Magnetic resonance imaging of soft tissue infection with iron oxide labeled granulocytes in a rat model.

OBJECT: We sought to detect an acute soft tissue infection in rats by magnetic resonance imaging (MRI) using granulocytes, previously labeled with superparamagnetic particles of iron oxide (SPIO). MATERIALS AND METHODS: Parasternal infection was induced by subcutaneous inoculation of Staphylococcus aureus suspension in rats. Granulocytes isolated from isogenic donor rats were labeled with SPIO. Infected rats were imaged by MRI before, 6 and 12 hours after intravenous injection of SPIO-labeled or unlabeled granulocytes. MR findings were correlated with histological analysis by Prussian blue staining and with re-isolated SPIO-labeled granulocytes from the infectious area by magnetic cell separation. RESULTS: Susceptibility effects were present in infected sites on post-contrast T2*-weighted MR images in all animals of the experimental group. Regions of decreased signal intensity (SI) in MRI were detected at 6 hours after granulocyte administration and were more pronounced at 12 hours. SPIO-labeled granulocytes were identified by Prussian blue staining in the infected tissue and could be successfully re-isolated from the infected area by magnetic cell separation. CONCLUSION: The application of SPIO-labeled granulocytes in MRI offers new perspectives in diagnostic specificity and sensitifity to detect early infectious processes

Macroscopic fluorescence imaging: a novel technique to monitor retention and distribution of injected microspheres in an experimental model of ischemic heart failure.

BACKGROUND:The limited effectiveness of cardiac cell therapy has generated concern regarding its clinical relevance. Experimental studies show that cell retention and engraftment are low after injection into ischemic myocardium, which may restrict therapy effectiveness significantly. Surgical aspects and mechanical loss are suspected to be the main culprits behind this phenomenon. As current techniques of monitoring intramyocardial injections are complex and time-consuming, the aim of the study was to develop a fast and simple model to study cardiac retention and distribution following intramyocardial injections. For this purpose, our main hypothesis was that macroscopic fluorescence imaging could adequately serve as a detection method for intramyocardial injections. METHODS AND RESULTS:A total of 20 mice underwent ligation of the left anterior descending artery (LAD) for myocardial infarction. Fluorescent microspheres with cellular dimensions were used as cell surrogates. Particles (5 × 10(5)) were injected into the infarcted area of explanted resting hearts (Ex vivo myocardial injetions EVMI, n = 10) and in vivo into beating hearts (In vivo myocardial injections IVMI, n = 10). Microsphere quantification was performed by fluorescence imaging of explanted organs. Measurements were repeated after a reduction to homogenate dilutions. Cardiac microsphere retention was 2.78 × 10(5) ± 0.31 × 10(5) in the EVMI group. In the IVMI group, cardiac retention of microspheres was significantly lower (0.74 × 10(5) ± 0.18 × 10(5); p<0.05). Direct fluorescence imaging revealed venous drainage through the coronary sinus, resulting in a microsphere accumulation in the left (0.90 × 10(5) ± 0.20 × 10(5)) and the right (1.07 × 10(5) ± 0.17 × 10(5)) lung. Processing to homogenates involved further particle loss (p<0.05) in both groups. CONCLUSIONS:We developed a fast and simple direct fluorescence imaging method for biodistribution analysis which enabled the quantification of fluorescent microspheres after intramyocardial delivery using macroscopic fluorescence imaging. This new technique showed massive early particle loss and venous drainage into the right atrium leading to substantial accumulation of graft particles in both lungs

Verification of SPIO-labeled granulocytes isolated from infected tissue.

<p>(A) Fraction of granulocytes isolated from infected tissue of rats treated with SPIO-labeled granulocytes. SPIO-labeled granulocytes (blue) were identified by Prussian blue staining. (B) fraction of granulocytes isolated from infected tissue of rats from the control group, which were treated with unlabeled granulocytes. Re-isolated granulocytes are completely negative for Prussian blue staining. The scale bar equals 20 µm.</p

Histological sections of the infected tissue.

<p>(A–C) Prussian blue staining of infected tissue samples from rats that received SPIO-labeled granulocytes. SPIO-labeled granulocytes (blue) accumulated within the infected tissue together with unlabeled granulocytes. The scale bar equals in (A) 250 µm, (B) 100 µm, (C) 25 µm. (D–F) Prussian blue staining of infected tissue samples from control rats. Massive infiltration of unlabeled granulocytes into the whole infected region. The scale bar equals in (D) 250 µm, (E) 100 µm, (F) 25 µm.</p

Transplantation of purified iPSC-derived cardiomyocytes in myocardial infarction

<div><p>Background</p><p>Induced pluripotent stem cells (iPSC) can be differentiated into cardiomyocytes and represent a possible autologous cell source for myocardial repair. We analyzed the engraftment and functional effects of murine iPSC-derived cardiomyocytes (iPSC-CMs) in a murine model of myocardial infarction.</p><p>Methods and results</p><p>To maximize cardiomyocyte yield and purity a genetic purification protocol was applied. Murine iPSCs were genetically modified to express a Zeocin^™ resistance gene under control of the cardiac-specific α-myosin heavy chain (α-MHC, MYH6) promoter. Thus, CM selection was performed during in vitro differentiation. iPSC-CM aggregates (“cardiac bodies”, CBs) were transplanted on day 14 after LAD ligation into the hearts of previously LAD-ligated mice (800 CBs/animal; 2-3x10⁶ CMs). Animals were treated with placebo (PBS, n = 14) or iPSC-CMs (n = 35). Myocardial remodeling and function were evaluated by magnetic resonance imaging (MRI), conductance catheter (CC) analysis and histological morphometry. <i>In vitro</i> and <i>in vivo</i> differentiation was investigated. Follow up was 28 days (including histological assessment and functional analysis). iPSC-CM purity was >99%. Transplanted iPSC-CMs formed mature grafts within the myocardium, expressed cardiac markers and exhibited sarcomeric structures. Intramyocardial transplantation of iPSC-CMs significantly improved myocardial remodeling and left ventricular function 28 days after LAD-ligation.</p><p>Conclusions</p><p>We conclude that iPSCs can effectively be differentiated into cardiomyocytes and genetically enriched to high purity. iPSC derived cardiomyocytes engraft within the myocardium of LAD-ligated mice and contribute to improve left ventricular function.</p></div

Detection of experimentally induced subcutaneous inflammatory process.

<p>T2*-weighted MRI axial images of the left pectoral area of infected rats. (A–C) Inflammatory area in the left pectoral region (ellipse) directly after inoculation of the staphylococcal suspension and (A) before injection of SPIO-labeled granulocytes, (B) 6 h after injection of the SPIO-labeled granulocytes including several spots with significant loss of signal intensity inside the inflamed area (white arrows). (C) Inflammatory area in the left pectoral region 12 hours after injection of SPIO-labeled granulocytes including multiple single spots (white arrows) with significant loss of signal intensity inside the inflamed area. (D–F) Inflammatory area in the left pectoral region (ellipse) of a control animal directly after inoculation of the staphylococcal suspension and (D) before injection of non-labeled granulocytes, (E) 6 h and (F) 12 h after injection of the non-labeled granulocytes. Areas of signal intensity loss could not be detected at any time in the control animals.</p

Histological verification of SPIO labeled granulocytes distribution.

<p>(A) Granulocytes were transfected <i>in vitro</i> with SPIO particles. Transfection efficiencies reached up to 90% as estimated by Prussian blue staining. Iron particle presence is identified as blue dots within cells. The scale bar equals 20 µm. (B) Unlabeled control granulocytes. Cells were counterstained with Nuclear Fast Red. The scale bar equals 20 µm.</p