*Purpose: Clinical assessments for inflammation and acute rejection (AR) require regular, invasive biopsies followed by staining protocols that provide static images of tissue. Despite such shortcomings, histopathology remains the gold standard for management of immunosuppression in Vascularized Composite Allotransplantation (VCA) . Thus, there is an unmet need for noninvasive, in vivo, real-time, sequential monitoring of inflammation in VCA. Because of their central role in innate and adaptive immune response, macrophages (MΦ) are an attractive target for noninvasive monitoring of inflammation. The recent development in Janjic lab of MΦ-targeted perfluorocarbon nanoemulsions (PFC-NE) enables in vivo, sequential, noninvasive imaging and tracking of MΦ using macrophage-specific near-infrared fluorescent (NIRF) probes. MMPSense680 on the other hand is a small molecule that is selectively cleaved by matrix-metalloproteinases, enzymes secreted by activated MΦ during inflammation. The aim of this study is to develop a in vivo non-invasive, real time imaging strategy for qualitative and quantitative assessment of the dynamics and kinetics of MΦ activation and migration into tissue sites of inflammation.

*Methods: Complete Freund’s adjuvant (CFA) was used to cause a lasting immune response and induce localized inflammation in rat hind limb. CFA injury simulates acute rejection as it induces significant, acute, sterile, inflammation at the site of injection that persists for 10 days. MMPSense680 was co-injected with CFA into one hind limb (to identify local macrophage activation), while NE was systemically injected (to allow the NIRF labeling of recipient MΦ migrating to the site of inflammation). Sequential clinical photography and NIRF imaging was performed in all animals (at 700nm for MMPSense680 and 800nm for NE) and signal intensity from the CFA limb was compared with the contralateral control limb (injected with PBS). Animals were followed for 15 days.

*Results: MMPSense signal peaked between 1- and 3-days post-injection. PFC-NE signal peaked between 2- and 6-days post-injection. The signals co-localized to the site of CFA injection, confirming both the migration and subsequent activation of systemic MΦ at the site of inflammation. The localized inflammation resolved at 15 days. The peak inflammatory signal and subsequent resolution was confirmed on clinical photography (e.g. erythema, edema) and correlated well with histopathologic and immunofluorescence findings (CD68 positivity and co-localization of NE inside macrophages along with MMP680 signal).

*Conclusions: Immune rejection presents a substantial barrier to successful patient outcomes in VCA. Presented multimodal non-invasive strategy allows real-time, reliable imaging for acute inflammation and holds promise in AR surveillance.

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