Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

The goal of our research is to understand the immune mechanisms underlying ischemia perfusion injury, and the inflammatory and tolerogenic responses after lung transplantation. We want to study the dynamic interactions between donor and recipient cells. We currently make use of several surgical and imaging techniques in the study of lung transplant immunology in the mouse.

These include orthotopic left lung transplantation and retransplantation, left pulmonary hilar clamping, non-invasive positron emission tomography, and two photon intravital imaging. Our research reveals unique immune responses in transplanted lungs. Using advanced imaging techniques, we discovered that recipient monocytes promote neutrophil infiltration in lung allografts via IL-1 beta.

We also found that FOXP3 regulatory T-cells cluster within these allografts and protect against antibody mediated rejection. Our technique is easy to set up with materials readily found in a microscopy lab, such as tissue glue and cover glasses. Additionally, it provides a larger field of view compared to other techniques.

Our lab is interested in understanding mechanisms that induce and maintain lung allograft tolerance. Intravital imaging will allow us to visualize interactions between immune cells during rejection and tolerance. To begin place an anesthetized mouse that has undergone lung transplantation in a right lateral decubitus position.

Swab the chest skin with 0.75%iodine and 70%ethanol mixture. Reopen the left thoracotomy through the third intercostal space. Now remove a portion of the skin and soft tissue over the left thoracotomy site and open the thoracotomy.

Clamp the ribs for 10 seconds to thrombose any microvasculature. Next, resect portions of the third to fifth ribs to create an imaging window. Use an electrocautery pen to stop any bleeding from the rib edges.

Now place a small bump of folded gauze under the right chest. With cotton swabs, lift the lung graft out of the chest and place the lower aspect of the lung onto a one by three centimeter wide strip of saline soaked gauze. To begin, place an intubated mouse with an open thoracotomy onto a base plate of the imaging chamber with the open left chest facing up.

With silk tape, secure the mouse over its face, front paws and tail. Apply glue to the bottom side of the cover glass attached to the top plate. Next, lower the top plate until the cover glass contacts the lung, allowing the glue to touch the lung surface.

Hold the cover glass against the lung. Inflate the lung for one to two seconds so that the area of glue adheres to the surrounding tissue. Place a thumb nut over each bolt and secure the top plate.

For two photon imaging, place the entire imaging chamber with the mouse onto a microscope stage. Use dichroic filters to separate the signals from the GFP reporter, Q dots and collagen generate SHG signal. Next, set a titanium sapphire femtosecond pulse laser to an excitation wavelength of 890 nanometers at the lowest possible power.

Apply one milliliter of water over the cover glass on the top plate. Now lower the water immersion objective into the water and focus on the surface of the lung. Turn on the base plate heater, keeping the temperature between 35 to 37 degrees Celsius.

Acquire image stacks with acquisition software of choice. At 24 hours post-transplant, there were more neutrophils in the lung relative to two hours post-transplant.