Yuanxin Chen

Enzyme-Sensitive Magnetic Resonance Imaging Targeting Myeloperoxidase Identifies Active Inflammation in Experimental Rabbit Atherosclerotic Plaques

Background— Inflammation undermines the stability of atherosclerotic plaques, rendering them susceptible to acute rupture, the cataclysmic event that underlies clinical expression of this disease. Myeloperoxidase is a central inflammatory enzyme secreted by activated macrophages and is involved in multiple stages of plaque destabilization and patient outcome. We report here that a unique functional in vivo magnetic resonance agent can visualize myeloperoxidase activity in atherosclerotic plaques in a rabbit model. Methods and Results— We performed magnetic resonance imaging of the thoracic aorta of New Zealand White rabbits fed a cholesterol (n=14) or normal (n=4) diet up to 2 hours after injection of the myeloperoxidase sensor bis-5HT-DTPA(Gd) [MPO(Gd)], the conventional agent DTPA(Gd), or an MPO(Gd) analog, bis-tyr-DTPA(Gd), as controls. Delayed MPO(Gd) images (2 hours after injection) showed focal areas of increased contrast (>2-fold) in diseased wall but not in normal wall (P=0.84) compared with both DTPA(Gd) (n=11; P<0.001) and bis-tyr-DTPA(Gd) (n=3; P<0.05). Biochemical assays confirmed that diseased wall possessed 3-fold elevated myeloperoxidase activity compared with normal wall (P<0.01). Areas detected by MPO(Gd) imaging colocalized and correlated with myeloperoxidase-rich areas infiltrated by macrophages on histopathological evaluations (r=0.91, P<0.0001). Although macrophages were the main source of myeloperoxidase, not all macrophages secreted myeloperoxidase, which suggests that distinct subpopulations contribute differently to atherogenesis and supports our functional approach. Conclusions— The present study represents a unique approach in the detection of inflammation in atherosclerotic plaques by examining macrophage function and the activity of an effector enzyme to noninvasively provide both anatomic and functional information in vivo.

Activated iron-containing microglia in the human hippocampus identified by magnetic resonance imaging in Alzheimer disease.

Although amyloid plaques and neurofibrillary pathology play important roles in Alzheimer disease (AD), our understanding of AD is incomplete, and the contribution of microglia and iron to neurodegeneration is unknown. High-field magnetic resonance imaging (MRI) is exquisitely sensitive to microscopic iron. To explore iron-associated neuroinflammatory AD pathology, we studied AD and control human brain specimens by (1) performing ultra-high resolution ex vivo 7 Tesla MRI, (2) coregistering the MRI with successive histologic staining for iron, microglia, amyloid beta, and tau, and (3) quantifying the relationship between magnetic resonance signal intensity and histological staining. In AD, we identified numerous small MR hypointensities primarily within the subiculum that were best explained by the combination of microscopic iron and activated microglia (p = 0.025), in contradistinction to the relatively lesser contribution of tau or amyloid. Neuropathologically, this suggests that microglial-mediated neurodegeneration may occur in the hippocampal formation in AD and is detectable by ultra-high resolution MRI.

Comparison of Gadofluorine-M and Gd-DTPA for Noninvasive Staging of Atherosclerotic Plaque Stability Using MRI

Background—Inflammation and neovascularization play critical roles in the stability of atherosclerotic plaques. Whole-body quantitative assessment of these plaque features may improve patient risk-stratification for life-threatening thromboembolic events and direct appropriate intervention. In this report, we determined the utility of the MR contrast agent gadofluorine-M (GdF) for staging plaque stability and compared this to the conventional agent Gd-DTPA. Methods and Results—Five control and 7 atherosclerotic rabbits were sequentially imaged after administration of Gd-DTPA (0.2 mmol/kg) and GdF (0.1 mmol/kg) using a T1-weighted pulse sequence on a 3-T MRI scanner. Diseased aortic wall could be distinguished from normal wall based on wall-to-muscle contrast-to-noise values after GdF administration. RAM-11 (macrophages) and CD-31 (endothelial cells) immunostaining of MR-matched histological sections revealed that GdF accumulation was related to the degree of inflammation at the surface of plaques and the extent of core neovascularization. Importantly, an MR measure of GdF accumulation at both 1 and 24 hours after injection but not Gd-DTPA at peak enhancement was shown to correlate with a quantitative histological morphology index related to these 2 plaque features. Conclusions—GdF-enhanced MRI of atherosclerotic plaques allows noninvasive quantitative information about plaque composition to be acquired at multiple time points after injection (within 1 and up to 24 hours after injection). This dramatically widens the imaging window for assessing plaque stability that is currently attainable with clinically approved MR agents, therefore opening the possibility of whole-body (including coronary) detection of unstable plaques in the future and potentially improved mitigation of cataclysmic cardiovascular events.

Clinical field-strength MRI of amyloid plaques induced by low-level cholesterol feeding in rabbits

Two significant barriers have limited the development of effective treatment of Alzheimers disease. First, for many cases the aetiology is unknown and likely multi-factorial. Among these factors, hypercholesterolemia is a known risk predictor and has been linked to the formation of β-amyloid plaques, a pathological hallmark this disease. Second, standardized diagnostic tools are unable to definitively diagnose this disease prior to death; hence new diagnostic tools are urgently needed. Magnetic resonance imaging (MRI) using high field-strength scanners has shown promise for direct visualization of β-amyloid plaques, allowing in vivo longitudinal tracking of disease progression in mouse models. Here, we present a new rabbit model for studying the relationship between cholesterol and Alzheimers disease development and new tools for direct visualization of β-amyloid plaques using clinical field-strength MRI. New Zealand white rabbits were fed either a low-level (0.125–0.25% w/w) cholesterol diet (n = 5) or normal chow (n = 4) for 27 months. High-resolution (66 × 66 × 100 µm3; scan time = 96 min) ex vivo MRI of brains was performed using a 3-Tesla (T) MR scanner interfaced with customized gradient and radiofrequency coils. β-Amyloid-42 immunostaining and Prussian blue iron staining were performed on brain sections and MR and histological images were manually registered. MRI revealed distinct signal voids throughout the brains of cholesterol-fed rabbits, whereas minimal voids were seen in control rabbit brains. These voids corresponded directly to small clusters of extracellular β-amyloid-positive plaques, which were consistently identified as iron-loaded (the presumed source of MR contrast). Plaques were typically located in the hippocampus, parahippocampal gyrus, striatum, hypothalamus and thalamus. Quantitative analysis of the number of histologically positive β-amyloid plaques (P < 0.0001) and MR-positive signal voids (P < 0.05) found in cholesterol-fed and control rabbit brains corroborated our qualitative observations. In conclusion, long-term, low-level cholesterol feeding was sufficient to promote the formation of extracellular β-amyloid plaque formation in rabbits, supporting the integral role of cholesterol in the aetiology of Alzheimers disease. We also present the first evidence that MRI is capable of detecting iron-associated β-amyloid plaques in a rabbit model of Alzheimers disease and have advanced the sensitivity of MRI for plaque detection to a new level, allowing clinical field-strength scanners to be employed. We believe extension of these technologies to an in vivo setting in rabbits is feasible and that our results support future work exploring the role of MRI as a leading imaging tool for this debilitating and life-threatening disease.

Ultra-high resolution in-vivo 7.0 T structural imaging of the human hippocampus reveals the endfolial pathway

The hippocampus is a very important structure in memory formation and retrieval, as well as in various neurological disorders such as Alzheimers disease, epilepsy and depression. It is composed of many intricate subregions making it difficult to study the anatomical changes that take place during disease. The hippocampal hilus may have a unique neuroanatomy in humans compared to that in monkeys and rodents, with field CA3h greatly enlarged in humans compared to that in rodents, and a white-matter pathway, called the endfolial pathway, possibly only present in humans. In this study we have used newly developed 7.0T whole brain imaging sequence, balanced steady-state free precession (bSSFP) that can achieve 0.4mm isotropic images to study, in vivo, the anatomy of the hippocampal hilus. A detailed hippocampal subregional segmentation was performed according to anatomic atlases segmenting the following regions: CA4, CA3, CA2, CA1, SRLM (stratum radiatum lacunosum moleculare), alveus, fornix, and subiculum along with its molecular layer. We also segmented a hypointense structure centrally within the hilus that resembled the endfolial pathway. To validate that this hypointense signal represented the endfolial pathway, we acquired 0.1mm isotropic 8-phase cycle bSSFP on an excised specimen, and then sectioned and stained the specimen for myelin using an anti-myelin basic protein antibody (SMI 94). A structure tensor analysis was calculated on the myelin-stained section to show directionality of the underlying fibers. The endfolial pathway was consistently visualized within the hippocampal body in vivo in all subjects. It is a central pathway in the hippocampus, with unknown relevance in neurodegenerative disorders, but now that it can be visualized noninvasively, we can study its function and alterations in neurodegeneration.

Application of dual 19F and iron cellular MRI agents to track the infiltration of immune cells to the site of a rejected stem cell transplant

Cellular MRI) was used to detect implanted human mesenchymal stem cells (hMSCs) and the resulting macrophage infiltration that occurs in response to xenotransplantation.

A multimodality imaging model to track viable breast cancer cells from single arrest to metastasis in the mouse brain

Cellular MRI involves sensitive visualization of iron-labeled cells in vivo but cannot differentiate between dead and viable cells. Bioluminescence imaging (BLI) measures cellular viability, and thus we explored combining these tools to provide a more holistic view of metastatic cancer cell fate in mice. Human breast carcinoma cells stably expressing Firefly luciferase were loaded with iron particles, injected into the left ventricle, and BLI and MRI were performed on days 0, 8, 21 and 28. The number of brain MR signal voids (i.e., iron-loaded cells) on day 0 significantly correlated with BLI signal. Both BLI and MRI signals decreased from day 0 to day 8, indicating a loss of viable cells rather than a loss of iron label. Total brain MR tumour volume on day 28 also correlated with BLI signal. Overall, BLI complemented our sensitive cellular MRI technologies well, allowing us for the first time to screen animals for successful injections, and, in addition to MR measures of cell arrest and tumor burden, provided longitudinal measures of cancer cell viability in individual animals. We predict this novel multimodality molecular imaging framework will be useful for evaluating the efficacy of emerging anti-cancer drugs at different stages of the metastatic cascade.

MRI and histopathologic study of a novel cholesterol-fed rabbit model of xanthogranuloma.

To develop a rabbit model of xanthogranuloma based on supplementation of dietary cholesterol. The aim of this study was to analyze the xanthogranulomatous lesions using magnetic resonance imaging (MRI) and histological examination.

CORRELATIVE MRI/OPTICAL/ELECTRON MICROSCOPY EVALUATION OF METAL DISTRIBUTION AND OXIDATIVE STATE IN THE ALZHEIMER’S HIPPOCAMPUS

USA; William S. Middleton Memorial Veterans Hospital, Madison, WI, USA; University of Wisconsin School of Medicine and Public Health, Madison, WI, USA; University of Wisconsin,Madison,Madison, WI, USA; Wisconsin Alzheimer’s Disease Research Center, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA; University of California-Irvine, Irvine, CA, USA; Geriatric Research Education and Clinical Center, Wm. S. Middleton Veterans Hospital, Madison, WI, USA. Contact e-mail: ksprecher@wisc.edu

Abstract 4000: Cellular MRI of human breast cancer cells labeled with gadofluorine M

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The ability to non-invasively locate and quantify tumor cell number at a metastatic site would be of considerable interest in pre-clinical and clinical metastasis research. To this end, our objective is to develop positive-contrast cellular magnetic resonance imaging (MRI) for use in quantifying tumor cell number at a metastatic site in vivo. The goals of the current study were to characterize the in vitro labeling of MDA-MB-231-luc-D3H2LN human breast cancer cells with a novel positive contrast MR agent Gadofluorine M (GdF), and to demonstrate the detectability of GdF-labeled cells in vitro at clinical strength MRI (1.5 and 3Tesla) scanners. Briefly, cells (1.5 × 107cells) were loaded by simple incubation for 24h with GdF at concentrations ranging from 25μM to 10mM. GdF-loading from 25μM to 1000μM did not result in significant changes in metabolic activity. Higher GdF-loading concentrations, 2500μM and 10mM, resulted in a 16% and 21% reduction in metabolic activity, respectively. The lowest GdF-loading concentration used (25μM) resulted in a significant (9-fold) increase in MR signal enhancement which then plateaued from 100μM to 1000μM. T1-relaxometry of cell pellets demonstrated a similar statistical trend in relaxation rates of cell pellets for all loading conditions, and at both field strengths. Inductively-coupled plasma atomic emission spectroscopy was performed on the cell pellet extracts to assess the average amount of GdF that had accumulated per cell at each incubating concentration. Since the relaxivity of an MR contrast agent can be affected by its intracellular location, we assessed whether internalized GdF-cc (GdF analog where the mannose moiety was replaced with carbocyanine) was cytosolic or compartmentalized into endosomes, via immunostaining for mannose-6-phosphate receptor. Using confocal fluorescence microscopy, we found that GdF is largely cytosolic and did not co-localize to late endosomes, an optimal situation for achieving maximum positive-contrast by T1-shortening since endosomal compartmentalization can lessen the enhancement seen in MR images. From these results, we have determined that the GdF loading concentration of 1000μM permits the highest payload for detection by cellular MRI in clinical field strength scanners. Information from this proof-of-principle study will support future work to develop positive-contrast cellular MRI as a new approach to quantitative imaging of metastatic cells in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4000.