Thank you for watching our videos. I am Susumu Minamisawa of the Jikei University School of Medicine Tokyo, Japan. Our opportunity on animal pulmonary hypertension due to rat heart disease which is classified as Group Two pulmonary hypertension.
The World Health Organization classifies pulmonary hypertension into five groups. The left atrial stenosis rat is an animal model group two pulmonary hypertension which is the most prevalent form of pulmonary hypertension and currently there is no approved therapies for this group of pulmonary hypertension. In this videos we are demonstrating the step by step procedure of how to perform left atrial stenosis surgery in rat.
Surgically created right atrial stenosis mimic mitral stenosis in human. When we use it to study the possibility and under of group two pulmonary hypertension. Let's get started.
Prepare the following equipment before the surgery, a small animal respirator and an anesthetic machine. An incubation kit composes of a pair of hemostat forceps, a tongue depressor and a 18 gauge angio-catheter. Surgical instruments include a pair of curved forceps, a pair of straight forceps, a needle driver, a chest retractor, a pair of scissors, a 5-0 monofilament suture, a clip applicator, a medium large clip and a 23 gauge chest tube.
Anesthetize the rat in an induction chamber with five percent isoflurane mixed with two meter per minute room air. Prior to intubation shave the chest hair. Apply hair removal cream to remove fine hair not removed by the shaver.
Hook the front teeth with a string and secure the strings to pins. After the string is secured open the mouth with the hemostat forceps and insert the tongue depressor into the mouth. Lift the tongue depressor to visualize the vocal chord.
Insert the 18 gauge angio-catheter as an endotracheal tube into the trachea and then quickly connect the catheter to the respirator. Set the tidal volume to 10 microliter per gram with a respiratory rate of 100 breaths per minute. Maintain anesthesia with two percent isoflurane mixed with two meter per minute room air.
Sterilize the surgical site with alternating scrubs of chlorhexidine and alcohol. Give Buprenorphine 01 milligram per kilogram subcutaneously. Cover the rat with a sterile drape.
Check pedal reflex to confirm successful anesthesia prior to surgery. Mark the incision site two centimeters below the armpit. Make a two centimeter left lateral chest wall incision with a pair of scissors.
Carefully separate the intercostal muscle between the fourth and the fifth rib using a straight and curved forceps until entering the chest cavity. Insert the chest retractor into the chest cavity. Use some force to open up the chest retractor to split the fourth and fifth rib apart by about one centimeter.
Lift the thymus with a pair of straight forceps. Carefully remove the thymus covering the heart with a pair of scissors. Carefully pass a 5-0 monofilament suture through the surface of the left ventricle right below the left atrial appendage.
Care should be taken to avoid passing the needle through the major coronary arteries. After the suture is in place and there is no significant bleeding tie a loose knot. Pull the suture thread up and forward to lift the heart out of the chest.
Once the heart is lifted outside of the chest quickly apply a medium large clip to the left atrium just above the mitral valve. Carefully remove the stay suture used to lift the heart. Begin close the chest with a 5-0 monofilament suture using simple interrupted pattern.
Insert a 23 gauge chest tube attached to a 10cc syringe into the chest cavity. And then proceed with closing the chest. Draw out any air, blood and pleurae effusion near the inserted chest tube using the attached 10cc syringe.
And then pull the tube when nothing is coming out. Close the skin area with 5-0 monofilament suture using simple interrupted pattern. Remove the surgical drape.
Give Buprenorphine 01 milligram per kilogram subcutaneously. Turn off the isoflurane. Excavate the rat when it starts to wake up.
Two weeks post op use echo cardiography to confirm the success of left atrial stenosis surgery. The effectiveness of the left atrial stenosis is confirmed using echo cardiography two weeks post op. Rats with left ventricle inflow velocity greater than two meters per second measured on four chamber view are considered to have developed significant stenosis.
10 weeks post left atrial stenosis surgery the rats in the LAS group show left atrial enlargement, pulmonary congestion, right ventricle pressure overload and increased pulmonary venous flow compared to the rats in the sham operated group. In addition there is significant increase of right ventricular systolic pressure in the left atrial stenosis group versus the sham operated group. However, the left ventricle systolic pressure remains the same in both groups.
Histological examination of long cross section stained with elastic Van Gieson shows increased pulmonary arterial and pulmonary venous medium sickness. An increased pulmonary venous dimension the left atrial stenosis group versus the sham operated group was a more of a smooth muscle acting in the staining shows increased number of smooth muscle cells in the pulmonary artery and the pulmonary vein of the left atrial stenosis group versus the control group. I hope you enjoyed watching this video.
Although the surgery is relatively simple care should be taken when applying the suture to the left ventricle to avoid hitting the major coronary arteries. When lifting the heart pull the suture up toward the head of the rat so the left atrium its running structure can be clearly visualized. By applying the clip to the left atrium the tightness of the clip should be sufficient to cause significant left atrial stenosis, but not too tight to induce acute pulmonary edema and right heart failure.
Thank you for watching this video. And good luck with your experiment.
