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This study was funded by the National Natural Science Foundation (82174407 to YJZ), Discipline Leader Training Program of Shanghai Pudong New Area Health Commission (PWRd2020-05),&#160;Pudong New Area Health Committee Discipline Leader Project (PWRq2020-56), and Shenzhen&#39;s Sanming Project of Medicine (No. SZZYSM202311006).

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). C57BL/6 male mice with a fracture model3 (6&#8211;8 weeks of age) were used in this study. The reagents and the equipment used are listed in the Table of Materials.&#160;
1. Preoperative care
<ol>
	<li>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.</li>
	<li>Place the mice on an animal heating pad and maintain a constant temperature of 37 &#176;C.</li>
	<li>Turn on the gas anesthesia machine and place the mouse in the induction chamber for 5 min at 2%&#8211;3% isoflurane.</li>
	<li>After induction, switch to 1%&#8211;2% isoflurane and maintain anesthesia via. a nose cone until the end of the procedure.&#160;Confirm that the mouse has lost its reflexes and that the muscles are fully relaxed. Verify the absence of a toe-pinch response and corneal reflex, and ensure that abdominal breathing is stable.&#160;Apply erythromycin ointment to both eyes to prevent corneal drying during anesthesia.</li>
</ol>
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.
<ol>
	<li>NIR-ICG imaging 
	NOTE: The step-by-step procedure for ICG near-infrared imaging is shown in Figure 1.
	<ol>
		<li>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.</li>
		<li>Turn on the imaging system and open FluoBeam v1.46. Adjust the camera height as required.</li>
		<li>Moisten a cotton swab with 75% ethanol and disinfect the plantar surface of the modeled hind paw.</li>
		<li>Using a 10 &#181;L microsyringe, aspirate 6 &#181;L 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.</li>
		<li>In the software, activate both the &#8220;camera&#8221; and &#8220;laser&#8221; functions. Click &#8220;modify&#8221; 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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>When the lymphatic vessels of the hind limb are clearly visible in the field of view, click &#8220;photo&#8221; to capture the images.</li>
		<li>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.</li>
		<li>Open the image analysis software.</li>
		<li>Import the fluorescence images by clicking File &#8594; 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).</li>
		<li>Select Oval, then click Edit &#8594; Selection &#8594; Specify; set Width = 30 and Height = 30, choose Oval, and click OK.</li>
		<li>Place the circle over a background area.</li>
		<li>Click Analyze &#8594; Measure, and record the Mean value in the Results window as fluorescence intensity at 0 h background (FI0hbg).</li>
		<li>Place the circle over the target observation site, click Analyze &#8594; Measure, and record the Mean value in the Results window as FI0h.</li>
		<li>Calculate the corrected fluorescence intensity at 0 h as Corrected FI0h = FI0h &#8722; FI0hbg.</li>
		<li>Open the matched 24 h image and repeat Steps 3&#8211;7 to obtain Corrected FI24h.</li>
		<li>Calculate the clearance rate using the background-corrected fluorescence intensity as follows: Clearance rate = [(FI0h &#8722; FI0hbg) &#8722; (FI24h &#8722; FI24hbg)] / (FI0h &#8722; FI0hbg) &#215; 100%.</li>
	</ol>
	</li>
	<li>B-mode ultrasonography 
	NOTE: The step-by-step procedure for ultrasound imaging and lymph node delineation is shown in Figure 2.
	<ol>
		<li>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.</li>
		<li>Apply ultrasound coupling gel to the modeled hind limb.</li>
		<li>Rotate the transducer so that its angle remains parallel to the longitudinal axis of the hind limb.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Export the images to 3D reconstruction software for three-dimensional image processing.</li>
		<li>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).</li>
		<li>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.</li>
		<li>Open the 3D reconstruction software.</li>
		<li>Click &#8220;Study&#8221; to enter the module, and select &#8220;Import.&#8221;</li>
		<li>Click the data folder, check the data to be imported, and double-click to open it.</li>
		<li>In the display mode, select &#8220;Image Processing&#8221; &#8594; &#8220;Load into 3D&#8221; &#8594; &#8220;Transverse View&#8221; &#8594; &#8220;Volume Measurement&#8221; &#8594; &#8220;Start.&#8221;</li>
		<li>Confirm that the slice orientation and step parameters (&#8220;voxel/spacing&#8221;) are consistent with the acquisition settings.</li>
		<li>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).</li>
		<li>Begin tracing on the first slice in which the PLN appears by left-clicking to start the contour.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Use the mouse wheel to move to the slice just before the PLN disappears, and repeat Step 10 to complete the third contour.</li>
		<li>Click &#8220;Finish&#8221; to generate the 3D segmented volume and output the volume measurement.</li>
		<li>Record the PLN volume (mm3) in the results panel.</li>
		<li>In the measurement results panel, select &#8220;Export&#8221; and export the results as a .csv or .xls file.</li>
		<li>Select the file type and export the image as TIFF Image Area (.tif).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Statistical analysis using statistical analysis software
	<ol>
		<li>Select New Table &#38; Graph, choose Grouped.</li>
		<li>Set the data-entry format to Enter replicate values, stacked into subcolumns.</li>
		<li>Enter the raw values for 1 / 7 / 14 and Sham / Fracture.-Open the graph.</li>
		<li>Change the graph type to Scatter (individual values) + mean + error bars.</li>
		<li>Format the axes: set the Y-axis and change the axis title.</li>
		<li>Click on Analyze and perform a two-way ANOVA.</li>
		<li>Compare Sham versus Fracture at each time point.</li>
		<li>Add the significance results to the graph.</li>
	</ol>
	</li>
</ol>
3. Post-operative care
<ol>
	<li>At the end of the experiment, place the mouse on a heating pad and allow it to recover from anesthesia.</li>
	<li>Monitor mice post-operatively for respiratory rate, nasal mucosa color, curling reflex, food intake, and behavioral changes. 
	NOTE: A normal respiratory rate is above 50 breaths per minute, and the nasal mucosa should remain pink; cyanosis or respiratory depression may indicate anesthetic overdose or residual anesthesia and should be managed by discontinuing medication, clearing the airway, elevating the head, and performing chest compressions or artificial respiration if necessary. Maintain body temperature at approximately 37 &#176;C using a heating pad to prevent hypothermia and promote anesthetic recovery. Administer meloxicam subcutaneously at 0.5&#8211;1 mg/kg every 24 hours for 3 days for post-operative analgesia, and assess any abnormalities with appropriate veterinary intervention.</li>
</ol>
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.
<ol>
	<li>NIR-ICG imaging
	<ol>
		<li>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 (%) = [(FI0h &#8211; FI24h) / (FI0h)] &#215; 100%</li>
		<li>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.</li>
	</ol>
	</li>
	<li>B-mode ultrasonography
	<ol>
		<li>Analyze B-mode and three-dimensional images using 3D reconstruction software to determine PLN volume.</li>
		<li>Assess changes in lymphatic drainage and lymph node volume over time by creating scatter plots with statistical analysis software.</li>
	</ol>
	</li>
	<li>Statistical analysis
	<ol>
		<li>Analyze repeated measurements obtained on days 1, 7, and 14 using two-way repeated-measures ANOVA.</li>
	</ol>
	</li>
</ol>

<ol><li>Zhang, S., Sheng, H., Xu, B., Lao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acute+external+iliac+artery+thrombosis+following+pelvic+fractures%3A+Two+case+reports%5BTitle%5D)+AND+%22Medicine+%28Baltimore%29%22%5BJournal%5D)">Acute external iliac artery thrombosis following pelvic fractures: Two case reports.</a> Medicine (Baltimore). 100 (6), e24710(2021).</li>
<li>Szczesny, G., Olszewski, W. L., Gewartowska, M., Zaleska, M., Gorecki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+healing+of+tibial+fracture+and+response+of+the+local+lymphatic+system%5BTitle%5D)+AND+%22J+Trauma%22%5BJournal%5D)">The healing of tibial fracture and response of the local lymphatic system.</a> J Trauma. 63 (4), 849-854 (2007).</li>
<li>Zheng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+therapy+for+fracture+healing+by+increasing+lymphatic+drainage%5BTitle%5D)+AND+%22J+Orthop+Translat%22%5BJournal%5D)">A novel therapy for fracture healing by increasing lymphatic drainage.</a> J Orthop Translat. 45, 66-74 (2024).</li>
<li>Thomas, C., Le, J. T., Benson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Managing+lymphedema+in+fracture+care%3A+Current+concepts+and+treatment+principles%5BTitle%5D)+AND+%22J+Am+Acad+Orthop+Surg%22%5BJournal%5D)">Managing lymphedema in fracture care: Current concepts and treatment principles.</a> J Am Acad Orthop Surg. 28 (18), 737-741 (2020).</li>
<li>Jiang, X., Nicolls, M. R., Tian, W., Rockson, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphatic+dysfunction%2C+leukotrienes%2C+and+lymphedema%5BTitle%5D)+AND+%22Annu+Rev+Physiol%22%5BJournal%5D)">Lymphatic dysfunction, leukotrienes, and lymphedema.</a> Annu Rev Physiol. 80, 49-70 (2018).</li>
<li>Biswas, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphatic+vessels+in+bone+support+regeneration+after+injury%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Lymphatic vessels in bone support regeneration after injury.</a> Cell. 186 (2), 382-397.e24 (2023).</li>
<li>Dimitriou, R., Tsiridis, E., Giannoudis, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+concepts+of+molecular+aspects+of+bone+healing%5BTitle%5D)+AND+%22Injury%22%5BJournal%5D)">Current concepts of molecular aspects of bone healing.</a> Injury. 36 (12), 1392-1404 (2005).</li>
<li>Arslan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+lymphedema+on+the+recovery+of+fractures%5BTitle%5D)+AND+%22J+Orthop+Sci%22%5BJournal%5D)">Effect of lymphedema on the recovery of fractures.</a> J Orthop Sci. 12 (6), 578-584 (2007).</li>
<li>Cheon, H., Kim, S. A., Kim, B., Jeon, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Investigation+of+optimizing+indocyanine+green+solution+for+in+vivo+lymphatic+research+using+near-infrared+fluorescence+indocyanine+green+lymphangiography%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Investigation of optimizing indocyanine green solution for in vivo lymphatic research using near-infrared fluorescence indocyanine green lymphangiography.</a> Sci Rep. 13 (1), 14966(2023).</li>
<li>Polomska, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minimally+invasive+method+for+the+point-of-care+quantification+of+lymphatic+vessel+function%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">Minimally invasive method for the point-of-care quantification of lymphatic vessel function.</a> JCI Insight. 4 (4), e126515(2019).</li>
<li>Wang, F., Weng, Y., Geng, J., Zhu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+narrative+review+of+indocyanine+green+near-infrared+fluorescence+imaging+technique%3A+A+new+application+in+thoracic+surgery%5BTitle%5D)+AND+%22Curr+Challenges+Thorac+Surg%22%5BJournal%5D)">A narrative review of indocyanine green near-infrared fluorescence imaging technique: A new application in thoracic surgery.</a> Curr Challenges Thorac Surg. 3, 35-35 (2021).</li>
<li>Zheng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphatic+platelet+thrombosis+limits+bone+repair+by+precluding+lymphatic+transporting+damps%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Lymphatic platelet thrombosis limits bone repair by precluding lymphatic transporting damps.</a> Nat Commun. 16 (1), 829(2025).</li>
<li>Kim, K. M., Kim, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphangiogenesis+after+nonvascularized+lymph+node+transplantation%3A+Lymphangiographic+findings+in+mice+and+minipigs%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Lymphangiogenesis after nonvascularized lymph node transplantation: Lymphangiographic findings in mice and minipigs.</a> PLoS One. 18 (12), e0295836(2023).</li>
<li>Zhang, P. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+pulmonary+lymphatic+drainage+is+necessary+for+inflammation+resolution+in+ARDS%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">Efficient pulmonary lymphatic drainage is necessary for inflammation resolution in ARDS.</a> JCI Insight. 9 (1), e173440(2024).</li>
<li>Kenney, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Persistent+popliteal+lymphatic+muscle+cell+coverage+defects+despite+amelioration+of+arthritis+and+recovery+of+popliteal+lymphatic+vessel+function+in+TNF-TG+mice+following+anti-TNF+therapy%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Persistent popliteal lymphatic muscle cell coverage defects despite amelioration of arthritis and recovery of popliteal lymphatic vessel function in TNF-TG mice following anti-TNF therapy.</a> Sci Rep. 12 (1), 12751(2022).</li>
</ol>

The authors have no conflicts of interest to declare.

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&#39;s lower limbs with coupling agent, as any uncovered areas will cause distortion in the resulting images when the probe scans those regi...

Traumatic fractures often cause significant tissue damage and swelling, increasing the risk of complications like infection, necrosis, and bone nonunion1,2. 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-&#945;, 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-&#945;, IL-1, and IL-6) and growth factors (TGF&#946;1 and PDGF) produced by immune cells at the damaged site are important regulatory factors for osteoblast apoptosis and BMSC proliferation8. H&#252;seyin 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 humans9,10,11. This technique allows for a quantitative evaluation of lymphatic vessel contractility, drainage velocity, and pathway integrity10,11. 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.

<table><tbody><tr><td>10 Microliter Microsyringe</td><td>Hamilton Company</td><td>80330/00</td><td>The Hamilton 80330/00 microsyringe is a precision instrument with a 10&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/70466/70466eq1.jpg&quot; style=&quot;margin: auto;&quot;/&gt;L 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.</td></tr><tr><td>3D Ultrasound Vevo 3100 Imaging System</td><td></td><td>Vevo 2100</td><td>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.</td></tr><tr><td>75% Ethanol</td><td>Shanghai Titan Scientific Co., Ltd.</td><td>G73537O</td><td>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.</td></tr><tr><td>Cotton Swab</td><td>Hebei Kangji Medical Equipment Co., Ltd.</td><td>20250303</td><td>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.</td></tr><tr><td>Coupling Agent</td><td>Tianjin Jinya Technology Development Co., Ltd.</td><td>20241029</td><td>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.</td></tr><tr><td>Graphpad Prism</td><td>GraphPad Software</td><td>Prism 10</td><td>A scientific graphing and statistical analysis software used for data analysis, visualization, and presentation.</td></tr><tr><td>Image J</td><td>National Institutes of Health</td><td>ImageJ 1.54d</td><td>A public-domain, Java-based image processing and analysis program widely used for biological and medical image analysis.</td></tr><tr><td>indocyanine green</td><td>Dandong Yichuang Pharmaceutical Co., Ltd.</td><td>8305128</td><td>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.</td></tr><tr><td>isoflurance&amp;nbsp;</td><td>RWD&amp;nbsp;</td><td>R650-IE</td><td>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.&amp;nbsp;</td></tr><tr><td>Microbalance</td><td>Shanghai Precision Scientific Instrument Co., Ltd.</td><td>FA1004B</td><td>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.</td></tr><tr><td>Near-Infrared Imaging System</td><td>Olympus</td><td>MVX10</td><td>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.</td></tr><tr><td>Vevo lab</td><td>\</td><td>Vevo LAB</td><td>An imaging analysis software package used for analyzing Vevo imaging data, including measurement, calculation, and report generation.</td></tr></tbody></table>

the assessment of lymphatic drainage using icg near-infrared imaging and ultrasound after fracture

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

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 &#181;L 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. &#160;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.&#160; 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.

On days 1, 7, and 14 after establishing the fracture model, the 24-h plantar ICG fluorescence intensity (FI24h) 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 &lt; 0.05) (Figure 4</strong...

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.

Traumatic fractures are often accompanied by impaired lymphatic drainage function. Reduced lymphatic drainage and lymph node enlargement after fracture ...

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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108 Views.  Shanghai University of Traditional Chinese Medicine. 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...

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

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