Minimally invasive quantification of lymph flow in mice and rats by imaging depot clearance of near-infrared albumin

Abstract

There is a lack of available methods to noninvasively quantify lymphatic function in small experimental animals, a necessity for studies on lymphatic system pathophysiology. We present a new method to quantify lymph flow in mice and rats, based on optically monitoring the depot clearance of near-infrared fluorescently labeled albumin and subsequent calculation of removal rate constants (k). BSA was conjugated with Alexa680 NHS ester and remained stable in protein-rich solutions without free dye dissociation. To assess lymph flow, mice or rats were imaged every 30 or 60 min during a 3- to 6-h period following an intradermal injection of 0.5 or 1 μl Alexa680-albumin. Mice were awake between measurements, whereas rats were anesthetized throughout the experiment. The k, a parameter defined as equivalent to lymph flow, was calculated from the slopes of the resultant log-linear washout curves and averaged -0.40 ± 0.03 and -0.30 ± 0.02%/min for control C57BL/6 and C3H mice, respectively. Local administration of the vasoconstrictor endothelin-1 in mice led to a significant reduction in k, whereas overhydration in rats increased k, reflecting the coupling between capillary filtration and lymph flow. Furthermore, k was 50% of wild type in lymphedema Chy mice where dermal lymphatics are absent. We conclude that lymph flow can be determined as its rate constant k by optical imaging of depot clearance of submicroliter amounts of Alexa680-albumin. The method offers a minimally invasive, reproducible, and simple alternative to assess lymphatic function in mice and rats.

Increased Interstitial Protein Because of Impaired Lymph Drainage Does Not Induce Fibrosis and Inflammation in Lymphedema

“…12 Skin inflammation was induced by topical application of oxazolone representing delayed-type hypersensitivity inflammatory reaction.…”

“…12 The intradermal injection of Albumin-Alexa 680 rendered only a small, hardly visible vesicle at the dorsal surface of the hind paw. After an initial distribution phase of 60 minutes, when little solute was cleared, we followed the washout of tracer for 6 hours ( Figure 6A).…”

State-of-the-Art Methods for Evaluation of Angiogenesis and Tissue Vascularization

“…164 Molecular clearance analysis, for example, using labeled albumin and interstitial tissue pressure, provides a complementary analysis of fluid homeostasis in tissues with compromised lymphatic vessels. 165 Lymphangiogenesis-blocking agents such as antibodies targeting VEGF-C, VEGFR3, or its coreceptor neuropilin-2, as well as soluble VEGFR3 and certain tyrosine kinase inhibitors, have been found to inhibit lymphatic metastasis in xenotransplanted tumors and in transgenic mice. Such experiments commonly use luciferase-or green fluorescent proteinexpressing tumor cells that can be imaged in the target tissues.…”

Quantitative measurement of lymphatic function in mice by noninvasive near-infrared imaging of a peripheral vein

“…Near-infrared (NIR) imaging has been especially helpful in this regard, as it has enabled quantitative measures in mice of lymphatic clearance from skin, collecting lymphatic vessel contractility, and flow to or through peripheral lymph nodes (3)(4)(5)(6). Translation to humans using clinically approved indocyanine green (ICG) is well underway, with many groups using this noninvasive imaging technique to map lymphatic flow routes or to measure collecting lymphatic vessel pumping pressure in superficial regions of the skin (7)(8)(9).…”