Fate of gadolinium in inflamed mouse brain: Release and phosphate interaction post-contrast agent administration

Mounting evidence for markedly increased cerebellar Gd retention under neuroinflammatory conditions after repeated linear gadolinium-based contrast agents (GBCA) administration in vivo, makes necessary a discrimination between Gd retained within the GBCA complex and forms dissociated form the complex and to characterize the chemical environment of the released Gd$^{3+}$. We employed electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopy, which enable direct detection of Gd$^{3+}$ release and evaluation of its molecular surroundings in intact cerebellar tissue from inflamed and non-inflamed brain sections exposed to either linear or macrocyclic GBCAs. The experiments were performed on sub-mm brain samples taken after administration of linear gadopentetate and macrocyclic gadobutrol in vivo in a murine multiple sclerosis model and ex vivo in organotypic hippocampal slice cultures under inflammatory conditions. EPR detected {\mu}M 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. Complementary mass spectrometry (MS) and MRI calibration experiments on Gd-spiked homogenized mouse brain tissue were used to assess the relaxation-active fraction of retained Gd evidencing binding to inorganic ligands. 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.