Animals were anesthetized with isofluorane and maintained at 37°C on a warming pad. In order to prevent fur from interfering with the fluorescent signal, animals were clipped 24 h before imaging. At each imaging session, dynamic NIRF imaging was performed immediately before and for up to 30 min after i.d. injection of 10 µl or 50 µl of 645 µM of ICG (Akorn, Inc., Lake Forest, IL) to the base of the mouse or rat tail, respectively, to image changes of lymphatic contractile function in response to dietary salt. Animals were imaged two to three times prior to the start of a HSD to establish baseline levels and every three to four days during a HSD for two weeks using a custom-built NIRF imaging system described elsewhere.7 Briefly, an animal was illuminated with 785 nm light from a laser diode outfitted with a convex lens, diffuser, and 785 nm bandpass filter to create a uniform excitation field. The 830 nm fluorescence was collected through holographic and bandpass filters placed prior to a 28 mm Nikon lens. The ears were also imaged using a macrolens following i.d. injection of 2 µl of 645 µM of ICG into the ear tip to image lymphatic capillary remodeling in response to a HSD. We used the concentration previously used in our animal studies.6,7,9 Animals in the control and experimental groups received the same ICG concentration and volume. The imaging data were analyzed with Matlab (The MathWorks, Inc., Natick, MA) and ImageJ (National Institutes of Health, Bethesda, MD) as described before.7 The same size of fixed regions of interest (ROIs) was defined along the entire fluorescent lymphatic vessels on fluorescence images. The averaged fluorescence intensity within each ROI in each fluorescence image was plotted as a function of distance and imaging time to generate a three-dimensional (3-D) spatio-temporal map. The number of pulses of ICG-laden lymph is an indication of lymphatic contractile activity and termed as contractions. The peak fluorescent intensities due to propagation of the fluorescent lymph along the lymphatic vessels were not influenced by respiration. The number of lymphatic contractions was measured for 5 min at 5 min after i.d. injection and the frequencies were calculated. The frequencies were normalized to the baseline, i.e., the data before any treatment. Fluorescent intensities in the draining inguinal LN (ILN), which can represent the extent of lymph flow, were also measured 10 mins after injection of ICG to mice. A circular ROI was selected over the inguinal region in control and treated mice and the averaged fluorescent intensity was measured. The ROI was the same size enabling comparison. Lymphatic diameters in rat ears after injection of 2 µl of ICG were measured from the images of the ear using ImageJ software, since there is little scatter and lymphatic vessels are located at the surface ( in depth). Values are presented as means the standard error of the mean (SEM). Statistical analysis was performed using SAS version 9.2 (SAS Institute Inc., Cary, NC). The data were analyzed using general linear model and the confidence interval for pairwise comparisons were calculated by Tukey’s studentized range test. The -value for each pairwise comparison was calculated from Fisher’s least significant difference. The significance level was set at .