Evaluation of functional status of lymph channels will be crucial for developing an understanding of lymphatic biology, evaluating the role of the lymphatics in health and disease, as well as diagnosing patients with disorders of the lymphatic system. Once a quantifiable imaging approach for assessing lymphatic function is developed and made available for clinical study, the opportunity to assess new therapeutic strategies and pharmacologic agents may spur the development of new treatments for patients suffering from lymphatic disorders. Fortunately for lymphatic imaging, NIR technology may be simple and economic to employ. As detailed below, NIR fluorescence imaging represents the only imaging modality to date sensitive enough for the direct imaging of propulsive lymph flow through lymphatic vessels deep within tissues.
The reason for the sensitivity arises from the penetration of NIR light, optimally in the wavelength range of 750–850 nm, and the ability to stably and repeatedly re-activate NIR-excitable dyes. To illustrate the sensitivity, compare a radiotracer (whether a gamma- or positron-emitter for the nuclear imaging techniques described above) and a typical NIR fluorophore. Whether produced in a cyclotron or generator, a radiotracer relaxes only once, emitting a single photon event before becoming spent. On the other hand, once a stable NIR fluorophore relaxes in tissues generating a fluorescent photon, it can be subsequently reactivated by propagating excitation light, enabling it to relax multiple times. Since the lifetime, or the mean time that the fluorophore resides in its activated state, is on the order of a nanosecond, theoretically there can be as many as one billion fluorescent photons emitted from a single fluorescent molecule per second. Realistically, low quantum efficiencies for fluorescent photon generation, high tissue attenuation of propagating excitation and emission photons, and other instrumentation factors prevent such enormous fluorescent photon output. Nonetheless, in a study of a dual-labeled NIR optical and gamma-emitter, Houston and co-workers83
found that the image quality of NIR optical imaging surpasses gamma scintigraphy in small animals, even though NIR imaging required only 800 ms of integration time while gamma scintigraphy required 15 min.
It is the high sensitivity of NIR optical imaging that allows image integration times to range from 50 ms to 800 ms, enabling rapid pharmacokinetic analysis of dye uptake75,84
from images as well as imaging of lymph trafficking. While pulsatile lymph flow has been quantified using intravital microscopy techniques in the mesentery of animals, there have been no previous reports of quantifiable lymphatic flow from non-invasive imaging. More recently, the ability to optically image propulsive lymph flow in swine,76
has been demonstrated. The common feature in these studies is the use of an image-intensifier which is sensitive in the NIR wavelength range, and a frame-transfer, 16-bit CCD camera which enables rapid and sensitive data acquisition with a low noise floor. The noise floor of fluorescence optical image acquisition arises because of autofluorescence as well as excitation light leakage through the rejection filters, which enable selective passage of fluorescent light to the image intensifier. For NIR imaging, the autofluorescence is virtually zero, but as one moves into the red excitation wavelengths using red exciting dyes, natural porphyrins and chlorophylls (from food) create a background that can obscure the signal from the fluorophore. In addition, the use of holographic rejection filters, which block 10 orders of magnitude of collimated, monochromatic excitation light, can also reduce the noise floor. Unfortunately, few commercially available instruments for small-animal imaging employ holographic filters. In addition, the success of holographic filters depends upon collimating the incident light and removing all possible stray light components. Not widely recognized is the fact that the use of broadband excitation sources, such as light-emitting diodes and lamps, reduce the effectiveness of holographic and interference filters, since these light sources are seldom monochromatic enough for effective blockage of excitation light. Finally, the availability of NIR-excitable dyes has increased in the past few years. IC-Green is approved in humans for assessing cardiac and hepatic function and for ophthalmologic studies and can be excited serendipitously at 780 nm, with a significant Stoke’s shift enabling fluorescent measurement at 830 nm and greater. Small Stoke’s shifts less than 30–40 nm can cause significant problems as they impede the ability to efficiently reject excitation light and collect the fluorescent signal. Unfortunately, IC-Green does not have a functional group and cannot be conjugated with a targeting peptide, protein, polysaccharide, or other molecule, but it can be used as a soluble dye which noncovalently associates with albumin to create a lymphotropic agent. A few commercially available NIR-excitable dyes with functional groups for creating molecular imaging agents are now available with sufficient Stoke’s shifts: IRDye800 CW (Licor, Inc., Lincoln NE), AlexaFluor 790 (Invitrogen, Carlsbad, CA), and Cy7 and Cy 7.5 (Amersham/GE Health-Science, Piscataway, NJ). Usually, as a general rule of thumb, it is important to excite NIR organic dyes with as narrow or as monochromatic source as possible in order to maximally reflect excitation light, and to use bandpass filters well away from the excitation line in order to collect as much emission as possible for enhanced sensitivity. With these caveats, as shown below, the use of NIR optical imaging of lymphatic function offers a new, economical, and significant research and diagnostic tool to the lymphatic research community. In the following, we briefly review the progress in dynamic imaging of lymphatic function in mice, swine, and humans as well as highlight the opportunities and challenges specific to each.
Lymph Imaging in Mice
Preclinical investigation of potential pro- and anti-lymphangiogenic agents are typically performed in rodent models, thereby necessitating the imaging of lymphatic function in these small-animal models. While their small size may facilitate imaging, the temporal and spatial resolution provides challenges for functional lymphatic imaging in small animals. As comprehensively reviewed by Kwon and Sevick-Muraca,84
lymphatic vessels in mice and rats have been imaged using (1) radiography to visualize mercury uptake in the two major lymph vessels in the mouse tail,85
and (2) micro-MR lymphangiography with Gd-laden dendrimers86–88
and (3) fluorescence video-microscopy to see the circumferential “honeycomb” lymphatic structure in the mouse tail.89–91
Lymphatic function has also been inferred from lymphoscintigraphy and microangiography by Slavin et al.
who reported that myocutaneous flap transfer restores lymphatic function. Several groups have demonstrated lymphatic transport of QDs to map out drainage patterns.72,93,94
Wunderbaldinger et al.95
used an enzyme-sensing optical probe with visible wavelength excited fluorophore, Cy5.5, for the detection of lymph nodes. To date there have been few, if any, studies that demonstrated contractile lymphatic motion that characterizes lymphatic function in larger animals. As shown in , Kwon and Sevick-Muraca84
recently demonstrated the ability to image IC-Green trafficking from the lymph plexus, through lymph vessels and lymphangions, to ischial nodes in the tail and to axillary nodes in mice. The intensity profile shows that the peak fluorescence occurs at an average of every 6.56 ± 1.14 s and the lymph flow velocity ranged from 0.28 mm/s to –1.35 mm/s. Lymph flow velocities from the propelled IC-Green packet in the major lymph vessels in the mouse tail ranged from 1.33 mm/s to –3.88 mm/s. While pulsatile lymph flow was detected in the deep lymph vessels, lymph propulsion was not visualized in the superficial lymphatic network of the tail. When imaging lymph flow to the axillary nodes, propulsive lymph flow was also detected.
FIGURE 11 NIR optical imaging enables detection of pulsatile lymph flow in mice. (A) Fluorescence image; (B) intensity profile as a function of time in a specific ROI along a lymph vessel after intradermal injection of 2 μL of 1.29 μM IC-Green in (more ...)
The ability to image contractile motion in mice provides new opportunities to (1) assess lymphatic function in transgenic mice models to better understand the role that specific gene expression has on the lymphatic function and to (2) investigate pharmacologic agents that stimulate the formation of functional lymphatics as well as stimulate the contractile apparatus of existing lymphatics.
Lymph Imaging in Swine
Yorkshire swine is the commonly preferred animal model for preclinical lymph-imaging investigations, as swine skin and lymph drainage pattern are similar to those of human skin.70,96
Similar to human dermis, swine dermis is characterized by open-ended lymphatic capillaries that take up particles and lymphotropic agents under a pressure gradient. Passively diffusing agents, such as soluble NIR fluorophores, are taken up in the initial lymphatics and then drain into afferent lymphatic channels that propel lymph to the lymph nodes. Recently, we reported results of quantitative imaging of afferent lymphatic function in swine.76
In addition to mapping lymphatic vasculature after intradermal injections of 100–200 μL of 32-μM IC-Green near mammary teats or on hindlimb, we conducted quantitative analysis of (1) the lymph flow velocities of transiting “packets” of IC-Green, and (2) their frequency of propulsion caused by lymphangion contractions. The “packets” of dye transited along lymph vessels 2–16 cm in length at velocities of 2.3–7.5 mm/s and frequencies of 0.5–3.3 pulses per minute. illustrates pulsatile lymph flow in a swine lymphatic. The figure represents images of a bolus of IC-Green (circle) transiting along a swine’s abdominal lymph channel from the injection site around the mammary teat to the inguinal lymph node at times 0, 2.6, and 5.2 s. The pulsatile lymph flow was also analyzed quantitatively to compute the period between the “packets” of dye transiting along a lymph channel. represents a plot of intensity profile at an ROI on an abdominal lymph channel as a function of imaging time after injection of 200 μL of 32-μM IC-Green around the mammary teats of swine. The peaks indicate a “bolus” of fluid transiting from the injection site to the inguinal lymph node on an average at every 72 s. Since manual lymphatic drainage (MLD) is an important component of complete decongestive therapy for lymphedema patients,97
we employed the noninvasive lymph-imaging technique to measure the efficacy of MLD in a healthy anesthetized swine. For instance, in one case, the NIR optical lymph-imaging technique enabled detection of change in lymph flow velocity in a swine abdominal lymphatic from 6.2 (±1.9) mm/s to 8.0 (±2.5) mm/s and change in the mean period between pulses from 48 (±37) to 69 (±40) s in response to massage. Noninvasive determination of pulsatile lymph flow and transport velocities via imaging can provide a tool to evaluate lymphatic function in response to existing therapies and may help assess function of newly regenerated lymph channels in response to novel therapeutic interventions, including VEGF-C or gene therapies.
Fluorescent images depict a bolus of IC-Green (circle) transiting along a swine’s abdominal lymph channel at (left) t = 0, (middle) t = 2.6, and (right) t = 5.6 s. The lymphatic channel drained to the swine’s inguinal region.
FIGURE 13 Plot of mean NIR fluorescent intensity profile of a ROI selected on a swine’s abdominal lymphatic vessel as a function of imaging time. Consistent peaks that appear on average every 72 s are observed 11 min after intradermal injection of 200 μL (more ...)
Lymph Imaging in Humans
The preclinical imaging studies in swine demonstrated the feasibility of performing deep tissue lymph imaging with microgram quantities of IC-Green. To translate the technique to the clinic we take advantage of the established safety record of IC-Green given in mg quantities. Sevick-Muraca et al.81
recently reported the feasibility of detecting sentinel lymph nodes with microgram administration of IC-Green in breast cancer patients. depicts a white-light and fluorescent image overlay of a 46-year-old African American breast cancer patient intradermally injected with 100 μL of 20 μg IC-Green in each quadrant of the peri-areolar region on her right breast. The injection sites were covered with an opaque plastic to prevent oversaturation of the imaging camera. Arrows indicate lymphatics pooling the dye to axilla. represents the pulsatile lymph flow in the breast lymphatics. The peaks in the plot of intensity profile at an ROI (oval in panel A) as a function of duration of image acquisition indicate that the lymph channel conducts “packets” of IC-Green from the injection site to the axilla on an average every 22.9 ± 7.8 s at a velocity of 2.2 (±0.6) mm/s. A fluorescent “hot-spot” in the axilla was resected and found to be a sentinel lymph node. IC-Green is now being used for human lymph imaging in an ongoing feasibility study to compare lymph flow of healthy subjects and lymphedema patients as next described.
FIGURE 14 NIR optical lymph imaging detects pulsatile flow in humans. (A) White-light and NIR fluorescent image overlay of a subject with breast cancer. Intradermal injection is performed with 100 μL of 20 μg IC-Green in the peri-areolar region (more ...)
Before aberrant lymphatic function can be identified through NIR imaging, baseline lymphatic function was first evaluated in healthy volunteers. shows white-light and fluorescent overlay images of a foot of a 46-year-old Caucasian female in a supine position during image acquisition. The subject was injected with 100 μL of 25 μg of IC-Green each in the first and second interdigital spaces (arrows) in the dorsum of the foot. The images are a combination of the white-light photograph of the foot overlain with corresponding fluorescent images at 0, 2, and 15 s. The circle identifies a “packet” of dye transiting at a velocity of 5.0 mm/s between lymphangions along the lymphatic channel, while the arrowheads identify the localized collection of fluorescent dye that demarks lymphangions. The period between two consecutive pulses observed during image acquisition was 164 s. is another white-light and fluorescent image overlay that illustrates the lymphatic vasculature of a human hand. The image represents a hand of a 22-year-old Caucasian male in a supine position at the time of imaging. The subject was injected with 100 μL of 25 μg of IC-Green in the four interdigital spaces. The velocity of a single packet transiting in one of the lymph vessels draining an injection site next to the thumb was 16.6 mm/s. The ability to safely and noninvasively map the lymphatic network, locate lymphangions, and quantify lymph function in healthy volunteers can be employed for a comparative assessment of lymph drainage in persons with lymphedema. In addition, the potential to detect pulsatile lymph flow noninvasively using microdose concentrations of IC-Green augurs well for molecular imaging of lymphatics.
FIGURE 15 White-light overlay with corresponding fluorescent images of lymphatic map of foot of a 46-year-old female who was injected with 100 μL of 25 μg of IC-Green in the first and second interdigital spaces (arrows) in the dorsum of the foot. (more ...)
FIGURE 16 A white-light and fluorescent overlay image of lymphatics in a human hand. A 22-year-old male was injected with 100 μL of 25 μg of IC-Green in the four interdigital spaces on the right hand. Five discrete lymphatics appear to drain the (more ...)