the hilar clamping of the left lung.
