Quantification of lymph flow in primary lymphedema mice using optical imaging

摘要: 正 The Chy mouse is a model of human primary lymphedema (Milroy's disease) due to an inactivating heterozygous Vegfr3 mutation. Despite the aplasia of dermal lymphatics, the swelling of the limbs is moderate. Lymphatics are present in the subcutis, however the functionality of these vessels is unknown. Lymph drainage can be quantified by measuring the rate of removal of a colloid from an area of interest. Our aim was to establish a noninvasive technique to quantify lymph flow in Chy lymphedema mice by studying the wash-out of a fluorescent probe visualized by optical imaging. Albumin was conjugated with Alexa-680 dye and purified by size exclusion chromatography and HPLC to exclude all free dye. The purity of the samples was confirmed by time-domain optical imaging analysis of the fluorescence lifetime of labeled albumin. We also verified with an in vitro phantom system that there was a linear relationship between the concentration of Alexa-albumin and the intensity of the signal via optical imaging. For studies of lymph transport, Chy and WT mice were imaged every hour for a 6 h period following injection of 0.5 μL Alexa-albumin intradermally in the paw or intramuscularly in the thigh. During imaging, mice were under isofluorane anesthesia whereas they were awake and freely moving between measurements. For calculations of removal rate constants (k), the natural logarithm of the fractional amount of counts remaining at the injection site was plotted against time. The curve was fitted with linear regression and k found as the slope of the curve for each mouse. The k for Chy mice paw averaged (0.16±0.03)%/min which was …