Longitudinal, Non-invasive Live Near-Infrared Fluorescence Imaging of Acute Vascularized Composite Allograft Rejection Validated by Ex Vivo 19F Magnetic Resonance Imaging and Histopathology.

Acute rejection remains a major cause of graft attrition in vascularized composite allotransplantation (VCA). Clinical assessment and superficial biopsy may fail to detect early or spatially heterogeneous inflammatory infiltration, particularly within deeper graft compartments. Because macrophages are among the earliest mediators of alloimmune activation, they represent an attractive target for noninvasive immune surveillance of skin-bearing grafts. We evaluated longitudinal near-infrared fluorescence (NIRF) imaging of macrophage infiltration using a perfluoro-15-crown-5 ether nanoemulsion (PCE-NE) labeled with a near-infrared fluorophore in a rat hindlimb transplantation model and a radial forearm flap model in non-human primates (NHPs). Single- and dual-dose administration strategies enabled serial visualization of macrophage recruitment and improved detection of evolving inflammatory responses during acute rejection. To capture the spatial heterogeneity of rejection, a multi-sampled region-of-interest analysis framework was developed for quantitative mapping of NIRF signal across donor and recipient tissues in rats and throughout transplanted flap regions in NHPs. To validate in vivo imaging findings, grafts were analyzed ex vivo using fluorine-19 (19F) magnetic resonance imaging, histopathology, and CD68 immunofluorescence. NIRF signal identified rejecting graft regions and demonstrated spatial patterns of inflammatory infiltration that corresponded to compartment-specific 19F MRI signal localization in ex vivo imaging. Imaging findings corresponded with Banff rejection grade and CD68-positive macrophage accumulation across Grade I-IV rejection. Importantly, increased NIRF and 19F MRI signals were detected during early Grade I-II rejection, when clinical manifestations remained limited. These findings establish a translational multimodal imaging strategy for longitudinal, noninvasive monitoring of acute VCA rejection and demonstrate concordance between in vivo NIRF imaging, ex vivo 19F MRI, histopathology, and immunofluorescence across rodent and non-human primate models.