In vivo visualization and quantification of collecting lymphatic vessel contractility using near-infrared imaging

Abstract

Techniques to image lymphatic vessel function in either animal models or in the clinic are limited. In particular, imaging methods that can provide robust outcome measures for collecting lymphatic vessel function are sorely needed. In this study, we aimed to develop a method to visualize and quantify collecting lymphatic vessel function in mice, and to establish an in vivo system for evaluation of contractile agonists and antagonists using near-infrared fluorescence imaging. The flank collecting lymphatic vessel in mice was exposed using a surgical technique and a near-infrared tracer was infused into the inguinal lymph node. Collecting lymphatic vessel contractility and valve function could be easily visualized after the infusion. A diameter tracking method was established and the diameter of the vessel was found to closely correlate to near-infrared fluorescence signal. Phasic contractility measures of frequency and amplitude were established using an automated algorithm. The methods were validated by tracking the vessel response to topical application of a contractile agonist, prostaglandin F2α, and by demonstrating the potential of the technique for non-invasive evaluation of modifiers of lymphatic function. These new methods will enable high-resolution imaging and quantification of collecting lymphatic vessel function in animal models and may have future clinical applications.

Figure 1. Visualization of a contractile CLV in mice after inguinal lymph node infusion of P20D680.

(a) Schematic of setup for inguinal lymph node infusion. (b) Prox1-GFP image of inguinal lymph node and efferent CLV. (c) NIR image after lymph node infusion with efferent lymphatic vessel perfused with P20D680. (d) Prox1-GFP signal of stages of contraction cycle of CLV at 63× magnification. (e) NIR signal of stages of contraction cycle at 63×.

Figure 2. Diameter tracking of CLVs.

(a) Signal profile of line bisecting a Prox1-GFP CLV. (b) Signal profile of line bisecting a P20D680 perfused CLV. (c) ROI analysis to determine appropriate threshold for CLV diameter tracking using NIR signals. (d) Contractility plot demonstrating results from diameter tracking using NIR signals of a CLV for a 3-min movie. (e) Corresponding contractility plot demonstrating fluorescent signal from region of interest over vessel.

Figure 3. Automated assessments of frequency and amplitude from contractility plots of diameter or fluorescence data.

(a) Visual output of Matlab algorithm using diameter tracking data to determine peaks and troughs (red circles), instantaneous mean (blue line in left plot) and instantaneous amplitude (blue line in right plot). Shaded regions represent data that are excluded from the quantification but are necessary for the algorithm to calibrate. (b) Visual output of Matlab algorithm using NIR signal data. (c) Quantification of frequency in contractions per min. (d) Quantification of amplitude expressed as percent of instantaneous mean. n = 15 mice, ***represents P < 0.001 (two-tailed Student’s t-test). Data are mean ± SD.

Figure 4. Representative NIR signal contractility plots of CLV response to topical treatment of PGF2α.

Eight-min movies were acquired with administration of 40 μL of either 0.1% DMSO vehicle control or 1 μmol/L, 10 μmol/L or 60 μmol/L PGF2α at t = 2 min. (a) Response to 0.1% DMSO. (b) Response to 1 μmol/L PGF2α. (c) Response to 10 μmol/L PGF2α. (d) Response to 60 μmol/L PGF2α.

Figure 5. Quantifications from NIR signal contractility plots of CLV response to topical treatment of PGF2α.

Quantifications from contractility plots from the post treatment period of t = 3 to 6 min were normalized to the baseline data from t = 0 to 2 min and expressed as a % of baseline. (a) Frequency of contractions. (b) Percent amplitude of contractions. (c) Pumping score (frequency x % amplitude). (d) Vessel tone. Number of vessels analyzed: n = 14 DMSO, n = 12 1 μmol/L PGF2α, n = 14 10 μmol/L PGF2α, n = 15 60 μmol/L PGF2α. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA with Dunnett’s multiple comparison test).

Figure 6. Non-invasive imaging of CLV contractility response to skin treatment of NO donor.

(a) Schematic indicating experimental design and a representative picture from non-invasive imaging of CLVs. Each mouse served as its own control with the NO donor (Rectogesic) treatment applied to the left leg and then the control (Linola fett) treatment applied to the right leg. Contractility was assessed in two vessels per leg (yellow ROIs). (b) Representative contractility plots from pre- and post-control treatment conditions. (c) Representative contractility plots from pre- and post-NO donor treatment conditions. The Y-axis scale is adjusted in each case such that the mean fluorescent intensity values fall in the center of the axis, in order to better visualize differences in percent amplitude. Quantifications from contractility plots from the post-treatment period were normalized to the pre-treatment data and expressed as a % of baseline. Raw values can be found in Table 1. (d) Frequency of contractions (e) Percent amplitude of contractions. (f) Pumping score (frequency x % amplitude). n = 6 C57BL/6J-Tyr^c-J albino mice were analyzed. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student’s t-test).