Date of Award

2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Bioengineering

First Advisor

Walter R. Witschey

Abstract

Reperfusion injury is additional injury that may occur following restoration of blood flow to ischemic tissue. A better understanding of the mechanistic process underlying reperfusion injury, and the development of non-invasive imaging tools to assess it, are necessary to mitigate this pathologic condition in patients suffering from a heart attack. In this thesis, I investigated the relationship between the duration of myocardial ischemia and reperfusion injury using magnetic resonance imaging (MRI). A fundamental principle of this investigation was the pathologic role of iron in reperfusion injury and its contribution to the MRI signal. Since iron has moderate volume magnetic susceptibility (χ), which is influenced in part by its redox state and ligand binding, pathologic iron accumulation can cause the tissue to become relatively more paramagnetic than non-ischemic myocardium. A unique aspect of my work was to map the magnetic susceptibility of ischemic myocardium using information obtained from the MRI signal phase. Altogether, these studies showed the spatial distributions of paramagnetic shifts of the infarcted myocardium in the acute, sub-acute and chronic phases of post-infarction injury, and to find their association with ischemia time and extent of iron deposition.

My research is supported by a wealth of studies using in vivo and tissue explant imaging, and molecular assays in animal models and in humans. I performed validation studies to elucidate the role of iron in reperfusion injury using histology, immunohistochemistry, inductively coupled plasma optical emission spectrometry (ICP-OES), and electron paramagnetic resonance (EPR) spectroscopy. Infarct iron concentration was elevated compared to healthy (remote) myocardium in reperfused and non-reperfused (permanently occluded) infarcts. Sources of iron came from the extracellular space including hemoglobin byproducts and the intracellular space including the cell’s labile iron pool. Iron handling and homeostasis at the cellular level was analyzed using real-time PCR, where mRNA analysis showed upregulation of ferritin which acts as an iron chelator and upregulation of heme oxygenase which is an enzyme that breaks down hemoglobin.

There are some important and novel findings to my work. To our knowledge, we were the first to detect iron in myocardial tissue by using magnetic susceptibility as an MR imaging biomarker both in pre-clinical and clinical studies. Experiments validating the presence of iron uncovered the dynamic wound healing process and the temporal evolution of iron, which declined from 3-days to 8-weeks post-infarction indicating the clearance of iron from the infarct region. It was discovered that increased ischemia duration caused a significant increase in infarct transmurality, microvascular obstruction, and iron content. Permanent coronary occlusion without reperfusion caused a completely transmural infarct. Despite permanent infarcts showing a lack of T2*-weighted hypointensity, there was a paramagnetic shift and elevated iron concentration in the permanent infarcts. This led us to further investigate sources of iron within the infarct region and discover a significant increase in labile iron concentration, which is expected to originate from intracellular myocytes.

While conventional cardiovascular MRI corroborates the findings of iron deposition, magnetic susceptibility imaging showed improved detection of tissue iron deposition and mitigates confounding factors such as edema and fibrosis. At high field strength (7 T) and high resolution, there was improved correspondence between MR imaging and infarct pathophysiology, where regions containing sparse iron deposition could be better detected compared to imaging at 3 T. Unlike conventional relaxation-based measurements, magnetic susceptibility showed independence of field strength. Myocardial infarction patients receiving reperfusion therapy showed magnetic susceptibility changes associated with hypokinetic myocardial wall motion and microvascular obstruction, demonstrating potential for clinical translation.

In this thesis I found that iron corresponded to a tissue paramagnetic shift. Both iron and magnetic susceptibility varied depending on the ischemia duration and post-infarction time point. Magnetic susceptibility showed improved specificity to iron deposition than conventional imaging approaches. Iron oxidation state and the type of protein-bound iron likely affect the magnetic susceptibility and change during wound healing which could be investigated in future work.

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