Gadolinium-based contrast agents (GBCAs) are frequently used in various magnetic resonance imaging (MRI) examinations and are generally considered to have an excellent safety profile.1 Based on the chemical structure, GBCAs can be divided into 2 classes—linear and macrocyclic agents. Both classes comprise ionic or nonionic compounds.2 The different GBCAs exhibit different thermodynamic and kinetic complex stabilities in vivo,3–5 imposing a higher risk of release of Gd3+ at physiological conditions upon linear GBCAs than upon macrocyclic agents.6
Recent publications have reported increased signal intensities on unenhanced T1-weighted (T1w) images in the dentate nucleus and globus pallidus (GP) regions of the brain of patients who received repeated contrast-enhanced MRI examination with linear GBCA gadodiamide,7–10 gadopentetate dimeglumine,7,11–13 and gadobenate dimeglumine.14 A correlation between these signal hyperintensities and the number of contrast-enhanced MRI examinations was first described by Kanda et al7 in 2014. Subsequent autopsy studies verified the accumulation of gadolinium in the enhanced brain structures.9,12 In 2 retrospective clinical studies, linear gadopentetate dimeglumine was directly compared with macrocyclic gadoterate meglumine11 and gadoteridol.12 Therein, a signal increase in the dentate nucleus was found only in patients receiving multiple administrations of the linear gadopentetate dimeglumine. This observation has been reproduced in a rat animal model after repeated administration of gadodiamide, but not after gadoterate meglumine.15 In addition, Radbruch et al16 showed in a recent clinical study no signal intensity increase in dentate nucleus and GP on unenehanced T1w images after multiple applications of the macrocyclic GBCA gadobutrol. The results of Radbruch et al are contrary to a prior published study of Stojanov et al,17 reporting that gadobutrol causes significant SI enhancement of these brain regions. However, the study design of this publication was critically discussed and does not convincingly support this conclusion.18,19
The mechanism of GBCA accumulation in these specific brain regions is yet unknown and raises several questions; these are as follows: (1) “By which pathway does gadolinium enter the brain?” (2) “How does it distribute itself within the brain to reach the 2 areas GP and CN, which are located widely apart from each other and from any potential BBB disruption?” and (3) “What is the chemical species of Gd?” One possible pathway might be the infiltration of GBCAs from blood into the cerebrospinal fluid (CSF), which is supported by a study on patients suspected of Ménière disease. This study described contrast enhancement of the subarachnoid space, the anterior eye segment, and the CSF in the internal auditory canal after intravenous administration of GBCA.20
The aim of this preclinical study was to reproduce the observed T1w signal intensity in humans in a rat model after repeated administration of linear and macrocyclic GBCAs. Comparable to the clinical observations, we performed MRI with a clinical 1.5 T scanner and evaluated the data with similar methods. Furthermore, the influence of both GBCA classes on T1w signal hyperintensity was determined by applying 3 linear (ionic, nonionic, protein-binding) and 2 macrocylic (ionic, nonionic) GBCAs. To evaluate the potential pathway of GBCA infiltration into CSF, we performed fluid-attenuated MR cisternography before and after intravenous GBCA application.
MATERIALS AND METHODS
Seventy-eight healthy Han-Wistar rats (Crl:WI; males; 200–225 g) were obtained from Charles River (Sulzfeld, Germany). The animals were kept under standard laboratory conditions at a temperature of 22°C and a dark/light rhythm of 12 hours. Standard rat chow and water were provided ad libitum. The animals were handled and treated according to German animal regulations. The GBCA administration and the MRI were performed in anesthesia initiated with 4% isofluorane and maintained with 1.5% isofluorane (Baxter GmbH, Unterschleißheim, Germany). During the MRI, the body temperature was maintained with a circulating warm water pad.
Signal Enhancement in the Brain
Sixty rats were randomly divided into a control and 5 GBCA groups (n = 10 per group). The animals received the linear GBCAs gadodiamide (Omniscan; GE Healthcare Buchler GmbH & Co KG, Braunschweig, Germany), gadopentetate dimeglumine (Magnevist; Bayer Vital GmbH, Leverkusen, Germany), gadobenate dimeglumine (Multihance; Bracco Imaging Deutschland GmbH, Konstanz, Germany), or the macrocyclic GBCAs gadobutrol (Gadovist; Bayer Vital GmbH) and gadoterate meglumine (Dotarem; Guerbet GmbH, Sulzbach/Taunus, Germany) or saline (control). Over a period of 2 weeks, the animals received 10 intravenous injections at a dose of 2.5 mmol Gd/kg body weight (b.w.), each on 5 consecutive days per week. All investigated GBCAs were applied by slow hand injection (∼0.8 mL/min) using their marketed formulation. The extended dose of 2.5 mmol Gd/kg b.w. per injection approximates about 4 times the human standard dose based upon body surface area normalization between rats and humans.21 One day before the first GBCA administration, as well as 3 days after the last injection (p.i.), a whole-brain MRI was performed for all animals. Subsequently, 5 randomly chosen animals per group were euthanized by exsanguination for dissection. The other 5 animals per group received a second brain MRI before dissection at 24 days p.i. All tissue samples were frozen for further detailed analysis (ongoing). An overview of the study setup is shown in Figure 1A.
Evaluation of CSF Spaces
Eighteen rats were randomly divided into 6 groups (n = 3 per group), which received the same GBCAs as in the first part of the study. After an MRC for anatomical reference and a fluid attenuated baseline brain scan, the animals received an intravenous injection of GBCA at a dosage of 1 mmol Gd/kg b.w. or volume adjusted saline. One minute after injection, a fluid attenuated sequence was started.
Magnetic resonance imaging was performed in a clinical 1.5 T MRI (Avanto; Siemens Healthcare, Erlangen, Germany) using a dedicated 2-channel rat brain coil (Rapid Biomedical GmbH, Rimpar, Germany).
For the evaluation of the signal enhancement, a 3-dimensional T1-weighted turbo spin echo sequence (repetition time (TR), 500 milliseconds; echo time 19 milliseconds) was adjusted to cover the whole rat brain with a resolution of 0.3 × 0.3 × 0.8 mm (field of view, 80 × 32.5 mm; 30 transversal slices). A turbo factor of 3 was used, the number of signal excitations was 1, and the acquisition time was 9:55 minutes.
For the evaluation of the CSF space, heavily T2-weighted MRC for anatomical references of the fluid space was followed by a precontrast and postcontrast heavily T2-weighted, fluid-attenuated inversion recovery (FLAIR) sequence. For the MRC, a variable flip angle 3-dimensional turbo spin echo sequence (TR, 4400 milliseconds; echo time, 553 milliseconds) with an initial refocusing flip angle of 180 degrees (decreased to a constant angle of 120 degrees for the refocusing echo train), a turbo factor of 79, and a magnetization restore pulse was used. The spatial resolution was 0.3 × 0.3 × 0.6 mm (field of view, 100 × 48 mm; 30 transversal slices), the number of signal excitations was 1.8, and the acquisition time was 7:57 minutes. Identical scan parameters were used for the FLAIR sequence. However, a nonselective inversion recovery pulse with an inversion time of 2250 milliseconds was included, and the TR was extended to 9000 milliseconds. The acquisition time was 16:14 minutes.
The image analysis was performed by 2 experienced readers, blinded to the experimental groups and time points, on an external workstation (MMWP; Siemens Healthcare, Erlangen, Germany). Manually defined region of interests (ROIs) were drawn around the deep cerebellar nuclei (CN), the pons, the GP, and the thalamus, respectively. The slice selection as well as the ROI positioning was guided by anatomical landmarks taken from a stereotactic rat brain atlas.22 Examples of the ROI placement are shown in Figure 1, B and C. The signal intensities of the left and right hemispheres were averaged for each region. The deep cerebellar nucleus-to-pons ratio (CN/Po) was calculated by dividing the mean signal intensity of the CN by that of the pons. The GP-to-thalamus signal intensity ratio (GP/Th) was calculated by dividing the mean signal intensity of the GP by that of the thalamus. The CN/Po and GP/Th signal intensity ratios of the 2 readers were averaged. The difference to baseline (% change) was calculated for both signal intensity ratios.
Statistical comparisons between the experimental groups and control group were performed at each time point with 2-way analysis of variance followed by the post hoc Bonferroni test for multiple comparison. The statistical evaluation of the signal ratios within experimental groups (ie, the time course) was performed with a paired t test after confirming Gaussian distribution with the Kolmogorov-Smirnov test. The calculations were performed with GraphPad Prism (GraphPad Software, La Jolla, CA) using a significance level of 5%.
Signal Enhancement in the Brain
All animals successfully completed the entire study. Increased signal intensities in the CN were observed 3 and 24 days p.i. of gadodiamide, gadobenate dimeglumine, and to a lesser extent of gadopentetate dimeglumine. No signal changes were visible after administration of gadobutrol, gadoterate meglumine, or saline at any time point. Representative images for each experimental group and time point are shown in Figure 2.
The results of the quantitative analysis of the CN/Po signal intensity ratio are summarized in Figure 3, showing the absolute CN/Po ratios for the 3 time points (Fig. 3A) and the respective percent change to baseline (Fig. 3B). Statistical comparison within the experimental groups revealed significantly increased CN/Po ratios for gadodiamide and gadobenate dimeglumine on day 3 and 24 p.i. compared with baseline. On day 24 p.i., gadodiamide exhibited the highest CN/Po ratio increase in 9.6% compared with baseline. No significant differences were found between 3 and 24 days p.i. in these groups. A moderately elevated, but not significant, increase in the CN/Po ratios was observed for gadopentetate dimeglumine only on day 24 p.i. No increased CN/Po ratios compared with baseline were observed in the MRI scans of rats that received gadobutrol, gadoterate meglumine, or saline at any time point. Instead, a slight decrease was determined 3 days p.i. with mean reductions of 1.8%, 2.6%, and 2.2% (% change to baseline) for gadobutrol, gadoterate meglumine, and saline, respectively (Fig. 3B). This reduction returned to the baseline CN/Po ratios at day 24 p.i.
For the GP, no signal increase was visible in the MRI scans for any group. The results of the quantitative evaluation of the of GP/Th signal intensity ratio are shown in Figure 4. No significant changes over time or between any experimental and the control group were found.
Evaluation of CSF Spaces
To examine a potential pathway for the infiltration of GBCAs into the brain, the penetration of the GBCA into the CSF was studied. Therefore, the change of the signal intensities after GBCA administration in fluid attenuated sequences was investigated. In Figure 5 (left row), MRC images represent the CSF cavities in 1 slice of the brain, which shows the dorsal and lateral arachnoid space, the aqueduct, and the internal auditory canal. Figure 5 (middle row, precontrast) illustrates the identical slice after the CSF signal has been fully attenuated by the inversion pulse. In Figure 5 (right row, postcontrast), qualitative evaluation of the CSF cavities shows signal enhancement after administration of all GBCAs in the CSF. All 3 animals per GBCA group demonstrated signal enhancement of the CSF. In contrast, after saline injection, no signal enhancement was measured in the CSF in the rat brains.
In this preclinical study, the signal enhancement on T1w images in the CN and GP was investigated in rats. For this purpose, the signal intensity ratios CN/Po and GP/Th were evaluated after repeated administration of extended doses of GBCAs (2.5 mmol Gd/kg b.w.) with different structural and physicochemical properties up to 24 days p.i. Two macrocyclic and 3 linear agents were systematically compared in this study. The groups that were administered with macrocyclic GBCAs (gadobutrol, gadoterate meglumine) and saline exhibited no elevated signal intensities in either the CN or GP. In contrast, the application of the linear GBCAs gadodiamide, gadobenate dimeglumine, and, to a lesser extent, gadopentetate dimeglumine resulted in increased and persistent CN/Po signal intensity ratios. Thus, the previously described differences between linear and macrocyclic GBCAs regarding signal enhancement in dentate nucleus were reproduced by comparing for the first time several commonly used GBCAs from each class.
Smaller differences between the linear GBCAs seem to exist as gadodiamide showed the most intense increase in CN/Po signal intensity ratio followed by gadobenate dimeglumine. Gadopentetate dimeglumine exhibited only a moderate increase in the CN/Po signal intensity ratio, which was statistically not significant. This is in contrast to clinical observations where patients showed significant hypersignal intensities of the dentate nucleus after serial applications of gadopentetate dimeglumine.7,11,12 The deviations of significantly increased signal intensity ratios in the clinical studies, and the nonsignificant increase in the present rat study may have several explanations. First of all, this study was conducted in rats. An animal model can approach, but is not able to replicate the clinical conditions in patients in all details. The rat model represented healthy animals at the same age and weight, and all animals have a similar genetic background. Conversely, in the clinical situation, the medical background of patients is diverse, and the disease process and treatment are highly variable. The second was the experimental setup, especially the high GBCA dose per injection (∼4 times based upon body surface area, but physically 25-fold of a single human standard dose). The influence of the osmolality of the administered GBCAs might become an issue with such a dosing regime because hyperosmolal solutions were sometimes used to open the blood-brain barrier in intra-arterial injections.23 However, in this study, a slow, intravenous injection (0.8 mL/min) was used, which should lead to sufficient dilution before the bolus reaches the brain. In this respect, it is remarkable that gadodiamide has the lowest osmolality (780 mOsm/kg, package insert) but the strongest increase in signal intensity, while gadobutrol (1603 mOsm/kg, package insert) and gadoterate meglumine (1350 mOsm/kg, package insert) have a much higher osmolality but did not evoke an increased signal intensity. In addition, the interval between the 10 injections and the time between the last injection and the MRI significantly differ from clinical studies. A possible explanation for the observed difference between gadopentetate dimeglumine and gadobenate dimeglumine might be the higher relaxivity of the latter and its weak binding to proteins resulting in stronger signal enhancement. A further important distinction between gadobenate dimeglumine and gadopentetate dimeglumine is their different pharmacokinetic profile and excretion pathway. Gadobenate dimeglumine is taken up by hepatocytes to a different extent in rats (∼50%) and humans (3%–5%),24 while gadopentetate dimeglumine is only excreted via the kidneys. Despite a more pronounced dual excretion pathway for gadobenate dimeglumine in rats, in this study, surprisingly higher signal intensities in the neuronal brain regions were observed for gadobenate dimeglumine than for gadopentetate dimeglumine. However, this discrepancy can only be resolved once the chemical species of the deposited gadolinium has been discovered.
The image acquisition and evaluation were performed in an analogous manner to the published clinical retrospective studies. A clinical 1.5 T MRI system was used, the whole brain was imaged with a standard T1w sequence, and the quantitative image evaluation was done using signal intensity ratios in a similar manner as described previously.7–11,14 A recently published study in rats by Robert et al15 focused on the cerebellum only and performed MRI on a dedicated preclinical system. In this approach, the whole deep CN (including the lateral cerebellar nucleus, which is equivalent to the dentate nucleus in humans) was used for the ROI-based analysis. In contrast to the present study, the cerebellum and not the pons was used as the reference region, which, however, resulted in an analogous difference between linear and macrocyclic agents. Interestingly, Robert et al observed a slight decrease in this signal intensity ratio for gadoterate meglumine and hyperosmolar saline, similar to the present study with slightly reduced CN/Po signal intensity ratios for gadoterate meglumine, gadobutrol, and saline on day 3 p.i. The reason for this decrease is unclear, but might be correlated to the repeated treatment under anesthesia because, in the present study, the signal intensity ratios returned to their baseline values in these groups on day 24 p.i. Potentially, this effect is also present in the linear GBCA groups but concealed by potential Gd deposition. For the GP, no elevated signal intensities were observed for any GBCA. The respective fluctuations of the GP/Th signal intensity ratios most likely reflect variations due to the experimental conditions. This is in contrast to clinical studies that reported a T1w signal increase for both regions (the dentate nucleus and the GP) after administration of gadodiamide and gadopentetate dimeglumine7,9,11 and indicates a possible limitation of the current animal model.
As the signal enhancement in the neuronal structures of the brain is prominent for linear but not for macrocyclic agents, these findings suggest that the differences could be attributed to differences in complex stabilities. It is tempting to draw parallels to the differences seen between linear and macrocyclic GBCA with respect to complex stability demonstrated in vitro and by long-term retention experiments in rats.6,25 However, the present study did not investigate the chemical species of Gd3+ in the brain, which would be necessary to learn more about the underlying mechanism. T1-weighted signal enhancement can result either by accumulation of intact GBCA or by accumulation of other chemical species, which incorporated the Gd3+ ion by transmetalation from the GBCA. If these not yet identified species do not precipitate in physiological environment, such as, Gd-phosphate, they may exhibit a significant r1-relaxivity and may thus also contribute to the observed signal intensity.
Although on T1w images no enhancement of CSF cavities was observed, a heavily T2-weighted FLAIR imaging sequence provided a clear enhancement of CSF cavities after the administration of all GBCAs, independently of their chemical structure. This FLAIR sequence is highly sensitive to concentrations of GBCA as low as 15.6 μM as shown in phantoms.26 Hence, the results demonstrate the presence of GBCA in the CSF at very low concentrations. An enhancement of the CSF in the internal auditory canal has been described before in patients with Ménière diseases undergoing MRI for the visualization of endolymphatic hydrops.27 However, very little knowledge exists about the impact of the physicochemical properties and the structure of GBCA on the distribution and on the intensity of enhancement of various CSF cavities. Most of the imaging studies of the endolymphatic hydrops were performed after intravenous injection of gadodiamide, but some also after gadoteridol, with both GBCAs giving similar findings.27 The time course of contrast enhancement in the CSF internal auditory canal was investigated in a small cohort of healthy volunteers with a maximum at 1.5 hours after administration of gadoteridol.28 Hence, the observed CSF signal enhancement in the present rat study may not represent the time point of maximal enhancement. In addition, the primary location of blood CSF infiltration still remains to be elucidated. The flow of the CSF is directed from its origin of formation, the choroid plexus, to the arachnoid granules where it is returned into the venous blood system. The observed widespread distribution of the GBCAs in the CSF cavity system may be explained by this CSF flow, induced by ependymal cilia movement, by choroid plexus pulsation, and by contraction and expansion of the ventricles.29 Cerebrospinal fluid is one of several compartments in the brain, which are usually well separated by highly specific barriers. Several publications reviewed by Brinker et al29 address exchange mechanisms of fluids and substances between these brain compartments. However, the infiltration of GBCAs into the CSF and the connection to the observed gadolinium deposition in the brain still remain obscure.
In summary, similar to recent retrospective clinical studies, an increased and persistent T1w signal intensity increase in the CN (which contains the dentate nucleus) quantified by the CN/Po signal intensity ratio was observed after multiple extended doses of linear GBCAs in rats. However, the reported signal enhancement in T1w images in the GP quantified by the GP/Th signal intensity ratio could not be reproduced in this animal model. No elevated signal intensities in either the CN or the GP were found after administration of macrocyclic agents. Thus, the previously described differences between linear and macrocyclic GBCAs regarding signal enhancement in the dentate nucleus were reproduced qualitatively by comparing for the first time several commonly used GBCAs from each class. A CSF signal enhancement on FLAIR images was observed for all GBCAs independent of their chemical structure. Thus, the GBCA passage from the blood into CSF might represent an initial pathway of GBCA infiltration into the brain. However, as of yet, no connection can be made between the CSF enhancement and the signal increase in the dentate nucleus and GP. Neither the underlying mechanism of GBCA exchange between different brain fluids nor the accumulation of Gd in the dentate nucleus and GP is known yet. A logical next step is the establishment of advanced analytical methods for gadolinium speciation in the dissected brain tissues. This would lead to a more detailed understanding of the observed signal enhancement and the role of the different GBCAs.
The authors thank Thomas Balzer, MD and Wolfgang Ebert, PhD for the critical reading of the manuscript, and Claudia Heyer, Ines Krause, David Hallmann, Robert Ivkic and Michael Hasbach for their excellent technical assistance.
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