Imaging lymphatic function and inflammation response through hypoxia via endogenous biomarker

Abstract

SIGNIFICANCE: The lymphatic system, crucial for immune function and fluid balance, is difficult to study due to its nearly invisible channels and passive movements. Despite its importance, real-time, noninvasive lymphatic imaging is limited and often relies on exogenous agents. A largely overlooked feature of the lymphatic system is its natural hypoxia, arising because lymphatic vessels and nodes are distant from oxygen-rich blood and serve as a waste reservoir and from amplifying factors such as inflammation. This hypoxia has been linked to lymphangiogenesis and cancer metastasis. AIM: We introduce a method for real-time macroscopic imaging of lymphatic function and response to inflammation, through the intrinsic hypoxia transients that occur in lymphatic structures. APPROACH: The naturally hypoxic environment of the lymphatic system was imaged in vivo in mice through the delayed fluorescence (DF) of metabolized endogenous protoporphyrin IX (PpIX), induced by 5-aminolevulinic acid. PpIX localizes to inflamed regions; when inflammation is cleared by lymphatics, the low oxygen conditions cause the DF signal to be amplified. DF imaging and lymphatic kinetics were characterized in wound, inflammation, and pancreatic tumor models. High-intensity popliteal and sentinel nodes, wounds, and tumors were excised for immunohistochemistry (IHC) and hematoxylin and eosin analysis. RESULTS: Lymphatic pumping frequency changed with increasing wound severity, and hypoxia appeared in sentinel nodes near tumors. Cyclical pumping occurred at edema sites and in wound and tumor-adjacent nodes. Uninjured anesthetized mice showed little contrast, whereas awake mice exhibited hypoxia localized to lymph nodes. Microscopy and IHC confirmed PpIX and hypoxia presence in nodes, tumors, and wounds, localized to macrophages and T cells. CONCLUSIONS: Unlike injection-based regional lymph node mapping, DF hypoxia imaging appears to provide a natural whole-body contrast mechanism, highlighting its potential for visualizing lymphatic function and associated hypoxia dynamics. This original documentation of lymphatic hypoxia has potential applications in surgical guidance, tracking of metastatic tumors, and immune response tracking.