Nearly all dietary lipids are transported from the intestine to venous circulation through the lymphatic system, yet the mechanisms that regulate this process remain unclear. Elucidating the mechanisms involved in the functional response of lymphatics to changes in lipid load would provide valuable insight into recent implications of lymphatic dysfunction in lipid related diseases. Therefore, we sought to develop an in situ imaging system to quantify and correlate lymphatic function as it relates to lipid transport. The imaging platform provides the capability of dual-channel imaging of both high-speed bright-field video and fluorescence simultaneously. Utilizing post-acquisition image processing algorithms, we can quantify correlations between vessel pump function, lymph flow, and lipid concentration of mesenteric lymphatic vessels in situ. All image analysis is automated with customized LabVIEW virtual instruments; local flow is measured through lymphocyte velocity tracking, vessel contraction through measurements of the vessel wall displacement, and lipid uptake through fluorescence intensity tracking of an orally administered fluorescently labelled fatty acid analogue, BODIPY FL C16. This system will prove to be an invaluable tool for scientists studying intestinal lymphatic function in health and disease, and those investigating strategies for targeting the lymphatics with orally delivered drugs to avoid first pass metabolism.
KEYWORDS: Lymphatic system, Luminescence, Near infrared, Tissues, Imaging systems, In vivo imaging, Video, Signal to noise ratio, Scattering, Visualization
Near-infrared imaging of lymphatic drainage of injected indocyanine green (ICG) has emerged as a new technology for clinical imaging of lymphatic architecture and quantification of vessel function, yet the imaging capabilities of this approach have yet to be quantitatively characterized. We seek to quantify its capabilities as a diagnostic tool for lymphatic disease. Imaging is performed in a tissue phantom for sensitivity analysis and in hairless rats for in vivo testing. To demonstrate the efficacy of this imaging approach to quantifying immediate functional changes in lymphatics, we investigate the effects of a topically applied nitric oxide (NO) donor glyceryl trinitrate ointment. Premixing ICG with albumin induces greater fluorescence intensity, with the ideal concentration being 150 μg / mL ICG and 60 g / L albumin. ICG fluorescence can be detected at a concentration of 150 μg / mL as deep as 6 mm with our system, but spatial resolution deteriorates below 3 mm, skewing measurements of vessel geometry. NO treatment slows lymphatic transport, which is reflected in increased transport time, reduced packet frequency, reduced packet velocity, and reduced effective contraction length. NIR imaging may be an alternative to invasive procedures measuring lymphatic function in vivo in real time.
Background - Near-infrared (NIR) imaging of lymphatic drainage of injected indocyanine green (ICG) has emerged as a
new technology for clinical imaging of lymphatic architecture and quantification of vessel function, offering better
spatial and temporal resolution than competing imaging modalities. While NIR lymphatic imaging has begun to be
reported in the literature, the technology is still in its infancy and its imaging capabilities have yet to be quantitatively
characterized. The objective of this study, therefore, was to characterize the parameters of NIR lymphatic imaging to
quantify its capabilities as a diagnostic tool for evaluating lymphatic disease.
Methods - An NIR imaging system was developed using a laser diode for excitation, ICG as a fluorescent agent, and a
CCD camera to detect emission. A tissue phantom with mock lymphatic vessels of known depths and diameters was
used as an alternative to in vivo lymphatic vessels due to the greater degree of control with the phantom.
Results and Conclusions - When dissolved in an albumin physiological salt solution (APSS) to mimic interstitial fluid,
ICG experiences shifts in the excitation/emission wavelengths such that it is maximally excited at 805nm and produces
peak fluorescence at 840nm. Premixing ICG with albumin induces greater fluorescence intensity, with the ideal
concentration being: 900μM (60g/L) albumin and 193.5μM (150μg/mL) ICG. ICG fluorescence can be detected as deep as 6mm, but spatial resolution deteriorates severely below 3mm, thus skewing vessel geometry measurements. ICG packet travel, a common measure of lymphatic transport, can be detected as deep as 5mm.
All dietary lipids are transported to venous circulation through the lymphatic system, yet the underlying mechanisms that
regulate this process remain unclear. Understanding how the lymphatics functionally respond to changes in lipid load is
important in the diagnosis and treatment of lipid and lymphatic related diseases such as obesity, hypercholesterolemia,
and lymphedema. Therefore, we sought to develop an in situ imaging system to quantify and correlate lymphatic
function as it relates to lipid transport. A custom-built optical set-up provides us with the capability of dual-channel
imaging of both high-speed bright-field video and fluorescence simultaneously. This is achieved by dividing the light
path into two optical bands. Utilizing high-speed and back-illuminated CCD cameras and post-acquisition image
processing algorithms, we have the potential quantify correlations between vessel contraction, lymph flow and lipid
concentration of mesenteric lymphatic vessels in situ. Local flow velocity is measured through lymphocyte tracking,
vessel contraction through measurements of the vessel walls and lipid uptake through fluorescence intensity tracking of a
fluorescent long chain fatty acid analogue, Bodipy FL C16. This system will prove to be an invaluable tool for both
scientists studying lymphatic function in health and disease, and those investigating strategies for targeting the lymphatic
system with orally delivered drugs.
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