Repeated Injection of Very Small Superparamagnetic Iron Oxide Particles (VSOPs) in Murine Atherosclerosis: A Safety Study.

Citrate-coated electrostatically stabilized very small superparamagnetic iron oxide particles (VSOPs) have been successfully tested as magnetic resonance angiography (MRA) contrast agents and are promising tools for molecular imaging of atherosclerosis. Their repeated use in the background of pre-existing hyperlipidemia and atherosclerosis has not yet been studied. This study aimed to investigate the effect of multiple intravenous injections of VSOPs in atherosclerotic mice. Taurine-formulated VSOPs (VSOP-T) were repeatedly intravenously injected at 100 µmol Fe/kg in apolipoprotein E-deficient (ApoE KO) mice with diet-induced atherosclerosis. Angiographic imaging was carried out by in vivo MRI. Magnetic particle spectrometry was used to detect tissue VSOP content, and tissue iron content was quantified photometrically. Pathological changes in organs, atherosclerotic plaque development, and expression of hepatic iron-related proteins were evaluated. VSOP-T enabled the angiographic imaging of heart and blood vessels with a blood half-life of one hour. Repeated intravenous injection led to VSOP deposition and iron accumulation in the liver and spleen without affecting liver and spleen pathology, expression of hepatic iron metabolism proteins, serum lipids, or atherosclerotic lesion formation. Repeated injections of VSOP-T doses sufficient for MRA analyses had no significant effects on plaque burden, steatohepatitis, and iron homeostasis in atherosclerotic mice. These findings underscore the safety of VSOP-T and support its further development as a contrast agent and molecular imaging tool.

Inorganic Phosphate-Induced Extracellular Vesicles from Vascular Smooth Muscle Cells Contain Elevated Levels of Hyaluronic Acid, Which Enhance Their Interaction with Very Small Superparamagnetic Iron Oxide Particles.

Patients with chronic kidney disease (CKD) have a high prevalence of hyperphosphatemia, where uremic toxins like inorganic phosphate (Pi) induce a cardiovascular remodeling. Related disorders like atherosclerosis bear the risk of increased morbidity and mortality. We previously found that Pi stimulates the synthesis and sulfation of the negatively charged glycosaminoglycans (GAGs) heparan sulfate and chondroitin sulfate in vascular smooth muscle cells (VSMC). Similar GAG alterations were detected in VSMC-derived exosome-like extracellular vesicles (EV). These EV showed a strong interaction with very small superparamagnetic iron oxide particles (VSOP), which are used as imaging probes for experimental magnetic resonance imaging (MRI). Hyaluronic acid (HA) represents another negatively charged GAG which is supposed to function as binding motif for VSOP as well. We investigated the effects of Pi on the amounts of HA in cells and EV and studied the HA-dependent interaction between VSOP with cells and EV. Rat VSMC were treated with elevated concentrations of Pi. CKD in rats was induced by adenine feeding. EV were isolated from culture supernatants and rat plasma. We investigated the role of HA in binding VSOP to cells and EV via cell-binding studies, proton relaxometry, and analysis of cellular signaling, genes, proteins, and HA contents. Due to elevated HA contents, VSMC and EV showed an increased interaction with VSOP after Pi stimulation. Amongst others, Pi induced hyaluronan synthase (HAS)2 expression and activation of the Wnt pathway in VSMC. An alternative upregulation of HA by iloprost and an siRNA-mediated knockdown of HAS2 confirmed the importance of HA in cells and EV for VSOP binding. The in vitro-derived data were validated by analyses of plasma-derived EV from uremic rats. In conclusion, the inorganic uremic toxin Pi induces HA synthesis in cells and EV, which leads to an increased interaction with VSOP. HA might therefore be a potential molecular target structure for improved detection of pathologic tissue changes secondary to CKD like atherosclerosis or cardiomyopathy using EV, VSOP and MRI.

Uremic Toxin-Induced Exosome-like Extracellular Vesicles Contain Enhanced Levels of Sulfated Glycosaminoglycans which Facilitate the Interaction with Very Small Superparamagnetic Iron Oxide Particles.

Uremic toxins exert pathophysiological effects on cells and tissues, such as the generation of a pro-calcifying subtype of exosome-like extracellular vesicles (EVs) in vascular cells. Little is known about the effects of the toxins on the surface structure of EVs. Thus, we studied the effects of uremic toxins on the abundance of sulfated glycosaminoglycans (GAGs) in EVs, and the implications for binding of ligands such as very small superparamagnetic iron oxide particles (VSOPs) which could be of relevance for radiological EV-imaging. Vascular cells were treated with the uremic toxins NaH2PO4 and a mixture of urea and indoxyl sulfate. Uremia in rats was induced by adenine feeding. EVs were isolated from culture supernatants and plasma of rats. By proton T1-relaxometry, magnetic particle spectroscopy, and analysis of genes, proteins, and GAG-contents, we analyzed the roles of GAGs in the ligand binding of EVs. By influencing GAG-associated genes in host cells, uremic toxins induced higher GAG contents in EVs, particularly of sulfated chondroitin sulfate and heparan sulfate chains. EVs with high GAG content interacted stronger with VSOPs compared to control ones. This was confirmed by experiments with GAG-depleted EVs from genetically modified CHO cells and with uremic rat-derived EVs. Mechanistically, uremic toxin-induced PI3K/AKT-signaling and expression of the sulfate transporter SLC26A2 in host cells contributed to high GAG contents in EVs. In conclusion, uremic conditions induce enhanced GAG contents in EVs, which entails a stronger interaction with VSOPs. VSOPs might be suitable for radiological imaging of EVs rich in GAGs.

Characterization of renal fibrosis in rats with chronic kidney disease by in vivo tomoelastography.

Chronic kidney disease (CKD) is characterized by structural changes, such as tubular atrophy, renal fibrosis, and glomerulosclerosis, all of which affect the viscoelastic properties of biological tissues. However, detection of renal viscoelasticity changes because diagnostic markers by in vivo elastography lack histopathological validation through animal models. Therefore, we investigated in vivo multiparametric magnetic resonance imaging (mp-MRI), including multifrequency magnetic resonance elastography-based tomoelastography, in the kidneys of 10 rats with adenine-induced CKD and eight healthy controls. Kidney volume (in mm3 ), water diffusivity (apparent diffusion coefficient [ADC] in mm2 /s), shear wave speed (SWS; in m/s; related to stiffness), and wave penetration rate (PR; in m/s; related to inverse viscosity) were quantified by mp-MRI and correlated with histopathologically determined renal fibrosis (collagen area fraction [CAF]; in %). Kidney volume (40% ± 29%, p = 0.009), SWS (11% ± 12%, p = 0.016), and PR (20% ± 15%, p = 0.004) were significantly increased in CKD, which was accompanied by ADC (-24% ± 27%, p = 0.02). SWS, PR, and ADC were correlated with CAF with R = 0.63, 0.75, and -0.5 (all p < 0.05), respectively. In the CKD rats, histopathology showed tubule dilation due to adenine crystal deposition. Collectively, our results suggest that collagen accumulation during CKD progression transforms soft-compliant renal tissue into a more rigid-solid state with reduced water mobility. We hypothesized that tubule dilation-a specific feature of our model-might lead to higher intraluminal pressure, which could also contribute to elevated renal stiffness. Tomoelastography is a promising tool for noninvasively assessing disease progression, detecting biomechanical properties that are sensitive to different pathologic features of CKD.

Chest computed tomography findings typical of COVID-19 pneumonia in Germany as early as 30 December 2019: a case report.

BACKGROUND: The first cases of coronavirus disease 2019 were officially confirmed in Germany and its European neighbors in late January 2020. In France and Italy, there is evidence that coronavirus disease 2019 was spreading as early as December 2019. CASE PRESENTATION: We report on a 71-year-old male patient from Germany who was admitted to our hospital on 30 December 2019 with pneumonia of unclear etiology and chest computed tomography findings typical of COVID-19 pneumonia. CONCLUSION: This case may indicate that coronavirus disease 2019 was already spreading in Germany as early as December 2019.

Visualization of Inflammation in Experimental Colitis by Magnetic Resonance Imaging Using Very Small Superparamagnetic Iron Oxide Particles.

Inflammatory bowel diseases (IBD) comprise mainly ulcerative colitis (UC) and Crohn´s disease (CD). Both forms present with a chronic inflammation of the (gastro) intestinal tract, which induces excessive changes in the composition of the associated extracellular matrix (ECM). In UC, the inflammation is limited to the colon, whereas it can occur throughout the entire gastrointestinal tract in CD. Tools for early diagnosis of IBD are still very limited and highly invasive and measures for standardized evaluation of structural changes are scarce. To investigate an efficient non-invasive way of diagnosing intestinal inflammation and early changes of the ECM, very small superparamagnetic iron oxide nanoparticles (VSOPs) in magnetic resonance imaging (MRI) were applied in two mouse models of experimental colitis: the dextran sulfate sodium (DSS)-induced colitis and the transfer model of colitis. For further validation of ECM changes and inflammation, tissue sections were analyzed by immunohistochemistry. For in depth ex-vivo investigation of VSOPs localization within the tissue, Europium-doped VSOPs served to visualize the contrast agent by imaging mass cytometry (IMC). VSOPs accumulation in the inflamed colon wall of DSS-induced colitis mice was visualized in T2* weighted MRI scans. Components of the ECM, especially the hyaluronic acid content, were found to influence VSOPs binding. Using IMC, co-localization of VSOPs with macrophages and endothelial cells in colon tissue was shown. In contrast to the DSS model, colonic inflammation could not be visualized with VSOP-enhanced MRI in transfer colitis. VSOPs present a potential contrast agent for contrast-enhanced MRI to detect intestinal inflammation in mice at an early stage and in a less invasive manner depending on hyaluronic acid content.

ADAMTS4-specific MR probe to assess aortic aneurysms in vivo using synthetic peptide libraries.

The incidence of abdominal aortic aneurysms (AAAs) has substantially increased during the last 20 years and their rupture remains the third most common cause of sudden death in the cardiovascular field after myocardial infarction and stroke. The only established clinical parameter to assess AAAs is based on the aneurysm size. Novel biomarkers are needed to improve the assessment of the risk of rupture. ADAMTS4 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 4) is a strongly upregulated proteoglycan cleaving enzyme in the unstable course of AAAs. In the screening of a one-bead-one-compound library against ADAMTS4, a low-molecular-weight cyclic peptide is discovered with favorable properties for in vivo molecular magnetic resonance imaging applications. After identification and characterization, it's potential is evaluated in an AAA mouse model. The ADAMTS4-specific probe enables the in vivo imaging-based prediction of aneurysm expansion and rupture.

Fibrin-targeting molecular MRI in inflammatory CNS disorders.

BACKGROUND: Fibrin deposition is a fundamental pathophysiological event in the inflammatory component of various CNS disorders, such as multiple sclerosis (MS) and Alzheimer's disease. Beyond its traditional role in coagulation, fibrin elicits immunoinflammatory changes with oxidative stress response and activation of CNS-resident/peripheral immune cells contributing to CNS injury. PURPOSE: To investigate if CNS fibrin deposition can be determined using molecular MRI, and to assess its capacity as a non-invasive imaging biomarker that corresponds to inflammatory response and barrier impairment. MATERIALS AND METHODS: Specificity and efficacy of a peptide-conjugated Gd-based molecular MRI probe (EP2104-R) to visualise and quantify CNS fibrin deposition were evaluated. Probe efficacy to specifically target CNS fibrin deposition in murine adoptive-transfer experimental autoimmune encephalomyelitis (EAE), a pre-clinical model for MS (n = 12), was assessed. Findings were validated using immunohistochemistry and laser ablation inductively coupled plasma mass spectrometry. Deposition of fibrin in neuroinflammatory conditions was investigated and its diagnostic capacity for disease staging and monitoring as well as quantification of immunoinflammatory response was determined. Results were compared using t-tests (two groups) or one-way ANOVA with multiple comparisons test. Linear regression was used to model the relationship between variables. RESULTS: For the first time (to our knowledge), CNS fibrin deposition was visualised and quantified in vivo using molecular imaging. Signal enhancement was apparent in EAE lesions even 12-h after administration of EP2104-R due to targeted binding (M ± SD, 1.07 ± 0.10 (baseline) vs. 0.73 ± 0.09 (EP2104-R), p = .008), which could be inhibited with an MRI-silent analogue (M ± SD, 0.60 ± 0.14 (EP2104-R) vs. 0.96 ± 0.13 (EP2104-La), p = .006). CNS fibrin deposition corresponded to immunoinflammatory activity (R2 = 0.85, p < .001) and disability (R2 = 0.81, p < .001) in a model for MS, which suggests a clinical role for staging and monitoring. Additionally, EP2104-R showed substantially higher SNR (M ± SD, 6.6 ± 1 (EP2104-R) vs. 2.7 ± 0.4 (gadobutrol), p = .004) than clinically used contrast media, which increases sensitivity for lesion detection. CONCLUSIONS: Molecular imaging of CNS fibrin deposition provides an imaging biomarker for inflammatory CNS pathology, which corresponds to pathophysiological ECM remodelling and disease activity, and yields high signal-to-noise ratio, which can improve diagnostic neuroimaging across several neurological diseases with variable degrees of barrier impairment.

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

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.

Investigating the Role of Sulfate Groups for the Binding of Gd3+ Ions to Glycosaminoglycans with NMR Relaxometry.

Glycosaminoglycans (GAGs) are highly negatively charged macromolecules with a large cation binding capacity, but their interaction potential with exogeneous Gd3+ ions is under-investigated. These might be released from chelates used as Gadolinium-based contrast agents (GBCAs) for clinical MR imaging due to transmetallation with endogenous cations like Zn2+ . Recent studies have quantified how an endogenous GAG sequesters released Gd3+ ions and impacts the thermodynamic and kinetic stability of some GBCAs. In this study, we investigate and compare the chelation ability of two important GAGs (heparin and chondroitin sulfate), as well as the homopolysaccharides dextran and dextran sulfate that are used as models for alternative macromolecular chelators. Our combined approach of MRI-based relaxometry and isothermal titration calorimetry shows that the chelation process of Gd3+ into GAGs is not just a long-range electrostatic interaction as proposed for the Manning model, but presumably a site-specific binding. Furthermore, our results highlight the crucial role of sulfate groups in this process and indicate that the potential of a specific GAG to engage in this mechanism increases with its degree of sulfation. The transchelation of Gd3+ ions from GBCAs to sulfated GAGs should thus be considered as one possible explanation for the observed long-term deposition of Gd3+ in vivo and related observations of long-term signal enhancements on T1 -weighted MR images.

Microscopic multifrequency magnetic resonance elastography of ex vivo abdominal aortic aneurysms for extracellular matrix imaging in a mouse model.

An abdominal aortic aneurysm (AAA) is a permanent dilatation of the abdominal aorta, usually accompanied by thrombus formation. The current clinical imaging modalities cannot reliably visualize the thrombus composition. Remodeling of the extracellular matrix (ECM) during AAA development leads to stiffness changes, providing a potential imaging marker. 14 apolipoprotein E-deficient mice underwent surgery for angiotensin II-loaded osmotic minipump implantation. 4 weeks post-op, 5 animals developed an AAA. The aneurysm was imaged ex vivo by microscopic multifrequency magnetic resonance elastography (µMMRE) with an in-plane resolution of 40 microns. Experiments were performed on a 7-Tesla preclinical magnetic resonance imaging scanner with drive frequencies between 1000 Hz and 1400 Hz. Shear wave speed (SWS) maps indicating stiffness were computed based on tomoelastography multifrequency inversion. As control, the aortas of 5 C57BL/6J mice were examined with the same imaging protocol. The regional variation of SWS in the thrombus ranging from 0.44 ± 0.07 to 1.20 ± 0.31 m/s was correlated fairly strong with regional histology-quantified ECM accumulation (R2 = 0.79). Our results suggest that stiffness changes in aneurysmal thrombus reflect ECM remodeling, which is critical for AAA risk assessment. In the future, µMMRE could be used for a mechanics-based clinical characterization of AAAs in patients. STATEMENT OF SIGNIFICANCE: To our knowledge, this is the first study mapping the stiffness of abdominal aortic aneurysms with microscopic resolution of 40 µm. Our work revealed that stiffness critically changes due to extracellular matrix (ECM) remodeling in the aneurysmal thrombus. We were able to image various levels of ECM remodeling in the aneurysm reflected in distinct shear wave speed patterns with a strong correlation to regional histology-quantified ECM accumulation. The generated results are significant for the application of microscopic multifrequency magnetic resonance elastography for quantification of pathological remodeling of the ECM and may be of great interest for detailed characterization of AAAs in patients.

Screening human lung cancer with predictive models of serum magnetic resonance spectroscopy metabolomics.

The current high mortality of human lung cancer stems largely from the lack of feasible, early disease detection tools. An effective test with serum metabolomics predictive models able to suggest patients harboring disease could expedite triage patient to specialized imaging assessment. Here, using a training-validation-testing-cohort design, we establish our high-resolution magic angle spinning (HRMAS) magnetic resonance spectroscopy (MRS)-based metabolomics predictive models to indicate lung cancer presence and patient survival using serum samples collected prior to their disease diagnoses. Studied serum samples were collected from 79 patients before (within 5.0 y) and at lung cancer diagnosis. Disease predictive models were established by comparing serum metabolomic patterns between our training cohorts: patients with lung cancer at time of diagnosis, and matched healthy controls. These predictive models were then applied to evaluate serum samples of our validation and testing cohorts, all collected from patients before their lung cancer diagnosis. Our study found that the predictive model yielded values for prior-to-detection serum samples to be intermediate between values for patients at time of diagnosis and for healthy controls; these intermediate values significantly differed from both groups, with an F1 score = 0.628 for cancer prediction. Furthermore, values from metabolomics predictive model measured from prior-to-diagnosis sera could significantly predict 5-y survival for patients with localized disease.

An NMR relaxometry approach for quantitative investigation of the transchelation of gadolinium ions from GBCAs to a competing macromolecular chelator.

Gadolinium-based contrast agents (GBCAs) have been used in clinical Magnetic Resonance Imaging (MRI) for more than 30 years. However, there is increasing evidence that their dissociation in vivo leads to long-term depositions of gadolinium ions in the human body. In vitro experiments provide critical insights into kinetics and thermodynamic equilibria of underlying processes, which give hints towards the in vivo situation. We developed a time-resolved MRI relaxometry-based approach that exploits distinct relaxivities of Gd3+ in different molecular environments. Its applicability to quantify the transmetallation of GBCAs, the binding of Gd3+ to competing chelators, and the combined transchelation process is demonstrated. Exemplarily, the approach is applied to investigate two representative GBCAs in the presence of Zn2+ and heparin, which is used as a model for a macromolecular and physiologically occurring chelator. Opposing indirect impacts of heparin on increasing the kinetic stability but reducing the thermodynamic stability of GBCAs are observed. The relaxivity of resulting Gd-heparin complexes is shown to be essentially increased compared to that of the parent GBCAs so that they might be one explanation for observed long-term MRI signal enhancement in vivo. In forthcoming studies, the presented method could help to identify the most potent Gd-complexing macromolecular species.

Assessment of Albumin ECM Accumulation and Inflammation as Novel In Vivo Diagnostic Targets for Multi-Target MR Imaging.

Atherosclerosis is a progressive inflammatory vascular disease characterized by endothelial dysfunction and plaque burden. Extracellular matrix (ECM)-associated plasma proteins play an important role in disease development. Our magnetic resonance imaging (MRI) study investigates the feasibility of using two different molecular MRI probes for the simultaneous assessment of ECM-associated intraplaque albumin deposits caused by endothelial damage and progressive inflammation in atherosclerosis. Male apolipoprotein E-deficient (ApoE-/-)-mice were fed a high-fat diet (HFD) for 2 or 4 months. Another ApoE-/--group was treated with pravastatin and received a HFD for 4 months. T1- and T2*-weighted MRI was performed before and after albumin-specific MRI probe (gadofosveset) administration and a macrophage-specific contrast agent (ferumoxytol). Thereafter, laser ablation inductively coupled plasma mass spectrometry and histology were performed. With advancing atherosclerosis, albumin-based MRI signal enhancement and ferumoxytol-induced signal loss areas in T2*-weighted MRI increased. Significant correlations between contrast-to-noise-ratio (CNR) post-gadofosveset and albumin stain (R2 = 0.78, p < 0.05), and signal loss areas in T2*-weighted MRI with Perls' Prussian blue stain (R2 = 0.83, p < 0.05) were observed. No interference of ferumoxytol with gadofosveset enhancement was detectable. Pravastatin led to decreased inflammation and intraplaque albumin. Multi-target MRI combining ferumoxytol and gadofosveset is a promising method to improve diagnosis and treatment monitoring in atherosclerosis.

Contribution of Tissue Inflammation and Blood-Brain Barrier Disruption to Brain Softening in a Mouse Model of Multiple Sclerosis.

Neuroinflammatory processes occurring during multiple sclerosis cause disseminated softening of brain tissue, as quantified by in vivo magnetic resonance elastography (MRE). However, inflammation-mediated tissue alterations underlying the mechanical integrity of the brain remain unclear. We previously showed that blood-brain barrier (BBB) disruption visualized by MRI using gadolinium-based contrast agent (GBCA) does not correlate with tissue softening in active experimental autoimmune encephalomyelitis (EAE). However, it is unknown how confined BBB changes and other inflammatory processes may determine local elasticity changes. Therefore, we aim to elucidate which inflammatory hallmarks are determinant for local viscoelastic changes observed in EAE brains. Hence, novel multifrequency MRE was applied in combination with GBCA-based MRI or very small superparamagnetic iron oxide particles (VSOPs) in female SJL mice with induced adoptive transfer EAE (n = 21). VSOPs were doped with europium (Eu-VSOPs) to facilitate the post-mortem analysis. Accumulation of Eu-VSOPs, which was previously demonstrated to be sensitive to immune cell infiltration and ECM remodeling, was also found to be independent of GBCA enhancement. Following registration to a reference brain atlas, viscoelastic properties of the whole brain and areas visualized by either Gd or VSOP were quantified. MRE revealed marked disseminated softening across the whole brain in mice with established EAE (baseline: 3.1 ± 0.1 m/s vs. EAE: 2.9 ± 0.2 m/s, p < 0.0001). A similar degree of softening was observed in sites of GBCA enhancement i.e., mainly within cerebral cortex and brain stem (baseline: 3.3 ± 0.4 m/s vs. EAE: 3.0 ± 0.5 m/s, p = 0.018). However, locations in which only Eu-VSOP accumulated, mainly in fiber tracts (baseline: 3.0 ± 0.4 m/s vs. EAE: 2.6 ± 0.5 m/s, p = 0.023), softening was more pronounced when compared to non-hypointense areas (percent change of stiffness for Eu-VSOP accumulation: -16.81 ± 16.49% vs. for non-hypointense regions: -5.85 ± 3.81%, p = 0.048). Our findings suggest that multifrequency MRE is sensitive to differentiate between local inflammatory processes with a strong immune cell infiltrate that lead to VSOP accumulation, from disseminated inflammation and BBB leakage visualized by GBCA. These pathological events visualized by Eu-VSOP MRI and MRE may include gliosis, macrophage infiltration, alterations of endothelial matrix components, and/or extracellular matrix remodeling. MRE may therefore represent a promising imaging tool for non-invasive clinical assessment of different pathological aspects of neuroinflammation.

Tailored Magnetic Multicore Nanoparticles for Use as Blood Pool MPI Tracers.

For the preclinical development of magnetic particle imaging (MPI) in general, and the exploration of possible new clinical applications of MPI in particular, tailored MPI tracers with surface properties optimized for the intended use are needed. Here we present the synthesis of magnetic multicore particles (MCPs) modified with polyethylene glycol (PEG) for use as blood pool MPI tracers. To achieve the stealth effect the carboxylic groups of the parent MCP were activated and coupled with pegylated amines (mPEG-amines) with different PEG-chain lengths from 2 to 20 kDa. The resulting MCP-PEG variants with PEG-chain lengths of 10 kDa (MCP-PEG10K after one pegylation step and MCP-PEG10K2 after a second pegylation step) formed stable dispersions and showed strong evidence of a successful reaction of MCP and MCP-PEG10K with mPEG-amine with 10 kDa, while maintaining their magnetic properties. In rats, the mean blood half-lives, surprisingly, were 2 and 62 min, respectively, and therefore, for MCP-PEG10K2, dramatically extended compared to the parent MCP, presumably due to the higher PEG density on the particle surface, which may lead to a lower phagocytosis rate. Because of their significantly extended blood half-life, MCP-PEG10K2 are very promising as blood pool tracers for future in vivo cardiovascular MPI.

Uraemic extracellular vesicles augment osteogenic transdifferentiation of vascular smooth muscle cells via enhanced AKT signalling and PiT-1 expression.

Extracellular vesicles (EV) function as messengers between endothelial cells (EC) and vascular smooth muscle cells (VSMC). Since chronic kidney disease (CKD) increases the risk for vascular calcifications, we investigated whether EV derived from uraemic milieu-stimulated EC and derived from uraemic rats impact the osteogenic transdifferentiation/calcification of VSMC. For that purpose, human EC were treated with urea and indoxyl sulphate or left untreated. Experimental uraemia in rats was induced by adenine feeding. 'Uraemic' and control EV (EVUR ; EVCTRL ) were isolated from supernatants and plasma by using an exosome isolation reagent. Rat VSMC were treated with a pro-calcifying medium (CM) with or without EV supplementation. Gene expressions, miRNA contents and protein expressions were determined by qPCR and Western blots, respectively. Calcifications were determined by colorimetric assays. Delivery of miRNA inhibitors/mimics to EV and siRNA to VSMC was achieved via transfection. EVCTRL and EVUR differed in size and miRNA contents. Contrary to EVCTRL , EC- and plasma-derived EVUR significantly increased the pro-calcifying effects of CM, including altered gene expressions of osterix, runx2, osteocalcin and SM22α. Further, EVUR enhanced the protein expression of the phosphate transporter PiT-1 in VSMC and induced a phosphorylation of AKT and ERK. Knock down of PiT-1 and individual inhibition of AKT and ERK signalling in VSMC blocked the pro-calcifying effects of EVUR . Similar effects were achieved by inhibition of miR-221/-222 and mimicking of miR-143/-145 in EVUR . In conclusion, EVUR might represent an additional puzzle piece of the complex pathophysiology of vascular calcifications in CKD.

Temperatures in Pigs During 3 T MRI Temperatures, Heart Rates, and Breathing Rates of Pigs During RF Power Deposition in a 3 T (128 MHz) Body Coil.

Exposure to radiofrequency (RF) power deposition during magnetic resonance imaging (MRI) induces elevated body-tissue temperatures and may cause changes in heart and breathing rates, disturbing thermoregulation. Eleven temperature sensors were placed in muscle tissue and one sensor in the rectum (measured in 10 cm depth) of 20 free-breathing anesthetized pigs to verify temperature curves during RF exposure. Tissue temperatures and heart and breathing rates were measured before, during, and after RF exposure. Pigs were placed into a 60-cm diameter whole-body resonator of a 3 T MRI system. Nineteen anesthetized pigs were divided into four RF exposure groups: sham (0 W/kg), low-exposure (2.7 W/kg, mean exposure time 56 min), moderate-exposure (4.8 W/kg, mean exposure time 31 min), and high-exposure (4.4 W/kg, mean exposure time 61 min). One pig was exposed to a whole-body specific absorption rate (wbSAR) of 11.4 W/kg (extreme-exposure). Hotspot temperatures, measured by sensor 2, increased by mean 5.0 ± 0.9°C, min 3.9; max 6.3 (low), 7.0 ± 2.3°C, min 4.6; max 9.9 (moderate), and 9.2 ± 4.4°C, min 6.1, max 17.9 (high) compared with 0.3 ± 0.3°C in the sham-exposure group (min 0.1, max 0.6). Four time-temperature curves were identified: sinusoidal, parabolic, plateau, and linear. These curve shapes did not correlate with RF intensity, rectal temperature, breathing rate, or heart rate. In all pigs, rectal temperatures increased (2.1 ± 0.9°C) during and even after RF exposure, while hotspot temperatures decreased after exposure. When rectal temperature increased by 1°C, hotspot temperature increased up to 42.8°C within 37 min (low-exposure) or up to 43.8°C within 24 min (high-exposure). Global wbSAR did not correlate with maximum hotspot. Bioelectromagnetics. 2021;42:37-50. © 2020 The Authors. Bioelectromagnetics published by Wiley Periodicals LLC on behalf of Bioelectromagnetics Society.

Microdistribution of Magnetic Resonance Imaging Contrast Agents in Atherosclerotic Plaques Determined by LA-ICP-MS and SR-µXRF Imaging.

PURPOSE: Contrast-enhanced magnetic resonance imaging (MRI) has the potential to replace angiographic evaluation of atherosclerosis. While studies have investigated contrast agent (CA) uptake in atherosclerotic plaques, exact CA spatial distribution on a microscale is elusive. The purpose of this study was to investigate the microdistribution of gadolinium (Gd)- and iron (Fe) oxide-based CA in atherosclerotic plaques of New Zealand White rabbits. PROCEDURES: The study was performed as a post hoc analysis of archived tissue specimens obtained in a previous in vivo MRI study conducted to investigate signal changes induced by very small superparamagnetic iron oxide nanoparticles (VSOP) and Gd-BOPTA. For analytical discrimination from endogenous Fe, VSOP were doped with europium (Eu) resulting in Eu-VSOP. Formalin-fixed arterial specimens were cut into 5-µm serial sections and analyzed by immunohistochemistry (IHC: Movat's pentachrome, von Kossa, and Alcian blue (pH 1.0) staining, anti-smooth muscle cell actin (anti-SMA), and anti-rabbit macrophage (anti-RAM-11) immunostaining) and elemental microscopy with laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and synchrotron radiation µX-ray fluorescence (SR-µXRF) spectroscopy. Elemental distribution maps of Fe, Eu, Gd, sulfur (S), phosphorus (P), and calcium (Ca) were investigated. RESULTS: IHC characterized atherosclerotic plaque pathomorphology. Elemental microscopy showed S distribution to match the anatomy of arterial vessel wall layers, while P distribution corresponded well with cellular areas. LA-ICP-MS revealed Gd and Fe with a limit of detection of ~ 0.1 nmol/g and ~ 100 nmol/g, respectively. Eu-positive signal identified VSOP presence in the vessel wall and allowed the comparison of Eu-VSOP and endogenous Fe distribution in tissue sections. Extracellular matrix material correlated with Eu signal intensity, Fe concentration, and maximum Gd concentration. Eu-VSOP were confined to endothelium in early lesions but accumulated in cellular areas in advanced plaques. Gd distribution was homogeneous in healthy arteries but inhomogeneous in early and advanced plaques. SR-µXRF scans at 0.5 µm resolution revealed Gd hotspots with increased P and Ca concentrations at the intimomedial interface, and a size distribution ranging from a few micrometers to submicrometers. CONCLUSIONS: Eu-VSOP and Gd have distinct spatial distributions in atherosclerotic plaques. While Eu-VSOP distribution is more cell-associated and might be used to monitor atherosclerotic plaque progression, Gd distribution indicates arterial calcification and might help in characterizing plaque vulnerability.

Molecular MR Imaging of Prostate Cancer.

This review summarizes recent developments regarding molecular imaging markers for magnetic resonance imaging (MRI) of prostate cancer (PCa). Currently, the clinical standard includes MR imaging using unspecific gadolinium-based contrast agents. Specific molecular probes for the diagnosis of PCa could improve the molecular characterization of the tumor in a non-invasive examination. Furthermore, molecular probes could enable targeted therapies to suppress tumor growth or reduce the tumor size.