The Assessment of Lymphatic Drainage Using ICG Near-Infrared Imaging and Ultrasound After Fracture
June 23rd, 2026
This protocol assesses lymphatic drainage after fracture by combining indocyanine green(ICG) near-infrared imaging with ultrasound to quantify plantar ICG clearance and popliteal lymph node enlargement in mice.
Our research evaluates lymphatic reflux after fractures using near-infrared imaging and the B-type ultrasound to monitor drainage, function, and lymph node swelling. Existing methods lack a standardized dynamic in vivo assessment, so our protocol compares the ICG near-imaging with the ultrasound for reproducible monitoring. To begin, weigh 0.2 milligrams of Indocyanine Green, or ICG.
Transfer it to a 1.5 milliliter microcentrifuge tube and dissolve it in one milliliter of PBS to prepare a 0.2 milligram per milliliter ICG solution. Wrap the tube with aluminum foil to protect it from light. Then turn on the imaging system and open FluoBeam version 1.46.
Adjust the camera height as required. After anesthetizing the C57 male mice with a fracture model, moisten a cotton swab with 75%ethanol and disinfect the plantar surface of the modeled hind paw. Using a 10 microliter microsyringe, aspirate six microliters of the ICG solution.
Insert the needle approximately one millimeter into the plantar surface and inject the solution at a constant rate until the full volume is delivered. In the software, activate both the Camera and Laser functions. Click Modify to create a new folder and set it as the save directory.
Place the mouse under the camera in the prone position with the modeled hind limb centered in the field of view. Set the exposure time to 30 milliseconds for both the zero and 24-hour images. Acquire images of the same plantar injection region of interest at zero and 24 hours post-injection to measure ICG signal intensity.
When the lymphatic vessels of the hind limb are clearly visible in the field of view, click Photo to capture the images. After image acquisition, export the data. Open the image analysis software and import the fluorescence images by clicking File and selecting Open.
Open the zero hour plantar image corresponding to the same animal and the same examination day. Then select Oval, click Edit, choose Selection, and select Specify. Set the Width and Height to 30, then choose Oval, and click OK.Place the circle over a background area.
Click Analyze and select Measure. Record the mean value in the Results window as fluorescence intensity at zero hours background. Then place the circle over the target observation site and record the mean value as demonstrated earlier.
Calculate the corrected fluorescence intensity at zero hours as FI0 hour. Open the matched 24-hour image and repeat the previous steps to obtain the corrected fluorescence intensity at 24 hours. Calculate the clearance rate using the background corrected fluorescence intensity as presented here.
Turn on the ultrasound system, connect the transducer, and position the mouse so that the hind limb is clearly visualized. Under continuous anesthesia, place the mouse in the prone position on the imaging stage with the head oriented away from the operator and the tail toward the operator. Flex the knee so that the popliteal fossa faces upward and slightly laterally.
Now, position the transducer over the depression of the popliteal fossa. Apply ultrasound coupling gel to the modeled hind limb and rotate the transducer so that its angle remains parallel to the longitudinal axis of the hind limb. Set the Scan Distance to four millimeters and the Step Size to 0.04 millimeters.
Identify the popliteal lymph node on the image according to its anatomical location. Stabilize the mouse by holding the tail and modeled hind limb with both hands, avoiding any movement during transducer scanning. Acquire images and identify the popliteal lymph node as a small oval or bean shaped structure with intermediate to low echogenicity.
Export the images to the three-dimensional reconstruction software for three-dimensional image processing. Record the animal identification, group code, and time point at zero hours or 24 hours. Open the three-dimensional reconstruction software.
Click Study to enter the module and select Import. Next, click the data folder, check the data to be imported, and double click to open it. In the Display mode, select Image Processing.
Then choose Load into 3D. Select Transverse View. Choose Volume Measurement and click Start.
Confirm that the slice orientation and step parameters are consistent with the acquisition settings. Then use the mouse wheel or slice slider to scroll from the proximal end to the distal end and identify the first slice in which the popliteal lymph node appears. Begin tracing on the first slice in which the popliteal lymph node appears by left-clicking to start the contour.
Trace a closed contour point by point along the lymph node edge, keeping it close to the outer capsule and excluding surrounding fat and vascular lumen. Use the mouse wheel to move to the slice showing the maximum cross-sectional area of the popliteal lymph node, and repeat the previous contour tracing step to complete the second contour. Next, move to the slice just before the popliteal lymph node disappears, and repeat the contour tracing step to complete the third contour.
Then click Finish to generate the three-dimensional segmented volume and output the volume measurement. Record the popliteal lymph node volume in cubic millimeters in the Results panel. In the Measurement Results panel, select Export, and export the results as a CSV or XLS file.
Select the file type and export the image as TIFF Image Area. At the end of the experiment, place the mouse on a heating pad and allow it to recover from anesthesia. On days one, seven, and 14 after fracture model establishment, 24-hour plantar ICG fluorescence intensity was consistently higher in the fracture group than in the sham surgery group.
The lymphatic ICG clearance rate in the fracture group was significantly lower than that in the sham surgery group. Popliteal lymph node volume in the fracture group mice was significantly higher than that in the control group on days one, seven, and 14. This protocol measures lymphatic drainage efficiency and the popliteal lymph node enlargement after fracture in mice.
The key challenge is maintaining consistent imaging parameters, injection accuracy, and the stable lamp positioning during ultrasound scanning. Future studies can combine immunological markers and the histology to validate lymphatic mechanisms during fracture healing.
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Overview
This article presents a method to assess lymphatic drainage function in mice following tibial fracture using near-infrared fluorescence imaging of indocyanine green (ICG) and ultrasound-based 3D reconstruction of popliteal lymph nodes. The approach enables dynamic, in vivo monitoring of lymphatic clearance and lymph node volume changes, offering a reproducible alternative to traditional techniques like histopathology or lymphoscintigraphy. Results show significantly reduced lymphatic clearance, increased ICG retention, and enlarged lymph nodes post-fracture, reflecting impaired lymphatic function during the acute inflammatory response.
Key Study Components
Area of Science
- Lymphatic biology
- Musculoskeletal injury
- Inflammation and immune response
Background
- Traumatic fractures are associated with impaired lymphatic drainage and lymph node enlargement.
- These changes reflect ongoing inflammatory processes and immune cell recruitment.
- Existing methods for assessing lymphatic function often lack in vivo, dynamic, and continuous monitoring capabilities.
- There is a need for reliable, reproducible techniques to study lymphatic reflux in fracture models.
Purpose of Study
- To develop and validate a method for quantifying lymphatic drainage function in mice after tibial fracture.
- To measure lymphatic clearance rate using ICG fluorescence and lymph node volume via ultrasound.
- To demonstrate the method’s superiority over traditional approaches in terms of stability and reproducibility.
Methods Used
- Intradermal injection of 6 µL of 0.2 mg/mL indocyanine green into the plantar region of mice.
- Near-infrared fluorescence imaging at 0 h and 24 h post-injection to calculate 24 h ICG clearance rate.
- Ultrasound imaging to 3D-reconstruct and measure volumes of popliteal lymph nodes.
- Comparison of lymphatic function between sham and tibial fracture surgery groups.
Main Results
- Lymphatic clearance rate was significantly decreased after fracture.
- Fluorescence intensity of ICG in the plantar region was markedly higher at 24 h post-fracture.
- Volume of the popliteal lymph nodes was significantly enlarged following tibial fracture.
- The method demonstrated high stability and reproducibility compared to histopathological or lymphoscintigraphy approaches.
Conclusions
- The described method enables dynamic, in vivo monitoring of lymphatic drainage and lymph node changes in fracture models.
- It provides a robust technical framework for studying lymphatic dysfunction in musculoskeletal injury.
- Impaired lymphatic reflux after fracture reflects active inflammatory and immune processes.
- This approach improves upon prior methods by allowing continuous, quantitative assessment of lymphatic function.
Frequently Asked Questions
Executive Industry Relevance
Assessing lymphatic drainage function provides critical insight into the inflammatory and immune response following musculoskeletal injury, a key consideration in preclinical models of fracture healing and immunomodulatory therapies. The described ICG-NIRF and ultrasound method enables dynamic, in vivo quantification of lymphatic clearance and lymph node remodeling, offering a reproducible biomarker for target validation in inflammation-driven pathways. This approach supports mechanistic de-risking by linking lymphatic dysfunction to pathophysiological processes relevant to drug discovery in trauma, inflammation, and tissue repair.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of lymphatic function as a biomarker of inflammatory response in fracture models, supporting target validation in immunomodulatory pathways.
- Operational Value: Provides quantitative, dynamic readouts of lymphatic clearance and lymph node volume changes for hypothesis testing in preclinical studies.
Screening & Assay Development
- Scientific Value: Generates standardized, reproducible measurements of lymphatic drainage function suitable for assay development in inflammation and immune modulation screening.
- Operational Value: Supports assay readiness through stable, continuous in vivo imaging that reduces variability compared to endpoint histopathological methods.
Translational & Preclinical Research
- Scientific Value: Facilitates translational continuity by enabling longitudinal monitoring of lymphatic recovery, linking acute injury response to preclinical validation of therapeutic interventions.
- Operational Value: Enhances predictive confidence in preclinical models by providing a clinically relevant, noninvasive biomarker of lymphatic dysfunction post-injury.
Pipeline & Workflow Integration
The method integrates into the discovery continuum from early target validation through preclinical efficacy testing, particularly in models where inflammation and immune modulation are central to mechanism of action.
- Discovery Biology: Supports hypothesis testing of lymphatic involvement in inflammatory pathways and immune cell recruitment following tissue injury.
- Screening: Enables assay standardization through quantifiable ICG clearance rates and lymph node volumetry for compound screening in immunomodulation.
- Analytics: Delivers quantitative, time-resolved outputs (clearance rate, fluorescence intensity, lymph node volume) that support statistical comparison across experimental groups.
- Translational Research: Connects dynamic lymphatic changes to preclinical validation by mirroring clinical observations of post-traumatic lymphatic dysfunction.
- Enterprise Reuse: Establishes a reusable imaging platform for studying lymphatic function across multiple injury and disease models.
Operational & Enterprise Impact
- Scientific Value: Improves target validation by reducing mechanistic ambiguity in inflammation-linked pathways through direct functional assessment of lymphatic drainage.
- Operational Value: Increases reproducibility and throughput in preclinical studies by replacing variable endpoint techniques with dynamic in vivo imaging.
- Strategic Value: Informs go/no-go decisions by providing early biomarker data on target engagement in immunomodulatory strategies.
- Portfolio Impact: Enables risk-adjusted prioritization of candidates based on effects on lymphatic function and inflammatory resolution.
Implementation Considerations
- Requires expertise in near-infrared fluorescence imaging and ultrasound-based 3D reconstruction for accurate lymphatic function assessment.
- Depends on access to NIRF imaging systems and high-frequency ultrasound probes capable of popliteal lymph node visualization in mice.
- Necessitates standardization of ICG injection volume, concentration, and anatomical injection site across study groups for reliable clearance rate calculation.
- Involves adaptation considerations when applying the method to different injury models or anatomical sites beyond tibial fracture and plantar injection.
- Limited by the need for anesthesia and imaging session scheduling, which may constrain longitudinal frequency in certain study designs.