two-photon imaging of a mouse lung graft for studying cellular dynamics and interactions in real-time

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.

Two-Photon Imaging of a Mouse Lung Graft for Studying Cellular Dynamics and Interactions in Real-Time

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's 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.

two-photon-imaging-mouse-lung-graft-for-studying-cellular-dynamics

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 crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

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

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.