Abstract
Iron (Fe) is an essential metal involved in a wide spectrum of physiological functions. Sub-cellular characterization of the size, composition, and distribution of ferritin(iron) can provide valuable information on iron storage and transport in health and disease. In this study we employ magnetic force microscopy (MFM), transmission electron microscopy (TEM), and electron energy loss spectroscopy (EELS) to characterize differences in ferritin(iron) distribution and composition across injured and non-injured tissues by employing a rodent model of spinal cord injury (SCI). Our biophysical and ultrastructural analyses provide novel insights into iron distribution which are not obtained by routine biochemical stains. In particular, ferritin(iron) rich lysosomes revealed increased heterogeneity in MFM signal from tissues of SCI animals. Ultrastructural analysis using TEM elucidated that both cytosolic and lysosomal ferritin(iron) density was increased in the injured (spinal cord) and non-injured (spleen) tissues of SCI as compared to naïve animals. In-situ EELs analysis revealed that ferritin(iron) was primarily in Fe3+ oxidation state in both naïve and SCI animal tissues. The insights provided by this study and the approaches utilized here can be applied broadly to other systemic problems involving iron regulation or to understand the fate of exogenously delivered iron-oxide nanoparticles.
Highlights
Iron (Fe) is an essential metal involved in a wide spectrum of physiological functions, including oxygen transport, enzymatic reactions, energy production, protein synthesis and DNA repair[1,2,3]
spinal cord injury (SCI) induced a shift in peripheral splenic macrophages to accumulate iron and ferritin, in a pattern similar to macrophages at the intraspinal injury site
Our in situ ultrastructural analysis revealed a significantly increased ferritin(iron) density in the cytoplasm and lysosomes of the macrophages present in the spleen and in the spinal cord of SCI rats as compared to naïve animals
Summary
Iron (Fe) is an essential metal involved in a wide spectrum of physiological functions, including oxygen transport, enzymatic reactions, energy production, protein synthesis and DNA repair[1,2,3]. Oxidation state of iron in tissue sections can be identified via Perls or Turnbull’s stains or by utilizing micro-focused X-ray beams[5,6] These basic histology approaches are useful in rapidly evaluating iron content in situ, their limited spatial resolution cannot resolve subcellular iron distribution, which can lead to misleading results such as confounding iron-rich macrophages with biogenic magnetite[7,8]. Analytical TEM approaches[10,14,15,16] have elucidated how the oxidation state of iron can differ in pathological vs physiological tissues by analyzing the iron-rich particles isolated from various pathologies[17,18]. Limited studies exist on subcellular mapping of the oxidation state of iron in situ in mammalian tissues These include the perfusion-Perls and Turnbull’s method coupled with TEM imaging, which is primarily applicable to animal www.nature.com/scientificreports/. Iron has been mapped in situ using magnetism-based microscopy[21,22], the effects of particle size, crystal structure, density and oxidation state[23] have not been adequately evaluated in these studies
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