Different Impact of Gadopentetate and Gadobutrol on Inflammation-Promoted Retention and Toxicity of Gadolinium Within the Mouse Brain

Abstract

Objectives: Using a murine model of multiple sclerosis, we previously showed that repeated administration of gadopentetate dimeglumine led to retention of gadolinium (Gd) within cerebellar structures and that this process was enhanced with inflammation. This study aimed to compare the kinetics and retention profiles of Gd in inflamed and healthy brains after application of the macrocyclic Gd-based contrast agent (GBCA) gadobutrol or the linear GBCA gadopentetate. Moreover, potential Gd-induced neurotoxicity was investigated in living hippocampal slices ex vivo.

Materials and methods: Mice at peak of experimental autoimmune encephalomyelitis (EAE; n = 29) and healthy control mice (HC; n = 24) were exposed to a cumulative dose of 20 mmol/kg bodyweight of either gadopentetate dimeglumine or gadobutrol (8 injections of 2.5 mmol/kg over 10 days). Magnetic resonance imaging (7 T) was performed at baseline as well as at day 1, 10, and 40 post final injection (pfi) of GBCAs. Mice were sacrificed after magnetic resonance imaging and brain and blood Gd content was assessed by laser ablation-inductively coupled plasma (ICP)-mass spectrometry (MS) and ICP-MS, respectively. In addition, using chronic organotypic hippocampal slice cultures, Gd-induced neurotoxicity was addressed in living brain tissue ex vivo, both under control or inflammatory (tumor necrosis factor α [TNF-α] at 50 ng/μL) conditions.

Results: Neuroinflammation promoted a significant decrease in T1 relaxation times after multiple injections of both GBCAs as shown by quantitative T1 mapping of EAE brains compared with HC. This corresponded to higher Gd retention within the EAE brains at 1, 10, and 40 days pfi as determined by laser ablation-ICP-MS. In inflamed cerebellum, in particular in the deep cerebellar nuclei (CN), elevated Gd retention was observed until day 40 after last gadopentetate application (CN: EAE vs HC, 55.06 ± 0.16 μM vs 30.44 ± 4.43 μM). In contrast, gadobutrol application led to a rather diffuse Gd content in the inflamed brains, which strongly diminished until day 40 (CN: EAE vs HC, 0.38 ± 0.08 μM vs 0.17 ± 0.03 μM). The analysis of cytotoxic effects of both GBCAs using living brain tissue revealed an elevated cell death rate after incubation with gadopentetate but not gadobutrol at 50 mM. The cytotoxic effect due to gadopentetate increased in the presence of the inflammatory mediator TNF-α (with vs without TNF-α, 3.15% ± 1.18% vs 2.17% ± 1.14%; P = 0.0345).

Conclusions: In the EAE model, neuroinflammation promoted increased Gd retention in the brain for both GBCAs. Whereas in the inflamed brains, efficient clearance of macrocyclic gadobutrol during the investigated time period was observed, the Gd retention after application of linear gadopentetate persisted over the entire observational period. Gadopentetate but not gadubutrol appeared to be neurotoxic in an ex vivo paradigm of neuronal inflammation.

FIGURE 1

Animal study design. A, Schematic illustration of the in vivo setup. A total of 24 HC and 29 EAE mice received 8 intravenous injections of either gadopentetate or gadobutrol at 2.5 mmol/kg BW (cumulative dose: 20 mmol/kg BW, 2-day pause after 4 consecutive applications). Mice underwent a baseline MRI before the first injection and further scans at day 1, 10, and 40 pfi of GBCAs. In EAE mice, baseline MRI started on day 12 to 13 postimmunization (peak of disease). After the MRI scans, 4 to 5 animals per group were sacrificed; brains were processed for LA-ICP-MS, and blood was collected for ICP-MS (n = 2/group). B, Clinical course of EAE animals based on the EAE score (mean ± SD). Red: gadopentetate-treated animals (n = 15), green: gadobutrol-treated animals (n = 14).

FIGURE 2

Schematic illustration of the ex vivo study design. Organotypic hippocampal slices were incubated for 12 days after initial preparation. On day 13 treatment with TNF-α at 50 ng/mL was started in half of the slices (total incubation time: 96 hours). On day 15, treatment with either gadopentetate or gadobutrol was initiated (total incubation time: 48 hours), whereas simultaneous incubation with TNF-α at 50 ng/mL was continued. Incubation with NMDA for 4 hours was performed in positive control slices only on day 16. On day 17, the chronic cultures were terminated, followed by live staining with PI and further fixation in PFA and sucrose. Cell viability was assessed using fluorescence microscopy; IMC was conducted to visualize Gd tissue content.

FIGURE 3

7 T MRI relaxometry in vivo. A, Representative longitudinal MRI scans (T1 map RARE-VTR sequence) of animals sacrificed 40 days pfi of GBCAs. Relaxometry revealed decreasing T1 relaxation times within the CN (red arrow) toward day 40 pfi of gadopentetate in EAE and HC animals. Gadobutrol-treated animals did not show prominent qualitative T1 shortening within the CN. B, T1 relaxation time change (%) differences between EAE and HC animals within the C, DN, and CN for both tested GBCAs. Mann-Whitney tests were computed for EAE versus HC after gadopentetate or gadobutrol treatment and the Bonferroni correction was applied for multiple testing over 3 consecutive time points. Data are displayed as mean ± SD.

FIGURE 4

Analysis of Gd distribution using LA-ICP-MS. A, On the left, H&E bright field image of cerebellar slice containing the CN corresponding to LA-ICP-MS tissue layer at ×2 magnification; CNs are additionally displayed at ×10 magnification. On the right, Allen brain atlas correlation image of CN formation. Nuclei from lateral to medial: DN, interposed nucleus (IP), vestibulocerebellar nucleus (VeCB), fastigial nucleus (FN). Scale bars: 1 mm. B, LA-ICP-MS images showing Gd distribution in cerebellar slices. Red areas display high and blue areas low Gd content (μM). Scale bars: 1 mm. C, Gd concentrations measured within the whole C, DN, or CN in μM of brain volume. Because of the low sample size, merely descriptive statistics are shown (mean ± SD).

FIGURE 5

Analysis of elemental colocalization using LA-ICP-MS. A, Laser ablation images of cerebellar Zn distribution. Red areas display high and blue areas low Zn content (μM). The EAE animals in both GBCA groups showed qualitatively higher Zn levels compared with HC. Scale bars: 1 mm. B, Demonstration of Zn levels within the whole C, the DN, and CN (μM). The EAE animals consistently presented higher elemental Zn levels than HC animals irrespective of the applied GBCA with a tendency to decrease toward day 40 pfi of GBCAs.

FIGURE 6

Toxicity of GBCAs on organotypic hippocampal slices. A, Representative fluorescence images of the DG; all nuclei in blue (DAPI), dead nuclei in red (PI). The DGs of slices coincubated with/without TNF-α (50 ng/mL) are displayed for all experimental conditions. The PI-positive cells appear in NMDA-treated (±TNF-α) and gadopentetate-treated slices at 50 mM (±TNF-α). Scale bars: 200 μm. B, Cell death rate (%) was elevated in NMDA (positive control)-treated slices and slices treated with 50 mM gadopentetate. Tumor necrosis factor α enhanced cell death rate significantly. Data were evaluated using Mann-Whitney tests (mean ± SD). C, IMC images of 10-μm-thick slices after gadopentetate or gadobutrol treatment at 10 mM and negative control. The DGs were surrounded by manually drawn ROIs (white), and thresholds were adjusted for optimal visualization. Scale bars: 200 μm.