Mayk Kresse

Superparamagnetic iron oxide-enhanced MRI of atherosclerotic plaques in Watanabe hereditable hyperlipidemic rabbits

RATIONALE AND OBJECTIVES Inflammatory atherosclerotic plaques are characterized by increased endothelial permeability and multiple macrophages. Blood-pool MRI contrast agents like superparamagnetic iron oxide (SPIO) have an affinity for the monocyte-macrophage system and thus, may label inflammatory plaques. The objective was to demonstrate SPIO uptake in plaques of atherosclerotic rabbits by MRI and histology. METHODS Aortas of anesthetized Watanabe hereditable hyperlipidemic rabbits were studied with a moderately T2*-weighted gradient-echo sequence at 1.5 T. Four groups of five animals each were studied: (1) without ultrasmall SPIO (carboxydextran coating; particle size, 25 nm; estimated plasma half-life, 6 hours) or with imaging after intravenous injection of SPIO at a dose (micromol Fe/kg) and postcontrast time delay (hours) of 50/8 (2), 50/24 (3), or 200/48 (4). In vivo MRI was compared with corresponding ex vivo histological iron stains. RESULTS Animals receiving 200 micromol Fe/kg demonstrated areas of focal signal loss clearly confined to the aortic wall on a mean of 24 +/- 9 (31% +/- 11%) of 76 +/- 5 images compared with 0 +/- 0 of 76 +/- 5 images in controls (P = 0.009). The number of images with this finding in groups 2 and 3 was not significantly different compared with controls. By microscopy, SPIO-iron was seen in the endothelial cells and subendothelial intimal macrophages of atherosclerosis-prone aortic wall segments. Atherosclerotic lesions demonstrating iron uptake also showed a high macrophage content. CONCLUSIONS SPIO accumulates in aortic plaques of atherosclerotic rabbits, producing a characteristic MRI finding. As SPIO accumulates in plaques with increased endothelial permeability and a high macrophage content, two established features of plaque inflammation, it may have a potential for noninvasive assessment of inflammatory atherosclerotic plaques.

Determination of plasma protein adsorption on magnetic iron oxides: sample preparation.

AbstractPurpose. The purpose of this study was to investigate the influence of the sample preparation on the plasma protein adsorption pattern of polysaccharide-stabilized iron oxide particles by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Methods. The iron oxide particles were incubated in vitro in human plasma for five minutes. Thereafter, four different methods for particle recovery, including adsorbed proteins from surplus plasma, were investigated: centrifugation, magnetic separation, gel filtration and membrane-based static microfiltration. Adsorbed proteins were desorbed from the particle surfaces by surfactants and analyzed by 2-D PAGE, as described elsewhere (1,2). Results. All the techniques investigated were able to separate small-size iron oxides (approx. 110 nm) and adsorbed proteins from excess plasma. The gels obtained by the different separation procedures displayed almost identical adsorption patterns. Major proteins identified were: fibrinogen, IgG, albumin and an unclassified protein of about 70 kDa with a pI value of 6.5−7.5. Conclusions. Centrifugation was regarded as the most suitable separation method due to its speed and ease of use. In contrast to gel filtration, any washing media can be used. The magnetic separation process is restricted to particles with high inducible magnetic saturation, in particular, to iron oxides with overall sizes > 50 nm.

The influence of the sample preparation on plasma protein adsorption patterns on polysaccharide-stabilized iron oxide particles and n-terminal microsequencing of unknown proteins

The in vivo organ distribution of i.v. injected drug carriers is strongly influenced by the adsorption of plasma proteins after i.v. injection, e.g. uptake by the mononuclear phagocytic system (MPS). 2-D PAGE could be established to analyze plasma protein adsorption patterns on polysaccharide-stabilized aqueous iron oxide dispersions used as contrast agents in Magnetic Resonance Imaging (MRI). After incubation in human plasma, centrifugation, a washing procedure and a solubilization step were carried out to obtain the proteins adsorbed onto these ultrasmall particles (65 nm in diameter). Patterns of adsorbed proteins were analyzed in dependence on the washing medium used, i.e. highly purified water, phosphate buffered saline and Krebs buffer pH 7.4. Conductivity and composition of the washing medium influenced the adsorption of IgG onto the particles, but had little effect on the other proteins present. IgG was strongly reduced when using the relatively high conductive buffers. The more stabilizing polysaccharide was desorbed the larger was the total amount of adsorbed proteins. Appearance of two unknown chains of spots in the range of appr. 92 kDa, accounting for appr. 10% and 2% of the overall detected protein amount, was observed only when using Krebs buffer during the washing process. Performing N-terminal microsequencing one unknown chain of spots could be identified as a dimer of fibrinogen gamma chains.

Intravenous MR lymphography with superparamagnetic iron oxide particles: experimental studies in rats and rabbits

The inter- and intralymphonodal distribution of IV-administered superparamagnetic iron oxide (SPIO) particles as a lymphographic contrast agent for MRI was studied in various animal models in rats and rabbits. In all animals a dosage of 200 μmol Fe/kg was tested. Imaging was done at 1.5 Tesla using proton-density-weighted spin-echo (PD-SE) and T2*-weighted gradient-echo (T2*-GRE) sequences. The time course of signal loss in popliteal lymph nodes of 21 rats was studied before and up to 72 h after IV injection of SPIO. Another 6 rats were dissected 24 h after IV injection of SPIO, all lymph nodes were embedded in agar gel and imaged ex vivo. Time course and pattern of lymph node signal loss was studied in 6 rabbits with reactive lymph node hyperplasia. The visualization of lymph node metastases was studied in 4 VX2 tumor-bearing rabbits. Most pronounced signal loss in lymph nodes was found 24 h after IV injection of SPIO with a decrease of signal in popliteal lymph nodes to 37 ± 15% (9 ± 5%) for rats and 56 ± 10% (16 ± 9%) for rabbits with the PD-SE (T2*-GRE) sequence. Ex vivo examinations of rat lymph nodes and in vivo examinations in rabbits with lymph node hyperplasia demonstrated marked variations in contrast agent accumulation between different lymph node groups. In VX2 tumor-bearing rabbits lymph node metastases could be well delineated in postcontrast MRI if a sufficient amount of contrast agent reached the lymph nodes (2 rabbits). Inhomogeneous signal loss as well as supersaturation impeded correct lymph node assessment (2 rabbits). We conclude that IV MR lymphography using SPIO may be an approach for non invasive tumor staging, but this new technique could be limited by variations in contrast agent distribution between different lymph node groups.

Magnetic resonance lymphography in rats: Effects of muscular activity and hyperthermia on the lymph node uptake of intravenously injected superparamagnetic iron oxide particles

RATIONALE AND OBJECTIVES We investigated the influence of muscular activity and regional body temperature changes on the accumulation of intravenously (i.v.) administered, dextran-coated superparamagnetic iron oxide (SPIO) particles in the lymph nodes of rats. METHODS Four groups of rats (N = 21) were used. Five rats were allowed to move freely after i.v. contrast administration (group 1). In another five rats, muscular inactivity (group 2) was induced during i.v. injection of SPIO particles and for up to 2 hr thereafter by anesthesia. In seven rats (likewise anesthetized), the contrast agent was administered while the extremities of one side of the body were warmed in a water bath for 2 hr (group 3). The rats in groups 1-3 received 100 mumol Fe/kg of the contrast agent. Four rats not given SPIO particles served as the control group (group 4). The lymph nodes of all animals were removed 24 hr after SPIO administration and were embedded in an agar matrix for magnetic resonance imaging at 1.5 T using a proton-density-weighted spin-echo (PD-SE) sequence and a T2*-weighted gradient-recalled echo (T2* GRE) sequence. RESULTS Signal loss varied widely among the different lymph nodes in group 1. A pronounced signal reduction was observed in the mesenteric (PD-SE = 20 +/- 6%, T2* GRE = 55 +/- 19%), iliac (PD-SE = 13 +/- 13%, T2* GRE = 44 +/- 24%), and popliteal (PD-SE = 24 +/- 7%, T2* GRE = 70 +/- 11%) lymph nodes and only a moderate reduction in the mandibular (PD-SE = 4 +/- 7%, T2* GRE = 42 +/- 15%), axillary (PD-SE = 0 +/- 4%, T2* GRE = 8 +/- 7%), and inguinal (PD-SE = 5 +/- 5%, T2* GRE = 34 +/- 18%) lymph nodes. The least pronounced signal loss occurred in the peripheral lymph nodes of group 2, ranging from 0 +/- 3% for PD-SE sequences and 10 +/- 11% for T2* GRE sequences to 13 +/- 15% for PD-SE sequences and 41 +/- 19% for T2* GRE sequences. In group 3, the uptake of contrast material in the peripheral lymph nodes of the hyperthermal side was significantly more pronounced than on the contralateral side (p < .01), and the contrast agent was distributed more evenly to the different lymph node groups than in group 1. CONCLUSION Muscular activity and regional hyperthermia markedly influence the accumulation of SPIO particles in different lymph node groups in rats. These findings must be considered in preclinical studies and in the clinical administration of MR lymphography.

Liver and spleen enhancement after intravenous injection of carboxydextran magnetite: effect of dose, delay of imaging, and field strength in an ex vivo model

It has been predicted that liver and spleen enhancement after administration of superparamagnetic contrast agents may be different, depending on the strength of the main magnetic field. With the use of anex vivo model, we investigated at 0.3, 0.5, and 1.5 T the effects on liver and spleen signal intensity of 5, 15, and 45 µmol/kg body weight of dextran magnetite (SHU 555A) in 54 rats. Nine rats served as controls. At different time delays since injection, the animals were killed, and after perfusion with saline, the liver, brain, and spleen were fixed in formalin. The specimens were embedded in an agar gel matrix and imaged with inversion recovery T1-weighted, proton density spin echo, and T2*-weighted gradient recalled echo (GRE) sequences. At each magnetic field strength, peak liver and spleen signal loss increased with increasing dose of the contrast medium. Signal loss was significantly more conspicuous after a dose of 15 than 5 µmol/kg body weight, but not after a dose of 45 compared with 15 µmol/kg. No signal change was observed in the brain. GRE images showed higher enhancement than proton density-weighted spin echo and inversion recovery images but were noisier. The enhancement showed a plateau between 30 min and 24 hours. Only the signal decrease of the liver after a low dose of contrast medium on GRE images was significantly higher (p<0.01) at 1.5 than at 0.5 and 0.3 T. Other differences in respect to the field strength were less significant (p<0.05) or nonsignificant. Differences in the spleen enhancement were nonsignificant. SHU 555A at a dose of 15 µmol/kg is an efficient intracellular contrast agent for liver and spleen at low, mid, and high field strength. Proton density spin echo images are probably the sequence of choice to exploit SHU 555A contrast effects and a wide time window for imaging after its intravenous injection does exist.

USPIO-enhanced direct thrombus MRI.

Endothelial dysfunction and macrophage infiltration are basic pathomechanisms of atherosclerosis and deep vein thrombosis. Ultrasmall superparamagnetic iron oxide particles (USPIO; charge DDM43/34, IDF, Berlin, Germany) were demonstrated to accumulate in inflammatory atherosclerotic plaques of Watanabe heritable hyperlipidemic rabbits through regionally increased endothelial permeability and uptake by macrophages (1). Initial clinical observations show that AMI227 (Sinerem®, Guerbet) was taken up in plaques of pelvic arteries in 7 of 19 patients (2). Inflammatory plaques are not only indicators of progression of atherosclerosis but may also trigger occluding thrombosis through endothelial ulceration and thus lead to myocardial infarction or cerebral infarction (vulnerable plaques) (3). Endothelial dysfunction is also present in fresh thrombi, where the endothelial cover is absent or incomplete (4). Only organized thrombi undergo complete endothelialization. During inflammatory organization, macrophages eliminate decomposition products of the thrombus through phagocytosis (siderophages). The aim of the present study was to investigate whether USPIO particles diffuse into nonendothelialized fresh thrombi or organized thrombi rich in macrophages owing to the high intravascular concentration of this blood pool contrast medium and thus allow for direct thrombus MR imaging.

Spio-Enhanced MR Lymphography

In patients with neoplastic disease, a possible metastatic spread in the regional and distant lymph nodes determines both the therapeutic concepts and prognosis.