Tom C.-C. Hu

Temporal and Noninvasive Monitoring of Inflammatory-Cell Infiltration to Myocardial Infarction Sites Using Micrometer-Sized Iron Oxide Particles

Micrometer‐sized iron oxide particles (MPIO) are a more sensitive MRI contrast agent for tracking cell migration compared to ultrasmall iron oxide particles. This study investigated the temporal relationship between inflammation and tissue remodeling due to myocardial infarction (MI) using MPIO‐enhanced MRI. C57Bl/6 mice received an intravenous MPIO injection for cell labeling, followed by a surgically induced MI seven days later (n = 7). For controls, two groups underwent either sham‐operated surgery without inducing an MI post‐MPIO injection (n = 7) or MI surgery without MPIO injection (n = 6). The MRIs performed post‐MI showed significant signal attenuation around the MI site for the mice that received an intravenous MPIO injection for cell labeling, followed by a surgically induced MI seven days later, compared to the two control groups (P < 0.01). The findings suggested that the prelabeled inflammatory cells mobilized and infiltrated into the MI site. Furthermore, the linear regression of contrast‐to‐noise ratio at the MI site and left ventricular ejection function suggested a positive correlation between the labeled inflammatory cell infiltration and cardiac function attenuation during post‐MI remodeling (r2 = 0.98). In conclusion, this study demonstrated an MRI technique for noninvasively and temporally monitoring inflammatory cell migration into the myocardium while potentially providing additional insight concerning the pathologic progression of a myocardial infarction. Magn Reson Med, 2010.

Monitoring dynamic alterations in calcium homeostasis by T1-weighted and T1-mapping cardiac manganese-enhanced MRI in a murine myocardial infarction model

Manganese has been used as a T1‐weighted MRI contrast agent in a variety of applications. Because manganese ions (Mn2+) enter viable myocardial cells via voltage‐gated Ca2+ channels, manganese‐enhanced MRI is sensitive to the viability and inotropic state of the heart. In spite of the established importance of Ca2+ regulation in the heart both before and after myocardial injury, monitoring strategies to assess Ca2+ homeostasis in affected cardiac tissues are limited. This study implements a T1‐mapping method to obtain quantitative information both dynamically and over a range of MnCl2 infusion doses. To optimize the current Mn2+ infusion protocols, we performed both dose‐dependent and temporal washout studies. A non‐linear relationship between infused MnCl2 solution dose and increase in left ventricular wall relaxation rate (ΔR1) was observed. Control mice also exhibited significant Mn2+ clearance over time, with a decrease in ΔR1 of ∼50% occurring in just 2.5 h. The complicated efflux time dependence possibly suggests multiple efflux mechanisms. With the use of the measured relationship between infused Mn2+ dose, ΔR1, and inductively coupled plasma mass spectrometry data analysis provided a means of estimating the absolute heart Mn concentration in vivo. We show that this technique has the sensitivity to observe or monitor potential alterations in Ca2+ handling in vivo because of the physiological remodeling after myocardial infarction. Left ventricular free wall ΔR1 values were significantly lower (P = 0.005) in the adjacent zone, surrounding the injured myocardial tissue, than in healthy tissue. This inferred reduction in Mn concentration can be used to estimate potentially salvageable myocardium in vivo for future treatment or evaluation of disease progression. Copyright

Assessing manganese efflux using SEA0400 and cardiac T1-mapping manganese-enhanced MRI in a murine model

The sodium–calcium exchanger (NCX) is one of the transporters contributing to the control of intracellular calcium (Ca2+) concentration by normally mediating net Ca2+ efflux. However, the reverse mode of the NCX can cause intracellular Ca2+ concentration overload, which exacerbates the myocardial tissue injury resulting from ischemia. Although the NCX inhibitor SEA0400 has been shown to therapeutically reduce myocardial injury, no in vivo technique exists to monitor intracellular Ca2+ fluctuations produced by this drug. Cardiac manganese‐enhanced MRI (MEMRI) may indirectly assess Ca2+ efflux by estimating changes in manganese (Mn2+) content in vivo, since Mn2+ has been suggested as a surrogate marker for Ca2+. This study used the MEMRI technique to examine the temporal features of cardiac Mn2+ efflux by implementing a T1‐mapping method and inhibiting the NCX with SEA0400. The change in 1H2O longitudinal relaxation rate, ΔR1, in the left ventricular free wall, was calculated at different time points following infusion of 190 nmol/g manganese chloride (MnCl2) in healthy adult male mice. The results showed 50% MEMRI signal attenuation at 3.4 ± 0.6 h post‐MnCl2 infusion without drug intervention. Furthermore, treatment with 50 ± 0.2 mg/kg of SEA0400 significantly reduced the rate of decrease in ΔR1. At 4.9–5.9 h post‐MnCl2 infusion, the average ΔR1 values for the two groups treated with SEA0400 were 2.46 ± 0.29 and 1.72 ± 0.24 s−1 for 50 and 20 mg/kg doses, respectively, as compared to the value of 1.27 ± 0.28 s−1 for the control group. When this in vivo data were compared to ex vivo absolute manganese content data, the MEMRI T1‐mapping technique was shown to effectively quantify Mn2+ efflux rates in the myocardium. Therefore, combining an NCX inhibitor with MEMRI may be a useful technique for assessing Mn2+ transport mechanisms and rates in vivo, which may reflect changes in Ca2+ transport. Copyright

Indirectly probing Ca(2+) handling alterations following myocardial infarction in a murine model using T(1)-mapping manganese-enhanced magnetic resonance imaging.

Prolonged ischemia causes cellular necrosis and myocardial infarction (MI) via intracellular calcium (Ca2+) overload. Manganese‐enhanced MRI indirectly assesses Ca2+ influx movement in vivo as manganese (Mn2+) is a Ca2+ analog. To characterize myocardial Mn2+ efflux properties, T1‐mapping manganese‐enhanced MRI studies were performed on adult male C57Bl/6 mice in which Ca2+ efflux was altered using pharmacological intervention agents or MI‐inducing surgery. Results showed that ( 1 ) Mn2+ efflux rate increased exponentially with increasing Mn2+ doses; ( 2 ) SEA0400 (a sodium–calcium exchanger inhibitor) decreased the rate of Mn2+ efflux; and ( 3 ) dobutamine (a positive inotropic agent) increased the Mn2+ efflux rate. A novel analysis technique also delineated regional features in the MI mice, which showed an increased Mn2+ efflux rate in the necrosed and peri‐infarcted tissue zones. The T1‐mapping manganese‐enhanced MRI technique characterized alterations in myocardial Mn2+ efflux rates following both pharmacologic intervention and an acute MI. The Mn2+ efflux results were consistent with those in ex vivo studies showing an increased Ca2+ concentration under similar conditions. Thus, T1‐mapping manganese‐enhanced MRI has the potential to indirectly identify and quantify intracellular Ca2+ handling in the peri‐infarcted tissue zones, which may reveal salvageable tissue in the post‐MI myocardium. Magn Reson Med, 2010.

Assessment of cell infiltration in myocardial infarction: A dose-dependent study using micrometer-sized iron oxide particles

Myocardial infarction (MI) is a leading cause of death and disabilities. Inflammatory cells play a vital role in the process of postinfarction remodeling and repair. Inflammatory cell infiltration into the infarct site can be monitored using T  2* ‐weighted MRI following an intravenous administration of iron oxide particles. In this study, various doses of micrometer‐sized iron oxide particles (1.1–14.5 μg Fe/g body weight) were injected into the mouse blood stream before a surgical induction of MI. Cardiac MRIs were performed at 3, 7, 14, and 21 days postinfarction to monitor the signal attenuation at the infarct site. A dose‐dependent phenomenon of signal attenuation was observed at the infarct site, with a higher dose leading to a darker signal. The study suggests an optimal temporal window for monitoring iron oxide particles‐labeled inflammatory cell infiltration to the infarct site using MRI. The dose‐dependent signal attenuation also indicates an optimal iron oxide dose of approximately 9.1–14.5 μg Fe/g body weight. A lower dose cannot differentiate the signal attenuation, whereas a higher dose would cause significant artifacts. This iron oxide‐enhanced MRI technique can potentially be used to monitor cell migration and infiltration at the pathological site or to confirm any cellular response following some specific treatment strategies. Magn Reson Med, 2011.

Regulatory Considerations Involved in Imaging

Today’s revolution in imaging technologies in the biomedical sciences has raised much needed hope for improved diagnostics, therapeutics, and the eventual cure of many debilitating illnesses. Imaging itself has become the seed technology that has fostered the development of many novel diagnostic approaches as well as helping point the way to witnessing the mechanism of action of drugs and biologics. The advancement of new drugs and biologics will be undertaken in the future with surrogate biomarkers, and many of these will be in the form of imaging. Imaging of pharmacodynamic responses to therapies such as changes in RECIST, cerebral glucose utilization, MRI BOLD changes reflecting neurologic activity, and many other novel approaches are opportunities for the imaging community to work with the regulatory community to contribute to the advancement of novel agents. As stated by Dr Steven Larson (2007) “We are experiencing a paradigm shift from anatomic towards biomarker (molecular imaging) as the primary means for assessing treatment response in oncology” and as such the regulatory environment for this to happen must be considered and developed to maximize the potential which imaging brings to medical diagnosis and to clinical decision making.

Magnetic Resonance as a Tool for Pharmaco-Imaging

Imaging technologies in the nonclinical laboratory have been greatly bolstered by the ever-improving methods available with magnetic resonance (MR) imaging. Small animal systems have been growing in capability even while becoming more amenable to use by biologists, revolutionizing how we can study pathophysiology and follow a drug or biologic therapy. MR’s ability to characterize many anatomical and physiological processes, based on their underlying influence on tissue magnetization properties, has led, for example, to discoveries in the psychopharmacology of attention deficit and cognitive drug therapies and in recording changes of oxygenation, blood flow and vessel permeability in acute studies, or the chronic remodeling of tissue water diffusion following therapy. This is a short and clearly abbreviated discussion of the applications of MRI in the nonclinical (and clinical) drug development laboratory, and it is meant to introduce the reader to the concepts and how this specific imaging modality likely offers the most versatile of all imaging modalities as well as being one with very high resolution.

TH‐D‐201C‐04: Quantification of Cellular Response in Myocardial Infarction Using Iron Oxide Particles‐Enhanced MRI

Purpose: To quantify the cellular response in myocardial infarction by using iron oxide particles cell labeling and MR imaging techniques. Method and Materials:Iron oxide particles can produce signal attenuation in MRI and were used to label either macrophages or mesenchymal stem cells. Macrophages were labeled via an intravenous administration of iron oxide particles whereas mesenchymal stem cells were labeled in vitro and then transplanted into the animal bone marrow. In the macrophage study varied doses (1.1–14.5 μg Fe/g body weight) were applied. After surgically inducing a myocardial infarction in the C57 mouse the labeled cells would mobilize to the infarction site attempting to repair the damaged tissue. To monitor the cellular response T2*‐weighted MRI was used to acquire short‐axis cardiac images at 3 7 14 and 21 days post‐infarction. Signal intensity normalized to the maximum signal from the ventricular blood was used to quantify labeled cells at infarction sites. Results: Cellular response during myocardial infarction was temporally and noninvasively monitored. In the macrophage study linear regression of normalized signal intensity at each time point revealed a linear relationship between the normalized signal intensity and iron oxide dose at 7 days (r2=0.92) and 14 days (r2=0.98) post‐infarction. An optimum dose as well as imaging time window for monitoring inflammatory cell response was also obtained. Either labeled macrophages or mesenchymal stem cells were evidenced at the infarction site in histology. Conclusions: A linear signal‐dose relationship at 7 and 14 days post‐infarction suggests that the number of labeled cells at the infarction site can be interpreted from the MR signal within this time window. This linear relationship may further apply to quantify labeled mesenchymal stem cells. Therefore the cell labeling and MR imaging technique have a potential to quantify cellular response in pathological processes as well as during cell therapy.

WE‐D‐303A‐04: Mircometer‐Sized Iron Oxide Particles (MPIO) Enhanced MRI with Granulocyte‐Colony Stimulating Factor (GCSF) Modulation in Murine Myocardial Infarction Model

Purpose: To monitor the MPIO and enhanced green fluorescenceprotein (eGFP) labeled mesenchymal stem cells (MSCs) infiltration into the myocardial infarction (MI) site using T 2 * ‐weighted MRI; To monitor the MRI contrast around the MI site post‐GCSF modulation. Methods: C57Bl/6 male mice (6–8 weeks old) were irradiated with an 8‐Gy dose. The labeled MSCs (3–7×105) were transplanted into the tibial medullary space 2 days post‐irradiation. The mice were divided into: 1) a sham‐operated group (Sham, n=7); 2) a MI group without GCSF injection (MI‐GCSF, n=7); and 3) a MI group with GCSF treatment (MI+GCSF, n=3). At 14 days post‐labeled MSCs transplantation, the two MI groups underwent surgery via permanent ligation of the left anterior descending coronary artery while the Sham group underwent open‐chest operation without perturbing the heart. The MI+GCSF group received subcutaneous GCSF injection 1 day post‐MI to enhance MSC mobilization. T 2 * ‐weighted short‐axis cardiac MRI was performed at baseline, 3, 7 and 14 days (D14) post‐surgery. Results: The MRI signal at the MI site was temporally attenuated for both MI groups, with more attenuated for MI+GCSF group (SNR 18.17±6.06 vs 11.37±1.01 at D14, p<0.05), but not for Sham group (30.63±5.69). The MI+GCSF group showed a trend of cardiac function improvement relative to MI‐GCSF group (left ventricular ejection function 45.55±7.52% vs 40.80±16.69% at D14), but it is insignificant possibly due to the small sample number. Dual‐labeled cells were fluorescently detected around the infarction site. Conclusions: Migration of MPIO‐labeled MSCs from bone marrow into the injured heart can be temporally monitored by MRI and additional signal attenuation caused by GCSF treatment can be differentiated. Results of this study suggest a potential approach in cell therapy to noninvasively monitor migration of labeled cells as well as the mobilization modulation produced by pharmaceuticals in the MI related events.

TU‐D‐332‐04: Modulating Mn2+ Efflux with SEA0400, Using Cardiac Manganese‐Enhanced MRI (MEMRI) T1‐Mapping in a Murine Model

Purpose: Ca 2+ is an important regulator of contractile function in the heart. Efflux mechanisms of the intracellular Ca 2+ concentration are regulated by the Na + / Ca 2+ exchanger (NCX) and plasma membrane Ca 2+ ‐ATPase (PMCA). During myocardial ischemic‐reperfusion intracellular Ca 2+ overloads via the reverse mode of the NCX, exacerbating myocardial injuries. Protocols that selectively inhibit this exchanger have shown potential therapeutic effects. Cardiac manganese‐enhanced MRI (MEMRI) can be implemented to quantify Mn 2+ concentration in vivo, where Mn 2+ has be sugested as a surrogate marker for Ca 2+ . This study introduces a potential technique to study cardiac Mn 2+ efflux by inhibiting the NCX using SEA0400. Method and Materials: Male C57Bl/6 mice (6–13 weeks) were separated into two groups to study the rate of Mn 2+ efflux; a control group and a group treated with SEA0400. Both groups were infused with a single dose of 190±2 nmoles/g BW Mn 2+ . The SEA0400 group were injected with 50 mg/kg SEA0400 one hour post‐ Mn 2+ infusion. Images were acquired on a horizontal 7.0 T Bruker BioSpec MRI spectrometer equipped with a micro imaging gradient. T1‐maps were acquired pre‐ Mn 2+ infusion and at various time points post‐ Mn 2+ infusion using an ECG‐gated, flow‐compensated Look‐Locker MRI pulse sequence. The change in relaxivity, ΔR1, in the left ventricular free wall (LV Wall), was calculated at different time points post‐infusion. Results: In the LV Wall 50% of the signal enhancement is attenuated within ∼3–4 hours post‐ Mn 2+ infusion. SEA0400 demonstrates the effectiveness of reducing the rate of Mn 2+ efflux. At a SEA0400 dose of 50 mg/kg the Mn 2+ efflux half‐life was approximately two times longer than the control group. Conclusion: This T1‐mapping technique can be used to quantify Mn 2+ efflux rates from the myocardium. By using a NCX inhibiting agent this technique can potentially be employed to interrogate individual Mn 2+ efflux mechanisms and rates in vivo.