How Well Do We Actually Measure Lymphatic Function in Lymphedema Animal Models?

Lymphedema, a chronic and progressive disease caused by impaired lymphatic transport, has long been studied in animal models, but a new mini-review argues that the field has been quietly measuring the wrong things. Researchers at Georgia Institute of Technology and Emory University, led by Shao-Yun Hsu, James Brandon Dixon, and Zhanna Nepiyushchikh, surveyed 70 primary studies across seven species and found a striking mismatch between the species most commonly used to model lymphedema and those in which the most informative measurements are technically possible. The review, published in Frontiers in Physiology, organizes lymphatic function assessment into three tiers and provides a practical cross-tabulation to help investigators pair the right animal model with the right measurement strategy.

The lymphatic system performs several interrelated jobs: it returns capillary ultrafiltrate and plasma proteins to the venous circulation, delivers antigens to draining lymph nodes for immune surveillance, absorbs dietary lipids through intestinal lacteals, and regulates interstitial pressure and macromolecular clearance. When lymphatic pumping capacity falls below the rate at which interstitial fluid is generated, fluid accumulates, vessels dilate and remodel, valves lose their normal structure and function, and the disease marches toward irreversible tissue fibrosis. The biologically meaningful variables are therefore those describing intrinsic pumping: contraction frequency, contraction amplitude, ejection fraction, intraluminal pressure, valve gating thresholds, and lymph flow rate.

Historically, direct measurement of these parameters has been technically formidable. Lymphatic vessels are small in caliber, optically transparent, and carry exceedingly low flow rates, which is why direct measurements were long restricted to large-animal cannulation studies or controlled ex vivo preparations such as pressure myography of isolated lymphangions. Mid-twentieth-century lymphangiography enabled in vivo visualization of lymphatic architecture, and glass micropipettes and pressure transducers later allowed the first direct measurements of hydrostatic pressure and peripheral lymph flow within individual vessels. Intravital microscopy subsequently revealed the lymphangion as the functional contractile unit, in which intrinsic smooth-muscle contractions and coordinated valve gating propel lymph against pressure gradients.

The review’s central contribution is a three-tier framework for assessing lymphatic function. Tier 1 covers morphological and indirect outcome measures, including volumetric quantification by water displacement, caliper or circumferential measurement, micro-CT, magnetic resonance imaging of soft-tissue and fat volumes, and immunohistochemical analysis with lymphatic endothelial markers such as LYVE-1, VEGFR-3, Prox-1, and podoplanin. These are the most widely adopted methods across species because they are technically accessible and reproducible, but they reliably capture only the downstream consequences of lymphatic insufficiency and are inherently limited in detecting early or subclinical dysfunction.

Tier 2 comprises imaging-based functional assessments, including near-infrared fluorescence lymphangiography with indocyanine green, radionuclide lymphoscintigraphy, magnetic resonance lymphangiography with gadolinium-based contrast agents, whole-body fluorescence platforms such as IVIS, and Evans blue dye lymphangiography. These modalities enable real-time or time-resolved visualization of lymphatic transport in vivo, bridging morphology and physiology. However, because the fraction of dye taken up by lymphatics from an interstitial injection cannot be measured directly, fluorescence intensity readouts remain relative rather than absolute, and conventional Tier 2 modalities do not yield true physical flow quantities.

Tier 3 consists of direct physiological assessments: ex vivo pressure myography with cannulated lymphangions, in situ lymphatic conductance measurement, interstitial fluid pressure quantification, transmural flow assays, and in vivo lymph cannulation. Pressure myography enables precise characterization of valve competence, back-leak pressure, gating thresholds, contraction frequency, amplitude, and ejection fraction at the lymphangion level. In larger animals, cannulation of thoracic ducts or peripheral collecting vessels enables direct measurement of lymph flow rate, intraluminal pressure dynamics, and lymph composition. The principal limitation is technical: these methods require specialized instrumentation and surgical expertise, frequently involve vessel transection, and restrict longitudinal monitoring within the same subject.

Surveying the 70 studies revealed sobering patterns at each tier. Among 62 surgical lymphedema model studies, only 7, or 11.3 percent, defined an explicit per-animal volumetric cutoff for model establishment, despite induction rates of roughly 60 to 80 percent for surgical models. Volumetric measurement itself was performed in most studies, approximately 96 percent of mouse, 88 percent of rat, and 88 percent of large-animal studies, but the deficit was not an absence of measurement; it was the absence of a pre-specified threshold. Histology was performed in the majority of studies across all species, yet marker choices and scoring criteria vary substantially between laboratories, with no consensus panel or standardized scoring system adopted.

Tier 2 imaging told a similar story of missed opportunity. Of 61 studies employing imaging, the large majority relied on pattern observation alone, such as stasis and dermal backflow, or on bulk clearance and intensity quantification, both of which capture downstream consequences rather than pumping function during disease development. Only 6 studies, or 10 percent, quantified collecting vessel contraction frequency, amplitude, and pumping score from real-time imaging, and all were conducted in rodents: four in mouse tail models and two in rat limb models. No rabbit or large-animal study achieved this level of functional resolution. Tier 3 was even sparser: direct in vivo measurement of physical lymphatic quantities appeared in only 7 of 70 studies, and no Tier 3 study conducted repeated direct measurements in the same animal over the course of disease progression.

The review frames this as a fundamental constraint: the most accessible endpoints tend to be the least informative about the disease process, while the most informative are the least accessible. Limb swelling and histological change are late manifestations, emerging only after pumping capacity has been overwhelmed for some time. Imaging mitigates this delay but depends on injected contrast agents for a physical reason: lymph is optically transparent and flows at velocities often below the detection threshold of label-free methods such as Doppler-based instrumentation. Emerging modalities, including Doppler optical coherence tomography, which has demonstrated label-free quantification of lymph flow velocity and volumetric flow rates in living animals, and photoacoustic imaging with lymphatic-absorbing contrast agents, may eventually enable more direct assessment, though their application to lymphedema models remains limited.

For investigators choosing a model, the authors offer pragmatic guidance. For acquired secondary lymphedema, the rat hindlimb model paired with quantitative longitudinal imaging and vessel-level analysis offers a balanced platform: it is large enough to permit quantitative imaging yet far more accessible than large-animal models. Genetic mouse models remain preferable for congenital lymphedema, where defined genetic backgrounds offer the closest match to human developmental mechanisms, while large-animal models are best reserved for microsurgical intervention and clinical-scale procedures, given their cost, space requirements, and lower throughput. Notably, porcine models have repeatedly failed to produce meaningful limb volume change despite extensive surgical disruption. Until techniques for longitudinal, non-disruptive measurement of lymphatic flow in diseased animals mature, the authors conclude, the pathophysiology of lymphedema as a disease of lymphatic transport is likely to remain incompletely characterized.

Subject of Research: Measurement methods for assessing lymphatic function in animal models of lymphedema

Article Title: Functional assessment of experimental lymphedema: a focused mini-review of animal models and measurement methods

Article References: Hsu, S.-Y., Dixon, J. B., & Nepiyushchikh, Z. (2026). Functional assessment of experimental lymphedema: a focused mini-review of animal models and measurement methods. Frontiers in Physiology, 17, Article 1912564. https://doi.org/10.3389/fphys.2026.1912564

Image Credits: AI Generated

DOI: 10.3389/fphys.2026.1912564

Keywords: lymphedema, lymphatic system, animal models, lymphatic pumping, near-infrared fluorescence imaging, lymphoscintigraphy, pressure myography, rat hindlimb model, mouse tail model, lymphangiography, fibrosis, lymphatic conductance