We demonstrate a simple method for creating and implementing an ex vivo lung setup from scratch. This allows for an affordable and modular setup that can be implemented for a variety of setups. The technique presented here is affordable, modular, and adaptable to the labs setting up the protocol.
The ex vivo operation can be used to complement in vivo models to help elucidate novel mechanisms. This DIY setup is designed to study the lungs in a controlled environment. It aligns with the main research focus of our lab, which is to investigate various approaches to improve the outcome after CPR.
However, the setup can be adapted to suit different research aims, making it highly versatile, The surgery requires a basic knowledge of respiratory anatomy and physiology and can quickly be mastered by following the protocol. Nonetheless, it is vital to diligently follow the steps described in our protocol, and most of all, protect the lungs, especially when removing the ribs. To perform a tracheostomy, pinch the skin of an anesthetized rat above the trachea with forceps and cut the skin.
Bluntly dissect the muscle and tissue to reach the trachea, ensuring no bleeding. Then pass curved forceps underneath the trachea and open them to allow room to pass 3-0 sutures underneath. Then pre-tie the sutures into a box knot.
Make a small incision between the cartilage rings of the trachea and insert the tracheal cannula. Tie off the suture to prevent air from escaping the tracheal incision and to ensure the cannula is not putting strain on the trachea. Once the tracheal cannula is secured, begin ventilating the rat.
Using large surgical scissors and forceps, remove the fur from the rat's abdomen. Grasp the xiphoid process with the forceps and make a small horizontal incision below the ribs, taking care to keep the diaphragm intact. Widen the horizontal cut to expose the entire diaphragm.
With a 22 gauge syringe, inject heparin into the inferior vena cava, being careful not to puncture the lungs. Grasp the xiphoid process with forceps and cut cranially along the sternum, constantly visualizing the lungs to prevent cutting them. Spread the rib cage apart using two large forceps and cut the inferior vena cava to euthanize the rat by exsanguination.
Trim any excess thymus to allow for easier visualization of the pulmonary vasculature. Locate the pulmonary artery and pass small curved forceps underneath. Again, pass a 3-0 suture underneath and pre-tie into a box knot.
Then make a small incision into the right ventricle of the heart and insert the PA cannula. Secure the cannula using the suture and begin perfusing at 1.5 milliliters per minute. Immediately excise the apex of the heart to prevent pressure buildup in the lungs.
Using small curved forceps, rupture the mitral valve and visually confirm that the forceps can enter the left atrium without obstruction. Tightly wrap a 3-0 suture around the heart below the atrium, and insert the PV cannula into the left atrium, ensuring that buffer can flow out of it before tying off the suture. Next, using blunt tip scissors, trim any excess tissue between the thoracic cavity and the tracheostomy incision to avoid damaging the trachea.
Ensure the trachea below the tracheal cannula and the entire heart-lung bloc are visible. To remove the heart-lung bloc and trachea, hold the tracheal cannula and use curved blunt tip scissors to excise the connective tissue behind the trachea. Lung viability after ischemia and reperfusion is presented.
The longest ischemia time to allow 150 minutes of reperfusion was eight minutes. It's crucial to properly cannulate the vasculature for the isolated lungs to remain viable throughout the experiment. The lungs remain intact after the procedure, allowing for basic measurements such as wet or dry weight or histology to be conducted post-experiment.
