Comprehensive Analysis of the Spatial Distribution of Gadolinium, Iron, Manganese, and Phosphorus in the Brain of Healthy Rats After High-Dose Administrations of Gadodiamide and Gadobutrol
Authors: Hummel Luis , Frenzel Thomas , Boyken Janina , Pietsch Hubertus , Seeliger Erdmann 
Published: December 2023

Objectives
After the administration of gadolinium-based contrast agents (GBCAs), residual gadolinium (Gd) has been detected in a few distinct morphological structures of the central nervous system (CNS). However, a systematic, comprehensive, and quantitative analysis of the spatial Gd distribution in the entire brain is not yet available. The first aim of this study is to provide this analysis in healthy rats after administration of high GBCA doses. The second aim is to assess the spatial distributions and possible Gd colocalizations of endogenous iron (Fe), manganese (Mn), and phosphorus (P). In addition, the presence of Gd in proximity to blood vessels was assessed by immunohistochemistry.

Materials and Methods
Male rats were randomly assigned to 3 groups (n = 3/group): saline (control), gadodiamide (linear GBCA), and gadobutrol (macrocyclic GBCA) with cumulative Gd doses of 14.4 mmol/kg of body mass. Five weeks after the last administration, the brains were collected and cryosectioned. The spatial distributions of Gd, Fe, Mn, and P were analyzed in a total of 130 sections, each covering the brain in 1 of the 3 perpendicular anatomical orientations, using laser ablation coupled with inductively coupled plasma mass spectrometry. Quantitative spatial element maps were generated, and the concentrations of Gd, Fe, and Mn were measured in 31 regions of interest covering various distinct CNS structures. Correlation analyses were performed to test for possible colocalization of Gd, Fe, and Mn. The spatial proximity of Gd and blood vessels was studied using metal-tagged antibodies against von Willebrand factor with laser ablation coupled with inductively coupled plasma mass spectrometry.

Results
After administration of linear gadodiamide, high Gd concentrations were measured in many distinct structures of the gray matter. This involved structures previously reported to retain Gd after linear GBCA, such as the deep cerebellar nuclei or the globus pallidus, but also structures that had not been reported so far including the dorsal subiculum, the retrosplenial cortex, the superior olivary complex, and the inferior colliculus. The analysis in all 3 orientations allowed the localization of Gd in specific subregions and layers of certain structures, such as the hippocampus and the primary somatosensory cortex. After macrocyclic gadobutrol, the Gd tissue concentration was significantly lower than after gadodiamide. Correlation analyses of region of interest concentrations of Gd, Fe, and Mn revealed no significant colocalization of Gd with endogenous Fe or Mn in rats exposed to either GBCA. Immunohistochemistry revealed a colocalization of Gd traces with vascular endothelium in the deep cerebellar nuclei after gadobutrol, whereas the majority of Gd was found outside the vasculature after gadodiamide.

Conclusions
In rats exposed to gadodiamide but not in rats exposed to gadobutrol, high Gd concentrations were measured in various distinct CNS structures, and structures not previously reported were identified to contain Gd, including specific subregions and layers with different cytoarchitecture and function. Knowledge of these distinct spatial patterns may pave the way for tailored functional neurological testing. Signs for the localization of the remaining Gd in the vascular endothelium were prominent for gadobutrol but not gadodiamide. The results also indicate that local transmetalation with endogenous Fe or Mn is unlikely to explain the spatial patterns of Gd deposition in the brain, which argues against a general role of these metals in local transmetalation and release of Gd ions in the CNS.