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

<meta charset="utf8"></head><body>小鼠肺移植中细胞动力学的实时监测

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.

<meta charset="utf8"></head><body>作者聚焦：通过活体成像推进肺移植免疫学

移植小鼠肺的活体双光子显微镜检查

小鼠肺移植物的双光子成像，用于实时研究细胞动力学和相互作用

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

Research

JoVE Journal

Medicine

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

62 次观看. 首先，将开胸手术的插管小鼠放在成像室的底板上，打开的左胸朝上。用丝带将鼠标固定在它的脸上、前爪和尾巴上。将胶水涂在连接到顶板的盖玻片的底部。接下来，降低顶板，直到盖玻片接触肺，让胶水接触肺表面。将盖玻片靠在肺上。给肺充气一到两秒钟，使胶水区域粘附在周围组织上。在每个螺栓上放置一个拇指螺母并固定顶板?...

视频: 小鼠肺移植物的双光子成像，用于实时研究细胞动力学和相互作用

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

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 ...