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Increased Retention of Gadolinium in the Inflamed Brain After Repeated Administration of Gadopentetate Dimeglumine

A Proof-of-Concept Study in Mice Combining ICP-MS and Micro– and Nano–SR-XRF

Wang, Shuangqing MD*,†; Hesse, Bernhard PhD‡,§; Roman, Marco PhD; Stier, Deborah MSc§; Castillo-Michel, Hiram PhD; Cotte, Marine PhD‡,¶; Suuronen, Jussi-Petteri PhD; Lagrange, Adrien MSc§,#; Radbruch, Helena PhD**; Paul, Friedemann MD††; Taupitz, Matthias MD‡‡; Schellenberger, Eyk PhD‡‡; Sack, Ingolf PhD‡‡; Infante-Duarte, Carmen PhD*

doi: 10.1097/RLI.0000000000000571
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Objectives The aim of this study was to determine in vivo if brain inflammation leads to increased gadolinium (Gd) retention in brain tissue after repeated applications of Gd-based contrast agents (GBCAs).

Materials and Methods Experimental autoimmune encephalomyelitis (EAE) was induced in female SJL/J mice (n = 6). Experimental autoimmune encephalomyelitis and healthy control mice (n = 4) received 2.5 mmol/kg Gd-DTPA over 10 days (8 injections, cumulated dose of 20 mmol/kg), starting at day 14 post immunization when EAE mice reached the maximal clinical disability. In a group of mice, T1-weighted 2-dimensional RARE images were acquired before the first GBCA injection and 1 day after the last injection. Mice were killed either 1 day or 10 days after the last Gd application. From each single animal, a brain hemisphere was used for Gd detection using inductively coupled plasma mass spectrometry, whereas the other hemisphere was processed for histology and synchrotron x-ray fluorescence spectroscopy (SR-XRF) analysis.

Results Gadolinium deposition in inflamed brains was mapped by SR-XRF 1 day after the last Gd-DTPA injections, although only mild signal hyperintensity was found on unenhanced T1-weighted images. In addition, using inductively coupled plasma mass spectrometry, we detected and quantified Gd in both healthy and EAE brains up to 10 days after the last injections. However, EAE mouse brains showed higher levels of Gd (mean ± SD, 5.3 ± 1.8 μg/g; range, 4.45–8.03 μg/g) with respect to healthy controls (mean ± SD, 2.4 ± 0.6 μg/g; range, 1.8–3.2 μg/g). By means of micro–SR-XRF, we identified submicrometric Gd hotspots in all investigated samples containing up to 5893 μg Gd/g tissue. Nano–SR-XRF further indicated that Gd small hotspots had an average size of ~160 nm diameter and were located in areas of high inflammatory activity.

Conclusions After repeated administrations of Gd-DTPA, ongoing inflammation may facilitate the retention of Gd in the brain tissue. Thus, neuroinflammation should be considered as a risk factor in the recommendation on use of linear GBCA-enhanced MRI.

From the *Institute for Medical Immunology, CharitéUniversitätsmedizin Berlin, Corporate Member of FreieUniversität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany

Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan, China

European Synchrotron Radiation Facility, Grenoble, France

§Xploraytion GmbH, Berlin, Germany

European Center for the Sustainable Impact of Nanotechnology, EcamRicert Srl, Padua, Italy

Laboratoire d'Archéologie Moléculaire et Structurale, Sorbonne Université, CNRS, Paris, France

#Materials Engineering, Berlin Institute of Technology

**Department of Neuropathology, CharitéUniversitätsmedizin Berlin

††NeuroCure Clinical Research Center and Experimental and Clinical Research Center, CharitéUniversitätsmedizin Berlin and Max Delbrueck Center for Molecular Medicine

‡‡Department of Radiology, CharitéUniversitätsmedizin Berlin, Berlin, Germany.

Received for publication December 14, 2018; and accepted for publication, after revision, March 11, 2019.

Shuangqing Wang and Bernhard Hesse contributed equally to this article.

Conflicts of interest and sources of funding: There are no conflicts of interest to declare. This work was supported by the European Synchrotron Radiation Facility and the German Research Foundation (Deutsche Forschungsgemeinschaft; SFB1340-1/B05, IN156/4-1, and GRK2260 [BIOQIC]).

Supplemental digital contents are available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.investigativeradiology.com).

Correspondence to: Carmen Infante-Duarte, PhD, Experimental Neuroimmunology, Institute for Medical Immunology, Charité–Universitätsmedizin Berlin, Campus Virchow Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: carmen.infante@charite.de.

Online date: April 29, 2019

Gadolinium (Gd)-based contrast agents (GBCAs) are widely used to enhance magnetic resonance imaging (MRI) by increasing the relaxation rate of water protons in tissue areas, in which they penetrated. Gadolinium-based contrast agents have been used over the past 3 decades with excellent safety records, and only rarely have serious adverse reactions been reported.1–3 However, during the last years, a body of reports points to a potential risk of Gd deposition in different tissues after multiple GBCA applications, including brain,4 bones,5 eyes,6 liver, lung, kidney, heart,7 and skin.8

Based on their molecular structure, GBCAs can be categorized into 2 classes, linear and macrocyclic. Compared with the macrocyclic, the open-chain and less stable linear GBCAs have increased Gd deposits in various organs,9–11 particularly in the brain.9,10,12–21 Gadolinium retention is enhanced in rats with partial renal failure22–24 and also in patients with deficient renal function.25 Application of linear GBCA is also associated with the development of nephrogenic systemic fibrosis (NSF) both in patients with advanced renal dysfunction26–30 and in experimental rat models.31,32 These facts led to suspensions or restrictions on linear agents by the European Medicament Agency (EMA) in November 2017 (EMA/625317/2017) and to the publication of new class warnings and recommendations by the US Food and Drug Administration. Particular caution is recommended in case of pregnancy or kidney problems and if patients did already have repeated scans with GBCA.33

In neurology, GBCAs are extensively used to diagnose and monitor diseases, such as multiple sclerosis (MS)34 that are characterized by alterations in the vascular density and/or permeability.35 Multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system (CNS) and is a major cause of neurological disability in young adults. Multiple sclerosis is considered to be an autoimmune disease in which self-reactive immune cells gain access to the CNS leading to myelin destruction and neuronal damage and the subsequent formation of multifocal lesions.36 Invasion of immune cells from peripheral blood into the CNS is facilitated by the disruption of the blood-brain barrier (BBB) function, which represents a hallmark of MS37 and is also characteristic for experimental autoimmune encephalomyelitis (EAE), the prototypical animal model for MS.38 In MS, BBB disruption is detected as parenchymal leakage of GBCA.39 Thus, enhanced MRI is commonly used for diagnostic purposes and to monitor treatment response in patients with MS.14 Consequently, MS patients commonly exposed to multiple lifetime doses within routine clinical care also show Gd deposition in the CNS.40 However, it remains unclear whether neuroinflammation and BBB disruption may affect the risk of Gd retention inside the CNS. To evaluate this, we used a paradigm of repeated applications of a linear Gd-DTPA in the animal model of MS. Gadolinium retention was evaluated by means of inductively coupled plasma mass spectrometry (ICP-MS) and micro– and nano–synchrotron x-ray fluorescence spectroscopy (SR-XRF).

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MATERIALS AND METHODS

Mouse Model of Experimental Autoimmune Encephalomyelitis and Study Design

All procedures were approved by the local Animal Welfare Department of the State Office of Health and Social Affairs Berlin (LAGeSo), in accordance with national and international guidelines to minimize discomfort to animals (86/609/EEC). Experimental autoimmune encephalomyelitis was induced as described previously.41 In brief, 6- to 8-week-old female SJL/J mice (Janvier Labs, France) were immunized subcutaneously with 250-μg proteolipid protein peptide 139 to 151 (purity 95%; Pepceuticals, Leicester, United Kingdom) and 800-μg Mycobacterium tuberculosis H37Ra (Difco, Franklin Lakes, NJ) emulsified in 100-μL complete Freund's adjuvant and 100-μL phosphate-buffered saline. Pertussis toxin (250 ng per mouse; List, Biological Laboratories, Campbell, CA) was injected intraperitoneally on the day of immunization (day 0) and again 2 days later (day 2). After immunization, mice were monitored daily for weight and clinical signs as follows: 0 = no disease; 1 = complete tail paralysis; 2 = hindlimb paresis; 3 = hindlimb plegia; 4 = paraplegia and forelimb weakness; and 5 = moribund or death due to EAE.

A total of 4 healthy mice and 6 EAE mice received repeated GD-DTPA injections as mentioned afterwards and were included in the experiments. T1-weighted MRI before the first injection and 1 day after the last injection were performed in 2 of the experimental animals, which were killed 1 day after the last injection for histology and SR-XRF. The additional 4 healthy control (HC) and 4 EAE mice were killed either 1 day (n = 2) or 10 days (n = 2) after the last injection, and tissues were processed for histology, ICP-MS, and SR-XRF. See the schematic illustration of the study design depicted in Figure 1A.

FIGURE 1

FIGURE 1

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Gadolinium-Based Contrast Agent (Gd-DTPA) Application

Healthy control and EAE mice were exposed for a total of 8 intravenous injections of a linear GBCA at 2.5 mmol/kg (Gd-DTPA; Magnevist, Bayer, Germany), which corresponded to a cumulated dose of 20 mmol/kg. Healthy control and EAE mice were exposed for 4 consecutive days to GBCA, followed by a 2-day pause and 4 additional consecutive daily injections. In EAE mice, GBCA application started on day 14 post immunization (p.i.), when the mice showed a disability peak (Fig. 1B).

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Magnetic Resonance Imaging

In vivo MRI coronal scans in 2 EAE mice were performed before the GD-DTPA applications and 1 day after the last GD-DTPA application, on a 7 T small-animal scanner (Bruker PharmaScan, Ettlingen, Germany), running ParaVision 5.1 software, with a 20-mm RF quadrature volume head coil (RAPID Biomedical GmbH, Rimpar, Germany) using a T1-weighted 2-dimensional RARE sequence (echo time = 11.5 milliseconds, repetition time = 975 milliseconds, number of average = 4, field of view = 25.6 mm, number of slices = 32, number of echoes = 2, slice thickness = 0.5 mm, scan time = 8 minutes and 19.2 seconds). Mice were anaesthetized with 1.5% to 2.0% isoflurane in 30% O2 and 70% N2O administered via face mask, with continuous respiration monitoring respiration using a pressure-sensitive pad placed on the thorax (Small Animal Instruments Inc, Stony Brook, NY). The animals were placed on a bed with circulating heated water to maintain constant body temperature.

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Tissue Processing and Histology

Mice were killed either 1 day or 10 days after the last Gd-DTPA application. Animals were deeply anesthetized with ketamine and transcardially perfused with PBS. Brains were cut sagittally into 2 symmetrical halves. Half of the brain was postfixed in 4% paraformaldehyde overnight at 4°C and then PBS washed, following 30% sucrose in PBS soaking until the tissue sunk to the bottom. Meanwhile, the other half of the brain was stored in liquid nitrogen for elements trace determination. Brains tissue for histology and micro–SR-XRF were embedded in Tissue Tek Optimal cutting temperature compound and frozen very fast in 2-methylbutane cooled with dry ice, then stored in −80°C. Tissue samples were cut in the horizontal plane in a cryostat, into 10-μm thick consecutive cryosections and stored at 4°C. In addition, 3 of the consecutive cryosections from each mouse were directly mounted between 2 ultralene foils (SPEX sample prep) for SR-XRF mapping. Brain frozen sections were stained with hematoxylin and eosin (H&E) to access inflammation, and images were acquired through a Zeiss Axio Observer microscope.

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Inductively Coupled Plasma Mass Spectrometry

First, the brains were dried to constant weight in vacuum at 20°C overnight, using a Concentrator Plus (Eppendorf, Hamburg, Germany) in V-AQ mode. Ten volumes of ultrapure grade concentrated HNO3 (65% vol/vol, Romil) were directly poured into each tube, then samples were placed into a thermostatted block (Falc) at 70°C for 8 hours to achieve complete mineralization of the tissue. After cooling, the digests were transferred into 15-mL tubes, pooled with triplicate rinse of the original tubes using 1 mL of Milli-Q water (18 MΩ cm−1 at 25°C) each, spiked with the internal standards Sc and Rh (Sc for K, P, Na, Ca, and Fe, Rh for Gd), to compensate for possible matrix effects and instrumental drifts, and brought to 10 mL with Milli-Q water (final concentration of Sc and Rh, 10 ng/g). The solutions were directly analyzed for determination of K, P, Na, Ca, Fe, and Gd, and further diluted with ultrapure HNO3 2% vol/vol (plus 10 ng/g of the internal standards) 1:50 for the determination of Ca and Fe or 1:1000 for the determination of K, P, and Na. Multielemental analyses were conducted by ICP-MS using an instrument Agilent 7900 equipped with ASX-500 autosampler (Cetac), glass cyclonic spray chamber thermostatted at 4°C, quartz torch, and Ni cones. The acquisition was performed in no-gas (for Na, Ca, and Gd) or He (4.3 mL/min, for K, P, and Fe) modes. Instrumental parameters were optimized for best sensitivity in the whole mass range, as well as minimum oxides (<2.5%) and double charges (<3%) levels. Quantification was obtained by external calibration with multielemental standards prepared in ultrapure HNO3 2% vol/vol from the certified level mother solutions ICP-MS calibration standards XXI and V from Analytical Technologies, and IV-Stock-26 from Inorganic Ventures. Three repeated acquisitions were performed for all elements within each analysis.

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Micro–Synchrotron X-ray Fluorescence Spectroscopy

Micro–SR-XRF maps were performed using the in-vacuum scanning x-ray spectroscopy setup at ID21 beamline at the European Synchrotron Radiation Facility in Grenoble, France.42 X-rays were generated by 3 undulators with the gaps being optimized for 7.3 keV. A Kohzu fixed exit double-crystal Si (111) monochromator was used in combination with a Ni-coated flat double-mirror rejecting high-energy harmonics. Downstream of the monochromator, the beam was focused down to ~0.6 × 0.8 μm2 (vertical × horizontal) using a fixed-curvature Kirkpatrick-Baez mirror system. The flux was ~5 × 1010 photons/s (~180 mA SR current in multibunch mode). A photodiode collecting the XRF from a thin Ti membrane inserted in the beam path was used to continuously monitor the incoming beam intensity. X-ray fluorescence spectroscopy and scattered radiation were collected with a dispersive energy silicon drift detector with an active area of 80 mm2 (SGX, Sensortech, Buckinghamshire, United Kingdom). Acquisition time per pixel was 100 milliseconds. The pixel size for collecting the XRF maps was set to 35 μm, 10 μm, 3 μm, or 0.5 μm (depending on the region of interest [ROI] size). Scans were performed in continuous mode. X-ray fluorescence spectroscopy normalization, spectral deconvolution, and quantification were done using the software PyMCA.43,44

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Nano–Synchrotron X-ray Fluorescence Spectroscopy

Complementary to the micro–SR-XRF analysis, one tissue section of mouse #3 (M3, EAE mouse killed 10 days after last GBCA application) was analyzed by means of nano-XRF at the ID16B, European Synchrotron Radiation Facility.45 A pink beam with an energy of 17.5 keV with ΔE/E = 1% was focused down to 70 × 65 nm2 (vertical × horizontal) using Kirkpatrick-Baez mirrors with a photon density of ~1.5 × 1011 photons/s. One seven-element silicon drift detector array (Mirion Technologies Inc, San Ramon, CA) was used. The nano-XRF map was recorded with a pixel size of 60 × 60 nm2 and 150 milliseconds of dwell time. In contrast to the setup installed at ID21, ID16B operates in air. Normalization and deconvolution of XRF spectra were performed using the software PyMCA. The given setup only allowed for a semiquantitative analysis (relative concentration between Gd and Fe). To derive absolute Gd concentrations in small subregions within the collected maps, the overall Fe concentration was normalized to the concentration of this region derived in the ID21 micro-XRF analyses. Based on this newly derived average Fe concentration, the detected counts of the Fe Ka XRF line could be attributed to mass fraction. The relative Gd-to-Fe ratio can be calculated from the XRF spectra, and thus the Gd absolute concentration can be estimated as well.

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RESULTS

Repeated Application of GBCA Leads to Slight T1 Signal Hyperintensity at 7 T MRI in EAE Brain and to Robust Gd Retention in Tissue

To assess whether MRI is sensitive enough to detect Gd retention in the brain of EAE mice, we exposed repetitively 2 EAE mice to Gd-DTPA at 2.5 mmol/kg, starting at day 14 p.i., as described in Methods. T1-weighted images were acquired before GBCA administration (baseline) and 1 day after the last application on a 7 T rodent MRI scanner. As shown in Figure 2A, slight hyperintense areas apparently in the choroid plexus of the fourth ventricle were observed at unenhanced T1-weighted MRI 1 day after the last application. Using Image J and taking the Allen Brain Atlas as reference, the signal intensity values of the cerebellar nuclei related to the background signal of the medulla were calculated. Relative signal intensity in the cerebellar nuclei increased slightly from 1.256 (before injections) to 1.361 (after injections).

FIGURE 2

FIGURE 2

The presence of Gd in the brain tissue of one animal was further evaluated with micro–SR-XRF in the post mortem brain. Micro-XRF images show a clear retention of Gd, particularly in the cerebellar areas (Fig. 2, B–D). Thus, Gd retention in the inflamed brain may not be excluded when enhancing signals are barely perceptible at MRI.

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Ongoing Brain Inflammation Leads to Enhanced Amount of Retained Gd in the Brain

The presence of Gd in the brains of HC and EAE mice was further investigated using ICP-MS, which allows detecting and quantifying elements at very low detection limits (ie, Gd, 0.04 μg/g; K, 0.02 mg/g; P, 0.2 mg/g; Na, 0.006 mg/g; Ca, 1 μg/g; and Fe, 3.5 μg/g). Four HC and 4 EAE mice were exposed to 8 repeated Gd-DTPA injections as described in the Methods. To assess short-term washout effects, half of the mice were killed 1 day and the other half 10 days after last injections. Brains were dissected into the 2 hemispheres; one of the hemispheres was then used to quantify Gd by ICP-MS. As reported in Table 1 and Supplementary Figure 1 (Fig. S1, Supplemental Digital Content 1, http://links.lww.com/RLI/A434), significant concentrations of Gd were detected in both HC and EAE mice ranging from 2 to 8 μg/g dry weight (to 0.4–1.9 μg/g wet weight). However, EAE brains showed higher levels of Gd (mean ± SD, 5.3 ± 1.8 μg/g; range, 4.2–8.03 μg/g) as compared with HCs (mean ± SD, 2.4 ± 0.6 μg/g; range, 1.8–3.2 μg/g). Moreover, although the small sample size does not allow a quantitative analysis, the preliminary data points to a mild washout of Gd in both HC (29% reduction) and EAE brains (26% reduction) over time (2.8 vs 2.0 in healthy and 6.1 vs 4.6 in EAE mice for day 1 and day10, respectively). All other major elements determined in the samples (K, P, Na, Ca, and Fe) matched the expected physiological concentrations and seemed to be unaffected by inflammation. Thus, Gd retention in brain tissue is enhanced under inflammatory conditions. Retention seems to be stable even 10 days after the last GBCA application.

TABLE 1

TABLE 1

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Gd Deposits Are Increased During Inflammation and Are Located in Areas of High Inflammatory Activity

Furthermore, we evaluated the impact of brain inflammation and BBB disruption on the short- and middle-term distribution of Gd in the brain tissue using the other brain hemisphere of the mice included in Table 1. Conventional H&E staining was used to identify inflammatory lesions in the EAE brains. The areas showing high inflammatory foci, mainly located perivascularly in the cerebellar region (indicated by a red arrow in the representative Fig. 3A), were then selected for micro–SR-XRF mapping of brain tissue obtained 1 day or 10 days after last GBCA application (Fig. 3B, bottom set of images). Corresponding control brain areas of the HC are shown in Figure 3B (top set of images). Interestingly, micro-XRF images at 35-μm pixel size indicate that although Gd retention was observed in both HC and EAE mice, there is an apparent accumulation of Gd in the inflamed cerebellum of the EAE. No obvious differences were observed in mice killed 1 day or 10 days after last application of GBCA.

FIGURE 3

FIGURE 3

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High-Resolution Micro–SR-XRF Images Indicate That Ongoing Brain Inflammation Leads to Increased Gd Retention

Based on the overview scans (35-μm pixel size), ROIs containing high amounts of Gd were selected to further analyze Gd distribution at the micrometer scale. Accumulation of Gd in small spots was visible at 3-μm step size (Fig. 3, D–E) and even more pronounced at 500-nm pixel size (Fig. 3, F–G) for both HC and EAE animals. Such representative images at high resolution further confirmed the broader accumulation of Gd into the brain tissue of the EAE mice compared with HC. In the fit of the average XRF spectrum, it can be seen that the Gd XRF L3 line is easily distinguished from the contribution of the other elements and of scattering peaks (Fig. 3C).

Interestingly, Figure 3F mapped from an immunized mouse but without clinical signs shows a prominent retention of Gd, especially in the DCN (Fig. 3D).

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Inside the Brain, Gd Accumulates in Submicron Hotspots

As shown previously, Gd deposits were observed in both HC and EAE mice. To investigate whether the 2 groups may differ in Gd hotspot size and content, we next calculated the Gd, P, S, and Fe elemental mass fraction maps in the spots detected in the cerebellar ROI using high-resolution XRF maps at 500-nm pixel size. Representative maps for HC and EAE animals are shown in Figure 4, A and B, respectively. A vertical profile was plotted in HC and EAE brain tissues, passing through the highest Gd value to estimate the size and Gd content of the hotspots. The mass fraction of Gd in HC and EAE brain peaked 2208 ppm and to 4322 ppm, respectively (Fig. 4, C and D). The size of these hotspot regions was smaller than 2 pixels in diameter. Considering that the x-ray spot size in the sample is slightly smaller than 1 μm, the spatial resolution only allows the conclusion that these hotspot regions are equal or smaller than 2 pixels in diameter, with 1 pixel corresponding to 500 nm. Table 2 and Supplementary Figure 2 (Fig. S2, Supplemental Digital Content 2, http://links.lww.com/RLI/A435) illustrate the average mass fractions determined in microgram per gram of tissue (ppm mass fraction) of Gd and the other investigated elements. In addition, Table 2 shows the maximal concentration of Gd found within the hotspots in a particular ROI that ranged between 1140 μg/g and 5893 μg/g. Gadolinium concentrations within the hotspots are similar in healthy and sick animals. Similarly, values obtained 10 days after the last application were also comparable to the values obtained 1 day after the last injection. Thus, although inflammation leads to increased number of Gd hotspots, the amount of Gd within the spots was extremely elevated in both HC and EAE brains, up to 10 days after the last GBCA application.

FIGURE 4

FIGURE 4

TABLE 2

TABLE 2

To further characterize the size of the spots, additional maps were obtained at 60-nm step size (nano-XRF) using brain tissue from EAE mouse #3 (10 days after last Gd injection). As illustrated in Figure 5, Gd accumulates in small hotspots with an average size of ~160 nm diameter, containing approximately 3000 to 5000 ppm of Gd. For the precise calculation of the spot size, the image of a selected region of Figure 5B (left) was segmented (binarized in 2 colors) as shown on the right panel of Figure 5B. This mask was subsequently used to calculate the size of the Gd spots. As shown in Figure 5B, the mask (right panel) corresponds very well with the gray-value image (left panel), indicating the accuracy of the size estimation. The mean size of the Gd pixel area was then computed into a diameter. The size distribution and its cumulative histogram are shown in Figure 5E. Figure 5C shows the spectrum of 2 different regions of the map depicted in Figure 5A. One spectrum was selected based on high Gd signals (A, in red), and the other one is the average spectrum of the full map (B, in black). The overlap of the 2 spectra indicates that the spectrum corresponding to Gd-rich regions (red spectrum) also reveals a higher Ca signal, when compared with the average Ca intensity (black spectrum; Fig. 5C). Thus, Ca levels seem to be increased in Gd-rich areas. This colocalization of Ca and Gd can be also observed in Figure 2D.

FIGURE 5

FIGURE 5

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DISCUSSION

It has been speculated that a disrupted BBB may support Gd deposits in the brain. To prove this, we have investigated in the animal model of MS whether brain inflammation may favor the retention of Gd in the tissue after repetitive administration of a linear GBCA in vivo. For that, we have used quantitative micro– and nano–SR-XRF to localize and quantify Gd deposits, combined with ICP-MS to assess the overall amount of Gd in the mouse brain.

We have demonstrated using ICP-MS that indeed in mice with EAE the concentration of retained Gd seems to be higher than that observed in healthy mice. As visualized by micro–SR-XRF, Gd accumulates in hotspots that contain up to several milligrams of Gd per gram of tissue. The size of those hotspots were further investigated by SR-nano XRF and found to be only ~160 nm in diameter.

Our data confirm that pronounced Gd retention could be observed also in the absence of BBB disruption as previously reported in humans46 and rats.9,12,17 On one hand, inflammation may enhance the passage of Gd by the disrupted BBB but also by a disrupted cerebrospinal fluid–blood barrier, which is indeed altered already during the early phases of EAE as reported by our group earlier.10 On the other hand, it has been demonstrated that inflammation leads to changes in the glycosaminoglycan (GAG) molecules that form the extracellular matrix, allowing the binding of iron oxide nanoparticles.47 Gadolinium released principally from less stable linear chelates of GBCA might be complexed in vivo by GAGs.48 Thus, inflammation-mediated GAG alteration may promote the Gd transchelation and its retention in the tissue.

Moreover, Gd could also bind to other macromolecules12 via the so-called transmetallation reaction, where Gd+3 is bumped out by another endogenous cation (such as Zn+2 or Ca+2). Dissociated free Gd+3 is less stable than chelated Gd in vivo, and these ions are prone to combine with other endogenous anions (such as phosphate, hydroxide, carbonate, or citrate) and deposit in the tissue.49 In line with previous reports pointing to an association of Gd deposits with calcifications in brain tumors4 and skin tissues from patients with NSF,50,51 we show here a colocalization of Gd with Ca. This may support the occurrence of a transchelation reaction as reported in skin samples of NSF patients.50,52 Those new Gd molecules generated by transmetallation processes in vivo may accumulate in the tissue, leading to toxic reactions53 and to histopathological alterations, as mentioned previously for the fibrotic skin or as suggested from in vitro studies.54,55 In addition, GBCAs are extracellular molecules under normal conditions, but the dissociated free Gd+3 that is kicked out by other cations could bind the phosphate acid from the cell wall and get internalized over time.56 Thus, Gd detected by means of micro–SR-XRF could be located extracellularly or intracellularly.

To assess the biological consequences or Gd retention, further studies are currently ongoing, in particular to clarify whether Gd deposits as an intact chelate or in dechelated or transchelated form.57

Gadolinium retention is particularly prominent in the DCN and also in the cerebellar granular layer. To precisely identify these regions, the Allen Brain Atlas (http://atlas.brain-map.org/) was used. This distribution confirmed previous observations in patients with different pathologies58–61 and also in experimental rat models.62 This particular distribution also points to potential chemical interactions of Gd with metals present in the cerebellum and, in particular, in the dentate nucleus—areas known as major repository of metals that are especially enriched in iron, zinc, and copper.63

Further, our data indicate that, after repetitive applications of GBCA, Gd could be detected in the brain tissue 1 day after the last injection of GBCA, although at 7 T unenhanced MRI, only slight hyperintense areas are visible within the fourth ventricle, most probably in the choroid plexus. We could not completely exclude that small parts of DCN were also hyperintense. Although, as pointed by Robert et al,64 and due to the limitations of the 7 T scanner mentioned later and the low resolution of the images, it is difficult to correctly allocate the diffuse hyperintensities, particularly to the small murine DCN. It should been considered that the relaxivity of Gd-DTPA decreases with field strength.65–67 Thus, the use of a high-field scanner may represent a limitation for revealing Gd retention in vivo. In this line, DCN hyperintensity was clearly visible at 1.5 T21 and 4.7 T MRI in healthy12 or nephrectomized rats24 injected with a cumulative dose of 12 mmol/kg linear GBCA and also in the previous reports of this research group on Gd deposition using a 2.35 T scanner.20,62 This is an important consideration because MRI is actually used to assess the deposition risk in patients with different pathologies that had received repetitive GBCA applications.68

Moreover, and although the small sample size does not allow a quantitative analysis, our preliminary data points to a mild washout of Gd over a short period (1 day compared with 10 days after last GBCA injection). These preliminary data are in line with previous studies using linear GBCA in rats.18,69 The observed washout effect of approximately 30% is also in agreement with the effect reported by Robert et al in the cerebellum of rats treated with a 12 mmol/kg cumulative dose of linear GBCA. In this study, an initial washout was observed 1 month after the last application; afterwards, however, the retained Gd remained constant for a period of 1 year.12 Thus, to assess the effect of inflammation on long-term Gd retention, an interval of at least 5 months between last injection of Gd and sampling should be considered as indicated recently by Robert et al.64

Of note, as proof of principle, we have applied in this study a linear GBCA known to be more prone to deposition when compared with the macrocyclic chelates.62,70 In this line, according to EMA PRAC decision 11/2017, macrocyclic agents should be clearly preferred in patients with intended repeated GBCA administration.71 It would be interesting as well to conduct a similar study comparing different linear and macrocyclic GBCA.

Moreover, ICP-MS quantifications demonstrated that the amount of retained Gd per gram tissue is increased during inflammation (means ± SD: EAE, 5.3 ± 1.8 μg/g; HC, 2.4 ± 0.6 μg/g). These values are in line with the reported Gd deposits observed in dentate nucleus of healthy rats 7 days9 or 8 weeks17 after the last GBCA application (cumulative dose of 50 mmol/kg).

However, high-resolution XRF revealed that Gd is in fact not at all homogeneously distributed, but rather concentrated in submicron hotspots that contain ~3 orders higher Gd concentration than the average background level at a millimeter scale. Further, nano-XRF suggested that these Gd containing accumulations are approximately 160 nm in diameter. Accumulation of Gd in spots containing up to 0.8 μg/g was also observed in the dentate nucleus of a patient who received at least one application of linear GBCA72 and in rats that received a cumulative dose of 50 mmol/kg Gd.17 Interestingly, using transmission electron microscopy, Lohrke et al17 identified deposits of Gd (apparently intraendothelial) principally in the dentate nucleus with a size of approximately 200 to 500 nm.

Thus, by means of SR-XRF, which enables submicron spatial resolution, high sensitivity, and elemental specify at the same time, we confirm previous reports on the magnitude and location of Gd deposits and indicate that ongoing inflammation facilitate Gd retention into the brain tissue.

A comprehensive risk assessment on the use of linear GBCAs should therefore consider a potentially increased risk of Gd retention for patients with chronic neuroinflammatory disorders.

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Limitations of the Study

The principal limitation of our study is the small number of animals included in our experiments that does not allows a quantitative analysis of the results. Unfortunately, the investigation of additional mice would be prohibitively time-consuming and unrealizable within our granted beam-time at the European Synchrotron Radiation Facility. Thus, our study has to be considered as a pilot investigation that, by means of exhaustive synchrotron-based elemental analysis, shows that ongoing CNS inflammation may favor Gd retention into the brain. In addition, the use of a 7 T scanner to monitor Gd deposits in vivo may limit the conclusiveness of our results, because it is well documented that the relaxivity of Gd-DTPA decreases with field strength.64–67

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ACKNOWLEDGMENTS

The authors thank Natascha Asselborn for the expert technical assistance. Shuangqing Wang acknowledges financial support from the China Scholarship Council.

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Keywords:

experimental autoimmune encephalomyelitis; gadolinium-based contrast agent; gadolinium deposition; inductively coupled plasma mass spectrometry; multiple sclerosis; neuroinflammation; synchrotron x-ray fluorescence spectroscopy

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