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The endothelin (ET) axis plays a pivotal role in cardiovascular diseases. Enhanced levels of circulating ET-1 have been correlated with an inferior clinical outcome after myocardial infarction (MI) in humans. Thus, the evaluation of endothelin-A receptor (ETAR) expression over time in the course of myocardial injury and healing may offer valuable information toward the understanding of the ET axis involvement in MI. We developed an approach to track the expression of ETAR with a customized molecular imaging probe in a murine model of MI. The small molecular probe based on the ETAR-selective antagonist 3-(1,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)methyl]-2(5H)-furanone (PD156707) was labeled with fluorescent dye, IRDye800cw. Mice undergoing permanent ligation of the left anterior descending artery (LAD) were investigated at day 1, 7, and 21 post surgery after receiving an intravenous injection of the ETAR probe. Cryosections of explanted hearts were analyzed by cryotome-based CCD, and fluorescence reflectance imaging (FRI) and fluorescence signal intensities (SI) were extracted. Fluorescence-mediated tomography (FMT) imaging was performed to visualize probe distribution in the target region in vivo. An enhanced fluorescence signal intensity in the infarct area was detected in cryoCCD images as early as day 1 after surgery and intensified up to 21 days post MI. FRI was capable of detecting significantly enhanced SI in infarcted regions of hearts 7 days after surgery. In vivo imaging by FMT localized enhanced SI in the apex region of infarcted mouse hearts. We verified the localization of the probe and ETAR within the infarct area by immunohistochemistry (IHC). In addition, neovascularized areas were found in the affected myocardium by CD31 staining. Our study demonstrates that the applied fluorescent probe is capable of delineating ETAR expression over time in affected murine myocardium after MI in vivo and ex vivo.

Aims Heart failure is associated with altered myocardial substrate metabolism and impaired cardiac energetics. Comorbidities like diabetes may influence the metabolic adaptations during heart failure development. We quantified to what extent changes in substrate preference, lipid accumulation, and energy status predict the longitudinal development of hypertrophy and failure in the non-diabetic and the diabetic heart.Methods and results Transverse aortic constriction (TAC) was performed in non-diabetic (db/+) and diabetic (db/db) mice to induce pressure overload. Magnetic resonance imaging, P-31 magnetic resonance spectroscopy (MRS), H-1 MRS, and F-18-fluorodeoxyglucose-positron emission tomography (PET) were applied to measure cardiac function, energy status, lipid content, and glucose uptake, respectively. In vivo measurements were complemented with ex vivo techniques of high-resolution respirometry, proteomics, and western blotting to elucidate the underlying molecular pathways. In non-diabetic mice, TAC induced progressive cardiac hypertrophy and dysfunction, which correlated with increased protein kinase D-1 (PKD1) phosphorylation and increased glucose uptake. These changes in glucose utilization preceded a reduction in cardiac energy status. At baseline, compared with non-diabetic mice, diabetic mice showed normal cardiac function, higher lipid content and mitochondrial capacity for fatty acid oxidation, and lower PKD1 phosphorylation, glucose uptake, and energetics. Interestingly, TAC affected cardiac function only mildly in diabetic mice, which was accompanied by normalization of phosphorylated PKD1, glucose uptake, and cardiac energy status.Conclusion The cardiac metabolic adaptations in diabetic mice seem to prevent the heart from failing upon pressure overload, suggesting that restoring the balance between glucose and fatty acid utilization is beneficial for cardiac function.

The tumor microenvironment is highly heterogeneous. For gliomas, the tumor-associated inflammatory response is pivotal to support growth and invasion. Factors of glioma growth, inflammation, and invasion, such as the translocator protein (TSPO) and matrix metalloproteinases (MMP), may serve as specific imaging biomarkers of the glioma microenvironment. In this study, noninvasive imaging by PET with [18F]DPA-714 (TSPO) and [18F]BR-351 (MMP) was used for the assessment of localization and quantification of the expression of TSPO and MMP. Imaging was performed in addition to established clinical imaging biomarker of active tumor volume ([18F]FET) in conjunction with MRI. We hypothesized that each imaging biomarker revealed distinct areas of the heterogeneous glioma tissue in a mouse model of human glioma. Tracers were found to be increased 1.4- to 1.7-fold, with [18F]FET showing the biggest volume as depicted by a thresholding-based, volumes of interest analysis. Tumor areas, which could not be detected by a single tracer and/or MRI parameter alone, were measured. Specific compartments of [18F]DPA-714 (14%) and [18F]BR-351 (11%) volumes along the tumor rim could be identified. [18F]DPA-714 (TSPO) and [18F]BR-351 (MMP) matched with histology. Glioma-associated microglia/macrophages (GAM) were identified as TSPO and MMP sources. Multitracer and multimodal molecular imaging approaches may allow us to gain important insights into glioma-associated inflammation (GAM, MMP). Moreover, this noninvasive technique enables characterization of the glioma microenvironment with respect to the disease-driving cellular compartments at the various disease stages. Cancer Res; 77(8); 1831–41. ©2017 AACR.

Stroke is the most common cause of death and disability from neurologic disease in humans. Activation of microglia and matrix metalloproteinases (MMPs) is involved in positively and negatively affecting stroke outcome. Novel, noninvasive, multimodal imaging methods visualizing microglial and MMP alterations were employed. The spatio-temporal dynamics of these parameters were studied in relation to blood flow changes. Micro positron emission tomography (μPET) using [F]BR-351 showed MMP activity within the first days after transient middle cerebral artery occlusion (tMCAo), followed by increased [F]DPA-714 uptake as a marker for microglia activation with a maximum at 14 days after tMCAo. The inflammatory response was spatially located in the infarct core and in adjacent (penumbral) tissue. For the first time, multimodal imaging based on PET, single photon emission computed tomography, and magnetic resonance imaging revealed insight into the spatio-temporal distribution of critical parameters of poststroke inflammation. This allows further evaluation of novel treatment paradigms targeting the postischemic inflammation.

Objectives In this study, we established and validated a time-resolved three-dimensional phase-contrast magnetic resonance imaging method (4D PC MRI) on a 9.4 T small-animal MRI system. Herein we present the feasibility of 4D PC MRI in terms of qualitative and quantitative flow pattern analysis in mice with transverse aortic constriction (TAC). Materials and methods 4D PC FLASH images of a flow phantom and mouse heart were acquired at 9.4 T using a four-point phase-encoding scheme. The method was compared with slice-selective PC FLASH and ultrasound using Bland–Altman analysis. Advanced 3D streamlines were visualized utilizing Voreen volume-rendering software. Results In vitro, 4D PC MRI flow profiles showed the transition between laminar and turbulent flow with increasing velocities. In vivo, 4D PC MRI data of the ascending aorta and the pulmonary artery were confirmed by ultrasound, resulting in linear regressions of R2 > 0.93. Magnitude- and direction-encoded streamlines differed substantially pre- and post-TAC surgery. Conclusions 4D PC MRI is a feasible tool for in vivo velocity measurements on high-field small-animal scanners. Similar to clinical measurement, this method provides a complete spatially and temporally resolved dataset of the murine cardiovascular blood flow and allows for three-dimensional flow pattern analysis. Electronic supplementary material The online version of this article (doi:10.1007/s10334-014-0466-z) contains supplementary material, which is available to authorized users.

Staphylococcus aureus causes very serious infections of vascular grafts. Knowledge of the molecular mechanisms of this disease is largely lacking because of the absence of representable models. Therefore, the aim of this study was to set up a mouse model of vascular graft infections that closely mimics the human situation. A catheter was inserted into the right carotid artery of mice, which acted as a vascular graft. Mice were infected i.v. using 8 different S. aureus strains, and development of the infection was followed up. Although all strains had varying abilities to form biofilm in vitro and different levels of virulence in mice, they all caused biofilm formation on the grafts. This graft infection was monitored using magnetic resonance imaging (MRI) and

Adverse cardiac remodeling after myocardial infarction ultimately causes heart failure. To stimulate reparative processes in the infarct, efficient delivery and retention of therapeutic agents is desired. This might be achieved by encapsulation of drugs in nanoparticles. The goal of this study was to characterize the distribution pattern of differently sized long-circulating lipid-based nanoparticles, namely micelles (similar to 15 nm) and liposomes (similar to 100 nm), in a mouse model of myocardial infarction (MI). MI was induced in mice (n = 38) by permanent occlusion of the left coronary artery. Nanoparticle accumulation following intravenous administration was examined one day and one week after surgery, representing the acute and chronic phase of MI, respectively. In vivo magnetic resonance imaging of paramagnetic lipids in the micelles and liposomes was employed to monitor the trafficking of nanoparticles to the infarcted myocardium. Ex vivo high-resolution fluorescence microscopy of fluorescent lipids was used to determine the exact location of the nanoparticles in the myocardium. In both acute and chronic MI, micelles permeated the entire infarct area, which renders them very suited for the local delivery of cardioprotective or anti-remodeling drugs. Liposomes displayed slower and more restricted extravasation from the vasculature and are therefore an attractive vehicle for the delivery of pro-angiogenic drugs. Importantly, the ability to non-invasively visualize both micelles and liposomes with MRI creates a versatile approach for the development of effective cardioprotective therapeutic interventions. (C) 2012 Elsevier B. V. All rights reserved.

The hyperlipidemic mouse model HypoE/SRBI−/− has been shown to develop occlusive coronary atherosclerosis followed by myocardial infarctions and premature deaths in response to high-fat, high-cholesterol diet (HFC). However, the causal connection between myocardial infarctions and atherosclerotic plaque rupture events in the coronary arteries has not been investigated so far. The objective of this study was to assess whether diet-induced coronary plaque ruptures trigger atherothrombotic occlusions, resulting in myocardial infarctions in HFC-fed HypoE/SRBI−/− mice. Methods: HypoE/SRBI−/− mice were characterized with respect to the individual dynamics of myocardial infarctions and features of infarct-related coronary atherosclerosis by serial noninvasive molecular and functional imaging, histopathology, and a pharmaceutical intervention. Detailed histologic analysis of whole mouse hearts was performed when spontaneously occurring acute myocardial infarctions were diagnosed by imaging. Results: Using the imaging-triggered approach, we discovered thrombi in 32 (10.8%) of all 296 atherosclerotic coronary plaques in 14 HFC-fed HypoE/SRBI−/− mice. These thrombi typically were found in arteries presenting with inflammatory plaque phenotypes. Acetylsalicylic acid treatment did not attenuate the development of atherosclerotic coronary plaques but profoundly reduced the incidence of premature deaths, the number of thrombi (7 in 249 plaques), and also the degree of inflammation in the culprit lesions. Conclusion: HFC-induced ruptures of coronary plaques trigger atherothrombosis, vessel occlusions, myocardial infarctions, and sudden death in these mice. Thus, the HypoE/SRBI−/− mouse model mimics major features of human coronary heart disease and might therefore be a valuable model for the investigation of molecular and cellular parameters driving plaque rupture-related events and the development of new interventional approaches.

Recently, integrated small-animal PET/MRI prototypes that provide isochronous and coregistered datasets of morphology and function through the simultaneous acquisition of PET and MRI data have been developed. However, the need for MRI compatibility can constrain the technical design of the PET components and may lead to a lower sensitivity and lower spatial and temporal resolutions. The aim of this study was to evaluate the suitability of a prototype preclinical PET/MRI system for the simultaneous assessment of cardiac metabolism and function in mice. A stand-alone high-resolution small-animal PET scanner using the same evaluation protocols was used as a reference. Methods: Simultaneous PET/MR images of an infarct mouse model (21 animals plus 3 controls) were acquired. The imaging performance of the MRI-compatible PET insert was evaluated with respect to count sensitivity; myocardium-to-background contrast; suitability for the analysis of global left ventricular function; and uptake difference in scar, border-zone, and remote regions. The radiotracer 18F-FDG was used to acquire cardiac gated PET data, applying retrospective coincidence sorting. The PET insert data were coregistered to the MR images by determination of the appropriate transformation matrix. Results: An optimal registration of PET and MR images from the integrated system was achieved, and the reconstructed images showed a good visual correspondence in infarct areas between PET and MRI data. As expected, the PET insert showed a poorer performance with respect to counting rate and myocardium-to-background ratio than did the high-resolution PET. Assessment of left ventricular volumes was possible with the current PET/MRI prototype. A good correlation was found between PET and MRI (R > 0.95). Local PET uptake was successfully determined for different tissue, and a differentiation among remote, border-zone, and scar tissue was possible. However, the uptake difference for the PET/MRI prototype was lower than that for the high-resolution stand-alone PET system. Conclusion: A hybrid PET/MRI prototype was successfully used to assess cardiac parameters in an infarct mouse model, although performance was reduced when compared with a high-resolution animal PET scanner. Future technical improvements are expected to result in comparable performance while providing higher registration accuracy.

Today, non-invasive imaging techniques are significantly contributing to the understanding of molecular processes in vivo. Positron emission tomography (PET) is a scintigraphic medical imaging modality that uses radiolabelled molecules (tracers), provides quantitative tomographic images and allows non-invasive assessment of the biodistribution of radioactive substances in vivo. The assessment of pathological glucose metabolism is the clinically best-established application of PET today; however, a multitude of different tracers are available to assess diverse physiological processes. The growing interest in pre-clinical imaging studies, in biological and medical basic research, as well as in pharmaceutical research, has fostered the recent growth in small-animal PET. Small-animal PET can be applied to enable the transfer from molecular findings in vitro to in vivo applications in humans, from bench to bed side.

To identify proteins involved in cardiac regeneration, a proteomics approach was applied. A total of 26 proteins, which displayed aberrant expression in mouse hearts infarcted through ligation of the left anterior descending coronary artery, were identified. These included the intermediate filament protein nestin, which was up-regulated in the infarct border zone. Corresponding changes were observed for its mRNA. Nestin mRNA was also up-regulated in hearts from 17 of 19 patients with end-stage heart failure, including 4 with acute myocardial infarction in comparison with 8 donor hearts. Immunofluorescence confocal laser scanning microscopy revealed that nestin is expressed, on the one hand, in small proportions of cardiomyocytes, endothelial cells, smooth muscle cells, neuronal cells, and fibroblasts. On the other hand, it was found to be coexpressed with the stem cell markers c-kit, Sca-1, Mdr-1, and Abcg2 in small interstitial cells. In infarcted hearts from chimeric mice transplanted with bone marrow from enhanced green fluorescent protein (EGFP) transgenic mice, less than 1% of nestin-positive cells coexpressed EGFP, although EGFP-positive cells were abundant in these. Consequently, enhanced expression of nestin in the injured myocardium might reflect spontaneous regenerative processes supposedly based on the differentiation of resident cardiac stem cells into diverse cardiac cell types.

Parkinson's disease is a slowly progressing neurodegenerative disorder caused by loss of dopaminergic neurons in the substantia nigra (SN), leading to severe impairment in motor and non-motor functions. Endogenous subventricular zone (SVZ) neural stem cells constantly give birth to new cells that might serve as a possible source for regeneration in the adult brain. However, neurodegeneration is accompanied by neuroinflammation and dopamine depletion, potentially compromising regeneration. We therefore employed in vivo imaging methods to study striatal deafferentation (N-ω-fluoropropyl-2β-carbomethoxy-3β-(4-[(123) I]iodophenyl)nortropane single photon emission computed tomography, DaTscan(™) ) and neuroinflammation in the SN and striatum (N,N-diethyl-2-(2-(4-(2-[(18) F]fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide positron emission tomography, [(18) F]DPA-714 PET) in the intranigral 6-hydroxydopamine Parkinson's disease mouse model. Additionally, we transduced cells in the SVZ with a lentivirus encoding firefly luciferase and followed migration of progenitor cells in the SVZ-olfactory bulb axis via bioluminescence imaging under disease and control conditions. We found that activation of microglia in the SN is an acute process accompanying the degeneration of dopaminergic cell bodies in the SN. Dopaminergic deafferentation of the striatum does not influence the generation of doublecortin-positive neuroblasts in the SVZ, but generates chronic astrogliosis in the nigrostriatal system.

The noninvasive imaging of matrix metalloproteinases (MMPs) activity in postischemic myocardial tissue holds great promise to predict cardiac function post-myocardial infarction. Consequently, development of MMP specific molecular imaging probes for noninvasive visualization and quantification of MMP activity is of great interest. A novel MMP imaging strategy is based on activatable cell-penetrating peptide probes (ACPP) that are sensitive to the proteolytic activity of MMP-2 and -9. The MMP-mediated activation of these ACPPs drives probe accumulation at the target site. The aim of this study was the development and characterization of radiolabeled MMP-2/9 sensitive ACPPs to assess MMP activity in myocardial remodeling in vivo. Specifically, a short and long-circulating MMP activatable cell-penetrating imaging probe (ACPP and Alb-ACPP, respectively; the latter is an ACPP modified with an albumin binding ligand that prolongs blood clearance) were successfully synthesized and radiolabeled. Subsequently, their biodistributions were determined in vivo in a Swiss mouse model of myocardial infarction. Both peptide probes showed a significantly higher uptake in infarcted myocardium compared to remote myocardium. The biodistribution for dual-isotope radiolabeled probes, which allowed us to discriminate between uncleaved ACPP and activated ACPP, showed increased retention of activated ACPP and activated Alb-ACPP in infarcted myocardium compared to remote myocardium. The enhanced retention correlated to gelatinase levels determined by gelatin zymography, whereas no correlation was found for the negative control: an MMP-2/9 insensitive non-ACPP. In conclusion, radiolabeled MMP sensitive ACPP probes enable to assess MMP activity in the course of remodeling post-myocardial infarction in vivo. Future research should evaluate the feasibility and the predictive value of the ACPP strategy for assessing MMP activity as a main player in postinfarction myocardial remodeling in vivo. © 2014 American Chemical Society.

Infective endocarditis (IE) is a severe and often fatal disease, lacking a fast and reliable diagnostic procedure. The purpose of this study was to establish a mouse model of Staphylococcus aureus-induced IE and to develop a MRI technology to characterize and diagnose IE. To establish the mouse model of hematogenous IE, aortic valve damage was induced by placing a permanent catheter into right carotid artery. 24 h after surgery, mice were injected intravenously with either iron particle-labeled or unlabeled S. aureus (strain 6850). To distinguish the effect of IE from mere tissue injury or recruited macrophages, subgroups of mice received sham surgery prior to infection (n = 17), received surgery without infection (n = 8), or obtained additionally injection of free iron particles to label macrophages (n = 17). Cardiac MRI was performed 48 h after surgery using a self-gated ultra-short echo time (UTE) sequence (TR/TE, 5/0.31 ms; in-plane/slice, 0.125/1 mm; duration, 12∶08 min) to obtain high-resolution, artifact-free cinematographic images of the valves. After MRI, valves were either homogenized and plated on blood agar plates for determination of bacterial titers, or sectioned and stained for histology. In the animal model, both severity of the disease and mortality increased with bacterial numbers. Infection with 105 S. aureus bacteria reliably caused endocarditis with vegetations on the valves. Cinematographic UTE MRI visualised the aortic valve over the cardiac cycle and allowed for detection of bacterial vegetations, while mere tissue trauma or labeled macrophages were not detected. Iron labeling of S. aureus was not required for detection. MRI results were consistent with histology and microbial assessment. These data showed that S. aureus-induced IE in mice can be detected by MRI. The established mouse model allows for investigation of the pathophysiology of IE, testing of novel drugs and may serve for the development of a clinical diagnostic strategy.

Despite treatments combining surgery, radiation-, and chemotherapy, patients affected by glioblastoma (GBM) have a limited prognosis. Addition of temozolomide (TMZ) to radiation therapy is the standard therapy in clinical application, but effectiveness of TMZ is limited by the tumor's overexpression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). The goal of this study was to use the highly specific and efficient RNA interference (RNAi) pathway to modulate MGMT expression to increase TMZ efficiency in chemotherapy resistant GBM. Using lentiviral-based anti-MGMT small hairpin RNA (shRNA) technology we observed a specific inhibition of the MGMT expression in GBM cell lines as well as in subcutaneous tumors. Tumor growth inhibition was observed following TMZ treatment of xenografts with low MGMT expression in contrast to xenografts with high MGMT expression. Bioluminescence imaging (BLI) measurements indicated that luciferase and shRNA-expressing lentiviruses were able to efficiently transduce the GBM xenografts in vivo. Treatment combining injection of a lentivirus expressing an anti-MGMT shRNA and TMZ induced a reduction of the size of the tumors, in contrast with treatment combining the lentivirus expressing the control shRNA and TMZ. Our data suggest that anti-MGMT shRNA therapy could be used in combination with TMZ chemotherapy in order to improve the treatment of resistant GBM.

UnlabelledAddition of temozolomide (TMZ) to radiation therapy is the standard treatment for patients with glioblastoma (GBM). However, there is uncertainty regarding the effectiveness of TMZ. Considering the rapid evolution of the disease, methods to assess TMZ efficacy early during treatment would be of great benefit. Our aim was to monitor early effects of TMZ in a mouse model of GBM using positron emission tomography (PET) with 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT).MethodsHuman glioma cells sensitive to TMZ (Gli36dEGFR-1) were treated with sub-lethal doses of TMZ to obtain cells with lower sensitivity to TMZ (Gli36dEGFR-2), as measured by growth and clonogenic assays. Gli36dEGFR-1 and Gli36dEGFR-2 cells were subcutaneously (s.c.) or intracranially (i.c.) xenografted into nude mice. Mice were treated for 7 days with daily injection of 25 or 50 mg/kg TMZ. Treatment efficacy was measured using [(18)F]FLT-PET before treatment and after 2 days. Computed Tomography (CT) or Magnetic Resonance Imaging (MRI) were used to determine tumor volumes before treatment and after 7 days.ResultsA significant difference was observed between TMZ and DMSO treated tumors in terms of variations of [(18)F]FLT T/B ratio as soon as day 2 in the i.c. as well as in the s.c. mouse model. Variations of [(18)F]FLT T/B uptake ratio between days 0 and 2 correlated with variations of tumor size between days 0 and 7 (s.c. model: n(tumor) = 17 in n(mice) = 11, PConclusionsOur results indicate that [(18)F]FLT-PET may be useful for an early evaluation of the response of GBM to TMZ chemotherapy in patients with glioma.

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in (1) and (2)). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells and in vivo in larger animals and humans. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation. In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics as confirmed by in vivo Magnetic Resonance Imaging (MRI): Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed. In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.