Method for constructing a lymphedema animal model based on fluorescence augmented reality guidance and uses thereof

Abstract

The embodiment of the present disclosure provides a lymphedema animal model construction method based on fluorescence augmented reality guidance and application thereof. The animal model construction method comprises: injecting a mixed tracer into a model animal, collecting a white light dissection image and an indocyanine green near-infrared fluorescence image; the mixed tracer comprises Evans blue and indocyanine green; removing target lymph nodes and the connected drainage lymphatic vessels of the model animal under the guidance of FAR, and collecting a postoperative fluorescence image to verify the completeness of the lymphatic drainage pathway blockage; closing the skin incision to obtain a lymphedema animal model. The animal model construction method of the embodiment can confirm the normal lymphatic drainage pathway from two dimensions of fluorescence imaging and white light field coloration, and can realize real-time visual positioning of the target lymph nodes and the drainage lymphatic vessels of the model animal for precise removal, and can realize non-invasive evaluation of the lymphatic return mode of the hind limbs of the model animal after operation.

Description

Technical Field

[0001] This disclosure relates to the field of medical animal experimental model technology, specifically to a method for constructing a lymphedema animal model based on fluorescence augmented reality guidance and its application. Background Technology

[0002] Secondary lymphedema is a common complication following tumor surgery and radiotherapy. Its pathological features include chronic local inflammation, abnormal extracellular matrix fibrosis, adipocyte deposition, and progressive sclerosis. These conditions further obstruct lymphatic flow and accelerate disease progression. Currently, complex anti-edema treatments and microsurgery are the main methods for improving the symptoms of secondary lymphedema, with lymphovenous anastomosis (LVA) being the most representative functional surgical treatment. Establishing reliable animal models is a prerequisite for in-depth research into the pathogenesis of secondary lymphedema and for evaluating the effectiveness of novel treatments.

[0003] Currently, the commonly used rodent secondary lymphedema model mainly involves surgically removing the inguinal and popliteal lymph nodes and draining lymphatic vessels to block lymphatic drainage. However, the above methods face the following main challenges in practice: (1) In rodents, the diameter of the lymphatic vessels collecting in the groin / popliteal region of rats is about 200-500 μm (average about 300 μm). Although they are thicker than those of mice (100-200 μm), they are still colorless and translucent with thin walls, making them difficult to identify reliably under conventional naked eye or white light surgical microscope; their capillary lymphatic vessels are only 5-30 μm, making them even more difficult to distinguish directly. Previous studies relied on staining with a single Evans blue dye. Although this method can stain lymph nodes and large lymphatic vessels, it has limited visibility of small capillary lymphatic vessels and drainage pathways. Moreover, the accuracy of localization decreases after the dye diffuses in the tissue. (2) Due to inaccurate lymphatic vessel localization, key lymphatic vessels are often left uncut during surgery, leading to the re-establishment of lymphatic drainage through collateral or residual pathways, resulting in a high failure rate of model formation. In addition, there are significant differences in operation between different surgeons, making it difficult to guarantee the stability and reproducibility of the model. (3) Traditional methods cannot immediately confirm whether all lymphatic drainage pathways have been completely interrupted after lymph node removal, leading to postoperative model evaluation relying on morphological observation several days to several weeks later, which delays the judgment of model quality. (4) Traditional model evaluation mainly relies on static indicators such as limb volume measurement and histopathological staining, which cannot dynamically, in real time and quantitatively evaluate the function of the lymphatic system. Furthermore, it requires animal sacrifice to obtain tissue specimens for analysis.

[0004] To address the problems of inaccurate pathological replication, lack of precise navigation during modeling, and difficulty in dynamically assessing postoperative lymphatic function in existing rodent secondary lymphedema models, fluorescence augmented reality technology can overlay indocyanine green (ICG) fluorescent images with white light fields of view, enabling non-invasive real-time visualization of lymph nodes and lymphatic vessels. This allows surgeons to simultaneously obtain anatomical morphology and lymphatic system information without adjusting their surgical posture.

[0005] Currently, near-infrared fluorescence (NIRF)-ICG technology has been used for lymphedema grading assessment and sentinel lymph node biopsy navigation. However, there are no relevant literature reports on the FAR method, which integrates fluorescence images and white light surgical field images, for precise intraoperative navigation, standardized operation guidance, and dynamic assessment of postoperative lymphatic drainage in rodent model construction. Summary of the Invention

[0006] To overcome the shortcomings of the existing technology, the purpose of this disclosure is to provide a method for constructing an animal model of lymphedema based on fluorescence augmented reality and its application.

[0007] To achieve the above objectives, the present disclosure proposes the following technical solutions: In a first aspect, embodiments of this disclosure propose a method for constructing an animal model of lymphedema based on fluorescence augmented reality guidance, the method comprising: The model animals were injected with a mixed tracer, and white light anatomical images and indocyanine green near-infrared fluorescence images were acquired; the mixed tracer included Evans blue and indocyanine green. Under FAR guidance, the target lymph nodes and their connected draining lymphatic vessels in the model animals were removed, and postoperative fluorescence images were acquired to verify the integrity of the lymphatic drainage pathway blockage. The skin incision was closed to obtain an animal model of lymphedema.

[0008] As one implementation method, the animal model construction method further includes a postoperative multidimensional dynamic assessment step, the assessment step including: At multiple pre-defined time points, perform at least one of the following non-invasive dynamic assessments on the model animals: Changes in limb volume, changes in limb circumference, indocyanine green lymphatic reflux pattern, Evans blue retention status, and histological verification.

[0009] As one implementation method, the change in limb volume is measured using the water displacement volume measurement method; the change in limb circumference is measured using the tape circumference measurement method.

[0010] In one implementation, the target lymph nodes include the popliteal lymph nodes and the inguinal lymph nodes.

[0011] In one embodiment, the volume ratio of Evans blue to indocyanine green in the mixed tracer is 1:1.

[0012] As one implementation method, the model animal is the Sprague-Dawley rat.

[0013] In one implementation, the excitation wavelength of the FAR is 770-790 nm, and the detection wavelength is >780 nm.

[0014] As one implementation method, the criteria for determining the success of the animal model include at least one of the following: Seven days post-surgery, the volume of the affected limb in the model animals increased by ≥20% compared to pre-surgery. Seven days post-surgery, the circumference of the affected limb in the model animals increased by ≥14% compared to pre-surgery. Seven days after surgery, images of the indocyanine green lymphatic drainage pattern and Evans blue retention on the affected side of the model animals were collected, showing that lymphatic drainage was blocked. Hematoxylin-eosin staining revealed fluid accumulation in the subcutaneous interstitial tissue of the affected hind limb after surgery.

[0015] Secondly, embodiments of this disclosure propose the use of the animal model construction method described in the first aspect in evaluating the efficacy of drugs for treating secondary lymphedema.

[0016] Thirdly, embodiments of this disclosure propose the use of the animal model construction method described in the first aspect in the study of the pathogenesis of secondary lymphedema or in the labeling and / or identification of the lymphatic system.

[0017] Compared with the prior art, the embodiments of this disclosure have at least the following beneficial effects: The animal model construction method provided in this embodiment combines indocyanine green (ICG) fluorescence imaging with Evans blue dye dual labeling, and uses FAR technology to acquire white light anatomical images and indocyanine green near-infrared fluorescence images. Based on this, the operator can simultaneously observe the indocyanine green fluorescence imaging and the appearance of Evans blue staining on the body surface. From both fluorescence imaging and white light surgical field color development, the complete and unobstructed normal lymphatic drainage pathways can be confirmed before surgery, and the preoperative baseline image acquisition and retention can be completed. At the same time, it enables precise real-time visualization and localization of the two sets of lymphatic drainage systems (superficial lateral and superficial medial) of the hind limbs of the model animal (rat), as well as the popliteal lymph nodes, inguinal lymph nodes, and lymphatic vessels during surgery. Among them, the above-mentioned lymph nodes are removed under FAR real-time navigation guidance, and immediate fluorescence images can be acquired after surgery. By comparing the preoperative and postoperative immediate fluorescence images, it is possible to directly, objectively, and in situ determine whether the lymphatic drainage pathways are completely severed, without relying on the long-term secondary edema phenotype for reverse inference, and to confirm that the occlusion of all drainage pathways is complete. Compared to the traditional single Evans blue staining method, this embodiment significantly improves the accuracy of lymph node localization, surgical efficiency, and model establishment success rate.

[0018] This embodiment employs a multi-dimensional longitudinal dynamic assessment system combining FAR lymphatic vessel fluorescence imaging (skin reflux pattern classification), limb volume measurement, limb circumference measurement, and histological staining postoperatively, achieving non-invasive monitoring of the entire process of lymphatic system functional recovery. In summary, this embodiment is suitable for research on the pathogenesis of secondary lymphedema and for preclinical efficacy evaluation of novel treatment methods (including surgery, drugs, and cell therapy), and has significant methodological promotion value.

[0019] Additional aspects and advantages of this application will be set forth in part in the description which follows, and will become apparent from the description or may be learned by practice of this application. Attached Figure Description

[0020] Figure 1 A schematic diagram of fluorescence augmented reality images under mixed tracers (EB:ICG) with different volume ratios is shown; Figure 2 A schematic diagram of fluorescence images of the superficial medial and superficial lateral lymphatic drainage pathways of the rat hind limbs and the popliteal and inguinal lymph nodes in the popliteal position is shown. Figure 3 A schematic diagram of the process for lymph node resection and in vivo / in vitro imaging verification under FAR guidance is shown. Figure 4 The diagram shows the limb changes, hindlimb reflux patterns, affected limb volume changes, affected limb circumference changes, and H&E staining results of rat skin 7 days after surgery. Detailed Implementation

[0021] The technical solutions in the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0022] The following explanations of some of the terms and materials used in this embodiment will help those skilled in the art to understand them.

[0023] FAR: Fluorescence Augmented Reality. In essence, Fluorescence Augmented Reality (FAR) is a visualization technology that combines Augmented Reality (AR) with fluorescence molecular imaging. Based on FAR technology, it can acquire anatomical images of tissues under visible light, clearly displaying the outlines of skin, subcutaneous tissue, lymph node, and Evans blue staining areas. Simultaneously, in the near-infrared excitation and reception path, ICG molecules are excited by a specific wavelength light source, capturing their emitted near-infrared fluorescence signals to specifically label lymphatic vessels, lymph nodes, and other lymphatic tissues. FAR technology can be modularly and independently adapted and integrated into various fluorescence imaging devices, without being limited to surgical microscopes. For example, it can be independently mounted on various non-microscope fluorescence imaging devices, including small animal in vivo fluorescence imaging devices, desktop near-infrared fluorescence imagers, handheld fluorescence navigation imaging devices, and open-system experimental fluorescence observation devices.

[0024] Indocyanine green (ICG) lymphatic return pattern: Using indocyanine green (ICG) as a lymphatic tracer, the fluorescence image morphology of ICG flowing and distributing within the lymphatic system, observed through fluorescence augmented reality (FAR) technology, serves as the visual classification standard in this embodiment for assessing the patency of lymphatic return and the severity of obstruction. Based on the continuity, morphology, and distribution range of the fluorescence images, the lymphatic return status is divided into four typical patterns (directly corresponding to obstruction from mild to severe): Linear type: Fluorescence shows continuous, clear linear lymphatic vessel shadows; Sputtering type: Fluorescence is distributed in a discontinuous, speckled sputtering pattern; Stardust type: The fluorescence appears as fine, scattered star-like dots, without a complete lymphatic vessel structure; Diffuse type: Fluorescence is uniformly diffused over a large area, and lymph fluid is significantly retained.

[0025] As the severity of lymphedema increases, it exhibits a shift from a linear lymphangiography pattern (showing only lymphatic vessels) to a diffuse pattern (showing leaking lymph fluid in surrounding tissues), specifically including the following grades: Grade 0: No skin reflux, only linear lymphatic vessel visualization pattern (normal without edema); Grade 1: Splash pattern appears around the lymph node dissection / resection site; Grade 2: The splash pattern extends to the entire limb, and a stardust pattern appears in the distal area; Grade 3: The stardust pattern extends towards the proximal end; Grade 4: A diffuse pattern appears, accompanied by a stardust pattern; Grade 5: No lymphatic drainage, i.e., black screen / no imaging mode.

[0026] It is understood that the procedures described in this embodiment, such as tracer injection, removal of target lymph nodes and drainage of lymphatic vessels, and closure of skin wounds, are all routine procedures and drug administration procedures in the fields of rodent model construction and pharmacology. The above-mentioned procedures are mature and the procedures are fixed. Those skilled in the art, based on common knowledge, can fully implement the relevant steps according to the technical content described in this embodiment. The operation process and final results will not differ significantly due to different operators, and the scheme has a high degree of repeatability.

[0027] The following will describe in detail the method for constructing an animal model of lymphedema based on fluorescence augmented reality and its application.

[0028] First, let me explain the method for constructing an animal model of lymphedema based on fluorescence augmented reality in the first aspect of this embodiment.

[0029] A method for constructing an animal model of lymphedema based on fluorescence augmented reality.

[0030] In current technology, lymphedema is a chronic, progressive disease caused by lymphatic system dysfunction leading to obstructed lymphatic drainage and accumulation in the interstitial spaces. Its pathogenesis is closely related to factors such as surgical injury, trauma, infection, and congenital lymphatic developmental abnormalities. It not only causes limb swelling, pain, and functional limitations but can also lead to complications such as skin fibrosis and recurrent infections, severely impacting quality of life. Currently, there is a lack of radical treatment options in clinical practice. Therefore, research on the pathogenesis, pathophysiological changes, and novel treatment strategies of lymphedema is of great significance for improving clinical diagnosis and treatment outcomes.

[0031] Currently, animal models are a core tool in basic and translational medicine research on lymphedema, and the quality of their construction directly affects the reliability and accuracy of research results. Existing methods for constructing lymphedema animal models mainly include surgical resection of lymph nodes / lymphatic vessels, lymphatic vessel ligation, radiation injury, and chemical induction. Among these, surgical resection is the most widely used method due to its direct operation and short modeling cycle. However, traditional surgical modeling methods have significant technical limitations: On the one hand, the lymphatic system has a delicate and complex anatomical structure. Traditional surgery relies on the operator's visual observation and anatomical experience to locate and remove lymph nodes, lacking precise visual guidance. This makes it difficult to clearly distinguish the boundaries between the target lymph node and its draining lymphatic vessels and surrounding normal tissues such as blood vessels and nerves, easily leading to incomplete removal of the target tissue or excessive damage to normal lymphatic tissue, resulting in incomplete lymphatic drainage blockage or abnormal model phenotype, significantly reducing the success rate of model establishment. On the other hand, current model establishment often uses a single tracer (such as Evans blue or indocyanine green alone) to assist in localization. However, single tracers have limitations in their effectiveness. Problems such as insufficient specificity of the imaging, large interference from tissue background, short fluorescence duration, or limited penetration depth make it impossible to achieve clear imaging of the entire lymphatic pathway, further exacerbating the blindness of intraoperative localization. In addition, traditional methods lack rapid and non-invasive means of verifying the blocking effect after surgery, and require long-term observation of the degree of limb edema in animals to indirectly judge the modeling effect. This is not only time-consuming, but also makes it difficult to detect modeling failure such as incomplete blocking in the early stage. At the same time, the inconsistency in the excision range due to differences in operator experience will result in large differences in the degree of edema between models and poor uniformity, which seriously affects the reliability and reproducibility of experimental data.

[0032] In view of this, this embodiment provides a method for constructing an animal model of lymphedema based on fluorescence augmented reality guidance. The method for constructing the animal model includes the following steps: S1. Inject the model animal with a mixed tracer and collect white light anatomical images and indocyanine green near-infrared fluorescence images; the mixed tracer includes Evans blue and indocyanine green.

[0033] The model animal was the Sprague-Dawley rat.

[0034] In step S1, a mixed tracer consisting of Evans blue and indocyanine green is injected into the model animal, causing the tracer to specifically accumulate in the target lymph nodes and draining lymphatic vessels along the lymphatic drainage pathway. Then, FAR technology is used to complete the preoperative fluorescence image acquisition, clearly marking the anatomical course, distribution location and adjacent relationships of the lymphatic system in advance, providing intuitive preoperative imaging basis for subsequent precise positioning of the surgical target area, planning of the surgical approach and resection range.

[0035] Specifically, the Evans blue used in this embodiment has lymphatic affinity and can specifically remain within the lumen of lymphatic vessels, macroscopically outlining the overall course of lymphatic vessels with high boundary recognition. Indocyanine green possesses near-infrared fluorescence imaging characteristics, strong tissue penetration, and minimal interference from autologous tissue background fluorescence, enabling precise visualization of deep micro-lymph nodes and fine micro-lymphatic vessel branches. After the two tracers are mixed and injected, they can migrate directionally along the physiological lymphatic return path with the interstitial fluid and specifically accumulate in lymph nodes and draining lymphatic vessels. Combined with FAR technology, weak fluorescence signals can be denoised, enhanced, and visualized in real time, suppressing noise interference from surrounding normal tissue, thereby acquiring high-definition preoperative complete lymphatic pathway fluorescence images.

[0036] Understandably, anesthetizing model animals before injecting the mixed tracer serves several purposes. First, it provides sedation and analgesia, restricts limb agitation and spontaneous movement, facilitates maintaining a standard body position, and ensures accurate tracer injection and stable imaging. Second, it reduces surgical stress in animals, minimizing the interference of pain and emotional agitation on the body's lymphatic physiological return rhythm. Third, it complies with ethical requirements for laboratory animals, reduces their pain during procedures, and creates stable and controllable experimental conditions for subsequent fluorescence imaging and surgical procedures.

[0037] Specifically, anesthetic drugs (such as inducing anesthesia with 4% isoflurane gas first, and then maintaining anesthesia with 1.5%-2% isoflurane) can inhibit the excitability of the central nervous system in model animals, block pain transmission, relax skeletal muscles, and bring the animals into a stable state of sedation, painlessness, and limb immobilization. At the same time, anesthesia can inhibit excessive excitation of the body's stress sympathetic nerves, maintain the normal physiological dynamics of lymphatic circulation, and avoid lymphatic vessel contraction and lymphatic reflux disorder caused by animal agitation and stress. This ensures that the subsequently injected Evans blue and indocyanine green mixed tracer can migrate and accumulate along the normal physiological lymphatic pathway without distribution shift or pathway distortion.

[0038] After anesthesia, the model animal can be shaved and skin prepared, covering the right hind limb and lower abdomen, to facilitate the subsequent injection of the mixed tracer. At the same time, after the mixed tracer is injected, the injection site can be gently massaged for 30 seconds to promote absorption.

[0039] In summary, step S1 utilizes a dual-tracer synergistic approach, balancing macroscopic imaging of lymphatic vessel contours with microscopic localization of deep lymph nodes. This effectively overcomes the limitations of single-tracer imaging, such as limited imaging range, unclear layers, and difficulty in identifying subtle lymphatic structures. Simultaneously, FAR technology enhances and optimizes fluorescence signals, significantly improving the contrast and clarity of lymphatic structure imaging and accurately distinguishing the boundaries between lymphatic tissue and surrounding blood vessels, nerves, and soft tissues. Preoperative visualization and documentation of lymphatic pathways avoids the shortcomings of traditional surgery that relies on blindly locating anatomical structures using visual experience, reducing the risk of intraoperative positioning errors and accidental damage to normal tissues. The construction method in this embodiment is non-invasive and simple to operate, with rapid imaging acquisition, and does not damage the original physiological and lymphatic anatomy of the model animal, ensuring the stability of subsequent modeling procedures and the uniformity of individual models.

[0040] S2. Under FAR guidance, the target lymph nodes and their connected draining lymphatic vessels in the model animal were removed, and postoperative fluorescence images were collected to verify the integrity of the lymphatic drainage pathway blockage.

[0041] In step S2, under the real-time visualization guidance of FAR technology, the target lymph nodes and their draining lymphatic vessels of the model animal are accurately located and completely removed, thus completely blocking lymphatic reflux in a specific area from an anatomical perspective. At the same time, by collecting postoperative fluorescence images, it is possible to intuitively determine whether the lymphatic drainage pathway is completely blocked, eliminate residual reflux channels, and ensure the effectiveness and reliability of the core steps of modeling, providing a key guarantee for constructing a stable lymphedema model.

[0042] Typically, before removing the target lymph node and its connected draining lymphatic vessels, a skin incision is made along the groin and popliteal fossa while the model animal is anesthetized. The incision edges are treated with monopolar electrocoagulation to interrupt the intradermal capillary lymphatic vessels and prevent postoperative lymphatic recanalization. Monopolar electrocoagulation relies on a high-frequency alternating current to generate a local thermal effect on the tissue at the incision edge, causing thermal coagulation and structural closure of the intradermal and subcutaneous micro-lymphatic endothelial tissue, directly interrupting the anatomical continuity of the intradermal capillary lymphatic vessels.

[0043] Under stable anesthesia, the skin surgical incisions are precisely made at the corresponding anatomical locations in the groin and popliteal fossa areas. At the same time, monopolar electrocoagulation is used to thermally coagulate the edges of the incisions, actively severing the intradermal capillary lymphatic network and artificially blocking the self-healing and regeneration channels of intradermal lymphatic vessels. This inhibits the formation of collateral lymphatic vessels and the recanalization of pathways after surgery at the wound level, ensuring that the lymphatic drainage blocking effect is long-lasting and irreversible, and improving the stability of the lymphedema animal model.

[0044] Among these advantages, the precise guidance of FAR technology not only shortens lymphatic vessel search time and reduces ineffective procedures, but also enables systematic resection of multiple target lymph nodes in a single surgery, effectively reducing the need for additional animals due to model failure. Furthermore, in subsequent non-invasive dynamic lymphatic function assessment systems, continuous longitudinal lymphangiography using FAR technology allows for dynamic tracking of lymphatic system function at multiple time points throughout the entire process on the same individual animal, overcoming the limitations of histological methods that require animal sacrifice and can only obtain static data at a single time point.

[0045] Understandably, the Evans blue and indocyanine green mixed tracer injected preoperatively in step S1 has specifically enriched in the target lymph nodes and draining lymphatic vessels. The FAR technology can precisely fuse the real-time fluorescence signal during the operation with the surgical field to form an augmented reality visualization image, clearly showing the anatomical boundaries between the target tissue and the surrounding blood vessels, nerves, and soft tissues, providing real-time navigation for the surgeon and ensuring that the resection operation is only aimed at the target lymphatic tissue. Then, when the fluorescence image is collected again after the operation, if the lymphatic drainage pathway is completely blocked, the tracer cannot flow back along the original pathway, and the fluorescence signal of the original lymphatic pathway in the image will be interrupted or disappear. Otherwise, there will be a continuous fluorescence signal, which serves as the basis for judging the blocking effect.

[0046] Specifically, step S2 uses FAR visualization technology to guide the precise resection of lymph nodes and draining lymphatic vessels, and immediately after surgery, visualizes and verifies the effect of lymphatic drainage pathway occlusion. Unlike existing technologies that rely on lagging, indirect evaluation methods such as limb swelling, changes in volume and circumference, and pathological examinations several days to weeks after surgery, step S2 aims to achieve immediate postoperative verification of the modeling effect. During the surgical window, the integrity of lymphatic pathway occlusion can be directly confirmed, ensuring thorough lymphatic drainage occlusion and uniform modeling standards for each animal model from the source, avoiding model failure and individual differences caused by lymphatic vessel residue or incomplete occlusion.

[0047] In step S2, intraoperative real-time observation of white light anatomical images and ICG near-infrared fluorescence images allows for precise resection based on clear identification of anatomical structures and lymphatic vessel pathways. Postoperatively, immediate fluorescence images can be acquired. Based on the characteristic of ICG specifically tracing lymphatic drainage pathways, preoperatively, continuous linear fluorescence signals of normal lymphatic vessels are observed. If the lymphatic pathway is completely blocked, the original continuous fluorescence pathway will be completely interrupted postoperatively, and pathological leakage images such as splashing, stardust, and diffusion may appear. By comparing preoperative and postoperative immediate fluorescence images, it is possible to directly, objectively, and in situ determine whether the lymphatic drainage pathway is completely severed, without relying on long-term secondary edema phenotypes for reverse inference.

[0048] Therefore, step S2 ensures the integrity of lymphatic pathway blockade. Real-time fluorescence imaging can clearly identify residual fine lymphatic vessels, incompletely severed drainage pathways, and hidden collateral drainage structures, promptly detecting incomplete blockade and ensuring complete interruption of the main lymphatic drainage pathway, fundamentally improving the accuracy and success rate of modeling.

[0049] In other words, the dual tracer imaging in step S1 provides precise navigation for step S2, avoiding missed or excessive removal of the target tissue during the procedure in step S2; and the postoperative fluorescence verification in step S2 directly relies on the mixed tracer injected in step S1, without the need for additional tracer substances, thus achieving non-invasive and rapid verification of the blocking effect, which can effectively solve the problem of the lag in traditional postoperative observation of edema phenotype.

[0050] Meanwhile, step S1 can clearly define the distribution of lymphatic pathways in advance, enabling the resection operation in step S2 to change from blind cutting based on experience to precise cutting based on visual navigation, effectively avoiding accidental damage to surrounding blood vessels, nerves and other normal tissues, while reducing technical differences between different operators and ensuring the standardization of the resection range.

[0051] In summary, step S2, guided by real-time FAR technology, enables precise and visualized resection of the target tissue. This effectively overcomes the limitations of traditional surgery, which relies on blind cutting based on anatomical experience, significantly reducing the risk of damage to normal blood vessels, nerves, and surrounding soft tissues, and improving the precision and safety of the surgical procedure. Specifically, step S2 allows for complete resection of the target lymph node and its connected draining lymphatic vessels, avoiding incomplete or excessive resection due to positioning errors in traditional surgery, ensuring the targeted and thorough blocking of lymphatic drainage. Furthermore, the postoperative fluorescence imaging verification method is non-invasive, rapid, and intuitive, allowing for immediate assessment of the blocking effect during modeling. Compared to the indirect assessment method of long-term postoperative observation of edema phenotypes, this method can detect incomplete blocking at an early stage, significantly reducing the modeling failure rate and improving model stability and repeatability.

[0052] S3. Close the skin incision to obtain an animal model of lymphedema.

[0053] In step S3, after the target lymph node and its connected draining lymphatic vessels are completely removed and the lymphatic drainage pathway is completely blocked by postoperative fluorescence imaging, the skin surgical incision is closed and sutured layer by layer to seal the surgical area, isolate the external environment, and protect the severed lymphatic anatomical structures from displacement and self-healing. At the same time, the local tissue physiological microenvironment is kept stable, which promotes the continuous accumulation and retention of lymph fluid in the interstitial space, induces the formation of a stable pathological phenotype, and finally prepares a standardized lymphedema animal model with complete structure, reliable phenotype, and can be used for subsequent experimental research.

[0054] Understandably, after the tracing and imaging in step S1 and the precise excision and pathway blockage verification in step S2, the local lymphatic return pathway of the model animal has been artificially severed. Then, by closing the skin incision with sutures, the subcutaneous soft tissue can be kept in its original anatomical position, limiting the traction, displacement and abnormal adhesion of wound tissue, and blocking the invasion of surface microorganisms into the surgical area to cause inflammatory interference. At the same time, the intact closed skin barrier can maintain the local tissue osmotic pressure and microenvironment homeostasis, avoiding wound exudation and external stimuli from interfering with the lymphatic circulation compensation process. This prevents the lymph fluid in the blocked area from establishing a normal return channel, and it can only continue to accumulate in the interstitial space, gradually inducing typical lymphedema pathological changes such as limb swelling, subcutaneous tissue hyperplasia and fibrosis, and finally naturally forming a stable animal model.

[0055] In step S1, the volume ratio of Evans blue to indocyanine green in the mixed tracer is 1:1.

[0056] In step S1, the volume ratio of Evans blue to indocyanine green in the mixed tracer is limited to 1:1. By optimizing the ratio of the two tracers, the synergistic matching of macroscopic lymphatic vessel contour staining and deep lymph node fluorescence imaging is achieved. At the same time, it adapts to the requirements of fluorescence imaging acquisition, balances the imaging intensity and distribution characteristics of the two reagents, and makes the preoperative lymphatic pathway imaging layers distinct and the boundaries clear, providing a standardized, stable and reliable imaging basis for subsequent intraoperative precise positioning, complete resection and verification of blocking effect.

[0057] Evans blue is primarily used for staining the macroscopic contours of lymphatic networks, clearly marking the overall course of superficial and main lymphatic vessels. A higher proportion is needed to ensure extensive imaging coverage. Indocyanine green, relying on near-infrared fluorescence properties, focuses on specific fluorescence imaging of deep, small lymph nodes and fine lymphatic vessels. An excessively high proportion can easily cause fluorescence diffusion and increased background interference. In this embodiment, a 1:1 volume ratio is used, with one part Evans blue ensuring complete contour outlining of lymphatic vessels and one part indocyanine green precisely providing fluorescence signals from deep lymphatic tissue. The appropriate ratio ensures that neither part obscures the imaging effect, allowing for simultaneous directional migration and enrichment along physiological lymphatic pathways. Furthermore, the fluorescence signal intensity effectively matches the signal enhancement and imaging recognition threshold of FAR technology, achieving a high degree of overlap and precise correspondence between the visible contours and the fluorescence signal.

[0058] This embodiment uses a mixed tracer with a specific ratio. This ratio provides moderate fluorescence signal intensity, which is suitable for the working range of FAR technology signal enhancement and noise reduction processing, resulting in higher imaging contrast and resolution. This facilitates accurate preoperative identification of lymphatic pathways and surrounding tissue boundaries. At the same time, it can fully leverage the advantages of the two tracers, ensuring both the complete and continuous blue staining outline of the main lymphatic vessels and clear imaging of weak fluorescence in deep lymph nodes, avoiding problems such as blurred imaging, signal overlap, or diffusion interference caused by imbalance in the ratio.

[0059] In summary, the above-mentioned specific ratio of mixed tracers can unify the tracer preparation standards, ensuring balanced and stable imaging and visual observation effects throughout the entire process of preoperative lymphatic pathway calibration, intraoperative surgical guidance, and postoperative drainage function verification. This effectively improves the accuracy of lymphatic pathway identification and the consistency of experimental operations, further enhancing the standardization of modeling and the reproducibility of experimental results.

[0060] In step S2, the target lymph nodes include the popliteal lymph nodes and the inguinal lymph nodes.

[0061] This embodiment explicitly uses the popliteal and inguinal lymph nodes as target lymph nodes for resection, identifying two core key relay nodes in the lower limb lymphatic drainage pathway. By precisely resecting these two lymph nodes and their connected draining lymphatic vessels, the main lymphatic drainage path from distal to proximal in the lower limb is directionally blocked. Combined with prior fluorescence tracing and FAR visualization guidance, the modeling target points are standardized and the resection sites are unified, stably constructing a lower limb lymphedema animal model with a pathological phenotype consistent with clinical findings.

[0062] Understandably, the popliteal lymph nodes are the primary relay hubs for lymphatic drainage in the lower leg region, responsible for collecting lymphatic drainage from the distal lower leg tissues; the inguinal lymph nodes are key gateways for the lower limb lymphatic system to flow into the trunk lymphatic system, undertaking the function of transporting lymphatic drainage from the thigh and lower limb. Physiologically, these lymph nodes constitute the core pathway for distal-proximal lymphatic drainage in the lower limbs. Selecting two lymph nodes as targets allows for the dual interruption of the main lymphatic drainage channels in the lower limbs from both upstream and downstream. Combined with the specific targeting and imaging of the two lymph nodes and associated lymphatic vessels using the mixed tracer in step S1, and then precise intraoperative resection guided by FAR in step S2 with fluorescence verification of the integrity of the blockage, postoperatively, the incision is closed in step S3 to inhibit the compensatory generation of collateral circulation, thereby stably inducing lymphatic fluid accumulation in the interstitial spaces of the lower limb tissues, forming typical pathological changes of lymphedema.

[0063] In summary, the popliteal and inguinal lymph nodes selected in this embodiment have fixed anatomical locations and clear anatomical landmarks, with minimal anatomical variation among individual animals. This facilitates standardized surgical positioning and enables the standardized construction of animal models in batches. Combined resection of both lymph nodes completely severs the main lymphatic drainage pathways of the lower limb, resulting in more thorough lymphatic drainage blockage compared to single lymph node resection. This reduces the likelihood of spontaneous compensatory collateral circulation and significantly improves the success rate of model establishment. Furthermore, the course of the two target lymph nodes and their draining lymphatic vessels is well-suited to the imaging properties of the Evans blue and indocyanine green mixed tracer, providing clear outlines and easily distinguishable boundaries under FAR technology. This reduces the difficulty of intraoperative dissection and minimizes accidental injury to surrounding blood vessels, nerves, and normal soft tissues. The model constructed based on the blockage of the popliteal and inguinal lymphatic pathways can simulate the pathogenesis and pathological characteristics of clinical secondary lymphedema of the lower limbs, exhibiting typical phenotypes such as limb swelling and skin fibrosis. This model is more suitable for research on the pathogenesis of lymphedema, drug screening, and evaluation of clinical treatment regimens.

[0064] In this embodiment, the excitation wavelength for FAR fluorescence imaging is set to 770-790 nm (e.g., 770 nm, 780 nm, 790 nm), and the fluorescence detection wavelength is set to greater than 780 nm.

[0065] Understandably, the near-infrared spectral response characteristics of indocyanine green are highly matched with the excitation band of 770-790 nm, allowing for efficient excitation and the output of a stable fluorescence signal. Simultaneously, selecting a detection wavelength greater than 780 nm in the long-wave near-infrared range effectively excludes the autofluorescence band of soft tissues such as skin, muscle, and blood vessels in experimental animals, optically filtering out background stray light and tissue background fluorescence noise, significantly improving the imaging signal-to-noise ratio.

[0066] This embodiment, by limiting the excitation and detection wavelengths, not only matches the inherent optical properties of indocyanine green but also fundamentally avoids fluorescence interference from animal autologous tissue. Simultaneously, it standardizes imaging timing parameters to ensure that the Evans blue and indocyanine green mixed tracer fully migrates and accumulates along the lymphatic pathway, completing preoperative fluorescence image acquisition within the optimal imaging window. Therefore, this embodiment achieves dual standardization in imaging optical parameters and image acquisition timing, providing stable and superior imaging conditions for precise lymphatic pathway imaging, intraoperative visualization guidance, and postoperative verification of lymphatic blockade effects.

[0067] As one implementation method, the animal model construction method further includes a postoperative multidimensional dynamic assessment step, the assessment step including: At multiple pre-defined time points, perform at least one of the following non-invasive dynamic assessments on the model animals: Changes in limb volume, changes in limb circumference, indocyanine green lymphatic reflux pattern, Evans blue retention status, and histological verification.

[0068] In the aforementioned non-invasive dynamic assessment, changes in limb volume and circumference are based on the principle of somatic morphology measurement. Lower limb circumference and volume parameters are measured quantitatively at regular intervals to directly reflect the degree of interstitial lymphatic fluid accumulation and the progression of edema over time. The indocyanine green lymphatic drainage pattern utilizes the principle of near-infrared fluorescence imaging, reintroducing indocyanine green tracer at preset postoperative time points to monitor in real-time whether lymphatic drainage pathways are reconstructed and whether collateral circulation is abnormally generated, thus verifying the long-term stability of lymphatic pathway blockage from a functional perspective. Evans blue retention is visualized and image acquired under white light surgical field. Evans blue is normally metabolized and cleared with lymphatic fluid. However, when lymphatic drainage pathways are blocked or lymphatic circulation is impaired, the dye cannot be properly cleared and will continuously accumulate in the subcutaneous soft tissue of the affected limb. Therefore, by observing the staining range, depth, and regression of Evans blue in the affected limb at different time points, the clearance capacity and degree of reflux obstruction of lymphatic fluid can be directly reflected, enabling dynamic and quantitative evaluation of the pathological progression of lymphedema. Histological verification involves staining pathological sections to observe the degree of fibrosis in the skin and subcutaneous tissue, the expansion of interstitial spaces, and lymphoid tissue hyperplasia at the microscopic level, achieving mutual corroboration between macroscopic phenotype and microscopic pathology. Simultaneously, the overall approach primarily employs non-invasive testing, avoiding invasive procedures that interfere with the natural disease course of the model, allowing for long-term, multi-timepoint continuous dynamic monitoring.

[0069] Based on this, this embodiment dynamically tracks the occurrence and development of edema, lymphatic pathway compensation, and histopathological changes in model animals from multiple levels, including macroscopic body surface morphology, lymphatic drainage function, and microscopic histopathology. This enables quantitative evaluation of modeling effect, screening of model quality, and analysis of disease progression, providing standardized data support for model effectiveness determination, subsequent mechanism research, and drug efficacy evaluation.

[0070] As one implementation method, the change in limb volume is measured using the water displacement volume measurement method; the change in limb circumference is measured using the tape circumference measurement method.

[0071] This embodiment explicitly defines the use of water replacement volume measurement for limb volume changes and tape circumference measurement for limb circumference changes, unifying standardized detection methods for postoperative dynamic assessment, standardizing measurement operation procedures and judgment criteria, and avoiding subjective errors and data deviations caused by traditional visual estimation and arbitrary measurement. Through two non-invasive and reproducible quantitative detection methods, the degree of limb edema in model animals is accurately quantified, providing objective and comparable quantitative data support for the dynamic evolution of lymphedema and the determination of model success.

[0072] Specifically, the water displacement volume measurement method is based on Archimedes' principle of water displacement. The test limb of the model animal is vertically immersed in a constant temperature sealed measuring cup. The volume of water displaced by the limb is equal to the actual volume of the limb. By accurately measuring the volume of the displaced fluid, the overall volume of the limb is calculated. By using the volume difference at different preset time points, the overall swelling of the limb caused by the accumulation of lymph fluid in the interstitial space is accurately characterized.

[0073] For example, on the 7th postoperative day, the volume of both hind limbs was objectively and quantitatively assessed using the water displacement volume measurement method. The specific procedure included: fixing the model animal in a prone position on the operating table, and sequentially immersing the affected and unaffected hind limbs vertically into a graduated cylinder filled with distilled water from a standard anatomical landmark (the fold of skin in the groin), recording the volume of displaced fluid (mL) as the limb volume. The volumes of the unaffected side (control group) and the operated side (model group) were calculated separately, and the rate of change compared to Day 0 was also calculated.

[0074] The tape circumference measurement method uses inelastic flexible tape to perform circumferential measurements at fixed anatomical landmarks of the limb (ankle joint, mid-calf, knee joint, mid-thigh, etc.). The measurement position, tightness, and measurement angle are fixed, and the circumference values ​​at each time point are recorded. The difference in circumference at different time points is used to quantitatively reflect the changes in the degree of local subcutaneous tissue thickening and edema.

[0075] For example, on postoperative day 7, the limb circumference (cm) of both hind limbs was measured using a non-elastic graduated tape measure at a standard anatomical location (2 cm proximal to the knee joint). Each measurement was repeated three times and the average was taken to reduce operational error. The circumference of the unaffected side (control group) and the operated side (model group) was calculated separately, and the rate of change from Day 0 was calculated. For histological verification, the model animals were sacrificed at predetermined time points, and the skin and subcutaneous tissue of the affected hind limb were precisely harvested. Hematoxylin-eosin staining was used to analyze skin thickness and the degree of tissue edema.

[0076] To validate the indocyanine green lymphatic drainage pattern, near-infrared fluorescence ICG lymphangiography images of the hind limbs of model animals can be collected weekly to establish a four-level standardized classification standard for lymphatic drainage patterns: linear, sputtering, stardust, and diffuse. This embodiment achieves qualitative and quantitative evaluation of the severity of lymphatic drainage obstruction by classifying, determining, and quantitatively counting the frequency of occurrence of each drainage pattern, dynamically tracking the postoperative lymphatic pathway obstruction status and collateral circulation compensation process, supplementing the functional assessment basis, and improving the multi-dimensional dynamic assessment system of the model.

[0077] Understandably, indocyanine green (ICG) can specifically enter superficial lymphatic vessels through the subcutaneous interstitial space. Under near-infrared imaging conditions with an excitation wavelength of 770-790 nm and a detection wavelength greater than 820 nm, it can clearly display the course of superficial lymphatic drainage and the distribution pattern of fluorescent dye. Under physiologically unobstructed conditions, ICG migrates regularly and linearly along lymphatic vessels, presenting a normal linear pattern. When lymphatic drainage is obstructed, lymphatic drainage is impaired, and dye spills out, presenting a sputtering, stardust, or diffuse pattern. When the main lymphatic pathway is completely blocked and there is no lymphatic drainage, a black screen / no imaging pattern is presented. By statistically analyzing the frequency of each type, the differences in lymphedema levels from Grade 1 to Grade 5 can be determined.

[0078] In summary, this embodiment establishes four distinct ICG lymphatic drainage classification criteria, providing an objective and unified basis for judgment. This effectively distinguishes itself from the shortcomings of traditional subjective judgments based on experience, achieving standardized grading and qualitative assessment of the degree of lymphatic drainage obstruction. Postoperative monitoring of lymphatic drainage patterns and statistical analysis of the frequency of each type can determine the degree of lymphedema.

[0079] As one implementation method, the criteria for determining the success of the animal model include at least one of the following: (1) The volume of the affected limb in the model animals increased by ≥20% 1-2 weeks after surgery compared with that before surgery; (2) The circumference of the affected limb in the model animals increased by ≥14% 1-2 weeks after surgery compared with that before surgery; (3) Seven days after the operation, images of the indocyanine green lymphatic reflux pattern and Evans blue retention on the affected side of the model animals were collected, showing that lymphatic drainage was blocked; (4) Hematoxylin-eosin staining revealed fluid accumulation in the subcutaneous interstitial tissue of the affected hind limb after surgery.

[0080] In the judgment criteria (1), this embodiment lists the increase in volume of the affected limb relative to the preoperative volume of the affected limb by no less than 20% one week after surgery as the criterion for model success. The animal's own normal preoperative state is used as a control, and a unified time node and quantitative threshold are set to establish an objective and quantifiable criterion for model success. The healthy limb is used as its own control to exclude the interference of temporary edema caused by surgical trauma, quickly distinguish between physiological postoperative swelling and pathological lymphedema, achieve standardized screening of qualified models, and eliminate individuals that do not meet the modeling standards.

[0081] One week after surgery, the acute inflammatory and edema stage has passed. The affected limb, whose lymphatic pathway was blocked, lost its normal lymphatic return route, and tissue fluid and lymph fluid continued to accumulate in the subcutaneous interstitium, causing the limb volume to increase continuously. Using the limb volume before modeling as a reference, when the volume increase reaches 20% or more, it proves that the limb has formed a stable and pathologically significant swelling change, which is consistent with the typical morphological characteristics of secondary lymphedema and meets the modeling conditions.

[0082] This embodiment employs a self-controlled preoperative model, completely avoiding judgment bias caused by individual animal size differences. The 20% increase threshold is clearly defined and standardized, and the judgment process is simple and efficient. Relying on the water replacement volume measurement method, numerical values ​​can be accurately obtained, resulting in high data accuracy and good experimental repeatability, making it suitable for large-scale unified screening of models. This morphological quantitative index can be used in conjunction with judgment criteria such as lymphatic drainage imaging and histopathological examination to further improve the multi-dimensional model evaluation system. At the same time, it provides an intuitive and reliable quantitative reference for subsequent studies on the degree of edema reduction and evaluation of the anti-edema efficacy of the test drugs.

[0083] In the judgment criteria (2), this embodiment includes the fact that the circumference of the affected limb increased by no less than 14% compared with the preoperative circumference one week after surgery as a criterion for successful model judgment. It establishes a quantitative evaluation standard for body surface morphology, effectively distinguishes between temporary inflammatory swelling caused by surgical trauma and persistent limb thickening caused by secondary lymphedema, and achieves rapid screening of qualified models.

[0084] Understandably, the acute inflammatory response period after surgery has passed in one week, and the acute inflammatory edema has gradually subsided. After modeling, the lymphatic drainage pathway of the affected limb is completely blocked, and tissue fluid and lymph cannot flow back to the center of gravity normally, resulting in continuous accumulation in the subcutaneous tissue and soft tissue spaces of the limb, causing a continuous increase in limb circumference. Using the model animal's own limb circumference before surgery as a control benchmark, when the circumference increase reaches 14% or more, it indicates that the limb has formed a stable pathological thickening change, which is consistent with the typical surface morphological characteristics of secondary lymphedema.

[0085] In summary, this embodiment employs a self-comparison method, which can eliminate the judgment error caused by individual animal body size differences. The 14% quantitative threshold standard is clear and unified. The limb circumference measurement is simple and convenient to operate, with high detection efficiency, requiring no complex instruments, and is suitable for rapid screening of large batches of experimental animals. This surface quantitative index can be combined with multiple judgment criteria such as limb volume changes, lymphatic drainage imaging, and histopathological examination to improve the multi-dimensional model evaluation system. At the same time, it can provide intuitive and accurate surface quantitative experimental data for subsequent exploration of disease development patterns and evaluation of the limb swelling reduction efficacy of anti-lymphedema drugs.

[0086] In the judgment criteria (3), this embodiment selects 7 days after surgery as the detection time node, and combines two imaging modes and two types of tracers for joint detection to exclude the interference of acute inflammatory edema caused by postoperative surgical trauma. The modeling effect is comprehensively judged from two dimensions: lymphatic circulation function and tissue dye metabolism. The core purpose is to objectively confirm that the target lymphatic drainage pathway is continuously obstructed and the lymphatic return function is pathologically impaired, accurately identify qualified secondary lymphedema models, unify the modeling judgment criteria, reduce subjective judgment errors, and effectively distinguish between temporary drainage abnormalities caused by surgical trauma and persistent pathological drainage obstructions caused by secondary lymphedema.

[0087] Evans blue has high lymphatic targeting, can be stably enriched in lymphatic tissue and rely on the complete lymphatic pathway to complete targeted drainage and metabolic clearance. In this method, the target lymph node and the connected draining lymphatic vessels on the affected side have been completely removed, and the normal lymphatic return channel has been completely blocked. Seven days after the operation, the acute damage of the surgical wound gradually repairs. At this time, the normal physiological drainage pathway still cannot be restored, and Evans blue cannot be transported and excreted normally to the proximal end. Under the observation of fluorescence augmented reality technology, the phenomenon of dye accumulation and drainage blockage that cannot flow normally can be clearly observed, thus confirming that the lymphatic drainage function has formed a persistent pathological damage.

[0088] In this embodiment, the observation period is one week after surgery, which can exclude interference factors such as short-term wound edema and inflammation after surgery, and the judgment results are accurate and reliable. The judgment basis (3) is based on the lymphatic targeting characteristics of two tracers, indocyanine green and Evans blue, and is combined with FAR to achieve joint verification. Indocyanine green can be specifically enriched in lymphatic vessels. Under FAR fluorescence imaging, the normal lymphatic pathway shows a continuous linear image. When lymphatic drainage is blocked, the original linear reflux pattern disappears and abnormal features such as fluorescence interruption, diffusion, and leakage appear. Evans blue relies on white light field observation. Under FAR white light surgical field, the dye distribution can be clearly observed. When the lymphatic circulation is normal, the dye will be gradually metabolized and cleared with the lymph fluid. If the drainage pathway is blocked, the dye cannot be discharged normally and will remain in the subcutaneous tissue of the affected limb for a long time. Among them, the acute inflammation caused by the surgery is basically subsided 7 days after surgery. At this time, the two imaging results show abnormalities simultaneously, which can confirm that there is a continuous blockage of lymphatic drainage.

[0089] In the judgment criteria (3), the lymphatic drainage imaging classification under indocyanine green near-infrared fluorescence is used as the judgment criteria. The normal physiological state is set as a linear drainage pattern. When observed 1 week after surgery, as long as the lymphatic drainage pattern of the affected limb deviates from the normal linear pattern and changes to any one of the abnormal imaging patterns of splash, stardust, or diffusion, it can be determined that lymphatic drainage is blocked. In this way, an intuitive and unified functional model judgment standard is established to eliminate the temporary abnormal drainage caused by acute inflammation during surgery and accurately screen qualified lymphedema animal models.

[0090] As those skilled in the art will know, in a normal body, the lymphatic pathways are intact and unobstructed, and indocyanine green can flow back along the lymphatic vessels in a directional and regular manner, presenting a continuous and regular linear image under FAR. After the removal of the target lymph node and the connected draining lymphatic vessels on the affected side using this method, the physiological lymphatic return pathway is disrupted and blocked, and the lymph fluid cannot achieve orderly centripetal flow, which easily leads to lymphatic leakage, local stasis and large-scale diffusion. The fluorescence image then changes regularly, successively showing a splash type of local extravasation, a stardust type of distal punctate stasis, and a diffuse type of large-scale diffuse infiltration. Therefore, as long as the return image no longer maintains the standard linear morphology, it proves that the dynamic lymphatic return pathway of the limb has formed a persistent pathological blockage, which is consistent with the functional pathological characteristics of secondary lymphedema.

[0091] In summary, this diagnostic method is characterized by clear classification and simple, straightforward logic. It uses deviation from the normal linear reflux pattern as the core criterion, ensuring standardized criteria that are easy for researchers to understand and implement. Utilizing in vivo fluorescence non-invasive observation, it allows for rapid screening of large-scale models without euthanizing animals. Furthermore, it can preliminarily differentiate the severity of lymphedema based on different abnormal imaging types. The observation period of one week post-surgery allows for sufficient resolution of temporary reflux disturbances caused by surgical trauma, ensuring reliable and accurate results. The aforementioned functional indicators can be combined with limb swelling volume indicators and subcutaneous interstitial fluid accumulation pathological indicators to construct a multi-level model evaluation system. Additionally, it allows for direct observation of the reversal process from abnormal reflux patterns to normal linear patterns in subsequent drug experiments, providing a direct and effective imaging basis for quantitatively evaluating the ability of drugs to improve lymphatic reflux.

[0092] In the judgment criterion (4), the pathological morphological changes of the subcutaneous tissue of the affected limb were observed by hematoxylin-eosin (HE) staining to verify the pathological changes caused by lymphatic drainage obstruction at the histological level, which was used to corroborate the successful construction of the animal secondary lymphedema model. Compared with macroscopic indirect indicators such as body surface appearance and volume measurement, histopathological examination can directly show the microscopic structural characteristics of interstitial edema, eliminate the interference of postoperative transient inflammatory edema, realize multi-level model verification from macroscopic phenotype to microscopic tissue changes, and improve the objectivity and rigor of model judgment.

[0093] Understandably, normal limbs have a dense subcutaneous tissue structure and narrow interstitial spaces, with no obvious accumulation of free fluid. When lymphatic drainage pathways are completely blocked, tissue fluid cannot be properly drained through the lymphatic system, continuously accumulating in the subcutaneous interstitial spaces. This leads to widening and loosening of the interstitial spaces, disordered tissue arrangement, and characteristic interstitial fluid accumulation. After hematoxylin-eosin staining, cells and connective tissue are specifically stained, while the accumulated tissue fluid appears as a blank, transparent area. Under a microscope, typical pathological features of lymphedema, such as loose subcutaneous interstitial edema, space dilation, and increased fluid cavities, can be clearly observed, thus confirming the organic tissue edema changes caused by lymphatic drainage obstruction.

[0094] Therefore, this embodiment enables gold standard verification at the microscopic histological level, providing a direct and accurate reflection of organic pathological changes caused by lymphatic drainage obstruction, thus overcoming the limitations of surface assessment which only observes macroscopic phenotypes. Furthermore, short-term postoperative swelling is primarily inflammatory infiltration caused by surgical trauma, mainly characterized by inflammatory cell infiltration; while lymphedema is characterized by interstitial fluid accumulation and interstitial space expansion, exhibiting significant pathological morphological differences, thus completely eliminating false-positive results caused by surgical stress. Finally, it improves the multi-dimensional model formation evaluation system, significantly enhancing the accuracy, completeness, and scientific rigor of model formation determination, and ensuring the stability of subsequent experimental results.

[0095] Next, the uses of the second aspect of this embodiment will be described.

[0096] Based on the animal model construction method of the first aspect, this embodiment also proposes the use of the above animal model construction method in evaluating the effects of drugs against secondary lymphedema.

[0097] It can be understood that the lymphedema animal model based on fluorescence-enhanced reality guidance constructed in this embodiment can be used for the efficacy screening, efficacy evaluation, mechanism of action exploration, and dosing regimen optimization of anti-secondary lymphedema candidate drugs, chemical drugs, biological agents, and traditional Chinese medicine preparations. The specific uses are as follows: For large-scale primary screening of the efficacy of anti-lymph edema drugs: The construction method of this embodiment uses FAR fluorescence visualization guidance, dual tracer precise imaging, and electrocoagulation to close the incision to inhibit lymphatic vessel recanalization. The constructed animal model has complete lymphatic reflux blockage, stable edema phenotype, small individual differences, and strong repeatability, avoiding the defects of inaccurate positioning, low modeling success rate, and strong self-healing ability of traditional modeling methods. It can provide a uniform and reliable evaluation carrier for different candidate drugs and is suitable for the preliminary screening of the anti-secondary lymphedema activity of large batches of compounds and natural extracts.

[0098] For multi-dimensional quantitative evaluation of the overall therapeutic effect of drugs: Relying on the multi-dimensional dynamic evaluation system supporting the model of this embodiment, the effect of drugs can be systematically evaluated from multiple levels. For example, through the water displacement method and tape circumference measurement method, the improvement effect of drugs on limb edema volume and circumference swelling degree can be quantitatively detected; through NIRF-ICG lymphangiography, the regulatory effect of drugs on lymphatic reflux pathway repair and reflux pattern reconstruction can be observed; through the observation of Evans blue dye retention 60 days after surgery, the long-term effect of drugs on improving lymphatic fluid retention and restoring tissue clearance ability can be evaluated, realizing the comprehensive quantitative evaluation of drug efficacy from macroscopic body surface phenotype to lymphatic function.

[0099] For the optimization study of drug dosing dose, dosing cycle, and intervention timing: The model of this embodiment supports dynamic non-invasive monitoring at multiple time points after surgery, and can continuously track the temporal changes of animal edema regression, lymphatic reflux recovery, and tissue pathological improvement under different dosing doses and different dosing courses, enabling the scientific determination of the optimal effective dose, optimal intervention time window, and course regimen of drugs, providing an experimental basis for the design of clinical drug use regimens.

[0100] For distinguishing the symptomatic relief and fundamental therapeutic effects of drugs: The model of this embodiment has a stable pathological basis for permanent lymphatic blockage, and can effectively distinguish whether the test drug only temporarily reduces inflammatory swelling or truly repairs lymphatic reflux function, inhibits the progression of fibrosis, and reverses the pathological process of lymphedema, avoiding misjudgment of drug efficacy caused by the self-healing of the model itself and improving the authenticity and reliability of preclinical evaluation results of drugs.

[0101] Suitable for comparative evaluation of single drugs and combination therapy: The animal model establishment process in this embodiment is standardized and the evaluation indicators are quantitative and objective. It can be used for horizontal comparison of the efficacy of various treatment regimens such as single drugs, different drug combinations, and drug combined with physical intervention, providing an ideal experimental platform for the research and optimization of comprehensive treatment strategies for secondary lymphedema.

[0102] The use of the third aspect of this embodiment will be described below.

[0103] Based on the animal model construction method in the first aspect, this embodiment also proposes the use of the above-mentioned animal model construction method in the study of the pathogenesis of secondary lymphedema or in the labeling and / or identification of the lymphatic system.

[0104] This embodiment utilizes a secondary lymphedema animal model constructed under fluorescence augmented reality guidance. The modeling mechanism closely resembles the pathological characteristics of postoperative and trauma-induced secondary lymphedema in clinical practice. It possesses advantages such as standardized modeling, stable phenotype, dynamic temporal monitoring, and quantifiable multi-dimensional pathology. It can be used to study the in-depth pathogenesis of the occurrence, development, prolongation, and irreversible progression of secondary lymphedema from multiple perspectives, including lymphatic function evolution, tissue morphology remodeling, pathological fibrosis, and lymphatic vessel compensatory remodeling. Specific applications are as follows: This animal model is used to replicate the pathological process of secondary lymphedema in clinical settings and serves as an ideal vehicle for mechanistic research. It precisely removes the popliteal fossa, inguinal core lymph nodes, and draining lymphatic vessels, combined with monopolar electrocoagulation of the incision to block the recanalization of intradermal capillary lymphatic vessels. This simulates the pathogenic factors of mechanical damage to lymphatic pathways caused by clinical surgery and trauma. After modeling, the edema phenotype is stable, with a low spontaneous healing rate. It highly replicates the complete pathogenesis of human secondary lymphedema, from early lymphatic fluid accumulation and progressive limb swelling to mid-to-late-stage tissue fibrosis and irreversible lesions, providing a reliable and standardized animal model for simulating the real clinical pathogenesis.

[0105] To elucidate the temporal evolution mechanism of lymphatic drainage dysfunction: In this embodiment, NIRF-ICG lymphangiography was performed 7 days postoperatively. By utilizing the dynamic progressive transformation patterns of four types of lymphatic drainage imaging modes—linear, sputtering, stardust, and diffuse—it was possible to continuously track the complete temporal changes in lymphatic drainage function after damage to the lymphatic pathway, from gradual attenuation to gradual interruption until complete cessation of lymphatic drainage function. The pathological evolution characteristics of lymphatic drainage dysfunction progressing from mild obstruction to severe obstruction were clearly identified, thereby clarifying that damage to lymphatic drainage function is a key factor in inducing the development of secondary lymphedema.

[0106] To explore the intrinsic link between lymphatic fluid retention and the development of limb edema: This embodiment uses water replacement volume measurement and tape circumference measurement to dynamically monitor changes in limb swelling. Combined with the residual Evans blue dye characteristics on the 60th day after surgery, it can systematically study the causal relationship between impaired interstitial fluid clearance after lymphatic pathway blockage, abnormal lymphatic fluid accumulation, and the formation, aggravation, and long-term maintenance of limb edema, and elucidate lymphatic retention as the core pathological basis for the continuous progression of edema.

[0107] To elucidate the pathological mechanisms of compensatory hyperplasia and abnormal remodeling of lymphatic vessels: This embodiment, combined with hematoxylin-eosin staining, clearly observes the typical pathological features of fluid accumulation in the subcutaneous interstitial tissue of the affected hind limb after surgery. Based on this, the intrinsic pathological mechanisms of compensatory hyperplasia and abnormal remodeling of lymphatic vessels can be systematically analyzed. This animal model, by severing the core lymphatic drainage pathway, causes continuous obstruction of lymphatic return in the limb, resulting in a large amount of lymph fluid accumulating in the subcutaneous interstitial tissue, forming a persistent fluid accumulation microenvironment. Long-term abnormal tissue fluid hydrostatic pressure stimulation and changes in the local pathological microenvironment will gradually induce the remaining local lymphatic vessels to initiate physiological compensatory hyperplasia first, making up for the deficiency in lymphatic drainage function. As interstitial fluid continues to accumulate and lymphatic return is obstructed... As the condition worsens, compensatory hyperplasia becomes insufficient to meet drainage needs, leading to pathological abnormal remodeling of lymphatic vessels, such as luminal malformation, disordered vessel wall structure, and chaotic course and arrangement. Hematoxylin-eosin staining can accurately determine the timing, degree of accumulation, and extent of diffusion of interstitial fluid accumulation, serving as a reference for pathological progression. It can simultaneously track the dynamic changes in the number, tissue structure, and spatial arrangement of lymphatic vessels at different stages of the disease, clarifying the causal relationship between lymphatic drainage obstruction leading to interstitial fluid accumulation, and subsequently triggering compensatory hyperplasia and pathological abnormal remodeling of lymphatic vessels. This fully elucidates the pathological mechanism of lymphatic vessel functional imbalance and abnormal tissue remodeling in the pathogenesis of secondary lymphedema.

[0108] Finally, this embodiment uses a mixture of Evans blue and indocyanine green as a tracer to specifically label the lymphatic system, which consists of lymphatic vessels and structures; then, it combines this with FAR technology to complete the identification of the lymphatic system. On the one hand, indocyanine green can achieve fluorescent labeling of lymphatic tissue, while Evans blue can achieve visible light labeling of drainage areas, thus completely outlining the lymphatic pathways; on the other hand, under FAR guidance, the anatomical location of lymphatic tissue can be identified intraoperatively to guide surgical operations, and postoperatively, changes in fluorescence signals and dye distribution can be used to determine whether lymphatic pathways are blocked or whether lymphatic drainage function is abnormal. This embodiment combines labeling and identification, resulting in stable imaging effects, and can be effectively applied to the visualization, structural identification, and functional status assessment of the lymphatic system.

[0109] The present application will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustration / explanation only and are not intended to limit the scope of the present application.

[0110] Unless otherwise specified, all materials, reagents and instruments used in the following embodiments are commercially available.

[0111] Below, we will first explain some of the raw materials involved in this embodiment to facilitate understanding by those skilled in the art.

[0112] Example 1: Construction of a rat hindlimb secondary lymphedema model.

[0113] 1.1 Laboratory Animals and Ethics: Male Sprague-Dawley (SD) rats, weighing 250–300 g (8 weeks old), were selected and supplied by an authorized vendor. All animal experimental procedures were reviewed and approved by the Institutional Animal Ethics Committee (IACUC) and followed the ARRIVE guidelines. Animals were acclimatized for one week prior to the experiment, with free access to food and water, 12-hour light and dark cycles, constant room temperature, and enrichment facilities provided in their cages.

[0114] 1.2 Preparation and screening of mixed tracers: Mix 3% w / v Evans blue (EB, Solarbio) solution with indocyanine green (ICG, 2.5 mg / mL distilled aqueous solution) at volume ratios of 2:1, 1:1, and 1:2, respectively. Prepare immediately before use, store away from light, and use within 4 hours.

[0115] Screening experiments with mixed tracers: Rats were anesthetized with 4% isoflurane and maintained with 1.5% isoflurane. After hair removal and skin preparation of the hind limbs, 50 μL of the mixed tracer was subcutaneously injected into the web between the 3rd and 4th toes and the ankle of the right hind limb (parallel experimental groups were set up, and the EB:ICG volume ratios were measured at 2:1, 1:1, and 1:2, respectively), gently for 30 seconds. Within 20 minutes after injection, fluorescence-enhanced reality images of the groin and popliteal fossa of the right hind limb were acquired using a laser lymphatic imaging system (equipped with FAR fluorescence technology, excitation wavelength 780 nm, detection wavelength 785 nm).

[0116] Please see Figure 1 , Figure 1 A schematic diagram of fluorescence augmented reality images under different volume ratios of mixed tracers (EB:ICG) is shown; wherein, Figure 1 Part a shows a schematic diagram of injecting tracer into the paw pad; Figure 1 The middle section shows fluorescence augmented reality images with EB:ICG volume ratios of 2:1, 1:1, and 1:2.

[0117] Based on the fluorescence augmented reality images, significant differences exist in the imaging characteristics among the groups: EB:ICG = 2:1 (lowest ICG ratio): Overall fluorescence signal intensity is weak, making it difficult to clearly distinguish the lymphatic vessels and target lymph nodes, resulting in poor imaging contrast and inability to effectively identify lymphatic drainage pathways. This may be due to insufficient fluorescence excitation efficiency, leading to a limited number of ICG molecules within the lymphatic vessels, which fails to generate sufficient near-infrared fluorescence signals; simultaneously, the image signal-to-noise ratio is low, and the signal difference between the lymphatic vessels and surrounding tissues is too small, making it difficult to distinguish from the background in FAR imaging; furthermore, lymph nodes, as tracer-rich sites, also fail to form clear, high-brightness images due to insufficient overall ICG dose.

[0118] EB:ICG = 1:2 (ICG predominance): Excessive fluorescence signal at the injection site creates a significant overexposed area, masking the fluorescence signal of the lymphatic vessels leading to the target lymph node. This results in a significant decrease in the contrast between the lymphatic vessels and the lymph node, along with numerous non-specific background noises, affecting the accuracy of structural identification. This may be due to: firstly, excessively high local ICG concentration at the injection site, creating an overexposed area that masks the weak signal of the distal lymphatic vessels; secondly, excessive ICG is prone to non-specific diffusion, leading to widespread staining of subcutaneous soft tissue and numerous background noises, severely interfering with the judgment of the actual lymphatic vessel course; and thirdly, the fluorescence signal within the lymphatic vessels is affected by high background noise, causing a break in the continuity of the pathway and making it impossible to completely trace to the target lymph node.

[0119] The EB:ICG 1:1 ratio group (equal volume ratio) showed moderate fluorescence intensity at the injection site, with neither significant overexposure nor insufficient signal. Lymphatic vessels were clearly and continuously visualized, and the target lymph nodes were fully visualized without significant background noise. This group provided the clearest imaging result simultaneously displaying lymphatic vessels and lymph nodes among the three groups. In this experimental group, sufficient ICG concentration produced stable and clear fluorescence signals for both lymphatic vessels and lymph nodes without reaching the overexposure threshold. Furthermore, the presence of EB effectively suppressed non-specific diffusion of ICG, reduced background noise, and improved the image signal-to-noise ratio. The moderate fluorescence signal intensity of this experimental group clearly displayed the lymphatic vessel course and fully presented the target lymph nodes, providing optimal visualization conditions for intraoperative fusion navigation and immediate postoperative verification.

[0120] In summary, when the concentration of ICG is too low, the signal is weak and cannot be effectively excited and detected; when the concentration of ICG is too high, fluorescence quenching and self-aggregation are likely to occur, while also leading to local background overexposure and reduced image contrast. While EB itself does not produce near-infrared fluorescence, it can suppress non-specific diffusion of ICG into surrounding tissues and reduce background interference by adjusting local osmotic pressure and tissue staining. Simultaneously, the blue staining of EB can provide an anatomical background under white light, complementing ICG fluorescence; however, excessively high EB ratios dilute the ICG concentration, weakening its tracing ability.

[0121] Under the conditions of this embodiment, the volume ratio of EB:ICG=1:1 achieves the best balance between fluorescence signal intensity, image contrast and background noise, and can clearly display the lymphatic vessels and target lymph nodes at the same time. It is the standard ratio used in all subsequent experiments. Therefore, the volume ratio of EB:ICG=1:1 is used for related experiments and effect verification in subsequent experiments.

[0122] 1.3 Preoperative baseline FAR lymphangiography: Five more rats were anesthetized with 4% isoflurane and maintained with 1.5% isoflurane. After shaving and preparing the skin of the hind limbs, 50 μL of a mixed tracer (EB:ICG=1:1) was subcutaneously injected into the web between the 3rd and 4th toes and the ankle of the right hind limb, followed by gentle massage for 30 seconds. Within 20 minutes after injection, fluorescence-enhanced reality images of the inguinal and popliteal regions of the right hind limb were acquired using a laser lymphatic imaging system (equipped with FAR fluorescence technology, excitation wavelength 780 nm, detection wavelength 785 nm). The superficial medial and superficial lateral lymphatic drainage pathways and the popliteal lymph node (PO) and inguinal lymph node (IN) locations were recorded (see [link to relevant documentation]). Figure 2 Simultaneously, the locations of lymph nodes and enlarged lymphatic vessels shown by Evans blue staining were recorded and compared with fluorescence images for verification, achieving dual localization confirmation.

[0123] The superficial lateral lymphatic drainage pathway originates in the network of the foot pad, then crosses to the lateral aspect of the lower third or middle of the lower leg, draining distally through the popliteal lymph nodes. The superficial medial lymphatic drainage pathway originates in the network above the ankle joint, extends into the thigh, and drains distally through the inguinal lymph nodes.

[0124] 1.4 FAR-guided surgery: Please see Figure 3 , Figure 3 This diagram illustrates the workflow for FAR-guided lymph node resection and in vitro / in vivo imaging verification; specifically including: Under maintained anesthesia, the surgical area was disinfected with 75% ethanol. Guided by FAR fluorescence combined with EB staining, the inguinal lymph nodes (IN) were located (fluorescently visible as bright oval structures, staining blue with Evans blue). A 1.5 cm inguinal incision was made, and the IN was excised under FAR guidance. The lymphatic vessels were ligated by bipolar electrocoagulation, and FAR confirmed the disappearance of fluorescence in the inguinal region in real time. The popliteal lymph nodes (PO) were located using the same method. The skin in the popliteal fossa was circumferentially excised, and the popliteal lymph nodes and surrounding fat pads were excised under FAR guidance. The lymphatic vessels were ligated by bipolar electrocoagulation, and FAR confirmed the disappearance of fluorescence in the popliteal fossa in real time. The leg was thoroughly rinsed with sterile saline to avoid wound contamination and infection. No active bleeding was ensured. The skin was sutured with 5-0 nylon sutures, leaving a 1-2 mm gap between the skin edges (gap suture) to prevent re-anastomosis of intradermal lymphatic vessels at the incision site. At the same time, the skin incision is circumferentially cauterized before suturing to further ensure the blockage of capillary lymphatic vessels.

[0125] 1.5 Postoperative analgesia and care: Immediately after surgery, mice were given an intramuscular injection of ketoprofen (1 mg / ml). They were then placed in separate cages for postoperative recovery, with free access to water and food. Mice were monitored daily for wound healing, pain, and signs of infection. If signs of infection were observed, antibiotic ointment was applied.

[0126] 1.6 Experimental Results: Seven days after surgery, changes in the limbs (hind limbs), skin reflux patterns, changes in the volume of the affected limb, and changes in the circumference of the affected limb were measured / tested (average values ​​were taken).

[0127] Please see Figure 4 , Figure 4 The diagram shows the limb changes, hindlimb reflux patterns, changes in the volume of the affected limb, and changes in the circumference of the affected limb in rats 7 days after surgery.

[0128] in, Figure 4 Part a shows a schematic diagram of the changes in the appearance of the rat's limbs 7 days after surgery; Figure 4 Part b shows representative images of skin reflux patterns in the control and model groups, specifically including linear, sputtering, stardust, and diffusion patterns. Figure 4 Part C is a schematic diagram showing the statistical results of various skin reflux patterns in the model group rats; Figure 4 Part d in the middle is a schematic diagram showing the changes in the volume of the affected limb in rats 7 days after surgery; Figure 4 Part e is a schematic diagram showing the changes in the circumference of the affected limb in rats 7 days after surgery; Figure 4 The middle part (f) is a schematic diagram of the H&E staining results of the rat's leg 7 days after surgery; Figure 4The middle g section is a schematic diagram of the H&E staining results of the rat's paw 7 days after surgery.

[0129] according to Figure 4 As shown in the figure, the hind limbs of the rats in the model group showed obvious swelling after modeling, which was in stark contrast to the normal limb morphology of the rats in the blank control group without any intervention. This directly reflects the abnormal swelling of the limbs caused by lymphatic obstruction. It is preliminarily determined that the modeling operation can successfully induce the characteristic surface lesions of secondary lymphedema in the rat limbs.

[0130] Meanwhile, 7 days post-surgery, FAR clearly identified the hindlimb skin return patterns, including linear, splash, stardust, and diffusion patterns. The control group (rats without any treatment) exhibited a linear pattern. Under physiological conditions, the lymphatic circulation in the blank control group was smooth and stable, showing only a regular, continuous linear lymphatic vessel imaging pattern. Lymph could complete directional centripetal return along the inherent lymphatic vessels, without lymph extravasation or skin reflux, representing the standard imaging manifestation of normal lymphatic drainage in a healthy organism. In contrast, the model group rats, after the lymphatic pathway structure was blocked, deviated from the normal linear lymphangiography pattern, completing the transition from simple lymphatic vessel imaging to lymphatic drainage into surrounding tissues. The pathological transformation of lymphatic leakage diffusion imaging was observed. The model group exhibited a shift from a linear lymphangiography pattern (showing only lymphatic vessels) to a diffuse pattern (showing leaked lymph in surrounding tissues), including sputtering (20%), stardust (40%), and diffuse (40%) patterns. Sputtering represented mild lymphatic vessel damage and localized leakage of small amounts of lymph, indicating mild lymphatic drainage obstruction. Stardust represented multiple obstructions in lymphatic drainage pathways, with lymph accumulating and spreading at multiple points, indicating moderate lymphatic reflux obstruction. Diffuse represented near-complete interruption of the main lymphatic pathway, with significant lymph leakage and extensive infiltration of surrounding soft tissues, indicating severe lymphatic drainage dysfunction. This classification distribution indicates that the vast majority of experimental rats developed moderate to severe lymphatic reflux dysfunction after modeling, with lymph unable to flow orderly along normal pathways and accumulating in large quantities in the soft tissues of the limbs, fully validating the effectiveness of lymphatic drainage pathway blockage from a functional perspective.

[0131] Further integration Figure 4 As shown, 7 days post-surgery, the volume of the affected limb increased by 23%, and the circumference increased by 14%. The 7-day post-surgery period was chosen as the detection point, as the initial acute inflammatory edema caused by the surgical wound had completely passed. At this time, the morphological data obtained represented stable pathological swelling resulting from long-term lymphatic fluid accumulation. Quantitative results showed that the volume of the affected limb in the model group rats increased by approximately 23% compared to the normal state, and the limb circumference increased by approximately 14%. All of these morphological indicators showed significant increases, with the limb volume increase being the largest. This numerically confirms that the rats' hind limbs have developed stable and irreversible pathological swelling, making the model establishment determination more standardized and data-driven.

[0132] Please continue reading. Figure 4 , Figure 4 The middle section (f) shows the H&E staining results of the rat's leg (hind limb) 7 days after surgery; Figure 4 Part g shows the H&E staining results of the rat paws (hind limbs) 7 days after surgery. Regarding the leg tissue, in the control group, the subcutaneous tissue structure was dense, with clear layers, a distinct boundary between the dermis and subcutaneous layer, neatly arranged collagen fibers, and narrow interstitial spaces, indicating no obvious fluid accumulation and uniform skin thickness. In contrast, in the model group, the total skin thickness was significantly increased (as shown by the arrows), and the dermis and subcutaneous layer were significantly thickened; the subcutaneous tissue spaces were significantly expanded and loosened, with numerous lightly stained, transparent areas, indicating tissue fluid retention / edema fluid accumulation due to obstructed lymphatic drainage; at the same time, the collagen fibers were disordered and separated by edema fluid, and the tissue density decreased, which are typical histological features of early lymphedema. Regarding the paw tissue, in the control group, the skin of the paws was thin, the epidermis was thin, the subcutaneous tissue was dense, the interstitial spaces were small, and there was no edema fluid accumulation. In the model group, the skin thickness of the claws was significantly increased, with both the epidermis and dermis thickening; the subcutaneous tissue was obviously loose and edematous, the interstitial spaces were enlarged, and a large number of lightly stained edematous areas were visible (as indicated by the arrows), accompanied by loose arrangement of collagen fibers; this change was particularly obvious in the distal extremities (claws), which is consistent with the clinical pathological pattern that the distal tissue fluid accumulation is more severe in lymphedema.

[0133] In summary, H&E staining results 7 days post-surgery showed that, compared with the control group, the model group rats exhibited significant subcutaneous tissue thickening, interstitial space expansion, fluid accumulation, and disordered collagen fiber arrangement in the leg and paw areas. These results confirm that the secondary lymphedema model constructed in this application can successfully induce typical pathological changes of lymphatic drainage obstruction, and that these changes exhibit tissue specificity and uniformity, indicating the successful establishment of the rat hindlimb lymphedema model.

[0134] In summary, the method for constructing an animal model of lymphedema based on fluorescence augmented reality provided in this embodiment uses a mixed tracer composed of Evans blue and indocyanine green. The optical imaging properties of the two tracers complement each other, enabling highly specific and high-contrast targeted imaging of lymph nodes and draining lymphatic vessels in the model animal, clearly outlining the complete course of the lymphatic system, and effectively overcoming the shortcomings of single tracers, such as large tissue background interference, blurred edges of lymphatic structures, and poor anatomical localization identification.

[0135] Secondly, the entire procedure is guided by fluorescence augmented reality (FAR) imaging technology. Preoperatively, real-time fluorescence images can be acquired to accurately predict the distribution and direction of lymphatic drainage pathways, providing intuitive imaging evidence for surgical incision planning and delineation of the target tissue resection area. During the procedure, the enhanced fluorescence reality visualization field allows for precise location of the target lymph node and its connected draining lymphatic vessels, enabling targeted and precise dissection and resection. This avoids the problem of damage to surrounding normal blood vessels, nerves, and soft tissues that is common with traditional blind resection methods, significantly improving the precision and safety of the surgical operation. Postoperatively, simultaneous acquisition of fluorescence images allows for non-invasive, intuitive, and rapid verification of the integrity of lymphatic drainage pathway blockage, timely detection of residual lymphatic return channels, and prevention of model failure due to incomplete blockage.

[0136] Meanwhile, under the precise guidance of fluorescence visualization, the extent and degree of resection of lymph nodes and lymphatic vessels can be standardized and controlled, resulting in a stable edema phenotype, good model uniformity, and strong reproducibility in the constructed lymphedema animal model. The modeling process in this embodiment is standardized and highly visualized, reducing reliance on the anatomical experience of operators and significantly improving the success rate of animal model construction. It can be stably applied to medical research scenarios such as exploring the pathogenesis of lymphedema, screening drug efficacy, and evaluating clinical intervention programs.

[0137] The technical solutions provided by the embodiments of this disclosure have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of this disclosure. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of this disclosure. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of this disclosure. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A method for constructing an animal model of lymphedema based on fluorescence augmented reality, characterized in that, The method for constructing the animal model includes: The model animals were injected with a mixed tracer, and white light anatomical images and indocyanine green near-infrared fluorescence images were acquired; the mixed tracer included Evans blue and indocyanine green. Under FAR guidance, the target lymph nodes and their connected draining lymphatic vessels in the model animals were removed, and postoperative fluorescence images were acquired to verify the integrity of the lymphatic drainage pathway blockage. The skin incision was closed to obtain an animal model of lymphedema.

2. The method for constructing an animal model of lymphedema based on fluorescence augmented reality as described in claim 1, characterized in that, The animal model construction method also includes a postoperative multidimensional dynamic assessment step, which includes: At multiple pre-defined time points, perform at least one of the following non-invasive dynamic assessments on the model animals: Changes in limb volume, changes in limb circumference, indocyanine green lymphatic reflux pattern, Evans blue retention status, and histological verification.

3. The method for constructing an animal model of lymphedema based on fluorescence augmented reality guidance according to claim 1, characterized in that, The changes in limb volume were measured using the water displacement volume measurement method; the changes in limb circumference were measured using the tape girth measurement method.

4. The method for constructing an animal model of lymphedema based on fluorescence augmented reality guidance according to claim 1, characterized in that, The target lymph nodes include the popliteal lymph nodes and the inguinal lymph nodes.

5. The method for constructing an animal model of lymphedema based on fluorescence augmented reality guidance according to claim 1, characterized in that, In the mixed tracer, the volume ratio of Evans blue to indocyanine green is 1:

1.

6. The method for constructing an animal model of lymphedema based on fluorescence augmented reality as described in claim 1, characterized in that, The model animal was the Sprague-Dawley rat.

7. The method for constructing an animal model of lymphedema based on fluorescence augmented reality guidance according to claim 1, characterized in that, The excitation wavelength of the FAR is 770-790 nm, and the detection wavelength is >780 nm.

8. The method for constructing an animal model of lymphedema based on fluorescence augmented reality as described in claim 1, characterized in that, The criteria for determining the success of the animal model include at least one of the following: Seven days post-surgery, the volume of the affected limb in the model animals increased by ≥20% compared to pre-surgery. Seven days post-surgery, the circumference of the affected limb in the model animals increased by ≥14% compared to pre-surgery. Seven days after surgery, images of the indocyanine green lymphatic drainage pattern and Evans blue retention on the affected side of the model animals were collected, showing that lymphatic drainage was blocked. Hematoxylin-eosin staining revealed fluid accumulation in the subcutaneous interstitial tissue of the affected hind limb after surgery.

9. The use of the animal model construction method according to any one of claims 1-8 in evaluating the efficacy of drugs for treating secondary lymphedema.

10. The use of the animal model construction method according to any one of claims 1-8 in the study of the pathogenesis of secondary lymphedema or in the labeling and / or identification of the lymphatic system.

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