EPR-based Assessment of Gadolinium-based Contrast Agent Retention and Gadolinium Transchelation in Inflamed Murine Brain

OBJECTIVES: In view of the mounting evidence for markedly increased cerebellar Gd retention under neuroinflammatory conditions after repeated linear GBCA administration in vivo , we aimed to discriminate between Gd retained within the GBCA complex and forms dissociated from the complex within the CNS and to characterize the chemical environment of the released Gd³⁺. For this purpose, we used electron paramagnetic resonance (EPR) spectroscopy, which enables direct detection of Gd³⁺ release and evaluation of its molecular surroundings in intact cerebellar tissue from inflamed and noninflamed brain sections exposed to either linear or macrocyclic GBCAs. MATERIALS AND METHODS: We performed EPR and electron-nuclear double resonance (ENDOR) experiments on sub-mm brain samples taken after administration of linear gadopentetate-dimeglumine and macrocyclic gadobutrol in vivo in a murine multiple sclerosis model and ex vivo in organotypic hippocampal slice cultures under inflammatory conditions. Complementary mass spectrometry (MS) and MRI calibration experiments on Gd-spiked homogenized mouse brain tissue were used to identify potential Gd bindings and assess the relaxation-active fraction of retained Gd. RESULTS: EPR detected µM range Gd levels in intact cerebellar biopsies and slices and distinguished between complex-bound and released Gd following linear GBCA administration in vivo . In biopsies, we detected by ENDOR phosphorus-containing molecules in the microenvironment of released Gd. Binding to inorganic ligands was evidenced using MS and MRI calibration experiments in homogenized mouse brain tissue. CONCLUSIONS: EPR and ENDOR proved to be sensitive methods for detecting Gd³⁺ release and characterizing retained Gd species within intact brain tissue. Our findings demonstrate inflammation-promoted Gd retention, underscore the importance of integrating in vivo and ex vivo analyses to unravel mechanisms of long-term Gd retention, and suggest that conventional MRI may underestimate the true extent of Gd accumulation, especially under neuroinflammatory conditions.