Visualization of Abscess Formation in a Murine Thigh Infection Model of Staphylococcus aureus by 19F-Magnetic Resonance Imaging (MRI)

Background: During the last years, 19 F-MRI and perfluorocarbon nanoemulsion (PFC) emerged as a powerful contrast agent based MRI methodology to track cells and to visualize inflammation. We applied this new modality to visualize deep tissue abscesses during acute and chronic phase of inflammation caused by Staphylococcus aureus infection. Methodology and Principal Findings: In this study, a murine thigh infection model was used to induce abscess formation and PFC or CLIO (cross linked ironoxides) was administered during acute or chronic phase of inflammation. 24 h after inoculation, the contrast agent accumulation was imaged at the site of infection by MRI. Measurements revealed a strong accumulation of PFC at the abscess rim at acute and chronic phase of infection. The pattern was similar to CLIO accumulation at chronic phase and formed a hollow sphere around the edema area. Histology revealed strong influx of neutrophils at the site of infection and to a smaller extend macrophages during acute phase and strong influx of macrophages at chronic phase of inflammation. Conclusion and Significance: We introduce 19 F-MRI in combination with PFC nanoemulsions as a new platform to visualize abscess formation in a murine thigh infection model of S. aureus. The possibility to track immune cells in vivo by this modality offers new opportunities to investigate host immune response, the efficacy of antibacterial therapies and th

In vivo labelling of resting monocytes in the reticuloendothelial system with fluorescent iron oxide nanoparticles prior to injury reveals that they are mobilized to infarcted myocardium

Aims To evaluate the feasibility of loading resting monocytes/macrophages by intravenous (i.v.) injection of fluorescent iron oxide nanoparticles prior to injury and tracking of these cells in the very same animal to myocardial infarction (MI) by magnetic resonance imaging (MRI) and optical imaging. Methods and results Rats were injected with fluorescent iron oxide nanoparticles (10 mg/kg) (n = 15) prior to injury. After disappearance of the nanoparticles from the blood, MI was induced. Monocytes/macrophages were then tracked in the very same animal by MRI and optical imaging. Control groups were (i) non-injected animals (n = 15), (ii) injected animals associated with a sham operation (n = 8), and (iii) animals treated with an anti-inflammatory agent (n = 6). The presence of iron-loaded cells can be detected by MRI in vivo in the infarcted myocardium. Here, we showed that the detection of inflammatory cells in vivo correlated well with ex vivo imaging (MRI and reflectance fluorescence) and histology. We also showed that the method is robust enough to depict changes in the inflammatory response. Conclusion This study demonstrates that resting monocytes/macrophages can be loaded in vivo by a simple i.v. injection of fluorescent superparamagnetic iron oxide nanoparticles prior to injury and then tracked, in the same animal, in a model of ischaemia-reperfusion leading to myocardial infarct. Although previous studies of macrophages infiltration following MI have labelled the macrophages after injury, this study, for the first time, has pre-load the resting monocytes with fluorescent iron oxide nanoparticle

Iron Oxide Nanoparticles for Visualization of Prostate Cancer in MRI

Prostate cancer (PCa) is one of the most common cancers in men. For detection and diagnosis of PCa, non-invasive methods, including magnetic resonance imaging (MRI), can reduce the risk potential of surgical intervention. To explore the molecular characteristics of the tumor, we investigated the applicability of ferumoxytol in PCa in a xenograft mouse model in two different tumor volumes, 500 mm3 and 1000 mm3. Macrophages play a key role in tumor progression, and they are able to internalize iron-oxide particles, such as ferumoxytol. When evaluating T2*-weighted sequences on MRI, a significant decrease of signal intensity between pre- and post-contrast images for each tumor volume (n = 14; p < 0.001) was measured. We, furthermore, observed a higher signal loss for a tumor volume of 500 mm3 than for 1000 mm3. These findings were confirmed by histological examinations and laser ablation inductively coupled plasma-mass spectrometry. The 500 mm3 tumors had 1.5% iron content (n = 14; σ = 1.1), while the 1000 mm3 tumors contained only 0.4% iron (n = 14; σ = 0.2). In vivo MRI data demonstrated a correlation with the ex vivo data (R2 = 0.75). The results of elemental analysis by inductively coupled plasma-mass spectrometry correlated strongly with the MRI data (R2 = 0.83) (n = 4). Due to its long retention time in the blood, biodegradability, and low toxicity to patients, ferumoxytol has great potential as a contrast agent for visualization PCa.SonderforschungsbereichDeutsche ForschungsgemeinschaftPeer Reviewe

In Vivo Cellular MRI In Experimental Traumatic Spinal Cord Injury

Spinal cord injury (SCI) remains one of the most devastating conditions in medicine; it is a complex medical condition with no cure currently available. Inflammation plays an important role in SCI as it can have both beneficial and detrimental effects. Cell therapy has emerged as a promising treatment for SCI due to the potential for stem cells, including multipotent mesenchymal stromal cells (MSC), for tissue regeneration and immunomodulation of the inflammatory cascade after the initial trauma. However, there are still important, unresolved questions regarding cell therapy that magnetic resonance imaging (MRI) can help to address by producing high-resolution images with exquisite soft tissue contrast in a non-invasive, non-destructive and three-dimensional (3D) manner, allowing a dynamic view of changing pathology and cellular events in vivo. In this thesis in vivo longitudinal imaging of SCI in mouse and rat models is presented using MRI. A resolution of 200μm in all three planes was achieved using a balanced steady state free precession (bSSFP) pulse sequence in a 3T whole-body clinical scanner. Using iron oxide particles as a contrast agent, cellular MRI was used to assess direct MSC transplantation in a mouse model and acute inflammation in a rat model. This was the first study to use cellular MRI for cell tracking in a mouse SCI model. We report on the use of cellular MRI to locate transplanted cells and monitor their overall distribution as well as to evaluate the delivery of transplanted cells to the target tissue in the early phase. Limitations of long-term cell tracking using iron oxide are also discussed. This is also the first study using cellular MRI to image in vivo cells associated with the inflammatory response within the lesion in a rat SCI model and the first demonstration of the use of bSSFP at 3T for rat body imaging. Having the tools for longitudinal in vivo cell monitoring in SCI will help gain a better understanding of both inflammation and response to cell therapy. As these tools are refined, they can be used to test different potential treatments for SCI and optimize them

IN VIVO LABELING OF IMMUNE CELLS FOR TRACKING USING MAGNETIC RESONANCE IMAGING

In the last two decades, iron oxide nanoparticles have been widely used to label cells in vivo for MRI-based tracking of cellular infiltration and inflammation in disease and injury. The goal of this thesis was to investigate the potential for in vivo labeling of immune cells in healthy mice. Healthy mice were administered iron based contrast agents intravenously (i.v) and MRI was performed longitudinally to detect and monitor signal changes in the liver, spleen, bone marrow and lymph nodes. Histology and flow cytometry were used to verify the presence of iron within cells. We show that cells in the bone marrow and spleen take up iron particles after the i.v administration of either superparamagnetic iron oxide (SPIO) or micron-sized iron oxide (MPIO) particles. This work sets the stage for future studies which aim to monitor the trafficking or recruitment of pre-labeled immune cells in models of inflammatory disease and immune disorders

MRI of myocardial infarction using lipid-based contrast agents

Ischemic heart disease is the leading cause of death worldwide. Occlusion of a coronary artery results in cardiac ischemia downstream, which leads to irreversible myocardial cell death. If patients survive the ischemic event, infarct healing and global cardiac remodeling take place. A dynamic cascade of events is initiated, which is characterized by four distinct phases: cell death, inflammation, the formation of granulation tissue and finally fibrosis (chapter 1). The main goal of this thesis was to develop and in vivo apply paramagnetic lipid-based contrast agents for contrast-enhanced MRI of murine myocardial infarction. The visualization of specific processes in myocardial infarction could give insight in pathophysiological mechanisms, predict outcome and provide readout of therapy efficacy. Furthermore, the nanoparticulate contrast materials are very promising as theranostics agents. Previously, several (targeted) contrast agents for the visualization of cell death, inflammation, angiogenesis and fibrosis have been developed. However, these are still not routinely applied. Moreover, MRI measurement protocols and sequences specifically aimed at contrast-enhanced MRI of the mouse heart have been developed. The current status of contrast-enhanced MRI of murine myocardial infarction is extensively reviewed in chapters 2 and 3. In chapter 4 the distribution and accumulation kinetics of paramagnetic micelles and liposomes in a mouse model of cardiac ischemia reperfusion (IR) injury was studied. In vivo T1-weighted MRI and high-resolution ex vivo fluorescence microscopy revealed that both types of nanoparticles accumulated specifically in the infarcted myocardium in both acute (day 1) and chronic (week 1 and 2) IR injury. Micelles displayed faster accumulation kinetics compared to liposomes, which is most probably related to their smaller size. Furthermore, liposomes sometimes co-localized with vessels and inflammatory cells, whereas this was not observed for micelles. Due to the specific accumulation in infarcted myocardium, the presented lipid-based nanoparticles are a promising platform for drug delivery to infarcted myocardium. Although reperfusion therapy limits infarct size, it also promotes apoptosis, resulting in adverse secondary IR injury. Visualization of apoptotic cells could aid in the detection of IR injury and the indication of potentially salvageable tissue. For this purpose, the potential of annexin A5 (anxA5)-functionalized liposomes was explored (chapter 5). AnxA5 is a protein that specifically binds to phosphatidylserine expressed by apoptotic cells. AnxA5-liposmes were injected in mice with IR injury and T1-weighted and cine MRI were performed 24 h later. Both anxA5-liposomes and non-functionalized liposomes accumulated in the infarcted myocardium, leading to similar signal intensities in the remote and infarct regions and a comparable distribution of enhanced pixels. Careful comparison of cine MR measurements and T1-weighted MR images revealed that anxA5-liposomes accumulated to a higher degree in less severely infarcted myocardium, whereas non-functionalized liposomes preferentially accumulated in severely infarcted myocardium. Ex vivo high-resolution microscopy confirmed these in vivo results. Therefore, anxA5-liposomes might be useful for drug delivery to potentially salvageable myocardial tissue. Inflammatory cells are key regulators in myocardial infarct healing and in adverse left ventricular remodeling. Therefore, a non-invasive method for imaging of inflammatory cells could provide information on infarct status and outcome. To this end paramagnetic phosphatidylserine (PS)-containing liposomes were developed. Inflammatory cells recognize PS expressed by apoptotic cells and subsequently engulf the dying cells. The association of liposomes to murine macrophages was determined in vitro (chapter 6). Liposomes containing 6 mol% of PS showed a higher association with macrophages compared to control-liposomes without PS. Furthermore, this association was Ca2+- and Mg2+-dependent and PS-containing liposomes were predominantly internalized by macrophages, whereas control-liposomes only bound to the macrophage cell membrane. Due to the enhanced in vitro uptake by macrophages, PS-containing liposomes might be suitable for in vivo visualization of macrophage content. Therefore, these liposomes were applied in a mouse model of IR injury (chapter 7). When PS-liposomes circulated for 2.5 h, the signal change on T1-weighted MR images was lower compared to mice injected with liposomes without PS. This is explained by the shorter blood-circulation half-life of PS-containing liposomes. Nevertheless, high-resolution ex vivo fluorescence microscopy revealed some co-localization of PS-liposomes and macrophages, while this was not observed for liposomes without PS. After 24 h of circulation both types of liposomes accumulated in the infarcted myocardium resulting in similar signal changes. These findings were confirmed by in vivo T1 mapping as well. Due to the specific association of PS-containing liposomes and inflammatory cells 2.5 h after administration, these nanoparticles might be suitable for drug delivery to inflammatory cells in the infarcted myocardium. Finally, chapter 8 concludes with a general discussion on the preceding chapters and some future perspective in the field of contrast-enhanced MRI of myocardial infarction

Non-invasive tracking of T cell recruitment to the tumor microenvironment in a murine glioma model by high field cellular magnetic resonance imaging

Gliomas are characterized by increased T cell exhaustion and poor T cell infiltration into the tumor as well as an overall highly immunosuppressive tumor microenvironment (TME). Response rates in preclinical glioma models and patients to promising new therapeutic approaches in the field of immunotherapies remain heterogenous. These include checkpoint blockade, peptide and mRNA vaccines and adoptive therapy with chimeric antigen receptor (CAR) or T cell receptor (TCR)-transgenic T cells. This demonstrates the need for non-invasive tracking of T cell recruitment to the TME in order to monitor immunotherapies, adapt therapeutic strategies and predict treatment outcome. Iron oxide nanoparticles (NP) can be visualized non-invasively by magnetic resonance imaging (MRI) and dedicated MRI sequences such as T2* mapping. Using isolated murine T cell cultures I show that labeling of T cells with iron oxide NPs as contrast agents is feasible and does not impair T cell viability and functionality as assessed by cytokine secretion and antigen-specific killing activity in vitro. I demonstrate that adoptively transferred T cells can be visualized intratumorally in a murine glioma model by high field MRI at 9.4 Tesla with high sensitivity and that T cells can be tracked non-invasively in a time course of at least two weeks. Correlative methods include immunohistochemistry, flow cytometry, tissue clearing and light sheet microscopy. Tumor relaxation times at an early time point after adoptive cell transfer (ACT) were a predictor for tumor response or resistance, which demonstrates that non-invasive quantification of spatial and temporal T cell dynamics in the TME can facilitate immune cell monitoring to assess immunotherapy efficacy

A nanoparticle ink allowing the high precision visualization of tissue engineered scaffolds by MRI

Hydrogels are widely used as cell scaffolds in several biomedical applications. Once implanted in vivo, cell scaffolds must often be visualized, and monitored overtime. However, cell scaffolds appear poorly contrasted in most biomedical imaging modalities such as magnetic resonance imaging (MRI). MRI is the imaging technique of choice for high-resolution visualization of low-density, water-rich tissues. Attempts to enhance hydrogel contrast in MRI are performed with “negative” contrast agents that produce several image artifacts impeding the delineation of the implant’s contours. In this study, a magnetic ink based on ultra-small iron oxide nanoparticles (USPIONs; <5 nm diameter cores) is developed and integrated into biocompatible alginate hydrogel used in cell scaffolding applications. Relaxometric properties of the magnetic hydrogel are measured, as well as biocompatibility and MR-visibility (T1-weighted mode; in vitro and in vivo). A 2-week MR follow-up study is performed in the mouse model, demonstrating no image artifacts, and the retention of “positive” contrast overtime, which allows very precise delineation of tissue grafts with MRI. Finally, a 3D-contouring procedure developed to facilitate graft delineation and geometrical conformity assessment is applied on an inverted template alginate pore network. This proof-of-concept establishes the possibility to reveal precisely engineered hydrogel structures using this USPIONs ink high-visibility approach

In Vivo Mapping of Vascular Inflammation Using Multimodal Imaging

Plaque vulnerability to rupture has emerged as a critical correlate to risk of adverse coronary events but there is as yet no clinical method to assess plaque stability in vivo. In the search to identify biomarkers of vulnerable plaques an association has been found between macrophages and plaque stability--the density and pattern of macrophage localization in lesions is indicative of probability to rupture. In very unstable plaques, macrophages are found in high densities and concentrated in the plaque shoulders. Therefore, the ability to map macrophages in plaques could allow noninvasive assessment of plaque stability. We use a multimodality imaging approach to noninvasively map the distribution of macrophages in vivo. The use of multiple modalities allows us to combine the complementary strengths of each modality to better visualize features of interest. Our combined use of Positron Emission Tomography and Magnetic Resonance Imaging (PET/MRI) allows high sensitivity PET screening to identify putative lesions in a whole body view, and high resolution MRI for detailed mapping of biomarker expression in the lesions.Macromolecular and nanoparticle contrast agents targeted to macrophages were developed and tested in three different mouse and rat models of atherosclerosis in which inflamed vascular plaques form spontaneously and/or are induced by injury. For multimodal detection, the probes were designed to contain gadolinium (T1 MRI) or iron oxide (T2 MRI), and Cu-64 (PET). PET imaging was utilized to identify regions of macrophage accumulation; these regions were further probed by MRI to visualize macrophage distribution at high resolution. In both PET and MR images the probes enhanced contrast at sites of vascular inflammation, but not in normal vessel walls. MRI was able to identify discrete sites of inflammation that were blurred together at the low resolution of PET. Macrophage content in the lesions was confirmed by histology.The multimodal imaging approach allowed high-sensitivity and high-resolution mapping of biomarker distribution and may lead to a clinical method to predict plaque probability to rupture