[⁸⁹Zr]Zr-DFO-EPO Imaging of Ischemia-Induced Cellular Stress

Hypoxia-inducible signaling drives the expression of cytoprotective genes, including genes for erythropoietin and erythropoietin receptor (EPOr), positioning EPOr targeting as a potential biomarker of cellular stress responses. In this study, we evaluate ischemic stroke tissue in vivo using ⁸⁹Zr-labeled recombinant human erythropoietin. Methods: Recombinant human erythropoietin was conjugated with desferrioxamine and radiolabeled with ⁸⁹Zr ([⁸⁹Zr]Zr-DFO-EPO), and its affinity for dimeric EPOr was confirmed in vitro. Longitudinal PET and MRI were performed daily for up to 72 h after stroke in 2 ischemic stroke models: the pertussis toxin-induced stroke in mice and transient middle cerebral artery occlusion in rats. Radiotracer accumulation and distribution in the stroke region were validated by autoradiography and hematoxylin and eosin histology. Specificity to erythropoietin receptors was evaluated by competitive displacement with excess unlabeled erythropoietin. Results: [⁸⁹Zr]Zr-DFO-EPO showed high radiochemical purity and accumulated in ischemic regions in both stroke models. Distinct spatial uptake patterns inside the stroke region were observed between models, with border zone-predominant accumulation in rats with transient middle cerebral artery occlusion and more homogeneous uptake in mice with pertussis toxin-induced stroke, reflecting the known differences in ischemia induction and vascular pathophysiology between the models. In mice, radiotracer uptake spatially correlated with hypoxia-inducible factor 1α and erythropoietin expression. In rats, tracer uptake was verified in the histologically delimited stroke region. Dose-dependent and specific EPOr binding was confirmed in rats ex vivo as well as in human ischemic tissue. In human ischemic stroke samples, radiotracer binding followed histologic morphology, with higher uptake in structurally preserved periinfarct regions and lower uptake in necrotic infarct cores. These findings support [⁸⁹Zr]Zr-DFO-EPO PET as a proof-of-concept approach for assessment of ischemia-associated EPOr-related stress signaling. Conclusion: Across 2 distinct ischemic stroke models, [⁸⁹Zr]Zr-DFO-EPO revealed model-dependent uptake patterns, with periinfarct enrichment confined to focal ischemia characterized by preserved tissue architecture. Specific and displaceable binding in human ischemic stroke tissue with comparable histomorphology underscores the translational relevance of EPOr imaging as a biomarker of hypoxia-associated cellular stress.