Intravital Two-Photon Microscopy of the Transplanted Mouse Lung
April 19th, 2024
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.
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.
Overview
This study presents a protocol for intravitally imaging the transplanted mouse left lung using two-photon microscopy. It aims to elucidate the immune mechanisms involved in ischemia perfusion injury and the responses following lung transplantation.
Key Study Components
Area of Science
- Neuroscience
- Immunology
- Transplantation Biology
Background
- Understanding immune mechanisms in lung transplantation is crucial.
- Ischemia perfusion injury affects transplant outcomes.
- Dynamic interactions between donor and recipient cells are significant.
- Advanced imaging techniques can reveal unique immune responses.
Purpose of Study
- To investigate the immune responses in transplanted lungs.
- To study the interactions between donor and recipient cells.
- To utilize advanced imaging techniques for real-time analysis.
Methods Used
- Orthotopic left lung transplantation and retransplantation.
- Left pulmonary hilar clamping.
- Non-invasive positron emission tomography.
- Two-photon intravital imaging.
Main Results
- Recipient monocytes promote neutrophil infiltration in lung allografts.
- IL-1 beta plays a key role in the immune response.
- Unique immune responses were observed in transplanted lungs.
- Advanced imaging techniques provided insights into cellular dynamics.
Conclusions
- The study enhances the understanding of lung transplant immunology.
- Real-time imaging is a valuable tool for studying cellular interactions.
- Further research could improve transplant outcomes.
Frequently Asked Questions
Executive Industry Relevance
Intravital two-photon microscopy of transplanted mouse lungs enables real-time visualization of immune cell dynamics critical to understanding graft rejection and tolerance. This capability addresses a key discovery-stage challenge by providing mechanistic insight into donor-recipient cell interactions and inflammatory pathways. The approach supports predictive confidence in preclinical models, informing risk-adjusted decisions for immunomodulatory strategies in transplantation research portfolios.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct observation of immune cell trafficking and interaction within transplanted tissue.
- Supports mechanistic de-risking by clarifying pathways involved in graft rejection and tolerance.
- Facilitates functional validation of immune targets such as monocytes, neutrophils, and regulatory T-cells.
- Provides dynamic evidence to prioritize or deprioritize immunomodulatory targets.
Screening & Assay Development
- Establishes validated in vivo imaging systems for quantitative assessment of immune responses.
- Supports reproducible measurement of cell infiltration and clustering in response to interventions.
- Enables standardization of imaging protocols for cross-study comparability.
- Prepares robust platforms for evaluating candidate therapeutics targeting immune pathways.
Translational & Preclinical Research
- Aligns preclinical models with disease-relevant immune mechanisms observed in transplantation.
- Enables longitudinal tracking of immune cell behavior post-transplant, supporting translational biomarker discovery.
- Provides continuity from mechanistic discovery to preclinical validation of immunotherapies.
- Informs risk-adjusted advancement of candidates based on in vivo immune modulation evidence.
Pipeline & Workflow Integration
This imaging technique integrates from early discovery through preclinical validation, bridging mechanistic studies and translational research in transplantation immunology.
- Discovery Biology: Supports hypothesis testing on immune cell roles in graft rejection and tolerance.
- Screening: Delivers quantitative, reproducible imaging outputs for immune cell infiltration and clustering.
- Analytics: Provides high-content, time-resolved data for comparative analysis of immune responses.
- Translational Research: Aligns preclinical findings with clinical immune mechanisms relevant to transplantation outcomes.
- Enterprise Reuse: Offers a reusable imaging platform adaptable to other pulmonary and immune-mediated disease models.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in immune target validation and mechanistic de-risking.
- Operational Value: Enhances standardization, reproducibility, and scalability of in vivo imaging workflows.
- Strategic Value: Improves go/no-go decisions and reduces late-stage biological risk in immunology portfolios.
- Portfolio Impact: Enables risk-adjusted prioritization of immunomodulatory strategies for transplantation and related indications.
Implementation Considerations
- Requires expertise in small animal surgery and advanced microscopy techniques.
- Demands access to two-photon microscopes, transgenic mouse lines, and imaging analysis software.
- Necessitates cross-team standardization of imaging protocols and data interpretation.
- Adaptable to other organ systems and disease models with appropriate technical modifications.
- Imaging depth and field of view are limited by tissue properties and instrumentation.
