Medicine
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The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats
Chapters
Summary March 8th, 2019
The rodent left pneumonectomy is a valuable technique in pulmonary hypertension research. Here, we present a protocol to describe the rat pneumonectomy procedure and postoperative care to ensure minimal morbidity and mortality.
Transcript
This protocol is significant because it produces the best possible model for pulmonary arterial hypertension. No other model produces the plexiform lesions found in human PAH patients. The main advantage is that we double the flow of blood in the right lung and pulmonary artery, causing endothelial damage and forcing the lungs to work twice as hard.
Our group has developed a highly efficient model of pulmonary arterial hypertension to develop therapeutics. Besides pulmonary artery hypertension, this method could be helpful in regenerative medicine and could give insight to other diseases with increased pulmonary flow like congenital heart defects or post-pneumonectomy patients. For someone who has never performed this technique, my advice is to follow the protocol exactly and to make sure the surgeon can see exactly what they are doing.
After anesthetizing the rat with 4%isoflurane, the animal is intubated using a fiber optic gooselight to illuminate the trachea. Insert a 16-gauge catheter into the larynx, and use a cold mirror to observe the condensation of the humidity in the exhaled breath from the catheter to confirm the intubation. Connect the rat to the ventilator and place the rat under the microscope in the right decubitus position on a 37 degree Celsius heating pad.
Secure the forelimbs with tape, and use clippers to shave the left thorax from behind the front leg to the end of the ribcage. Clean the exposed skin with three sequential 10%povidone iodine solution and 70%ethanol wipes, and place a pulse-oximeter onto the rat's foot to monitor the heart rate and oxygen saturation throughout the surgery. Now, adjust the microscope and bring the surgical view into focus.
Then, place a sterile drape over the rat's body and the instrument tray to create a sterile environment. And finally, place sterile instruments on the sterile instrument tray. To perform a left pneumonectomy, use Cooley-Mayo scissors and Gerald tissue forceps to cut a two to three centimeter hole in the surgical drape, and use a surgical blade to make a two centimeter-long lateral incision in the left thorax.
Use Cooley-Mayo scissors to cut each layer of tissue until the ribs and intercostal muscles are exposed. Use a mosquito to make a hole through the muscle of the third intercostal space. Using a double-ended probe, move the lung to visualize the pulmonary artery, and use McPherson-Vannas Iris scissors to open the intercostal muscles to an about one to two centimeter width.
Place a small self-retaining retractor into the left pleura to hold the ribs and muscles open, and move the left lung lower in the abdomen to allow access to the pulmonary artery and bronchus. For ligation of the left main bronchus and pulmonary artery, first, load a medium Hemoclip into a ligating clip applicator and use the Wangensteen atraumatic forceps to carefully lift the upper portion of the left lung to expose the pulmonary artery. Close the clip and applicator around the artery, taking care not to close or rupture the left azygos vein.
And open the incision further using the tonsil to separate the muscle fibers. After loading another medium Hemoclip into the ligating clip applicator, use the atraumatic forceps and standard tissue forceps to lift the lower portion of the lung out of the incision until the left main bronchus and left pulmonary vein are visualized. Carefully ligate the left main bronchus and pulmonary vein together with the second Hemoclip, and use scissors to remove the lung without cutting or tearing the clip.
Then, use a small piece of sterile gauze to absorb any blood and to stem any bleeding. Use the needle holder and a 4-0 prolene suture to close the ribs and intercostal muscles. Before closing the intercostal muscles, insert a 16-gauge catheter into the thoracic cavity away from the surgical incision and into the seventh intercostal space, and immediately remove the needle, leaving the catheter in place.
Place a 5-0 suture into the skin and around the chest tube so that when the chest tube is removed, the hole will be tied shut, and tie the sutures to close the ribs. Close the skin and subcutaneous space with a running 5-0 suture, and use a three milliliter syringe to evacuate the air from the pleural cavity through the catheter to restore the normal negative pressure in the thorax. Use a needle holder to immediately clamp the catheter to prevent air from going back into the thoracic cavity.
Remove the catheter and tie the suture to close the hole. In the pneumonectomy and monocrotaline group, severe pulmonary hypertension develops by day 21, as evidenced by an increase in mean pulmonary artery pressure compared to control untreated sham animals. In the pneumonectomy plus Sugen group, the mean pulmonary artery pressure is three times higher than in the control group.
The right ventricular systolic pressure is also much higher in both the monocrotaline-and Sugen-treated animals, compared to the control group. In hematoxylin and eosin stained normal rat lung tissue sections, spaces between the alveoli and the alveolar structures are apparent, and the vessels are clear and of a normal thickness. In the pulmonary artery hypertension lung, there is evidence of remodeling, thickening of the vessel walls, severe constriction of the vessels, and inflammation and focal pulmonary arteritis.
The animal has to compensate for losing a lung, so it's important to be patient during the recovery period. Monitor the vital signs and give additional oxygen or lung volume as necessary. Sugen creates endothelial apoptosis which is clinical relevant sign of pulmonary arterial hypertension.
The left pneumonectomy model in rodents has paved the way for scientists to study flow abnormalities and develop novel treatments for pulmonary arterial hypertension. Due to the use of isoflurane, Sugen, and/or MCT in this model, personal protective equipment must be worn at all times.
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