Vasculocis

All mice underwent weekly micro-computed tomography (µ-CT) scans of both hindlimbs for repetitive volume assessments. Smallanimal single-photon emission computed tomography (SPECT)/CT was performed as in an earlier described model [19] . In brief, mice were scanned on a Siemens INVEON multimodality preclinical scanner (Siemens Preclinical Solutions, Knoxville, TN, USA). The projection was set to 1200 ms and the transaxial field of view was 44 mm. Before every imaging session, mice were anesthetized with a mixture of 1.5-2.0% isoflurane and 100% oxygen and placed in prone position on a heated SPECT/CT animal bed.
Two mice per group (six in total) underwent Technetium 99 m human serum albumin ( 99 Tc-HSA) lymphoscintigraphy at weeks 1 and 6 to assess the lymphatic clearance. Mice were injected subcutaneously with a bolus injection of 0.02 mL 99m Tc-HSA between each hindlimb's second and third toe. Doses were approximately 14.9 ± 1.9 MBq for left hindlimb and 15.1 ± 1.5 MBq for right hindlimb (Vasculocis, CIS Bio International, Paris, France) administered using a 0.5 mL 30-gauge syringe (Covidien, Medtronic Minneapolis, USA). The tracer uptake was expected to have reached steady state after 45 min [21] (link). The SPECT/CT was performed after 45 min and 4 h after injection of 99m Tc-HSA and moved around freely between imaging sessions.

CT and single-photon emission computed tomography (SPECT)-lymphoscintigraphy was performed following a previously described model [13] with minor modifications. Briefly, the scans were performed on a Siemens INVEON multimodality pre-clinical scanner (Siemens pre-clinical solutions, Knoxville, TN, US). The minor modifications included the projection which was set to 1200 ms (950 ms previously) and the transaxial field of view which was 44 mm (38 mm previously). Through each imaging session, the animals were anesthetized with a mixture of 1.5-2% isoflurane and 100% oxygen and placed front feet first in a prone position on a heated SPECT/CT animal bed (38 mm wide).
The animals received an injection of 0.02 ml 99m Tc-HSA subcutaneously between the second and third toe to measure the lymphatic removal rate. The mean radioactive activity was approximately 14.9 ± 1.9 MBq for the left hindlimb and 15.1 ± 1.5 for the right MBq (Vasculocis, CIS Bio International) and administered using a 0.5 ml 30 G insulin syringe (Covidien) under general anesthesia with a mixture of 1.5-2% isoflurane and 100% oxygen.
After 45 min the tracer was assumed to have reached a steady flow with a constant removal rate [18] . Mice underwent SPECT-lymphoscintigraphy at 45 min and 4 h after injection of 99m Tc-HSA and moved around freely between image sessions.