shows an example of a fluorescent image frame from Media 1
in the ventrolateral view of a nude mouse displaying the lymphatic trafficking of ICG from the base of the tail, where ICG was intradermally injected, to the ILN, and subsequently to the ALN. The lymphatic vessels on the lower left ventral side and contralateral regions were also visualized. In order to determine lymphatic contractile function, a series of 66 ROIs were selected along the entire fluorescent collecting lymphatic vessel as shown in .
Fig. 1 NIR fluorescence image (A, Media 1) of the lymphatics in a nude mouse and quantification of lymphatic contractile function after selection of 66 ROIs along the lymphatic vessel (B). A 3-D plot of fluorescent intensity as a function of time and distance (more ...)
shows a generated, three-dimensional (3-D) spatial-temporal map of fluorescent intensity as a function of time and length, showing coordinated propagation of 19 contraction waves. The frequency of propagating contractions averaged 6.24/min. We also observed lower frequency of contractions (1.33/min) in inguinal afferent lymphatic vessels and asynchronous propulsion of ICG between afferent (pre-nodal) and efferent (post-nodal) collecting lymphatic vessels. The different lymph pump activities between the afferent and efferent lymphatic vessels in the LN were observed previously in isolated lymphatic vessels [19
To quantify the apparent propagation velocity of contraction waves, two ROIs were selected over the lymphatic vessel. As an example, shows fluorescent intensity as a function of time for 10 s for ROI 1 and ROI 66 (i.e., the first and the last ROI as shown in , respectively). The transit time was determined by indentifying the multiple fluorescent intensity peaks in both ROIs and was found to average 6.78 ± 2 s during 19 propagating contractions. Since the distance between two ROIs was 1.8304 cm, the averaged apparent propagation velocity was calculated to be 2.96 ± 1.02 mm/s. Unlike the contractile frequency, there was no apparent difference in propagation velocities between afferent and efferent lymphatic vessels. Propulsive lymph velocities and contraction frequencies ranged from 2.11 ± 0.74 to 8.13 ± 2.96 mm/s and 1.35 to 6.32/min, respectively, in 6 nude mice.
In addition to the difference in lymphatic function between inguinal afferent and efferent lymphatic vessels, we also observed differences in lymphatic contractility along the lymphatic vessel.
As shown in
, an analysis of fluorescent intensity versus time and length along the entire efferent lymphatic vessel shows (i) four contraction waves starting propagations approximately at ROI 30 and (ii) the different patterns of lymphatic propulsion, which are likely mediated by the different lymphangions in which the ROI’s were selected. In order to elucidate the different modes of lymphatic contraction and relaxation, four ROIs (i.e. ROIs 5, 18, 30, and 66) were selected and fluorescent intensities within each ROI were plotted as a function of time as shown in . Fluorescent intensity profiles in ROI 5 and ROI 18 illustrate no propulsive flow and a slightly pulsatile flow, respectively.
Fig. 2 A 3-D plot of fluorescent intensity as a function of time and distance (B) after selecting 66 ROIs along the lymphatic vessel (A) shows propagating contraction waves in an orthograde direction (arrow). (C) Plot of fluorescent intensity profiles for four (more ...)
also shows the rapid dynamic decrease of fluorescent intensities due to the lymphatic contraction and the subsequent slow increase in the intensities due to the lymphatic relaxation and lymph “filling” within ROI 30. In addition, the passage of a packet of ICG-laden lymph through ROI 66 to the ALN is represented by intensity peaks as shown in , reflecting the contractile assembly upstream emptying (contraction) and the subsequent downstream filling (relaxation). We observed the propagation velocity of 8.13 ± 2.96 mm/s between ROIs 30 and 66.
Intravital fluorescence imaging was also conducted after a midline incision of the abdominal skin from the inguinal to the axillary regions to confirm that ICG was picked up by the initial lymphatics and drained to LNs through the contractile, collecting lymphatic vessels.
shows an overlay of white light and fluorescent images that depict lymphatic trafficking of ICG along the branched internodal collecting lymphatic vessels. shows magnified images in the circle shown in . As shown in , the collecting lymphatic vessels run parallel to the blood vessels. After intravital fluorescence imaging, 10 μL of EBD was intradermally injected at the base of the tail, but several millimeters away from the previous ICG injection site for direct visualization of the lymphatics. Similar to ICG, EBD also binds to tissue macromolecules and is selectively taken up by the initial lymphatic vessels [21
]. shows the lymphatic drainage of EBD along the lymphatic vessels, which is the same lymphatic flow pathway depicted by ICG fluorescence in . As demonstrated in , non-invasive whole-body fluorescence imaging data correlated with intravital fluorescence and EBD images, demonstrating that both ICG and EBD were taken up by the initial lymphatics and drained from the ILN, through the contractile collecting lymphatic vessels, to the ALN.
Fig. 3 Overlay of white light and fluorescent images acquired by in vivo (A) and intravital (B) NIR fluorescent imaging after injection of ICG and a color image (C) after injection of EBD to a nude mouse. Asterisk denotes ILN. Arrows in A-C denote collecting (more ...)
More importantly, propulsive flow acquired from in vivo fluorescence imaging occurs along the lymphatic vessels.
is an example from a C57BL/6 mouse, the background common to most transgenic mouse models. shows similar lymphatic architecture and function as described in nude mice. We observed the propagation velocity of 8.06 ± 0.74 mm/s in the inguinal efferent collecting lymphatic vessel. Propulsive lymph velocities and contraction frequencies ranged from 2.33 ± 0.51 to 8.06 ± 0.74 mm/s and 1.66 to 4.34/min, respectively, in 6 C57BL/6 mice.
Fig. 4 Overlay (A) of white light and fluorescent images in a C57BL/6 mouse and a 3-D plot of the fluorescent intensity as a function of time and distance (B) showing the propagation of the fluorescent packet of lymph represented by the peak in fluorescence (more ...)
We also imaged PROX1+/− mice using dynamic NIR fluorescent imaging following i.d. injection of ICG. It has been observed that most Prox1+/− adult mice were obese compared with their wild-type counterparts [3
shows abnormal lymphatic drainage patterns from the injection site on the base of the tail. Diffused dye patterns as well as tortuous and ruptured lymphatic vessels were visualized as shown in . This phenomenon was also reported using an invasive method with injection of EBD [3
]. However, lymphatic contractile function in Prox1+/− mice has not been investigated.
Overlay of white light and fluorescent images in Prox1+/− mice depicting abnormal lymphatic drainage patterns.
From our dynamic fluorescence imaging, we observed abnormal lymphatic contractile function as shown in Media 2
shows another example of overlay of white light and fluorescent images () from Media 2
depicting a tortuous fluorescent collecting lymphatic vessel indicated by a double arrow. A 3-D spatio-temporal map of fluorescent intensities in an inguinal efferent collecting lymphatic vessel indicates orthograde (arrow) and retrograde (broken arrow) propagation of contraction waves as shown in . To quantify propagation velocity, four ROIs were selected. As shown in , if we define Δt1 and Δt2 as the time delays between the fluorescence maxima or the onset of contraction of upstream and downstream regions of interest (i.e. Δt1 = tpeak
(ROI 30) - tpeak
(ROI 1) and Δt2 = tpeak
(ROI 91) - tpeak
(ROI 40)), we find negative values of Δt1 over the entire period of imaging, indicating that the contractions in ROI 30 preceded those in upstream ROI 1. The averaged Δt1 is −1.74 ± 0.16 s and the propagation velocity in the retrograde direction was found to be 6.56 ± 0.6 mm/s. We also observed positive values of Δt2, indicating propagation of contraction waves in the orthograde direction.
Fig. 6 Overlay (A) of white light and fluorescent images (B, Media 2) and quantification of abnormal lymphatic contractile function of Prox1+/− mice. Double arrows denote a tortuous collecting lymphatic vessel. (C) A 3-D plot of the fluorescent intensity (more ...)
The averaged Δt2 is 2.81 ± 0.09 s and the propagation velocity in the orthograde direction was found to be 5.82 ± 0.2 mm/s. Persistent retrograde lymph flow, taken together with tortuous and mispatterned lymphatic vessels, may contribute to adult-onset obesity caused by Prox1 haploinsufficiency. Using isolated and cannulated lymphatics, investigators have demonstrated contractile propagation in the orthograde and retrograde directions [22
]. Dixon et al
] showed from intravital microscopic imaging that retrograde flow occurred in rat mesenteric lymphatics ranging from 2 to 18% of the time during imaging procedures. We observed the asymmetric contractility at junctions of branched lymphatic vessels and retrograde propagation of contractions in normal mice using ICG fluorescence (not shown) similar to that reported using invasive methods by others [23