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13.01.2014

Mri iron supplements, shoulder girdle pain - Try Out

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To test the capability of MRI to detect ferritin overexpression, T2 relaxation times were measured in suspensions of wild-type and ferritin-transduced C2C12 cells cultured with and without ferric citrate supplementation (1 mM for 48 hours).
Correlation in graft size measurements assessed by MRI was compared to histologic evaluation (embryonic skeletal myosin staining) using Pearson correlation coefficient. The ferritin-overexpressing cells were readily detectable by MRI in vitro, yielding significant changes in T2 compared to wild-type cells.
To validate the hypothesis that ferritin overexpression is suitable as an MRI reporter for noninvasive imaging of grafted cells, we transplanted wild-type and ferritin-overexpressing myoblasts into the infarcted hearts of syngeneic C3H mice.
The area of signal hypointensity was measured in each MRI at the short-axis plane of the heart. The present study demonstrates the feasibility of using the MRI reporter ferritin for noninvasive imaging of cardiac grafts in the mouse heart. We propose that the natural iron storage protein ferritin will be useful for noninvasive long-term monitoring of transplanted cells into the infarcted heart. In this study, we overexpressed MRI reporter ferritin in a model cell type (C2C12) and visualized the transgenic cells in vitro and in vivo after transplantation into the infarcted hearts of syngeneic C3H mice.
Ferritin overexpression provided sufficient MRI contrast to make transduced cells detectable in vitro and in vivo in murine hearts.
When transplanted cells divide, any intracellular label, including iron oxide particles and the ferritin-stored iron would be diluted. In summary, this is the first use of MRI for detection of ferritin gene expression in cardiac grafts. Control and transduced cells were supplemented with (A) 0.2 mM ferric citrate from 24 h post transduction or (B) with 2 mM for 96 h prior to measurement. Overexpression of the MRI Reporter Genes Ferritin and Transferrin Receptor Affect Iron Homeostasis and Produce Limited Contrast in Mesenchymal Stem Cells. Our goal was to explore if overexpression of ferritin, a nontoxic iron-binding protein, can be used for noninvasive magnetic resonance imaging (MRI) of cells transplanted into the infarcted heart.
We compared the growth of wild-type and HA-ferritin C2C12 cells both with and without ferric citrate supplementation (1 mM). For in vitro imaging, wild-type and transgenic C2C12 cells were grown in maxi dishes, washed with PBS to remove excess iron, and then trypsinized and imaged alive in Eppendorf tubes. This approach ensured identification of hypointense MRI signals in transgenic grafts and distinguished true signal from motion and flow artifacts. Given that transverse MRI relaxivity of ferritin is much higher than its longitudinal relaxivity, we were able to detect ferritin-tagged cells using T2*-weighted bright- and black-blood image sequences in a clinical 3 T scanner.


For example, although DNA and protein degradation occur rapidly after cell death,21 there are no data as to how rapidly ferritin complexes undergo degradation after the death of transplanted cells and for how long the MRI signal from iron persists. In this regard, continuous production of ferritin in daughter cells offers a significant advantage for cell tracking by an MRI-detectable gene reporter over a particle-based cell labeling method.
Iron-oxide labeling and outcome of transplanted mesenchymal stem cells in the infarcted myocardium. Ferritin as an endogenous MRI reporter for noninvasive imaging of gene expression in C6 glioma tumors. MagA is sufficient for producing magnetic nanoparticles in mammalian cells, making it an MRI reporter. A single-lead electrocardiogram (ECG) was recorded from needle subcutaneous electrodes attached to the animal's extremities and was used to trigger the MRI acquisitions using commercial software (Small Animal Monitoring and Gating System, SA Instrument Inc., Stony Brook, NY). Supplementation of growth medium by ferric citrate caused additional shortening of T2 with further amplification of the difference between wild-type and transgenic cells. The presence of transgenic grafts in the infarcted mouse heart was detected by T2*-weighted MRI as areas of hypointensity caused by accumulation of iron in overexpressed ferritin complexes.
Imaging-based cell-tracking methods can potentially evaluate the short-term distribution of infused cells (using iron oxide particles),4–7 their long-term survival and proliferation (using the gene reporter approach25), and cardiac differentiation (using reporter genes26). It will be important to determine how quickly ferritin can be produced by daughter cells and how quickly ferritin complexes can bind a sufficient amount of iron from the extracellular environment to be detected by MRI. B, Quantification of changes in T2 relaxation time of WT C2C12 and cells transduced by pcDNA3-HA-ferritin with and without iron citrate supplementation.
A and B, Graft detection in vivo by MRI and ex vivo by embryonic myosin staining 3 weeks after transplantation of 500,000 transgenic C2C12 cells overexpressing ferritin. The overexpression of TfR1 was well tolerated by the cells but Fth1 was found to affect the cell’s iron homeostasis, leading to phenotypic changes in the absence of iron supplementation and an upregulation in transcript and protein levels of the cell’s endogenous transferrin receptor. In summary, these data show that overexpression of ferritin in C2C12 cells can be detected by MRI in vitro. Cine MRI techniques did not detect any contractile activity of the area containing skeletal muscle grafts, consistent with previous reports of their lack of electromechanical coupling.18,19 MRI signal void in the graft area was detected in vivo 3 weeks after transplantation of transgenic cells overexpressing ferritin (see Figure 4E).
C and D, Prussian blue staining indicates iron accumulation in C2C12 cells transduced by pcDNA3-HA-ferritin plasmids (D), but not in WT cells (C). Neither the sole overexpression of Fth1 nor TfR1 resulted in significant increases in intracellular iron content, although significant differences were seen when the two reporter genes were used in combination, in the presence of high concentrations of iron. Stem cell labeling with superparamagnetic iron oxide nanoparticles (SPIO) allows labeled cells to be detected by magnetic resonance imaging (MRI) and is commonly used to track stem cell engraftment.4,5 However, the MRI signal hypointensity caused by those particles does not reflect the actual cell number after several rounds of cell division owing to particle dilution.


In this assay, the presence of iron was indicated by a bright blue color in a granular cytoplasmic distribution. To facilitate iron loading, cell medium in all wells was supplemented with 1 mM ferric citrate. C and D, In vivo MRI and histology of the mouse heart 3 weeks after engraftment of 500,000 wild-type (WT) C2C12 cells. Increase in the TE makes MRIs more T2*-weighted and thus makes a cellular graft more visible in the mouse heart (arrow).
The supplementation of the culture medium with iron sources was a more efficient means to obtain contrast than the use of reporter genes, where high levels of intracellular iron were reflected in transverse (T2) relaxation.
Transgenic grafts were detected in vivo 3 weeks after transplantation into infarcted hearts of syngeneic mice as areas of hypointensity caused by iron accumulation in overexpressed ferritin complexes. Prussian blue staining is a sensitive technique for iron detection; it confirmed significant accumulation of iron in the cytoplasm of transduced cells after iron supplementation of the media, whereas no blue cells were observed in iron-supplemented wild-type controls (Figure 1, C and D). We noticed that supplementation of cell medium with iron citrate in high doses (1 mM) reduced the expansion of both wild-type C2C12 and transduced cells. The feasibility of imaging iron-supplemented cells by MRI is shown using a 3R-compliant chick embryo model.
Thus, significant overexpression of ferritin was achieved with increased iron storage capacity. Our studies demonstrated the feasibility of ferritin overexpression in mouse skeletal myoblasts and the successful detection of transgenic cells by MRI in vitro and in vivo after transplantation into the infarcted mouse heart.
These experiments lay the groundwork for using the MRI gene reporter ferritin to track stem cells transplanted to the heart. Supplementation of cell medium with low concentrations of ferric citrate (1 μM) did not affect cell growth in either wild-type or transgenic cells (data not shown). In summary, ferritin overexpression did not interfere with cell viability, proliferation, or differentiation in mouse skeletal myoblasts, and ferritin increased resistance to iron toxicity.



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