Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

The overall goal of this procedure is to establish an literative bronchiolitis model in mirroring orthotopic lung transplantation. This is accomplished by first harvesting the donor lung from a C 57 BL 10 mouse. The second step is to prepare the cuff for the bronchus pulmonary artery and vein for anastomosis.

Next, the prepared donor lung is transplanted into recipient C five seven BL six mouse. The final step is to observe the transplanted mouse for 21 or 28 days after transplantation. Ultimately, histological analysis is used to show literative bronchiolitis lesions in the transplanted lung.

The main advantage of of this model as compared to the mirroring heterotopic airway transplant model is that the current model mimics the human condition. Generally, individuals new to this method will struggle because the higher structures in mice are very delicate and more susceptible to injury because masses only a 10th of the mass of the rat Place the donor mouse in a sealed holding chamber and induce anesthesia with 5%isof fluorine. Next, use the 20 gauge intravenous catheter to intubate the mouse or Trae, and then place it on a rodent ventilator.

Maintain anesthesia with one to 2%isof fluorine. After making sure the mouse is fully anesthetized by unresponsiveness to tail flick and toe pinch, place it in a supine position and prep the abdomen with 70%alcohol. Now, cut away the skin overlying the abdomen with a pair of scissors and expose the abdominal musculature.

Next, perform a lapar sternotomy by first making a midline incision through the abdominal wall, and then by cutting transversely across the sternum, move the intestines away and locate the inferior vena cava, which is just below the liver. Inject heparin at 100 microliters per kilogram into the IVC. Next, pull down the falciform ligament of the liver and cut the diaphragm along the ventral causal attachment toward the spine.

Cut both sides of the chest wall to the neck in order to expose the thoracic cavity. Cut the rib cage bilaterally while protecting the lung and remove the thymus. Next, cut the intrathoracic IVC at the level of the diaphragm.

Now locate the heart and excise the right atrial appendage. Then make a transverse incision at the root of the pulmonary artery trunk and use the incision to flush the lungs with two milliliters of cooled lactated ringers and 0.1 milliliters of heparin. Expose the trachea and then cut it away along with the esophagus.

Now arrest ventilation at two thirds of end tidal inflation. Excise the heart long block and store it on ice. Place gauze on the excised lung to protect it from light.

Incise the pulmonary ligament up to the pulmonary vein or pv. Remove the esophagus and aorta to localize the hilum. Find the pulmonary artery or pa, which is located at the most cranial aspect of the hilum and the attached main bronchus.

Carefully dissect the pulmonary artery away from the bronchus. Now, make a PA cuff from a 24 gauge IV catheter by cutting it to 0.5 millimeters in length with an extension of 0.7 millimeters. The surface of the cuff is AB braided.

In order to provide a surface for anastomosis, make a cuff for the bronchus using similar material to the PA cuff only using a 20 gauge catheter cut to a length of one millimeter with an extension of 0.7 millimeters. The PV cuff size varies with the weight of the donor mouse. For mice weighing 24 to 27 grams.

The cuff size is 22 gauge with 0.7 millimeter length and 0.7 millimeter extension for mice weighing 27 to 32 grams. The lengths are the same, but a 20 gauge catheter is used. Now, insert the cuffs into the distal lens of the pulmonary artery pulmonary vein and bronchus securing each with nine oh silk suture.

After the cuffs have all been properly attached. Wrap the prepared donor lung in lactated ringers soaked gauze, and place it on ice until transplantation. To prepare the recipient mouse induce anesthesia and place on mechanical ventilation in the same manner that was used for the donor.

Shave the left chest wall and prep with 70%alcohol. Begin by making a thoracotomy incision in the left third intercostal space. Extend the incision dorsally close to the spine.

Make sure the surgical field is draped in a sterile manner and place a micro vessels clamp on the left pulmonary vessels and bronchus adjacent to the heart. The pulmonary artery can be visualized at the cranial aspect, the pulmonary vein at the coddle end of the hilum and the bronchus between them. Next, use gentle traction on a hemostat to cause mild tension on the pa, PV and bronchus.

Leave the hilar structures clamped and carefully resect the left lung. Isolate the pulmonary artery pulmonary vein and bronchus, and then place a oh suture loosely around each structure. Dissect the PA completely from its adventitial sheath, and then make a small transverse incision of approximately one fourth of the vessel circumference into the anterior wall.

While leaving the continuation of the dorsal part of the artery intact, obtain the donor lung, which is wrapped in cold lactated ringers soaked cotton gauze, and position it into the thoracic cavity. Next, insert the cuffs into the recipient pa, PV and bronchus to create anastomosis and ligate these with nano suture. Next, remove the recipient lung.

Then remove the hilar cross clamp, which allows for reperfusion and ventilation. Position the transplanted lung back into the recipient thorax and close the thoracotomy incision Using five oh suture, the mouse should now recover from anesthesia. Buprenorphine can be given for pain relief after the mouse has regained consciousness.

This table shows the histologic scores of acute rejection and prevalence of literative bronchiolitis or OB.Post-transplant scoring of acute rejection is referred to as an A score. The allograft combinations show considerably more acute or chronic rejection pathology when compared to the ISO graft. Additionally, prevalence of literative bronchiolitis was significantly higher in the allograft combinations than in the ISO graft at day 21 and 28 post-transplant.

The macro findings and histopathology at 28 days post lung transplantation are shown here. These images represent macro findings and H and E stained ISO graft lung, and right naive lung. These images represent h and e and masen trireme stained BL 10 lung allograft transplanted into BL six mouse recipient, which developed OB and non OB respectively.

The white arrow in one B identifies the OB lesions. This panel shows BL six lung allograft transplanted into BL 10 mouse recipient Once mastered. This technique can be done in almost one half hour if it is performed properly.

The utility of this technique is that it can be utilized to answer mechanistic questions regarding primary graft dysfunction, acute rejection, and ablative bronchiolitis post lung transplantation.