Optical tracer size differences allow quantitation of active pumping rate versus Stokes-Einstein diffusion in lymphatic transport

Abstract

Lymphatic uptake of interstitially administered agents occurs by passive convective–diffusive inflow driven by interstitial concentration and pressure, while the downstream lymphatic transport is facilitated by active propulsive contractions of lymphatic vessel walls. Near-infrared fluorescence imaging in mice was used to measure these central components of lymphatic transport for the first time, using two different-sized molecules––methylene blue (MB) and fluorescence-labeled antibody immunoglobulin G (IgG)-IRDye 680RD. This work confirms the hypothesis that lymphatic passive inflow and active propulsion rates can be separated based upon the relative differences in Stokes–Einstein diffusion coefficient. This coefficient specifically affects the passive-diffusive uptake when the interstitial volume and pressure are constant. Parameters such as mean time-to-peak signal, overall fluorescence signal intensities, and number of active peristaltic pulses, were estimated from temporal imaging data. While the mean time to attain peak signal representative of diffusion-dominated flow in the lymph vessels was 0.6±0.2??min for MB and 8±6??min for IgG, showing a size dependence, the active propulsion rates were 3.4±0.8??pulses/min and 3.3±0.5??pulses/min, respectively, appearing size independent. The propulsion rates for both dyes decreased with clearance from the interstitial injection-site, indicating intrinsic control of the smooth muscles in response to interstitial pressure. This approach to size-comparative agent flow imaging of lymphatic function can enable noninvasive characterization of diseases related to uptake and flow in lymph networks.

Fig. 1

Uptake of MB from the injection site in the paw to an axillary lymph node (white arrow).

Fig. 2

Active and passive components of lymphatic uptake of MB. Inset shows representative ROI locations (circles were enlarged to be visible, actual ROI size was $\sim 2\mathrm{mm}$ for nodes and 0.5 mm for lymph vessels). ROI circles are color coded to match the uptake curves. Raw data (light solid lines) and smoothed curves (dotted lines) are shown. 5-mm scale bar is shown on inset.

Fig. 3

Comparison of passive uptake components of MB and IgG-IRDye shown in a representative lymph node (solid) and its corresponding afferent lymph vessel (dotted). MB clears rapidly from the node owing to its small size. Inset shows a time-expanded view. Note that curves were smoothed, so active lymph vessel propulsion is not evident here. The second peak (indicated by red asterisk in the main plot) of the double peak seen in the IgG-IRDye uptake in the lymph node was always found to be higher in signal intensity that the first and was chosen for peak time and intensity estimation. Color-coded asterisks have been used to indicate peak locations.

Fig. 4

(a)–(e) Comparison of various measured parameters for MB ($N=8$) and MIgG-IRDye 680RD ($N=5$) cohorts. (f) shows comparison bar graphs of transient versus steady-state propulsion rate in all animals. * indicates significant difference with $p$ $\text{value}<0.009$.