Yijen L. Wu

Longitudinal Tracking of Recipient Macrophages in a Rat Chronic Cardiac Allograft Rejection Model With Noninvasive Magnetic Resonance Imaging Using Micrometer-Sized Paramagnetic Iron Oxide Particles

Background— Long-term survival of heart transplants is hampered by chronic rejection (CR). Studies indicate the involvement of host macrophages in the development of CR; however, the precise role of these cells in CR is unclear. Thus, it is important to develop noninvasive techniques to serially monitor the movement and distribution of recipient macrophages in chronic cardiac allograft rejection in vivo. Methods and Results— We have employed a rat heterotopic working-heart CR model for a magnetic resonance imaging experiment. Twenty-one allograft (PVG.1U→PVG.R8) and 9 isograft (PVG.R8→PVG.R8) transplantations were performed. Recipient macrophages are labeled via intravenous injection of micron-sized paramagnetic iron oxide particles (0.9 &mgr;m in diameter) at a dose of 4.5 mg Fe per rat 1 day before transplantation. Serial in vivo magnetic resonance images were acquired for up to 16 weeks. The migration of labeled recipient cells in our CR model, in which cardiac CR is evident at 3 weeks and most extensive by 16 weeks after transplantation, can be assessed with the use of in vivo magnetic resonance imaging for >100 days after a single micron-sized paramagnetic iron oxide injection. The location and distribution of labeled recipient cells were confirmed with magnetic resonance microscopy and histology. Conclusions— This approach may improve our understanding of the immune cells involved in CR and the management of heart transplantation. Moreover, this study demonstrates the feasibility of noninvasively observing individual targeted cells over long time periods by serial in vivo magnetic resonance imaging.

Noninvasive Evaluation of Cardiac Allograft Rejection by Cellular and Functional Cardiac Magnetic Resonance

OBJECTIVES We sought to use cardiac magnetic resonance (CMR) to establish sensitive and reliable indexes for noninvasive detection of acute cardiac allograft rejection. BACKGROUND Appropriate surveillance for acute allograft rejection is vitally important for graft survival. The current gold standard for diagnosing and staging rejection after organ transplantation is endomyocardial biopsy, which is not only invasive but also prone to sampling errors. The motivation of this study is to establish a CMR-based alternative that is noninvasive and sensitive for early detection of allograft rejection before irreversible damage occurs. METHODS We employed a noninvasive 2-pronged approach to detect acute cardiac allograft rejection using a rodent working heart and lung transplantation model. We used CMR to detect immune-cell infiltration at sites of rejection by monitoring the accumulation of dextran-coated ultra-small superparamagnetic-iron-oxide-labeled immune cells (in particular macrophages) in vivo. Simultaneously, we used CMR tagging and strain analysis to detect regional myocardial function loss resulting from acute rejection. RESULTS Immune cells infiltration, mainly macrophages and monocytes, could be identified with CMR by in vivo labeling with ultra-small superparamagnetic-iron-oxide. Our data show that immune-cell infiltration in cardiac allograft rejection was highly heterogeneous. Thus, it is not surprising to find inconsistencies between rejection and endomyocardial biopsy results because of the limited number and small samples available. Tagged CMR and strain analysis showed that, as with immune-cell infiltration, ventricular functional loss was also heterogeneous. Although changes in global systolic function were generally not observed until the later stages of rejection, our data revealed that a functional index derived from local strain analysis correlated well with rejection grades, which may be a more sensitive parameter for detecting early rejection. CONCLUSIONS CMR is noninvasive and provides a 3-dimensional, whole-heart perspective of the rejection status, potentially allowing more reliable detection of acute allograft rejection.

Decreased reticuloendothelial system clearance and increased blood half-life and immune cell labeling for nano- and micron-sized superparamagnetic iron-oxide particles upon pre-treatment with Intralipid.

BACKGROUND Superparamagnetic iron-oxide nanoparticles are useful as contrast agents for anatomical, functional and cellular MRI, drug delivery agents, and diagnostic biosensors. Nanoparticles are generally cleared by the reticuloendothelial system (RES), in particular taken up by Kupffer cells in the liver, limiting particle bioavailability and in-vivo applications. Strategies that decrease the RES clearance and prolong the circulation residence time of particles can improve the in-vivo targeting efficiency. METHODS Intralipid 20.0%, an FDA approved nutritional supplement, was intravenously administered in rats at the clinical dose (2g/kg) 1h before intravenous injection of ultra-small superparamagnetic iron-oxide (USPIO) or micron-sized paramagnetic iron-oxide (MPIO) particles. Blood half-life, monocyte labeling efficiency, and particle biodistribution were assessed by magnetic resonance relaxometry, flow cytometry, inductively-coupled plasma MS, and histology. RESULTS Pre-treatment with Intralipid resulted in a 3.1-fold increase in USPIO blood half-life and a 2-fold increase in USPIO-labeled monocytes. A 2.5-fold increase in MPIO blood half-life and a 5-fold increase in MPIO-labeled monocytes were observed following Intralipid pre-treatment, with a 3.2-fold increase in mean iron content up to 2.60pg Fe/monocyte. With Intralipid, there was a 49.2% and 45.1% reduction in liver uptake vs. untreated controls at 48h for USPIO and MPIO, respectively. CONCLUSIONS Intralipid pre-treatment significantly decreases initial RES uptake and increases in-vivo circulation and blood monocyte labeling efficiency for nano- and micron-sized superparamagnetic iron-oxide particles. GENERAL SIGNIFICANCE Our findings can have broad applications for imaging and drug delivery applications, increasing the bioavailability of nano- and micron-sized particles for target sites other than the liver.

Tracking T-cells in vivo with a new nano-sized MRI contrast agent

UNLABELLED Non-invasive in vivo tracking of T-cells by magnetic resonance imaging (MRI) can lead to a better understanding of many pathophysiological situations, including AIDS, cancer, diabetes, graft rejection. However, an efficient MRI contrast agent and a reliable technique to track non-phagocytic T-cells are needed. We report a novel superparamagnetic nano-sized iron-oxide particle, IOPC-NH2 series particles, coated with polyethylene glycol (PEG), with high transverse relaxivity (250 s(-1) mM(-1)), thus useful for MRI studies. IOPC-NH2 particles are the first reported magnetic particles that can label rat and human T-cells with over 90% efficiency, without using transfection agents, HIV-1 transactivator peptide, or electroporation. IOPC-NH2 particles do not cause any measurable effects on T-cell properties. Infiltration of IOPC-NH2-labeled T-cells can be detected in a rat model of heart-lung transplantation by in vivo MRI. IOPC-NH2 is potentially valuable contrast agents for labeling a variety of cells for basic and clinical cellular MRI studies, e.g., cellular therapy. FROM THE CLINICAL EDITOR In this study, a novel PEG coated superparamagnetic nano-sized iron-oxide particle was investigated as a T-cell labeling agent for MRI studies. The reported particles can label T-cells with over 90% efficiency, without using transfection agents, HIV-1 transactivator peptide, or electroporation, therefore may enable more convenient preclinical call labeling studies.

MRI Investigations of Graft Rejection Following Organ Transplantation Using Rodent Models

Publisher Summary This chapter examines the magnetic resonance imaging (MRI) investigations of graft rejection following organ transplantation using rodent models. The emerging noninvasive MRI methodologies for evaluating acute allograft rejection as potential alternatives for surveillance biopsy are described. In the renal transplantation model, most of the allogeneic kidney grafts show mild acute rejection on postoperative day (POD) 3, with light interstitial edema and some foci of lymphoplasmacytic infiltration in the perivascular space and in the surrounding interstitium, but the glomeruli are within the normal range. On POD 4, the allografts undergo moderate acute rejection with lymphoplasmacytic in filtration found over the entire interstitium. On POD 5, extensive interstitial lymphoplasmacytic in filtration is found with hemorrhage. The combined information criterion is used to select the order of the autoregression (AR) model, and the AR coefficients are estimated by Burgs method, which is based on the minimization of the sum of the forward and backward squared prediction errors. The results shows that the two-pronged approach to detect the initiation of graft rejection and to monitor the function of a transplanted organ during various stages of the graft rejection process using the noninvasive MRI methodology is very attractive and merits further development.

Magnetic Resonance Imaging Investigation of Macrophages in Acute Cardiac Allograft Rejection After Heart Transplantation

Background—Current immunosuppressive therapy after heart transplantation either generally suppresses the recipient’s entire immune system or is mainly targeting T-lymphocytes. Monocytes/macrophages are recognized as a hallmark of acute allograft rejection, but the roles that they play are not well characterized in vivo, because the tools for accessing in situ macrophage infiltration are lacking. In this study, we used MRI to investigate the role of macrophages in acute heart allograft rejection by cellular and functional MRI with selectively depleted systemic macrophages without affecting other leukocyte population, as well as to explore the possibility that macrophages could be an alternative therapeutic target. Methods and Results—A rodent heterotopic working heart–lung transplantation model was used for studying acute allograft rejection. Systemic macrophages were selectively depleted by treating recipient animals with clodronate-liposomes. Macrophage infiltration in the graft hearts was monitored by cellular MRI with in vivo ultrasmall superparamagnetic iron oxide particles labeling. Graft heart function was evaluated by tagging MRI followed by strain analysis. Clodronate-liposome treatment depletes circulating monocytes/macrophages in transplant recipients, and both cellular MRI and pathological examinations indicate a significant reduction in macrophage accumulation in the rejecting allograft hearts. In clodronate-liposome–treated group, allograft hearts exhibited preserved tissue integrity, partially reversed functional deterioration, and prolonged graft survival, compared with untreated controls. Conclusions—Cardiac cellular and functional MRI is a powerful tool to explore the roles of targeted immune cells in vivo. Our results indicate that macrophages are essential in acute cardiac allograft rejection, and selective depletion of macrophages with clodronate-liposomes protects hearts against allograft rejection, suggesting a potential therapeutic avenue. Our findings show that there is a finite risk of forming an intraventricular mass, presumably from the cellular debris or lipid material. Further optimization of the dosing protocol is necessary before clinical applications.

Automatic Detection of Regional Heart Rejection in USPIO-Enhanced MRI

Contrast-enhanced magnetic resonance imaging (MRI) is useful to study the infiltration of cells in vivo. This research adopts ultrasmall superparamagnetic iron oxide (USPIO) particles as contrast agents. USPIO particles administered intravenously can be endocytosed by circulating immune cells, in particular, macrophages. Hence, macrophages are labeled with USPIO particles. When a transplanted heart undergoes rejection, immune cells will infiltrate the allograft. Imaged by T2*-weighted MRI, USPIO-labeled macrophages display dark pixel intensities. Detecting these labeled cells in the image facilitates the identification of acute heart rejection. This paper develops a classifier to detect the presence of USPIO-labeled macrophages in the myocardium in the framework of spectral graph theory. First, we describe a USPIO-enhanced heart image with a graph. Classification becomes equivalent to partitioning the graph into two disjoint subgraphs. We use the Cheeger constant of the graph as an objective functional to derive the classifier. We represent the classifier as a linear combination of basis functions given from the spectral analysis of the graph Laplacian. Minimization of the Cheeger constant based functional leads to the optimal classifier. Experimental results and comparisons with other methods suggest the feasibility of our approach to study the rejection of hearts imaged by USPIO-enhanced MRI.

A New Method for Preparing Mesenchymal Stem Cells and Labeling with Ferumoxytol for Cell Tracking by MRI

Mesenchymal stem cells (MSCs) are among the major stem cells used for cell therapy and regenerative medicine. In-vivo cell-tracking by magnetic resonance imaging (MRI) is crucial for regenerative medicine, allowing verification that the transplanted cells reach the targeted sites. Cellular MRI combined with superparamagnetic iron-oxide (SPIO) contrast agents is an effective cell-tracking method. Here, we are reporting a new “bio-mimicry” method by making use of the “in-vivo environment” of MSCs to prepare native MSCs, so that (i) the phagocytic activity of cultured MSCs can be recovered and expanded MSCs can be ex-vivo labeled with Ferumoxytol, which is currently the only FDA approved SPIO nanoparticles for human use. Using our new method, 7-day cultured MSCs regain the capability to take up Ferumoxytol and exhibit an intracellular iron concentration of 2.50 ± 0.50 pg/MSC, comparable to that obtained by using Ferumoxytol-heparin-protamine nanocomplex; and (ii) cells can be re-sized to more native size, reducing from 32.0 ± 7.2 μm to 19.5 ± 5.2 μm. Our method can be very useful for expanding MSCs and labeling with Ferumoxytol, without the need for transfection agents and/or electroporation, allowing cell-tracking by MRI in both pre-clinical and clinical studies.

Mapping stain distribution in pathology slides using whole slide imaging

Background: Whole slide imaging (WSI) offers a novel approach to digitize and review pathology slides, but the voluminous data generated by this technology demand new computational methods for image analysis. Materials and Methods: In this study, we report a method that recognizes stains in WSI data and uses kernel density estimator to calculate the stain density across the digitized pathology slides. The validation study was conducted using a rat model of acute cardiac allograft rejection and another rat model of heart ischemia/reperfusion injury. Immunohistochemistry (IHC) was conducted to label ED1 + macrophages in the tissue sections and the stained slides were digitized by a whole slide scanner. The whole slide images were tessellated to enable parallel processing. Pixel-wise stain classification was conducted to classify the IHC stains from those of the background and the density distribution of the identified IHC stains was then calculated by the kernel density estimator. Results: The regression analysis showed a correlation coefficient of 0.8961 between the number of IHC stains counted by our stain recognition algorithm and that by the manual counting, suggesting that our stain recognition algorithm was in good agreement with the manual counting. The density distribution of the IHC stains showed a consistent pattern with those of the cellular magnetic resonance (MR) images that detected macrophages labeled by ultrasmall superparamagnetic iron-oxide or micron-sized iron-oxide particles. Conclusions: Our method provides a new imaging modality to facilitate clinical diagnosis. It also provides a way to validate/correlate cellular MRI data used for tracking immune-cell infiltration in cardiac transplant rejection and cardiac ischemic injury.

Improved Subspace Estimation for Low-Rank Model-Based Accelerated Cardiac Imaging

Sparse sampling methods have emerged as effective tools to accelerate cardiac magnetic resonance imaging (MRI). Low-rank model-based cardiac imaging uses a predetermined temporal subspace for image reconstruction from highly undersampled (k, t)-space data and has been demonstrated effective for high-speed cardiac MRI. The accuracy of the temporal subspace is a key factor in these methods, yet little work has been published on data acquisition strategies to improve subspace estimation. This paper investigates the use of non-Cartesian k-space trajectories to replace the Cartesian trajectories that are omnipresent but are highly sensitive to readout direction. We also propose “self-navigated” pulse sequences that collect both navigator data (for determining the temporal subspace) and imaging data after every RF pulse, allowing for even greater acceleration. We investigate subspace estimation strategies through analysis of phantom images and demonstrate in vivo cardiac imaging in rats and mice without the use of ECG or respiratory gating. The proposed methods achieved 3-D imaging of wall motion, first-pass myocardial perfusion, and late gadolinium enhancement in rats at 74 frames/s, as well as 2-D imaging of wall motion in mice at 97 frames/s.