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.
Highlights
Direct measurements of vessel diameter[9,10,11]
After optimization of this procedure, we found that a bolus infusion over 15 s of 500 nL of 10 μmol/L P20D680 lymphatic tracer into the lymph node led to consistent and reliable perfusion of the efferent CLV with no evident leakage from the vessel (Fig. 1c, Supplementary Movie 1)
We have demonstrated the suitability of this method to visualize valve function and tonic and phasic changes in lymphatic contractility
Summary
Direct measurements of vessel diameter[9,10,11]. In vivo, the frequency of contractions has been quantified using non-invasive imaging in several previous studies using either manual counting or semi-automated measures[8,12,13,14]. We hypothesized that NIR fluorescent imaging could be used to estimate both the amplitude (or strength) and frequency of spontaneous lymphatic contractions Such a system could allow non-invasive quantification of lymphatic contractility after intradermal injection of near-infrared tracers with potential translation to the clinic. The methods were successfully demonstrated non-invasively by measuring the effect of topical skin administration of a nitric oxide (NO) donor on the underlying collecting lymphatic vessel contractility. These results reveal that CLV contractile function and the response of these vessels to contractile modifiers can be imaged and quantified in mice using NIR fluorescent imaging
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