Histology and Gadolinium Distribution in the Rodent Brain After the Administration of Cumulative High Doses of Linear and Macrocyclic Gadolinium-Based Contrast Agents

Abstract

Objectives: Retrospective studies in patients with primary brain tumors or other central nervous system pathologies as well as postmortem studies have suggested that gadolinium (Gd) deposition occurs in the dentate nucleus (DN) and globus pallidus (GP) after multiple administrations of primarily linear Gd-based contrast agents (GBCAs). However, this deposition has not been associated with any adverse effects or histopathological alterations. The aim of this preclinical study was to systematically examine differences between linear and macrocyclic GBCAs in their potential to induce changes in brain and skin histology including Gd distribution in high spatial resolution.

Materials and methods: Fifty male Wistar-Han rats were randomly allocated into control (saline, n = 10 rats) and 4 GBCA groups (linear GBCAs: gadodiamide and gadopentetate dimeglumine, macrocyclic GBCAs: gadobutrol and gadoteridol; n = 10 rats per group). The animals received 20 daily intravenous injections at a dose of 2.5 mmol Gd/kg body weight. Eight weeks after the last GBCA administration, the animals were killed, and the brain and skin samples were histopathologically assessed (hematoxylin and eosin; cresyl violet [Nissl]) and by immunohistochemistry. The Gd concentration in the skin, bone, brain, and skeletal muscle samples were analyzed using inductively coupled plasma mass spectroscopy (ICP-MS, n = 4). The spatial Gd distribution in the brain and skin samples was analyzed in cryosections using laser ablation coupled with ICP-MS (LA-ICP-MS, n = 3). For the ultra-high resolution of Gd distribution, brain sections of rats injected with gadodiamide or saline (n = 1) were assessed by scanning electron microscopy coupled to energy dispersive x-ray spectroscopy and transmission electron microscopy, respectively.

Results: No histological changes were observed in the brain. In contrast, 4 of 10 animals in the gadodiamide group but none of the animals in other groups showed macroscopic and histological nephrogenic systemic fibrosis-like skin lesions. The Gd concentrations observed in the skin/brain samples (in nanomole Gd per gram of tissue) for each agent were as follows: gadodiamide: 1472 ± 115/11.1 ± 5.1, gadopentetate dimeglumine: 80.8 ± 6.2/13.1 ± 7.3, gadobutrol: 1.1 ± 0.5/0.7 ± 0.4, and gadoteridol: 1.7 ± 0.8/0.5 ± 0.2. The average detected residual Gd concentration in the brain was approximately 15-fold higher for linear than for macrocyclic GBCAs. The highest amounts of Gd found in brain corresponded to less than 0.0002% of the injected dose per gram of tissue. Using LA-ICP-MS, high Gd concentrations in the deep cerebellar nuclei and in the granular layer of the cerebellar cortex were detected only for linear gadodiamide and gadopentetate dimeglumine but not for gadoteridol or gadobutrol. The energy dispersive x-ray spectroscopy analysis revealed Gd-containing spots in the skin of animals administered gadodiamide and gadopentetate dimeglumine. Transmission electron microscopy revealed several Gd-containing spots in the region of the dentate nuclei in the brain of 1 animal injected with gadodiamide.

Conclusions: After repeated high dosing, nephrogenic systemic fibrosis-like macroscopic and histopathological lesions of the skin were observed only in some of the gadodiamide-treated animals. No histopathological findings were detected in the rodent brain. The administration of linear GBCAs was associated with significantly higher Gd concentrations in the brain and skin compared with macrocyclic GBCA administration. The results of LA-ICP-MS demonstrated local accumulation of Gd within the deep cerebellar nuclei and the granular layer only after the administration of linear agents. In summary, the detected low Gd concentrations in the skin and brain were well correlated with the higher kinetic stability of macrocyclic GBCA.

FIGURE 1

Schematic illustration of the study design. Healthy male Wistar-Han rats were randomly allocated into 1 control and 4 GBCA groups n = 10 (saline, gadodiamide, gadopentetate dimeglumine, gadobutrol, gadoteridol). Each animal received 20 daily intravenous injections at a dose of 2.5 mmol Gd/kg body weight. Eight weeks after the last application, the animals were killed, the histology and Gd distribution (LA ICP-MS and SEM-EDX and TEM-EDX) were investigated, and ICP-MS analyses were conducted (* Samples were prepared from FFPE sections from the histology group).

FIGURE 2

Despite the repeated high-dose administration of either linear or macrocyclic GBCAs (gadodiamide, gadopentetate dimeglumine, gadoteridol, gadobutrol, and control group), no histopathological changes in the brain could be detected at microscopic examination of slides stained with (A and B) H&E or (C) cresyl violet (Nissl stain). Immunohistochemistry using (D) GFAP to assess the astrocyte number and (E) morphology, and a microglial marker (Iba1) did not reveal any differences between animals administered linear or macrocyclic GBCAs compared with vehicle controls.

FIGURE 3

Transmission electron microscopy tissue localization of Gd-containing spots in the region of the lateral (dentate) cerebellar nuclei in the brain after the repeated high-dose application of gadodiamide (20 × 2.5 mmol Gd/kg body weight). TEM evaluation showed several positive signals: (A) 1 single focus (original magnification, ×18,900) and (B) several electron-dense signals occurring as multiple roundish nodules with variable diameter (original magnification, ×18,800). The location indicates intracellular deposition within endothelial cell of blood vessels; 1 signal appeared to be adjacent to an endothelial cell on the adluminal side. The EDX analysis showed, compared with the control area (C), Gd-positive signals (D, arrows). No positive signals could be detected in neurons or in the neuropil.

FIGURE 4

A, Macroscopic skin appearance and (B) an overview of the skin tissue and (C and D) enlarged H&E skin sections of animals administered saline, gadodiamide, gadopentetate dimeglumine, gadoteridol, and gadobutrol. Representative H&E-stained FFPE sections of animals, each injected with 20 injections at a dose of 2.5 mmol Gd/kg body weight at 8 weeks after the last injection. All animals in the gadodiamide group showed fibrosis and mononuclear cell infiltration and an increase in dermal cellularity compared with the saline group and all other investigated groups.

FIGURE 5

Scanning electron microscopy coupled to energy dispersive x-ray (SEM-EDX) spectroscopy for the detection of elemental composition, including Gd presence in the skin after the application of 20 injections (2.5 mmol Gd/kg body weight) of the linear agents gadodiamide and gadopentetate dimeglumine. A, An overview of the skin tissue (original magnification, ×50), (B) an enlarged skin section (original magnification, ×1000), and (C) the presence of Gd-containing domains in the connective tissue of the subcutis. The gadodiamide-injected rat (upper row) showed approximately 3 to 4 times more Gd domains in the skin than the gadopentetate dimeglumine-administered rat (lower row).

FIGURE 6

A, Eight weeks after the last injection (20 × 2.5 mmol Gd/kg body weight), high Gd concentrations were measured for the linear agents gadodiamide and gadopentetate dimeglumine in the skin and bone. B, The average concentration (mean ± SD) of residual Gd in the brain was approximately 100-fold lower compared with the skin in gadodiamide-injected rats and approximately 15-fold higher for linear than for macrocyclic GBCAs. Low Gd concentrations were measurable when both classes of GBCAs were used.

FIGURE 7

A, Seven different brain sections (brain I-VII) were investigated. The Gd concentrations in the 7 dissected brain samples showed no significant preferential anatomical location of Gd, such as the GP (brain II) or lateral (dentate) cerebellar nucleus (brain VI). B, Interestingly, the highest Gd concentration in the brain was detected in the olfactory bulb (brain I) decreasing in the posterior direction in animals administered gadopentetate dimeglumine, whereas the concentration of Gd was similar in all 7 investigated brain regions of animals administered with gadodiamide. All values are given as mean ± SD.

FIGURE 8

Tissue distribution after repeated high-dose administration of saline, gadodiamide, gadopentetate dimeglumine, gadobutrol, and gadoteridol measured using laser ablation ICP-MS. The highest Gd signals were detected in the (A) skin, (B) GP region, and (C) deep cerebellar nuclei region after the administration of the linear GBCAs gadodiamide and gadopentetate dimeglumine. In addition to the specific localization in the deep cerebellar nuclei, substantial Gd signals could also be visualized in other brain regions, such as the granular layer of the cerebellar cortex and in the facial nuclei in the pons. No local accumulation of Gd was observed within the deep cerebellar nuclei and the granular layer after the administration of macrocyclic gadobutrol and gadoteridol.