The constant pressure within the fixation device maintains the lungs in a reasonably inflated state generating a histological lung specimen suitable for evaluating mild cigarette smoke induced emphysema. The main advantage of this model is that it can fix many lungs at the same time using the same constant pressure without lung collapse or deflation. Demonstrating the procedure will be Naoko Arano a graduate student from my laboratory.
After confirming a lack of response to reflex motion make an incision in the skin and muscle tissue along the medial line of the mouse, aiming for the cephalic region, and cut laterally to create a wider working space. After exposing the diaphragm layer puncture the tissue with forceps and open the thoracic space. Cut the sternal area allowing the lungs and heart to be visualized.
And cut the left atrium and right ventricle. Insert a 24 gauge cannula connected to a perfusion pump into the right ventricle area and direct the cannula to the cephalic area until it reaches the pulmonary artery. Then, turn on the pump and perfuse the tissue with approximately 200 milliliters of PBS for one hour until all of the lung tissue has changed to a white color.
At the end of the perfusion cut the connective tissue surrounding the trachea, lungs and heart, and use a suture to tie off the right main bronchus. Next, harvest all of the individual lobes of the right lung and place the heart and lobes of the left lung into individual 10 milliliter syringes containing an appropriate fixative. Retract the plunger creating a vacuum in the syringe to inflate the lungs and remove the lung from the syringe.
Insert a 20 gauge cannula into the trachea and use a suture to secure the cannula in place. Connect a one milliliter syringe filled with fixative to the cannula and fill the lung with additional fixative to check for leaks. Be sure that the insertion site was secured to prevent the lung specimen from detaching from the pulmonary board during the fixation.
Then transfer the tissue to the lung fixation pressure equipment, removing the sample and tying off the trachea with a knot at the end of the fixation period. The lower component of the lung fixation pressure equipment into which the lung sample is inserted is connected via 20 gauge cannula to the tip of the formalin flow with a three-way stopcock. Pressure is generated from the different surface levels of the fixative between the lower and upper compartments.
The pressure difference is 25 centimeters of water but the height adjustment knob can be used to adjust the pressure to between 25 and 30 centimeters of water. A pump connects the lower and upper containers, preserving a 25 centimeter difference in the fixative surface height, and directs the flow of the agent. After 48 hours of fixation the lungs from SMP30 knockout mice exposed to ambient air do not exhibit a marked airspace enlargement.
Significant airspace enlargement and alveolar wall destruction, however, are observed in SMP30 knockout mice exposed to chronic cigarette smoke. In addition, the mean linear intercept and destructive index of the lung specimens are significantly greater in the smoke exposed SMP30 knockout mice than in the air exposed animals. Our lab uses 10%formalin but researchers can use other fixation agents according to their experimental needs.
As we have reported using the demonstrated fixation system lung samples can be harvested from various mouse emphysema models for their morphometric evaluation. As fixing agents can be hazardous always wear the appropriate personnel protective equipment and work in a well-ventilated room.
