In-vivo tracking of deuterium metabolism in mouse organs using LC-MS/MS.

Mass spectrometry-based metabolomics with stable isotope labeling (SIL) is an established tool for sensitive and precise analyses of tissue metabolism, its flux, and pathway activities in diverse models of physiology and disease. Despite the simplicity and broad applicability of deuterium (2H)-labeled precursors for tracing metabolic pathways with minimal biological perturbations, they are rarely employed in LC-MS/MS-guided metabolomics. In this study, we have developed a LC-MS/MS-guided workflow to trace deuterium metabolism in mouse organs following 2H7 -glucose infusion. The workflow includes isotopically labeled glucose infusion, mouse organ isolation and metabolite extraction, zwitterion-based hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry, targeted data acquisition for sensitive detection of deuterated metabolites, a spectral library of over 400 metabolite standards, and multivariate data analysis with pathway mapping. The optimized method was validated for matrix effects, normalization, and quantification to provide both tissue metabolomics and tracking the in-vivo metabolic fate of deuterated glucose through key metabolic pathways. We quantified more than 100 metabolites in five major mouse organ tissues (liver, kidney, brain, brown adipose tissue, and heart). Furthermore, we mapped isotopologues of deuterated metabolites from glycolysis, tricarboxylic acid (TCA) cycle, and amino acid pathways, which are significant for studying both health and various diseases. This study will open new avenues in LC-MS based analysis of 2H-labeled tissue metabolism research in animal models and clinical settings.

Noninvasive assessment of metabolic turnover during inflammation by in vivo deuterium magnetic resonance spectroscopy.

Background: Inflammation and metabolism exhibit a complex interplay, where inflammation influences metabolic pathways, and in turn, metabolism shapes the quality of immune responses. Here, glucose turnover is of special interest, as proinflammatory immune cells mainly utilize glycolysis to meet their energy needs. Noninvasive approaches to monitor both processes would help elucidate this interwoven relationship to identify new therapeutic targets and diagnostic opportunities. Methods: For induction of defined inflammatory hotspots, LPS-doped Matrigel plugs were implanted into the neck of C57BL/6J mice. Subsequently, 1H/19F magnetic resonance imaging (MRI) was used to track the recruitment of 19F-loaded immune cells to the inflammatory focus and deuterium (2H) magnetic resonance spectroscopy (MRS) was used to monitor the metabolic fate of [6,6-2H2]glucose within the affected tissue. Histology and flow cytometry were used to validate the in vivo data. Results: After plug implantation and intravenous administration of the 19F-containing contrast agent, 1H/19F MRI confirmed the infiltration of 19F-labeled immune cells into LPS-doped plugs while no 19F signal was observed in PBS-containing control plugs. Identification of the inflammatory focus was followed by i.p. bolus injection of deuterated glucose and continuous 2H MRS. Inflammation-induced alterations in metabolic fluxes could be tracked with an excellent temporal resolution of 2 min up to approximately 60 min after injection and demonstrated a more anaerobic glucose utilization in the initial phase of immune cell recruitment. Conclusion: 1H/2H/19F MRI/MRS was successfully employed for noninvasive monitoring of metabolic alterations in an inflammatory environment, paving the way for simultaneous in vivo registration of immunometabolic data in basic research and patients.

Biodegradable polyphosphoester micelles act as both background-free 31P magnetic resonance imaging agents and drug nanocarriers.

In vivo monitoring of polymers is crucial for drug delivery and tissue regeneration. Magnetic resonance imaging (MRI) is a whole-body imaging technique, and heteronuclear MRI allows quantitative imaging. However, MRI agents can result in environmental pollution and organ accumulation. To address this, we introduce biocompatible and biodegradable polyphosphoesters, as MRI-traceable polymers using the 31P centers in the polymer backbone. We overcome challenges in 31P MRI, including background interference and low sensitivity, by modifying the molecular environment of 31P, assembling polymers into colloids, and tailoring the polymers' microstructure to adjust MRI-relaxation times. Specifically, gradient-type polyphosphonate-copolymers demonstrate improved MRI-relaxation times compared to homo- and block copolymers, making them suitable for imaging. We validate background-free imaging and biodegradation in vivo using Manduca sexta. Furthermore, encapsulating the potent drug PROTAC allows using these amphiphilic copolymers to simultaneously deliver drugs, enabling theranostics. This first report paves the way for polyphosphoesters as background-free MRI-traceable polymers for theranostic applications.

High-throughput screening of caterpillars as a platform to study host-microbe interactions and enteric immunity.

Mammalian models of human disease are expensive and subject to ethical restrictions. Here, we present an independent platform for high-throughput screening, using larvae of the tobacco hornworm Manduca sexta, combining diagnostic imaging modalities for a comprehensive characterization of aberrant phenotypes. For validation, we use bacterial/chemical-induced gut inflammation to generate a colitis-like phenotype and identify significant alterations in morphology, tissue properties, and intermediary metabolism, which aggravate with disease progression and can be rescued by antimicrobial treatment. In independent experiments, activation of the highly conserved NADPH oxidase DUOX, a key mediator of gut inflammation, leads to similar, dose-dependent alterations, which can be attenuated by pharmacological interventions. Furthermore, the developed platform could differentiate pathogens from mutualistic gastrointestinal bacteria broadening the scope of applications also to microbiomics and host-pathogen interactions. Overall, larvae-based screening can complement mammals in preclinical studies to explore innate immunity and host-pathogen interactions, thus representing a substantial contribution to improve mammalian welfare.

Diabetes disturbs functional adaptation of the remote myocardium after ischemia/reperfusion.

Diabetes mellitus type 2 is associated with adverse clinical outcome after myocardial infarction. To better understand the underlying causes we here investigated sarcomere protein function and its calcium-dependent regulation in the non-ischemic remote myocardium (RM) of diabetic mice (db/db) after transient occlusion of the left anterior descending coronary artery. Before and 24 h after surgery db/db and non-diabetic db/+ underwent magnetic resonance imaging followed by histological and biochemical analyses of heart tissue. Intracellular calcium transients and sarcomere function were measured in isolated cardiomyocytes. Active and passive force generation was assessed in skinned fibers and papillary muscle preparations. Before ischemia and reperfusion (I/R), beat-to-beat calcium cycling was depressed in diabetic cardiomyocytes. Nevertheless, contractile function was preserved owing to increased myofilament calcium sensitivity and higher responsiveness of myocardial force production to ß-adrenergic stimulation in db/db compared to db/+. In addition, protein kinase C activity was elevated in db/db hearts leading to strong phosphorylation of the titin PEVK region and increased titin-based tension of myofilaments. I/R impaired the function of whole hearts and RM sarcomeres in db/db to a larger extent than in non-diabetic db/+, and we identified several reasons. First, the amplitude and the kinetics of cardiomyocyte calcium transients were further reduced in the RM of db/db. Underlying causes involved altered expression of calcium regulatory proteins. Diabetes and I/R additively reduced phospholamban S16-phosphorylation by 80% (P < 000.1) leading to strong inhibition of the calcium ATPase SERCA2a. Second, titin stiffening was only observed in the RM of db/+, but not in the RM of db/db. Finally, db/db myofilament calcium sensitivity and force generation upon ß-adrenergic stimulation were no longer enhanced over db/+ in the RM. The findings demonstrate that impaired cardiomyocyte calcium cycling of db/db hearts is compensated by increased myofilament calcium sensitivity and increased titin-based stiffness prior to I/R. In contrast, sarcomere function of the RM 24 h after I/R is poor because both these compensatory mechanisms fail and myocyte calcium handling is further depressed.

Multiparametric MRI identifies subtle adaptations for demarcation of disease transition in murine aortic valve stenosis.

Aortic valve stenosis (AS) is the most frequent valve disease with relevant prognostic impact. Experimental model systems for AS are scarce and comprehensive imaging techniques to simultaneously quantify function and morphology in disease progression are lacking. Therefore, we refined an acute murine AS model to closely mimic human disease characteristics and developed a high-resolution magnetic resonance imaging (MRI) approach for simultaneous in-depth analysis of valvular, myocardial as well as aortic morphology/pathophysiology to identify early changes in tissue texture and critical transition points in the adaptive process to AS. AS was induced by wire injury of the aortic valve. Four weeks after surgery, cine loops, velocity, and relaxometry maps were acquired at 9.4 T to monitor structural/functional alterations in valve, aorta, and left ventricle (LV). In vivo MRI data were subsequently validated by histology and compared to echocardiography. AS mice exhibited impaired valve opening accompanied by significant valve thickening due to fibrotic remodelling. While control mice showed bell-shaped flow profiles, AS resulted not only in higher peak flow velocities, but also in fragmented turbulent flow patterns associated with enhanced circumferential strain and an increase in wall thickness of the aortic root. AS mice presented with a mild hypertrophy but unaffected global LV function. Cardiac MR relaxometry revealed reduced values for both T1 and T2 in AS reflecting subtle myocardial tissue remodelling with early alterations in mitochondrial function in response to the enhanced afterload. Concomitantly, incipient impairments of coronary flow reserve and myocardial tissue integrity get apparent accompanied by early troponin release. With this, we identified a premature transition point with still compensated cardiac function but beginning textural changes. This will allow interventional studies to explore early disease pathophysiology and novel therapeutic targets.

Dynamic monitoring of vital functions and tissue re-organization in Saturnia pavonia (Lepidoptera, Saturniidae) during final metamorphosis by non-invasive MRI.

Magnetic resonance imaging (MRI) is the key whole-body imaging technology for observing processes within a living object providing excellent resolution and contrast between soft tissues. In the present work, we exploited the non-destructive properties of MRI to track longitudinally the dynamic changes that take place in developing pupae of the Emperor Moth (Saturnia pavonia) during the last days before eclosion. While in diapause pupae, body fluid was almost homogeneously distributed over the internal compartments, as soon as wings, legs, flight muscles and the head region were fully developed, a significant redistribution of water levels occurred between thoracic and abdominal regions. During the last two days before eclosion, the developing moths transferred substantial amounts of liquid into the gut and the labial gland, and in case of females, into developing eggs. Concomitantly, the volume of the air sacs increased drastically and their expansion/compression became clearly visible in time-resolved MR images. Furthermore, besides ventilation of the tracheal system, air sacs are likely to serve as volume reservoir for liquid transfer during development of the moths inside their pupal case. In parallel, we were able to monitor noninvasively lipid consumption, cardiac activity and haemolymph circulation during final metamorphosis.

Multi-targeted 1H/19F MRI unmasks specific danger patterns for emerging cardiovascular disorders.

Prediction of the transition from stable to acute coronary syndromes driven by vascular inflammation, thrombosis with subsequent microembolization, and vessel occlusion leading to irreversible myocardial damage is still an unsolved problem. Here, we introduce a multi-targeted and multi-color nanotracer platform technology that simultaneously visualizes evolving danger patterns in the development of progressive coronary inflammation and atherothrombosis prior to spontaneous myocardial infarction in mice. Individual ligand-equipped perfluorocarbon nanoemulsions are used as targeting agents and are differentiated by their specific spectral signatures via implementation of multi chemical shift selective 19F MRI. Thereby, we are able to identify areas at high risk of and predictive for consecutive development of myocardial infarction, at a time when no conventional parameter indicates any imminent danger. The principle of this multi-targeted approach can easily be adapted to monitor also a variety of other disease entities and constitutes a technology with disease-predictive potential.

Phagocytosis of a PFOB-Nanoemulsion for 19F Magnetic Resonance Imaging: First Results in Monocytes of Patients with Stable Coronary Artery Disease and ST-Elevation Myocardial Infarction.

Fluorine-19 magnetic resonance imaging (19F MRI) with intravenously applied perfluorooctyl bromide-nanoemulsions (PFOB-NE) has proven its feasibility to visualize inflammatory processes in experimental disease models. This approach is based on the properties of monocytes/macrophages to ingest PFOB-NE particles enabling specific cell tracking in vivo. However, information on safety (cellular function and viability), mechanism of ingestion and impact of specific disease environment on PFOB-NE uptake is lacking. This information is, however, crucial for the interpretation of 19F MRI signals and a possible translation to clinical application. To address these issues, whole blood samples were collected from patients with acute ST-elevation myocardial infarction (STEMI), stable coronary artery disease (SCAD) and healthy volunteers. Samples were exposed to fluorescently-labeled PFOB-NE and particle uptake, cell viability and migration activity was evaluated by flow cytometry and MRI. We were able to show that PFOB-NE is ingested by human monocytes in a time- and subset-dependent manner via active phagocytosis. Monocyte function (migration, phagocytosis) and viability was maintained after PFOB-NE uptake. Monocytes of STEMI and SCAD patients did not differ in their maximal PFOB-NE uptake compared to healthy controls. In sum, our study provides further evidence for a safe translation of PFOB-NE for imaging purposes in humans.

Dissociation of 19F and fluorescence signal upon cellular uptake of dual-contrast perfluorocarbon nanoemulsions.

OBJECTIVE: Perfluorocarbon nanoemulsions (PFCs) tagged with fluorescence dyes have been intensively used to confirm the in vivo 19F magnetic resonance imaging (MRI) localization of PFCs by post mortem histology or flow cytometry. However, only limited data are available on tagged PFCs and the potential dissociation of fluorescence and 19F label after cellular uptake over time. MATERIALS AND METHODS: PFCs were coupled to rhodamine (Rho) or carboxyfluorescein (Cfl) and their fate was analyzed after in vitro uptake by J774, RAW and CHO cells by flow cytometry and 19F MRI. In separate in vivo experiments, the dual-labelled emulsions were intravenously applied into mice and their distribution was monitored in spleen and liver over 24 h. In a final step, time course of fluorescence and 19F signals from injected emulsions were tracked in a local inflammation model making use of a subcutaneous matrigel depot doped with LPS (lipopolysaccharide). RESULTS: Internalization of fluorescence-labelled PFCs was associated with a substantial whitening over 24 h in all macrophage cell lines while the 19F signal remained stable over time. In all experiments, CflPFCs were more susceptible to bleaching than RhoPFCs. After intravenous injection of RhoPFCs, the fluorescence signal in spleen and liver peaked after 30 min and 2 h, respectively, followed by a successive decrease over 24 h, whereas the 19F signal continuously increased during this observation period. Similar results were found in the matrigel/LPS model, where we observed increasing 19F signals in the inflammatory hot spot over time while the fluorescence signal of immune cells isolated from the matrigel depot 24 h after its implantation was only marginally elevated over background levels. This resulted in a massive underestimation of the true PFC deposition in the reticuloendothelial system and at inflammatory hot spots. CONCLUSION: Cellular uptake of fluorescently tagged PFCs leads to a dissociation of the fluorescence and the 19F label signal over time, which critically impacts on interpretation of long-term experiments validated by histology or flow cytometry.