surgical excision of a mouse lung graft for two-photon imaging

Real-Time Monitoring of Cellular Dynamics in Mouse Lung Transplantation

Lung transplantation is the final option for many patients suffering from end-stage pulmonary disease; however, long-term survival after lung transplantation is poor compared to other solid organ transplants. Survival at 5 years is only ~60%-70%1, compared to 80%-90% in hearts2 and 85%-90% in kidneys3. Many complications after lung transplantation, such as primary graft dysfunction, antibody-mediated rejection, and chronic lung allograft dysfunction, are due to the host immune response to the allograft. For example, our group has shown that neutrophils are rapidly recruited into the lung allograft following transplant-induced ischemia-reperfusion injury and form dynamic clusters surrounding blood monocytes4. Crosstalk between donor and recipient cells is responsible for deleterious alloimmune responses5,6,7, and the ability to study these dynamic cell interactions in a live animal model is invaluable.
Two-photon microscopy allows for high-resolution intravital imaging for depths up to several hundred microns with minimal photobleaching of tissues8,9. It is used in a variety of tissues and anatomic sites, including the neocortex10,11, skin12,13, and kidney14,15. More recently, it has been adapted to non-static organs such as the lung and heart4,16,17. In this protocol, we describe a technique to image stabilized, ventilated, and perfused pulmonary grafts following murine orthotopic left lung transplantation. A key benefit of the transplant model is the ability to genetically manipulate the donor and recipient separately. Individual cell populations can be visualized with transgenic mouse strains with knock-in fluorescent protein expression, adoptive transfer of fluorescently-labeled cells5, or intravenous injection of fluorescently-labeled antibodies to bind cell-specific markers4,16,17,18.
In order to stabilize the lung during imaging, this protocol involves gluing the lung to a coverglass, while other groups have described suction stabilization using a custom-made reversible vacuum device19. Our protocol has several advantages, including a larger area of imaging and relative ease of setup using commonly available materials in a microscopy lab (including coverglass and glue). Since this gluing technique constrains the lung at the upper surface, it is expected to decrease ventilatory motion and allow for deeper imaging. This intravital imaging technique allows for detailed observation of immune cell behavior and interactions in real-time, which contributes to the study of immune mechanisms that regulate inflammatory versus tolerogenic responses following lung transplantation.

Author Spotlight: Advancing Lung Transplant Immunology Through Intravital Imaging

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

The goal of our research is to understand the immune mechanisms underlying ischemia reperfusion 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.

Surgical Excision of a Mouse Lung Graft for Two-Photon Imaging

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.

Research

JoVE Journal

Medicine

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...