实验概要
The procedure presented below describes a method for perfusing rat lung.
主要试剂
Phosphate Buffered Saline
主要设备
1.Dissecting Microscope or Eye Magnifier
2.Q-Tip
3.Surgical Scissors or Scalpel
4.Two 10 mL Syringes with a stopcock and a small gauge needle (25 or 27 ga) attach
实验步骤
1. Rat should be fully anesthetized (e.g., unresponsive to toe pinch).
2. Euthanize the rat by exsanguination. The easiest way to do this is by cutting the abdominal aorta. Avoid cardiac puncture, as the puncture site will leak during the perfusion procedure.
3. Open the chest widely, taking care not to puncture the lungs. Cut through the entire sternum at the midline; lifting the sternum with forceps and keeping the scissor tips horizontal will lessen the chance of inadvertent lung puncture. Next, make lateral incisions in the chest wall in both directions from the midline incision. Retract one side of the chest wall laterally, bluntly dissect the pleura to free up the lung, gently push the lung away from the chest wall with a Q-tip, and cut the chest wall vertically along the spinal processes. Repeat for the other half of the chest wall. Be careful not to puncture the lung with the sharp edges of the cut ribs.
4. Place vascular clamps or tie ligatures on both the superior and inferior vena cavae to prevent backflow of perfusion buffer into the systemic venous system. It helps to use magnification (dissecting microscope or eye magnifiers) for the remaining steps.
5. With a scalpel or small scissors, make an incision in the LEFT ventricle of the heart, making certain that the heart chamber is open to the outside.
6. Prepare a 10 ml syringe filled with PBS, with a stopcock and a small gauge needle (25 or 27 ga) attached. Have a second PBS-filled syringe available in case it is needed. Insert the needle into the RIGHT ventricle, and gently push the PBS into the pulmonary circulation. You should see the right heart and pulmonary artery swell a little bit, and fluid should immediately begin to flow from the left ventricle incision. Continue to push PBS through until the lungs are white and the fluid coming out of the left ventricle is completely clear- this usually takes 10–15 ml of buffer. The small needle will prevent you from pushing in the buffer too rapidly, and also will have fewer problems with leaks than with larger needles.
7. Another option that works well for the perfusion, but that takes more time, is to cannulate the pulmonary artery. This can be done by introducing a plastic catheter through the right heart and into the pulmonary artery, and ligating the pulmonary artery to hold the catheter in place. With this method it is not necessary to clamp the vena cavae. Buffer may be pushed into the catheter with a syringe, taking care not to create excessive pressure in the pulmonary system, or with a gravity feed of buffer not more than about 10 cm above heart level.