Pharmakovigilanz von Gadolinium-basierten MRT-Kontrastmitteln in der Ratte: Neurobiologische, histologische und biochemische Beobachtungen

Gadolinium-based contrast agents (GBCAs) are widely used in clinical magnetic resonance imaging (MRI) since the mid-1980s. Recently, concerns have been raised since abnormal signal increases on magnetic resonance images as well as trace amounts of gadolinium (Gd) were detected in the brain even years after GBCA application. The genesis, form of the retained Gd, and reversibility of this accumulation of Gd within the deep cerebellar nuclei and the Globus pallidus (GP) of the central nervous system (CNS) are still not fully explained. Especially clinically relevant consequences and potential comorbidity of the phenomenon are of greatest interest to assess the safety of the routinely used compounds. The American Food and Drug Administration (FDA) as well as the European Medicines Agency (EMA) prompted all manufacturers of GBCAs as well as the scientific community to thoroughly investigate the phenomenon and to conduct vigilance studies. Following this official call, we assessed intermediate and long-term (5 – 52 weeks after the last injection) effects of multiple administrations of macrocyclic- (gadobutrol) and linear-chelated (gadodiamide) GBCAs in a rat model and studied neuro-histopathology as well as gene expression, and distribution of retained Gd in combination with a comprehensive behavioral phenotyping. As an established preclinical model, healthy male Wistar-Han rats received either 8 injections of 0.6, 1.2 or 1.8 mmol/kg BW gadobutrol, gadodiamide or saline as a control. Using a hypothesis-free study design, a broad range of behavioral domains including general health and metabolic status, motor function, emotion, sensorimotor function, nociception and cognition were investigated longitudinally for up to 52 weeks post GBCA administration (p.i.). Tissue samples were taken 11, 35 and 54 weeks p.i. for further assessment of the neuropathology. Mirroring the clinical situation, laser ablation inductively coupled plasma-mass spectrometry (LA-ICP-MS) revealed retention of Gd in the Nucleus dentatus (DN) of animals treated with gadodiamide. This long-term outcome was associated with an early intermittent and reversible reduction of the startle reaction to acoustic stimuli around 7 weeks p.i. Grooming behavior of gadodiamide animals, measured in the OpenField test 5 weeks p.i., was increased due to dermatitis-like skin lesions and loss of fur, known to be occurring with high doses of linear GBCAs. In general, conducted tests for motor function, cognition, nociception as well as the metabolic and emotional status of the animals were not affected by exposure to GBCAs. Gene expression profiling by RNA-sequencing as well as the stereologically assessed number of NeuN-positive cells in the deep cerebellar nuclei revealed no apparent differences 11 weeks p.i. Subcellular fractionation of brain tissue revealed, that Gd seems to co-enrich especially in synaptosomes of animals treated with gadodiamide. The detected amount of Gd found in synaptosomes of rats with exposure to gadobutrol was smaller, and in addition the intact form of gadobutrol was detected exclusively. In contrast, in synaptosomes extracted from brain tissue of gadodiamide animals insoluble Gd as well as high molecular weight species were found in addition to the intact GBCA. These results point to some form of dechelation of the less stable GBCA gadodiamide in vivo with (subsequent) binding to certain molecules prone to act as polyanionic acceptors for Gd3+ cations, eventually resulting in high-relaxivity complexes that are able to cause the visible hyperintensities on MR images. New, and to some extend surprising, expression sites of the extracellular matrix and perineuronal net (PNN) protein aggrecan, representing a polyanionic acceptor for (dissociated) Gd3+, partially overlapped with sites of deposited Gd, as revealed by combined application of immunohistology and LA-ICP-MS. Taken together, this study revealed that repeated GBCA application of gadodiamide at clinically relevant dosage induce a transient reduction in acoustic startle response in rats. Sites of GBCA retention overlap partially with PNN proteins but retention of Gd in the DN was neither associated with a general pathway deregulation nor neuronal cell loss. The findings described in this work have been partially published in in the peer-reviewed journal Scientific Reports by Habermeyer et al. (Habermeyer et al., 2020). Our preclinical studies yield a caveat for the clinical use of linear GBCAs and should challenge further research in the direction of PNN-GBCA interactions as well as the effect of GBCAs on the (peripheral) sensory nervous system.Gadolinium-basierte Kontrastmittel (GBKM) sind ein wichtiges Hilfsmittel in der diagnostischen Bildgebung mittels Magnetresonanztomographie (MRT) und werden seit Mitte der 1980er Jahre eingesetzt. Im Jahr 2014 wurde eine ungewöhnlich hohe Signalintensität in bestimmten Hirnregionen auf nicht-kontrastmittel verstärkten MRT-Bildern mit der Gabe von GBKM in der Krankengeschichte von Patienten in Verbindung gebracht. Folgeuntersuchungen belegten, dass sich das paramagnetische Element Gadolinium (Gd) der GBKM vor allem nach wiederholter Gabe von linearen GBKM in spezifischen Hirn-Regionen ablagert und so zu den abnormen und persistierenden Signalverstärkungen auf MRT-Bildern führt. Der genaue Ablagerungsmechanismus, die Form der Ablagerung sowie die mögliche Reversibilität dieser für lange Zeit nachweisbaren Retention vor allem im zerebellaren Nucleus dentatus (DN) und dem Globus pallidus (GP) sind noch nicht vollständig geklärt. Von besonderem Interesse sind hierbei vor allem die potenziell klinisch-relevanten Folgen dieser Gd-Ablagerungen, die im Falle ihrer Bestätigung eine Neubewertung der Sicherheit der GBKM nach sich ziehen würden. Sowohl die europäische Arzneimittel-Agentur (EMA) als auch die American Food and Drug Administration (FDA) haben deshalb die Hersteller der GBKM sowie die allgemeine Forschungsgemeinschaft dazu aufgefordert das Phänomen gründlich zu untersuchen, um die offenen Fragen zu beantworten. In der vorliegenden Studie wurden die mittel- und langfristigen neurobiologischen Folgen der wiederholten Gabe eines makrozyklischen (Gadobutrol) sowie eines linearen (Gadodiamid) GBKM in einem Rattenmodell untersucht. Es wurden sowohl eine umfassende Verhaltenscharakterisierung als auch Untersuchungen neuro-histopathologischer Parameter, der Genexpression in den betroffenen Hirngebieten und der subzellulären Verteilung der Ablagerungen durchgeführt. Gesunden, männlichen Wistar-Han Ratten wurde 8 Mal entweder 0,6, 1,2 oder 1,8 mmol/kg KG Gadobutrol, Gadodiamid oder Saline als Kontrolle verabreicht. In einem Hypothesen-freien Studienaufbau wurde das Verhalten der Tiere longitudinal über einen Zeitraum von 52 Wochen nach der letzten Injektion einschließlich folgender Domänen untersucht: Genereller Gesundheitszustand, Motorfunktion, emotionaler Status, Sensorimotorfunktion, Nozizeption, Metabolismus und kognitive Fähigkeiten. 11, 35 und 54 Wochen nach der letzten Injektion wurden Gewebeproben entnommen und molekularbiologisch untersucht. Genau wie in der klinischen Situation konnte auch im Rattenmodel die Ablagerung von Gd im Gewebe nach der Verabreichung von Gadodiamid durch Massenspektrometrie mit induktiv gekoppelter Plasma- und Laserablation (LA-ICP-MS) nachgewiesen werden. Darüber hinaus war eine frühe, aber reversible Reduktion der Sensormotorkopplung (Muskelkontraktion auf einen Stimulus hin/Startle-response) auf akustische Signale 7 Wochen nach der letzten Injektion zu beobachten. Tiere, die die höchste Dosis Gadodiamid erhalten hatten, zeigten eine gesteigerte Fellpflege im OpenField-Test, die auf bereits beschriebene, Dermatitis-ähnliche Hautläsionen und Fellverlust zurückzuführen waren. Die Messung der Motorfunktion, Kognition, Nozizeption sowie des metabolischen Zustands war hingegen für alle Tiere vergleichbar. Die Analyse der Genexpression mittels RNS-Sequenzierung sowie die stereologische Quantifizierung NeuN-positiver Zellen im DN ergab keine Auffälligkeiten. Die subzelluläre Fraktionierung des Hirngewebes ergab eine deutliche Anreicherung von Gd in Synaptosomen von Tieren der Gadodiamid-Gruppe. Die Gabe von Gadobutrol führte zu deutlich weniger Ablagerung von Gd, das zudem nur in Form des intakten Chelat-Komplexes nachgewiesen werden konnte. Im Gegensatz dazu wurde in Proben der Gadodiamid-behandelten Tiere zusätzlich noch unlösliches Gd detektiert, was auf eine Herauslösung des Gd-Ions aus dem weniger stabilen, linearen Chelat hinweist. Überraschend war dabei die mittels einer Kombination von immunhistologischer Färbung mit LA-ICP-MS erhaltene Beobachtung einer überlappenden Verteilung des extrazellulären Matrixproteins Aggrecan mit der Ablagerung von Gd im DN. Aggrecan agiert als Poly-Kationen- Akzeptorprotein und könnte potenziell dissoziierte Gd-Ionen binden. Zusammenfassend ist eine transiente Reduktion der akustisch-induzierten Sensorimotorkopplung der Tiere nach Gabe klinisch-relevanter Dosen Gadodiamide festzuhalten. Die Ablagerungen von Gd im Hirngewebe zeigen eine teilweise Überlappung mit der Verteilung des perineuralen Netzproteins Aggrecan, während weder eine generelle Veränderung der Genexpression in den betroffenen Hirnregionen noch ein neuronaler Zellverlust festgestellt werden konnten. Wesentliche Ergebnisse der hier beschriebenen Studie wurden teilweise in Habermeyer et al. publiziert (Habermeyer et al., 2020). Auf Basis dieser präklinischen Ergebnisse sollte eine genauere Untersuchung im klinischen Kontext erfolgen sowie besondere Sorgfalt bei der Wahl des Typs von GBKM (linear vs. makrozyklisch) vor der Applikation im Patienten nach sich ziehen. Zusätzlich dient die Studie als Grundlage für weitere Analyse der potenziellen Interaktion zwischen GBKM und perineuralen Netzen sowie der GBKM-induzierten Veränderungen des (peripheren), sensorischen Nervensystems

Comprehensive phenotyping revealed transient startle response reduction and histopathological gadolinium localization to perineuronal nets after gadodiamide administration in rats

Gadolinium based contrast agents (GBCAs) are widely used in clinical MRI since the mid-1980s. Recently, concerns have been raised that trace amounts of Gadolinium (Gd), detected in brains even long time after GBCA application, may cause yet unrecognized clinical consequences. We therefore assessed the behavioral phenotype, neuro-histopathology, and Gd localization after repeated administration of linear (gadodiamide) or macrocyclic (gadobutrol) GBCA in rats. While most behavioral tests revealed no difference between treatment groups, we observed a transient and reversible decrease of the startle reflex after gadodiamide application. Residual Gd in the lateral cerebellar nucleus was neither associated with a general gene expression pathway deregulation nor with neuronal cell loss, but in gadodiamide-treated rats Gd was associated with the perineuronal net protein aggrecan and segregated to high molecular weight fractions. Our behavioral finding together with Gd distribution and speciation support a substance class difference for Gd presence in the brain after GBCA application

Silhouette-Length-Scaled Gait Parameters for Motor Functional Analysis in Mice and Rats

Gait analysis of transgenic mice and rats modeling human diseases often suffers from the condition that those models exhibit genotype-driven differences in body size, weight, and length. Thus, we hypothesized that scaling by the silhouette length improves the reliability of gait analysis allowing normalization for individual body size differences. Here, we computed video-derived silhouette length and area parameters from a standard markerless gait analysis system using image-processing techniques. By using length- and area-derived data along with body weight and age, we systematically scaled individual gait parameters. We compared these different scaling approaches and report here that normalization for silhouette length improves the validity and reliability of gait analysis in general. The application of this silhouette length scaling to transgenic Huntington disease mice and Parkinson´s disease rats identifies the remaining differences reflecting more reliable, body length-independent motor functional differences. Overall, this emphasizes the need for silhouette-length-based intra-assay scaling as an improved standard approach in rodent gait analysis

Techno-economic optimization of a new Biomass-to-liquid concept

The investigation of 2nd generation biofuels has gained in importance in recent years. Mostly because they hold the potential of reducing the greenhouse gas emissions of the transport sector on a larger scale. Inter alia, these alternative fuels can be produced via a Biomass-to-Liquid (BtL) process based on Fischer-Tropsch (FT) synthesis. The produced synthetic hydrocarbons can directly be used as drop-in fuels in the existing infrastructure. Though, the market launch of this technology depends strongly on its economic feasibility. The EU-project COMSYN (Compact Gasification and Synthesis process for Transport Fuels) aims to scratch at this economic limitation by combining a number of compact and highly efficient process steps. Besides the new gasification concept (developed by VTT) and the subsequent hot gas filtration, a FT-microreactor (developed from INERATEC) is attempting to achieve the ambiguous goal of reducing the biofuel production costs by up to 35 % compared to alternative routes. Within the project the DLR is assigned with the techno-economic assessment and optimization of the process concept. The necessary flowsheet model is set up in Aspen Plus®. Various different process setups are evaluated technically, economically as well as ecologically with the DLR in-house software tool TEPET. In dependence of several technical and economic boundary conditions, the techno-economically most favorable results can be determined and will be presented. Since the project also contains an experimental demonstration of the whole process at the test site of VTT, the flowsheet model and the subsequent evaluation can rely on experimental data of the project partners of VTT and INERATEC. The evaluation and optimization approach of this new BtL concept in dependence of both, economic and technical boundary conditions constitute an extension of previous work of DLR (Albrecht et al)

Table_1_Gene-dosage- and sex-dependent differences in the prodromal-Like phase of the F344tgHD rat model for Huntington disease.pdf

In Huntington disease (HD) the prodromal phase has been increasingly investigated and is currently in focus for early interventional treatments. Also, the influence of sex on disease progression and severity in patients is under discussion, as a sex-specific impact has been reported in transgenic rodent models for HD. To this end, we have been studying these aspects in Sprague Dawley rats transgenic for HD. Here, we took up on the congenic F344tgHD rat model, expressing a fragmented Htt construct with 51 CAG repeats on an inbred F344 rat background and characterized potential sexual dimorphism and gene-dosage effects in rats during the pre-symptomatic phase (1–8 months of age). Our study comprises a longitudinal phenotyping of motor function, emotion and sensorimotor gating, as well as screening of metabolic parameters with classical and automated assays in combination with investigation of molecular HD hallmarks (striatal cell number and volume estimation, appearance of HTT aggregates). Differences between sexes became apparent during middle age, particularly in the motor and sensorimotor domains. Female individuals were generally more active, demonstrated different gait characteristics than males and less anxiolytic-like behavior. Alterations in both the time course and affected behavioral domains varied between male and female F344tgHD rats. First subtle behavioral anomalies were detected in transgenic F344tgHD rats prior to striatal MSN cell loss, revealing a prodromal-like phase in this model. Our findings demonstrate that the congenic F344tgHD rat model shows high face-validity, closely resembling the human disease’s temporal progression, while having a relatively low number of CAG repeats, a slowly progressing pathology with a prodromal-like phase and a comparatively subtle phenotype. By differentiating the sexes regarding HD-related changes and characterizing the prodromal-like phase in this model, these findings provide a foundation for future treatment studies.</p

Video_1_Gene-dosage- and sex-dependent differences in the prodromal-Like phase of the F344tgHD rat model for Huntington disease.MP4

In Huntington disease (HD) the prodromal phase has been increasingly investigated and is currently in focus for early interventional treatments. Also, the influence of sex on disease progression and severity in patients is under discussion, as a sex-specific impact has been reported in transgenic rodent models for HD. To this end, we have been studying these aspects in Sprague Dawley rats transgenic for HD. Here, we took up on the congenic F344tgHD rat model, expressing a fragmented Htt construct with 51 CAG repeats on an inbred F344 rat background and characterized potential sexual dimorphism and gene-dosage effects in rats during the pre-symptomatic phase (1–8 months of age). Our study comprises a longitudinal phenotyping of motor function, emotion and sensorimotor gating, as well as screening of metabolic parameters with classical and automated assays in combination with investigation of molecular HD hallmarks (striatal cell number and volume estimation, appearance of HTT aggregates). Differences between sexes became apparent during middle age, particularly in the motor and sensorimotor domains. Female individuals were generally more active, demonstrated different gait characteristics than males and less anxiolytic-like behavior. Alterations in both the time course and affected behavioral domains varied between male and female F344tgHD rats. First subtle behavioral anomalies were detected in transgenic F344tgHD rats prior to striatal MSN cell loss, revealing a prodromal-like phase in this model. Our findings demonstrate that the congenic F344tgHD rat model shows high face-validity, closely resembling the human disease’s temporal progression, while having a relatively low number of CAG repeats, a slowly progressing pathology with a prodromal-like phase and a comparatively subtle phenotype. By differentiating the sexes regarding HD-related changes and characterizing the prodromal-like phase in this model, these findings provide a foundation for future treatment studies.</p

Image_1_Gene-dosage- and sex-dependent differences in the prodromal-Like phase of the F344tgHD rat model for Huntington disease.tiff

In Huntington disease (HD) the prodromal phase has been increasingly investigated and is currently in focus for early interventional treatments. Also, the influence of sex on disease progression and severity in patients is under discussion, as a sex-specific impact has been reported in transgenic rodent models for HD. To this end, we have been studying these aspects in Sprague Dawley rats transgenic for HD. Here, we took up on the congenic F344tgHD rat model, expressing a fragmented Htt construct with 51 CAG repeats on an inbred F344 rat background and characterized potential sexual dimorphism and gene-dosage effects in rats during the pre-symptomatic phase (1–8 months of age). Our study comprises a longitudinal phenotyping of motor function, emotion and sensorimotor gating, as well as screening of metabolic parameters with classical and automated assays in combination with investigation of molecular HD hallmarks (striatal cell number and volume estimation, appearance of HTT aggregates). Differences between sexes became apparent during middle age, particularly in the motor and sensorimotor domains. Female individuals were generally more active, demonstrated different gait characteristics than males and less anxiolytic-like behavior. Alterations in both the time course and affected behavioral domains varied between male and female F344tgHD rats. First subtle behavioral anomalies were detected in transgenic F344tgHD rats prior to striatal MSN cell loss, revealing a prodromal-like phase in this model. Our findings demonstrate that the congenic F344tgHD rat model shows high face-validity, closely resembling the human disease’s temporal progression, while having a relatively low number of CAG repeats, a slowly progressing pathology with a prodromal-like phase and a comparatively subtle phenotype. By differentiating the sexes regarding HD-related changes and characterizing the prodromal-like phase in this model, these findings provide a foundation for future treatment studies.</p

Image_4_Gene-dosage- and sex-dependent differences in the prodromal-Like phase of the F344tgHD rat model for Huntington disease.TIFF

In Huntington disease (HD) the prodromal phase has been increasingly investigated and is currently in focus for early interventional treatments. Also, the influence of sex on disease progression and severity in patients is under discussion, as a sex-specific impact has been reported in transgenic rodent models for HD. To this end, we have been studying these aspects in Sprague Dawley rats transgenic for HD. Here, we took up on the congenic F344tgHD rat model, expressing a fragmented Htt construct with 51 CAG repeats on an inbred F344 rat background and characterized potential sexual dimorphism and gene-dosage effects in rats during the pre-symptomatic phase (1–8 months of age). Our study comprises a longitudinal phenotyping of motor function, emotion and sensorimotor gating, as well as screening of metabolic parameters with classical and automated assays in combination with investigation of molecular HD hallmarks (striatal cell number and volume estimation, appearance of HTT aggregates). Differences between sexes became apparent during middle age, particularly in the motor and sensorimotor domains. Female individuals were generally more active, demonstrated different gait characteristics than males and less anxiolytic-like behavior. Alterations in both the time course and affected behavioral domains varied between male and female F344tgHD rats. First subtle behavioral anomalies were detected in transgenic F344tgHD rats prior to striatal MSN cell loss, revealing a prodromal-like phase in this model. Our findings demonstrate that the congenic F344tgHD rat model shows high face-validity, closely resembling the human disease’s temporal progression, while having a relatively low number of CAG repeats, a slowly progressing pathology with a prodromal-like phase and a comparatively subtle phenotype. By differentiating the sexes regarding HD-related changes and characterizing the prodromal-like phase in this model, these findings provide a foundation for future treatment studies.</p

Image_6_Gene-dosage- and sex-dependent differences in the prodromal-Like phase of the F344tgHD rat model for Huntington disease.TIFF

In Huntington disease (HD) the prodromal phase has been increasingly investigated and is currently in focus for early interventional treatments. Also, the influence of sex on disease progression and severity in patients is under discussion, as a sex-specific impact has been reported in transgenic rodent models for HD. To this end, we have been studying these aspects in Sprague Dawley rats transgenic for HD. Here, we took up on the congenic F344tgHD rat model, expressing a fragmented Htt construct with 51 CAG repeats on an inbred F344 rat background and characterized potential sexual dimorphism and gene-dosage effects in rats during the pre-symptomatic phase (1–8 months of age). Our study comprises a longitudinal phenotyping of motor function, emotion and sensorimotor gating, as well as screening of metabolic parameters with classical and automated assays in combination with investigation of molecular HD hallmarks (striatal cell number and volume estimation, appearance of HTT aggregates). Differences between sexes became apparent during middle age, particularly in the motor and sensorimotor domains. Female individuals were generally more active, demonstrated different gait characteristics than males and less anxiolytic-like behavior. Alterations in both the time course and affected behavioral domains varied between male and female F344tgHD rats. First subtle behavioral anomalies were detected in transgenic F344tgHD rats prior to striatal MSN cell loss, revealing a prodromal-like phase in this model. Our findings demonstrate that the congenic F344tgHD rat model shows high face-validity, closely resembling the human disease’s temporal progression, while having a relatively low number of CAG repeats, a slowly progressing pathology with a prodromal-like phase and a comparatively subtle phenotype. By differentiating the sexes regarding HD-related changes and characterizing the prodromal-like phase in this model, these findings provide a foundation for future treatment studies.</p

Image_7_Gene-dosage- and sex-dependent differences in the prodromal-Like phase of the F344tgHD rat model for Huntington disease.TIFF

In Huntington disease (HD) the prodromal phase has been increasingly investigated and is currently in focus for early interventional treatments. Also, the influence of sex on disease progression and severity in patients is under discussion, as a sex-specific impact has been reported in transgenic rodent models for HD. To this end, we have been studying these aspects in Sprague Dawley rats transgenic for HD. Here, we took up on the congenic F344tgHD rat model, expressing a fragmented Htt construct with 51 CAG repeats on an inbred F344 rat background and characterized potential sexual dimorphism and gene-dosage effects in rats during the pre-symptomatic phase (1–8 months of age). Our study comprises a longitudinal phenotyping of motor function, emotion and sensorimotor gating, as well as screening of metabolic parameters with classical and automated assays in combination with investigation of molecular HD hallmarks (striatal cell number and volume estimation, appearance of HTT aggregates). Differences between sexes became apparent during middle age, particularly in the motor and sensorimotor domains. Female individuals were generally more active, demonstrated different gait characteristics than males and less anxiolytic-like behavior. Alterations in both the time course and affected behavioral domains varied between male and female F344tgHD rats. First subtle behavioral anomalies were detected in transgenic F344tgHD rats prior to striatal MSN cell loss, revealing a prodromal-like phase in this model. Our findings demonstrate that the congenic F344tgHD rat model shows high face-validity, closely resembling the human disease’s temporal progression, while having a relatively low number of CAG repeats, a slowly progressing pathology with a prodromal-like phase and a comparatively subtle phenotype. By differentiating the sexes regarding HD-related changes and characterizing the prodromal-like phase in this model, these findings provide a foundation for future treatment studies.</p