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

The technique at the heart of the study is spatial transcriptomics, a technology that maps gene expression directly onto intact tissue sections rather than requiring cells to be dissolved into a suspension first. The approach has been applied successfully to tumors, lung, ureter and skin, but until now it had never been established for a whole-limb vascularized composite allograft. The obstacle was not the analysis platform itself but the pre-analytical handling of large, heterogeneous tissue. A rat hindlimb contains compartments with sharply different mechanical properties and densities, and mishandling at any stage of harvest, freezing, embedding, sectioning or permeabilization can destroy RNA integrity, distort morphology or blur spatial resolution.

Writing in Frontiers in Molecular Biosciences, Marie Dreck, Elena Grabis and colleagues describe a refined workflow that spans every step from surgery to sequencing. The team used Lewis rats as limb donors and transgenic Wistar Kyoto rats carrying a human CD68 promoter driving green fluorescent protein as recipients, which allowed them to visualize host-derived macrophages as they infiltrated the graft. Tissue was harvested between 18 and 90 days after transplantation, with ischemia time kept as short as possible to protect the fragile RNA molecules inside the cells.

Several critical design decisions emerged from the team’s trial and error. First, the tibia and fibula were removed by careful periosteal elevation before embedding. Early cryosectioning trials showed that retained bone mechanically sheared and fragmented the adjacent soft tissue, producing uneven sections unsuitable for either histology or spatial analysis. Second, the freezing method mattered enormously. Rapid immersion in liquid nitrogen degraded RNA and introduced severe freezing artifacts, so the researchers instead used controlled freezing in an isopropanol bath on dry ice, which cooled the tissue more gradually at around minus 78 degrees Celsius and yielded uniformly preserved, intact sections with excellent adhesion to the slide.

Formalin fixation and paraffin embedding, the standard preservation method in most pathology laboratories, proved fatal for the workflow. When the team compared FFPE specimens with cryopreserved ones at multiple quality control checkpoints, the FFPE samples showed collapsed ribosomal RNA peaks and an accumulation of short fragments, indicating extensive degradation. Their cDNA profiles were fragmented and weak, and their sequencing libraries contained little detectable material in the 200 to 600 base pair range needed for the Visium platform. The cryopreserved samples, by contrast, showed strong 18S and 28S ribosomal peaks, broad cDNA distributions between 200 and 9,000 base pairs, and a clean library peak near 482 base pairs. Cryopreservation, the authors conclude, is essential because it maintains the intact poly(A) tails that the spatial barcoded oligonucleotides on the capture slide require to hook released messenger RNA.

RNA quality also depended on the extraction chemistry. RNA isolated from sections using the conventional phenol-guanidine thiocyanate-chloroform method yielded RNA Integrity Number values between 3.7 and 4.2, while a column-based Qiagen RNeasy approach pushed values up to between 6.0 and 6.9. That is still below the threshold of 7 recommended by 10x Genomics, the manufacturer of the Visium platform, yet every sample passed the downstream quality controls, with median log10 unique molecular identifiers per spot between 2.95 and 3.85, acceptable transcript complexity and low mitochondrial read percentages. The finding suggests that conventional RIN cutoffs may be overly restrictive for structurally complex composite tissue, and that RNA integrity measured after extraction does not always reflect the true biological state of the tissue.

Permeabilization, the step in which cell membranes are dissolved so that messenger RNA can diffuse to the capture spots, required its own careful calibration. Because permeabilization efficiency is tissue-dependent, the team tested incubation times of 30, 33, 36, 42 and 48 minutes on hindlimb cross-sections placed on tissue optimization slides. After reverse transcription with fluorescently labeled nucleotides, the fluorescence-positive area was quantified in ImageJ using an identical intensity threshold across conditions. The 33-minute sample showed the highest fluorescence-positive area at 32.4 percent, compared with 9.2 percent at 36 minutes and 17.4 percent at 42 minutes, indicating that this timing released RNA efficiently while preventing overpermeabilization and signal diffusion.

The immunofluorescence side of the workflow required separate ingenuity. The endogenous GFP signal in the hCD68 reporter rats survived only a brief fixation protocol: 15 minutes in pre-warmed 4 percent paraformaldehyde, followed by washes and blocking in goat serum with Tween-20. Even then the native fluorescence was faint, so the researchers added an anti-GFP antibody to amplify the signal, enabling reliable visualization of recipient-derived monocytes and macrophages infiltrating skin, muscle and connective tissue. Consecutive 10 micrometer cryosections were stained in parallel with the spatial transcriptomics runs, so the protein-level maps serve as complementary validation of the transcriptional data.

The payoff is visible in the downstream analysis of the representative sample. Unsupervised clustering identified seven spatially organized transcriptional compartments that corresponded cleanly to known anatomy: activated stroma, inflammatory myeloid regions, adipose tissue, B-cell and plasma cell zones, extracellular-matrix-remodeling stroma, keratinizing epithelium and peripheral glial structures. Histologically annotated vessels matched spatial enrichment of endothelial markers, and a vessel-distance map computed in the SPATA2 software confirmed the spatial correspondence. Cell-type composition was further refined by integrating the spatial data with a published Rattus norvegicus single-cell reference atlas using the cell2location and CytoSPACE algorithms.

The authors are candid about trade-offs. Removing the bone improved sectioning but excluded the osseous compartment from transcriptomic analysis, and immunofluorescence and spatial transcriptomics were performed on adjacent rather than identical sections, so precise transcript-to-protein colocalization remains out of reach. Yet the framework’s generality is its most exciting feature. The same principles of controlled freezing, bone removal, optimized permeabilization and GFP-preserving fixation can be transferred to other large, complex tissues, from musculoskeletal specimens to peripheral nerve and organ transplants. For a field in which rejection biology has long been read through macroscopic graft changes and conventional histology, the ability to watch immune cells arrive, localize and change their transcriptional programs across an entire living limb composite promises to reshape how acute and chronic rejection are understood, and ultimately how they are treated.

Subject of Research: Optimized spatial transcriptomics and immunofluorescence protocol for whole rat vascularized composite allograft hindlimb sections

Article Title: Preserving RNA integrity and morphology in whole rat allograft hindlimb sections: an optimized protocol for spatial transcriptomics and immunofluorescence

Article References: Dreck, M., Grabis, E., Schneider, L., Lind, M., Heinrichs, P., Christmann, T., Neidert, N., Brose, T. Z., Kreuzaler, S., Pankratz, F., Heiland, D. H., Eisenhardt, S. U., & Thomé, J. (2026). Preserving RNA integrity and morphology in whole rat allograft hindlimb sections: an optimized protocol for spatial transcriptomics and immunofluorescence. Frontiers in Molecular Biosciences, 13, Article 1910122. https://doi.org/10.3389/fmolb.2026.1910122

Image Credits: AI Generated

DOI: 10.3389/fmolb.2026.1910122

Keywords: spatial transcriptomics, vascularized composite allotransplantation, rat hindlimb model, RNA integrity, immunofluorescence, cryopreservation, Visium, graft rejection, macrophages, GFP reporter rats, cryosectioning, reconstructive surgery