The Assessment of Lymphatic Drainage Using ICG Near-Infrared Imaging and Ultrasound After Fracture

Summary

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.

Abstract

Traumatic fractures are often accompanied by impaired lymphatic drainage function. Reduced lymphatic drainage and lymph node enlargement after fracture indicate ongoing, dynamic processes of the acute inflammatory response and immune cell recruitment. This article describes a method in which 6 µL of 0.2 mg/mL indocyanine green was injected intradermally into the plantar region of mice undergoing sham or tibial fracture surgery. Near-infrared fluorescence imaging quantified plantar ICG signal at 0 h and 24 h to compute the 24 h clearance rate as a readout of murine lymphatic drainage. Ultrasound was used simultaneously to 3D-reconstruct the popliteal lymph nodes (PLNs), compare their volumes, and assess fracture-induced enlargement.  The results demonstrated that after fracture, the lymphatic clearance rate significantly decreased, the fluorescence intensity of ICG in the plantar region was markedly higher, and the volume of the PLNs was significantly enlarged.  Compared with previous methods of measuring the lymphatic system, such as histopathological sections or lymphoscintigraphy, which often fail to achieve in vivo, dynamic, and continuous observation, this method demonstrates high stability and reproducibility. Notably, it enables dynamic in vivo. monitoring of lymphatic drainage function, providing robust technical support for investigating changes in lymphatic reflux in fracture models.

Introduction

Traumatic fractures often cause significant tissue damage and swelling, increasing the risk of complications like infection, necrosis, and bone nonunion^1,2. Grzegorz Szczesny et al. showed that lymphatic drainage dysfunction, leading to tissue edema, is linked to delayed healing³. Studies indicate that during the fracture healing process, in addition to the repair of bone tissue, the lymphatic system at the fracture site also plays an important role. Lymphatic vessels are responsible not only for draining interstitial fluid and preventing swelling⁴, but also for regulating immune cell migration and clearing metabolic waste. However, in the early stages of fracture, pro-inflammatory factors such as TNF-α, IL-1, IL-6, and Macrophage Colony-Stimulating Factor (MCSF) can damage lymphatic endothelial cells⁵, leading to pathological changes such as degeneration, edema, and fibrosis⁶. Relevant studies have shown the presence of lymphatic endothelial cells in bone and periosteum, as well as surrounding adipose and muscle tissues. Furthermore, lymphatic drainage also participates in regulating the microenvironment of hematoma after fracture; effective lymphatic drainage facilitates the survival of osteoblasts and the proliferation of bone marrow mesenchymal stem cells (BMSCs)⁷. During a fracture, the pro-inflammatory factors (TNF-α, IL-1, and IL-6) and growth factors (TGFβ1 and PDGF) produced by immune cells at the damaged site are important regulatory factors for osteoblast apoptosis and BMSC proliferation⁸. Hüseyin Arslan and other scholars have confirmed the negative impact of lymphatic edema on osteoblasts. These research findings highlight the important role of lymphatic drainage in fracture healing. Recent advancements in ICG near-infrared (NIR) fluorescence imaging have facilitated noninvasive, real-time observation of lymphatic flow dynamics in both animal models and humans^9,10,11. This technique allows for a quantitative evaluation of lymphatic vessel contractility, drainage velocity, and pathway integrity^10,11. A study using murine models of arthritis and inflammation has shown that ICG-NIR imaging can differentiate between acute and chronic lymphatic responses, highlighting distinct drainage patterns during tissue injury and recovery. Furthermore, NIR lymphatic imaging has been shown to be effective for monitoring functional recovery following manual lymphatic drainage and for evaluating the efficacy of lymphatic-targeted therapies. Ultrasonography provides a complementary quantitative modality for assessing the morphology and volume of draining lymph nodes, as well as tissue edema associated with impaired lymphatic drainage. The integration of NIR fluorescence imaging with ultrasound enables concurrent assessment of lymphatic function and anatomical changes, providing a multimodal approach for investigating lymphatic dynamics in vivo. Despite growing recognition of the critical role of lymphatic dysfunction in post-traumatic healing, standardized and reproducible methods for assessing lymphatic drainage after fractures remain lacking. Many current imaging methods are invasive, lack adequate spatial resolution, or fail to dynamically monitor lymph flow. Therefore, developing a protocol that combines ICG-NIR and ultrasound imaging is anticipated to address this methodological shortcoming and provide an effective tool for assessing lymphatic recovery and its contribution to bone repair.

This study presents an innovative framework combining ICG-NIR fluorescence imaging with high-resolution ultrasound to assess lymphatic drainage after tibial fractures in mice. This approach enables semi-quantitative analysis of drainage efficiency and measures the volume of lymph nodes during the fracture healing process. It also has some limitations. Firstly, ICG-NIR is primarily used to evaluate superficial lymphatic vessels, and its imaging capability for deeper structures is limited. Secondly, although changes in lymph node volume provide some information, they lack specificity and cannot fully replace direct functional measurements. This methodology has significant translational potential and improves our understanding of lymphatic function in bone regeneration.

Protocol

The animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai Model Organisms Center (Approval No. NMB-SMP-IACUC-01-0002-R02-03). C57 male mice with a fracture model (6–8 weeks of age) were used in this study. The reagents and the equipment used are listed in the Table of Materials.

1. Preoperative care

1. Acclimate the mice for at least 1 week with ad libitum. access to food and water. Randomly assign the animals to the fracture or sham group using a computer-generated randomization list.
2. Place the mice on an animal heating pad and maintain a constant temperature of 37 °C.
3. Turn on the gas anesthesia machine and place the mouse in the induction chamber for 5 min at 2%–3% isoflurane.
4. After induction, switch to 1%–2% isoflurane and maintain anesthesia via. a nose cone until the end of the procedure.

2. Experimental procedure

NOTE: Standardize anesthesia settings, ICG dose and volume, injection site and depth, exposure time, ROI definition, and ultrasound scan parameters across all sessions to minimize inter-animal variability.

1. NIR-ICG imaging
   NOTE: The step-by-step procedure for ICG near-infrared imaging is shown in Figure 1.
   1. Weigh 0.2 mg of indocyanine green (ICG), transfer it to a 1.5 mL microcentrifuge tube, and dissolve it in 1 mL PBS to prepare a 0.2 mg/mL ICG solution. Wrap the tube with aluminum foil to protect it from light.
   2. Turn on the imaging system and open FluoBeam v1.46. Adjust the camera height as required.
   3. Moisten a cotton swab with 75% ethanol and disinfect the plantar surface of the modeled hind paw.
   4. Using a 10 µL microsyringe, aspirate 6 µL of the ICG solution. Insert the needle approximately 1 mm into the plantar surface and inject the solution at a constant rate until the full volume is delivered.
   5. In the software, activate both the “camera” and “laser” functions. Click “modify” to create a new folder and set it as the save directory. Keep the exposure time identical for image acquisition at 0 h and 24 h.
   6. Place the mouse under the camera in the prone position with the modeled hind limb centered in the field of view. Ensure that the modeled hind limb is centered in the field of view.
   7. Set the exposure time to 30 ms and keep it identical for both the 0 h and 24 h images. Acquire images of the same plantar injection ROI at 0 h and 24 h post-injection to measure ICG signal intensity. Keep the exposure time identical at 0 h and 24 h.
   8. When the lymphatic vessels of the hind limb are clearly visible in the field of view, click “photo” to capture the images.
   9. After image acquisition, export the data and use image analysis software to quantify fluorescence intensity at 0 h and 24 h, and then calculate the fluorescence clearance rate.
   10. Open the image analysis software.
   11. Import the fluorescence images by clicking File → Open, and open the 0 h plantar image corresponding to the same animal and the same examination day (then open the matched 24 h image afterward).
   12. Select Oval, then click Edit → Selection → Specify; set Width = 30 and Height = 30, choose Oval, and click OK.
   13. Place the circle over a background area.
   14. Click Analyze → Measure, and record the Mean value in the Results window as fluorescence intensity at 0 h background (FI0hbg).
   15. Place the circle over the target observation site, click Analyze → Measure, and record the Mean value in the Results window as FI0h.
   16. Calculate the corrected fluorescence intensity at 0 h as Corrected FI0h = FI0h − FI0hbg.
   17. Open the matched 24 h image and repeat Steps 3–7 to obtain Corrected FI24h.
   18. Calculate the clearance rate using the background-corrected fluorescence intensity as follows: Clearance rate = [(FI0h − FI0hbg) − (FI24h − FI24hbg)] / (FI0h − FI0hbg) × 100%.
2. B-mode ultrasonography
   NOTE: The step-by-step procedure for ultrasound imaging and lymph node delineation is shown in Figure 2.
   1. 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, and position the transducer over the depression of the popliteal fossa.
   2. Apply ultrasound coupling gel to the modeled hind limb.
   3. Rotate the transducer so that its angle remains parallel to the longitudinal axis of the hind limb.
   4. Set the scan distance to 4 mm and the step size to 0.04 mm. Identify the popliteal lymph node on the image according to its anatomical location.
   5. Stabilize the mouse by holding the tail and the modeled hind limb with both hands. Do not move the animal while the transducer is scanning.
   6. Acquire images. Identify the popliteal lymph node as a small oval or slightly bean-shaped nodule with intermediate-to-low echogenicity, slightly lower than that of the surrounding fat, and clearly demarcated from the adjacent darker, striated muscle.
   7. Export the images to 3D reconstruction software for three-dimensional image processing.
   8. After completing the 3D scan on the ultrasound imaging system console, save the scan as a 3D dataset, and record the animal ID, group code, and time point (0 h / 24 h).
   9. Export the raw data from the acquisition console to the analysis computer, keeping the original folder structure unchanged; use one folder for each animal at each time point whenever possible.
   10. Open the 3D reconstruction software.
   11. Click “Study” to enter the module, and select “Import.”
   12. Click the data folder, check the data to be imported, and double-click to open it.
   13. In the display mode, select “Image Processing” → “Load into 3D” → “Transverse View” → “Volume Measurement” → “Start.”
   14. Confirm that the slice orientation and step parameters (“voxel/spacing”) are consistent with the acquisition settings.
   15. 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 PLN appears (it usually appears as an oval or bean-shaped structure, slightly hypoechoic relative to the surrounding fat, with a relatively clear boundary).
   16. Begin tracing on the first slice in which the PLN appears by left-clicking to start the contour.
   17. Trace a closed contour point by point along the edge of the lymph node, keeping the contour as close as possible to the outer capsule and avoiding inclusion of the surrounding fat or vascular lumen.
   18. Use the mouse wheel to move to the slice showing the maximum cross-sectional area of the PLN, and repeat Step 10 to complete the second contour.
   19. Use the mouse wheel to move to the slice just before the PLN disappears, and repeat Step 10 to complete the third contour.
   20. Click “Finish” to generate the 3D segmented volume and output the volume measurement.
   21. Record the PLN volume (mm³) in the results panel.
   22. In the measurement results panel, select “Export” and export the results as a .csv or .xls file.
   23. Select the file type and export the image as TIFF Image Area (.tif).
   24. Maintain blinding during analysis by displaying only the animal code and time point, without showing the group assignment (fracture/sham), until all volume extraction has been completed and the groups are decoded for statistical analysis.
3. Statistical analysis using statistical analysis software
   1. Select New Table & Graph, choose Grouped.
   2. Set the data-entry format to Enter replicate values, stacked into subcolumns.
   3. Enter the raw values for 1 / 7 / 14 and Sham / Fracture.-Open the graph.
   4. Change the graph type to Scatter (individual values) + mean + error bars.
   5. Format the axes: set the Y-axis and change the axis title.
   6. Click on Analyze and perform a two-way ANOVA.
   7. Compare Sham versus Fracture at each time point.
   8. Add the significance results to the graph.

3. Post-operative care

1. At the end of the experiment, place the mouse on a heating pad and allow it to recover from anesthesia.
2. Monitor its condition closely, and if no abnormalities are observed, return it to the cage for continued housing.

4. Evaluation

NOTE: Have an investigator blinded to group allocation perform image acquisition and quantitative analyses, and keep all datasets coded until statistical analyses are completed.

1. NIR-ICG imaging
   1. Use image analysis software to perform relative quantitative analysis of fluorescence intensity in ICG images collected from mouse footpads.
      NOTE: The calculation formula for the clearance rate of ICG in mouse footpads is as follows:
      Clearance rate (%) = [(FI_0h – FI_24h) / (FI_0h)] × 100%
   2. Use the same method to record the clearance rate for each measurement, and analyze the relationship between lymphatic clearance rate and time using statistical analysis software.
2. B-mode ultrasonography
   1. Analyze B-mode and three-dimensional images using 3D reconstruction software to determine PLN volume.
   2. Assess changes in lymphatic drainage and lymph node volume over time by creating scatter plots with statistical analysis software.
3. Statistical analysis
   1. Analyze repeated measurements obtained on days 1, 7, and 14 using two-way repeated-measures ANOVA.

Results

On days 1, 7, and 14 after establishing the fracture model, the 24-h plantar ICG fluorescence intensity (FI_24h) in the fracture group was consistently higher than that in the sham surgery group (Figure 3). Relative quantitative analysis of ICG fluorescence intensity from collected plantar images showed that the lymphatic ICG clearance rate in the fracture group was significantly lower than that in the sham surgery group (p < 0.05) (Figure 4). Meanwhile, three-dimensional B-mode ultrasonography revealed that the popliteal lymph node volume in the fracture group mice was significantly higher than that in the control group on days 1, 7, and 14 (p < 0.05) (Figure 5). These results demonstrated a high correlation between lymphatic clearance rate and lymph node enlargement.

Figure 1: Step-by-step images of using ICG near-infrared imaging. (A) Indocyanine green (ICG) powder is weighed using a microbalance. (B) A 10 µL microsyringe is used to aspirate 6 µL of the ICG solution. (C,D) The needle is inserted approximately 1 mm into the paw, and the solution is injected at a constant rate until the full volume is delivered. (E) The mouse is positioned under the camera. (F)The modeled hind limb is centered in the field of view. Please click here to view a larger version of this figure.

Figure 2: Step-by-step images of using ultrasound. (A) The anesthetized mouse is placed in the prone position on the imaging platform. (B,C) Ultrasound coupling gel is applied to the modeled hind limb. (D) The transducer is rotated so that it remains parallel to the longitudinal axis of the hind limb. (E) Ultrasound images are acquired. (F) The lymph node is delineated for analysis. Please click here to view a larger version of this figure.

Figure 3: Plantar ICG fluorescence intensity in the fracture group and sham group at different time points. At days 1, 7, and 14 after fracture model establishment, the 24-h plantar ICG fluorescence intensity (FI_{24 h}) in the fracture group was significantly higher than that in the sham group. Scale bars: 5 mm. Please click here to view a larger version of this figure.

Figure 4: ICG clearance rates in lymphatic vessels of the fracture group and sham group. Relative quantitative analysis of plantar ICG fluorescence images showed that the lymphatic ICG clearance rates in the fracture group were consistently lower than those in the sham group on days 1, 7, and 14 after fracture model establishment (p < 0.05). Please click here to view a larger version of this figure.

Figure 5: Three-dimensional ultrasound images of the popliteal lymph nodes in the fracture group and the sham group. On days 1, 7, and 14 after fracture model establishment, the three-dimensional ultrasound results showed that the volume of the popliteal lymph nodes in the fracture group was significantly larger than that in the sham group (p < 0.05), indicating the impact of fractures on lymph node enlargement. Please click here to view a larger version of this figure.

Discussion

The protocol includes several easily overlooked details. For instance, the exposure time is not fixed and must be determined based on the specific fluorescence intensity. However, the exposure time for the same group of mice at 0 h and 24 h must remain consistent; otherwise, the results may be unreliable. When using ultrasound for observation, it is essential to fully cover the mouse's lower limbs with coupling agent, as any uncovered areas will cause distortion in the resulting images when the probe scans those regions. During scanning, the mouse's lower limbs should remain still. The operator can stabilize the mouse by gently pulling the lower limbs with one hand while holding the tail with the other. This step is particularly challenging yet crucial for accurate ultrasound examination.

ICG-NIR and ultrasound are widely used in various research and clinical contexts, but it is important to note that their parameters can vary depending on the specific indicators being observed^12,13. ICG-NIR can be used to dynamically assess lymphatic drainage velocity and pathway integrity, whereas ultrasound serves as a complementary modality for evaluating the morphology and volume of draining lymph nodes. When using ultrasound to examine tissues, pre-localization of their anatomical position prior to the experiment can significantly improve efficiency. For instance, the popliteal lymph node is located between the biceps femoris tendon, semimembranosus tendon, and gastrocnemius muscle, adjacent to the knee joint capsule. During observation, oval-shaped dark shadows resembling the popliteal lymph node may appear. When identification is uncertain, moving the probe can help confirm the shadow's location.

One of the limitations of this study is that ICG-NIR is primarily suited for assessing superficial lymphatic vessels and cannot effectively visualize deeper structures. Due to the limited tissue penetration of NIR fluorescence (on the order of a few millimeters), ICG-NIR imaging primarily reports superficial lymphatic drainage and does not directly capture deeper collecting vessels or intranodal inflow dynamics^14,15. Although it can detect changes in lymph node volume, it does not provide direct information on lymph node function. Variations in ICG injection efficiency, depth of anesthesia, and other uncontrollable factors, such as differences between mice, may introduce biases. Future studies should combine immunological markers and histological evidence to validate these mechanisms and explore the potential of ICG-NIR in assessing deeper lymphatic vessels and node function.

Despite these limitations, this study has notable strengths. Compared with microsurgical injury, genetic/inflammatory lymphedema models, or deep-functional techniques, our protocol prioritizes noninvasive longitudinal monitoring, enabling multi-time-point evaluations without harming the animals and making it easily reproducible. The calculation of the ICG clearance rate is simple and intuitive, supported by a standardized image-processing workflow that reduces the likelihood of error. Additionally, ultrasound imaging complements the limitations of ICG-NIR by enabling the detection of deeper lymph nodes. The combined application of these two techniques provides a comprehensive assessment of lymphatic drainage following fractures, offering valuable tools for future clinical and research applications.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 Microliter Microsyringe	Hamilton Company	80330/00	The Hamilton 80330/00 microsyringe is a precision instrument with a 10L capacity, featuring a 26s gauge needle, 51mm length, and point style 2. It is designed for accurate delivery of small volumes in chromatography, sample injection, and analytical chemistry applications.
3D Ultrasound Vevo 3100 Imaging System		Vevo 2100	The Vevo 2100 is a high-frequency ultrasound imaging system for preclinical research, providing real-time 3D visualization of small animals. It offers superior resolution for cardiovascular, oncology, and developmental biology studies, integrated with Vevo LAB software for data analysis.
75% Ethanol	Shanghai Titan Scientific Co., Ltd.	G73537O	75% ethanol is a disinfectant solution widely used in laboratories for surface sterilization, equipment cleaning, and antisepsis. It effectively kills bacteria, viruses, and fungi through protein denaturation and is commonly applied in biological and clinical settings.
Cotton Swab	Hebei Kangji Medical Equipment Co., Ltd.	20250303	Cotton swabs are sterile, absorbent applicators used for precise sample collection, application of solutions, or cleaning in medical, diagnostic, and laboratory procedures. They feature wooden or plastic sticks with cotton tips for hygienic handling.
Coupling Agent	Tianjin Jinya Technology Development Co., Ltd.	20241029	Coupling agent, commonly known as ultrasound gel, is a water-based medium used to enhance acoustic transmission between the ultrasound transducer and skin. It eliminates air pockets for clear imaging, is non-irritating, and supplied in a 250mL container for medical and diagnostic use.
Graphpad Prism	GraphPad Software	Prism 10	A scientific graphing and statistical analysis software used for data analysis, visualization, and presentation.
Image J	National Institutes of Health	ImageJ 1.54d	A public-domain, Java-based image processing and analysis program widely used for biological and medical image analysis.
indocyanine green	Dandong Yichuang Pharmaceutical Co., Ltd.	8305128	Indocyanine green is a water-soluble anionic dye used in medical diagnostics for determining cardiac output, hepatic function, liver blood flow, and ophthalmic angiography. It is supplied in 25mg vials for intravenous administration and exhibits fluorescence in the near-infrared spectrum for imaging purposes.
isoflurance	RWD	R650-IE	Isoflurane is an inhaled general anesthetic. It has a small blood/gas partition coefficient. When used for animal anesthesia, it can induce anesthesia smoothly, rapidly and comfortably, with a quick recovery, good muscle relaxation, and no excitatory effect on the sympathetic nervous system. The metabolic rate of isoflurane in the liver is low, so it has little toxicity to the liver, and there are no obvious side effects even with repeated use. To avoid the adverse effects of anesthetic waste gas on the environment and laboratory personnel, it is recommended to use it together with a gas recovery system.
Microbalance	Shanghai Precision Scientific Instrument Co., Ltd.	FA1004B	The FA1004B microbalance is a high-precision analytical instrument designed for accurate weighing of small samples, offering readability up to 0.0001g and suitable for laboratory applications requiring precise measurements in research and quality control.
Near-Infrared Imaging System	Olympus	MVX10	The Olympus MVX10 is a macro zoom fluorescence microscope system optimized for near-infrared imaging. It offers high-resolution visualization of large specimens with a wide zoom range, advanced optics for deep tissue penetration, and compatibility with fluorescence techniques in biological and materials research.
Vevo lab	\	Vevo LAB	An imaging analysis software package used for analyzing Vevo imaging data, including measurement, calculation, and report generation.