Key refinements to a large animal model for measurement of real-time lymphatic transport

BACKGROUND: The inability to directly measure real-time lymphatic transport hinders our understanding of lymphatic disorders and effective monitoring of therapeutic interventions. Here, we report three refinements to the use of novel near-infrared fluorophores in a large animal model for assessment of lymphatic function using optical fluorescence with the ultimate goal of direct clinical translation. METHODS: A lymphadenectomy and lymphovenous bypass was performed in the left groin. Two different near-infrared (NIR) fluorophores composed of the identically same scaffold were injected into the respective hind limbs of female swine. Transit from injection sites into the lymphatic system and systemic circulation was assessed over three hours of NIR imaging immediately following injections. Albumin-conjugated fluorophores with 700 nm and 800 nm emission, respectively, were delivered with manual intradermal injection. Blood and urine samples were collected at standard time points. Continuous NIR imaging of the lymphovenous bypass and the superficial epigastric vein and its adjacent skin was also performed at standard time points. Blood and urine samples were collected to analyze systemic concentrations and renal excretion of both fluorophores, respectively. NIR imaging was performed to analyze real-time lymphatic transport of the fluorophores through the lymphovenous bypass and in the central circulation. RESULTS: Fluorophore pharmacokinetics with albumin conjugation and manual intradermal injections produced inconsistent results. Three modifications to the study protocol were designed. Fluorophores were modified from albumin conjugation to polyethylene glycol (PEG) conjugation. Fluorophores were delivered with VAX-ID ® devices instead of manual intradermal injections. Additionally, a novel correction factor was developed to account for differences in NIR fluorescence between the fluorophores. CONCLUSION: The refinement of a large animal model to assess real-time lymphatic function represents a crucial step toward clinical translation. The optimized fluorophore composition and delivery mechanism captures dynamic changes in lymphatic function.