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Imaging of Macrophages in Soft-Tissue Infection in Rats: Relationship between Ultrasmall Superparamagnetic Iron Oxide Dose and MR Signal Characteristics¹

¹ From the Institute of Diagnostic Radiology (A.M.L., D.W., K.G., B.M.) and Division of Cellular and Molecular Pathology (B.S.), University Hospital Zurich, Rämistrasse 100, 8091 Zurich, Switzerland; Novartis Pharma, Basel, Switzerland (E.P.); Froehlich Pharma Consulting, Zurich, Switzerland (J.F.); Institute of Virology, University of Zurich, Switzerland (J.G.); and Institute of Radiology, Hospital Im Schachen, Aarau, Switzerland (A.H.K.). Received July 25, 2003; revision requested October 3; final revision received April 6, 2004; accepted May 24. Address correspondence to D.W. (e-mail: dominik.weishaupt@dmr.usz.ch).

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To describe dose-dependent signal intensity (SI) characteristics of experimentally induced soft-tissue abscesses on 1.5-T T1- and T2*-weighted magnetic resonance (MR) images obtained 24 hours after administration of ultrasmall superparamagnetic iron oxide (USPIO) and to describe the relationship between SI and amount of USPIO uptake and macrophage iron content.

MATERIALS AND METHODS: Local institutional review committee on animal care approved the experiments, which were performed according to the guidelines of the National Institutes of Health and the committee on animal research at our institution. Unilateral calf muscle abscesses were induced in 21 rats with an injection of a Staphylococcus aureus suspension. The rats were divided into three groups of seven animals each: low USPIO dose (50 µmol of iron per kilogram of body weight), high USPIO dose (150 µmol Fe/kg), and control (saline solution). All rats were imaged before and 24 hours after USPIO administration at 1.5 T (transverse T1-weighted spin-echo, T2*-weighted fast gradient-echo, and short inversion time inversion-recovery sequences). Images were analyzed quantitatively and qualitatively with regard to SI and signal pattern. Temporal variation of calculated contrast-to-noise ratios was analyzed with the Wilcoxon signed rank test. MR findings were correlated with histopathologic findings, including those of electron microscopy.

RESULTS: Twenty-four hours after USPIO administration in the high-dose group, susceptibility effects were present in abscess periphery on postcontrast T2*-weighted images (P = .04), and SI enhancement was noted on postcontrast T1-weighted images within both abscess wall and abscess center (P = .04 for both). In the low-dose group, SI enhancement was noted in entire abscess on T1-weighted postcontrast images (P = .03). Neither significant SI loss (P = .09) nor susceptibility effects were detected in periphery or center of any abscess on postcontrast T2*-weighted images. There was no obvious difference in total amount of macrophages among the groups, but there was a clear difference with regard to individual iron content of iron-positive macrophages between the USPIO dose groups.

CONCLUSION: At 1.5 T, SI characteristics of abscesses on T1- and T2*-weighted images obtained 24 hours after USPIO injection strongly depend on administered dose of the contrast agent. At low doses, T1 effects were stronger than T2* effects.

© RSNA, 2005

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The role of magnetic resonance (MR) imaging for detection of macrophage phagocytic activity is evolving. Several in vitro and in vivo studies have demonstrated the feasibility and potential clinical applications of macrophage-specific MR imaging following intravenous administration of ultrasmall superparamagnetic iron oxide (USPIO) in animals and humans (1–15).

The experience with USPIO for imaging of soft-tissue infection is limited (16). Recently, Kaim et al (17,18) have demonstrated the feasibility of USPIO-enhanced MR imaging in detection of phagocytic-active macrophages in soft-tissue infection at a 4.7-T magnetic field.

An important consideration with regard to the clinical implementation of USPIO-enhanced MR imaging for the assessment of soft-tissue infection is the relationship between the dose of the intravenously administered USPIO and the resulting MR signal changes with T1- and T2*-weighted pulse sequences.

So far, authors of most of the studies, including Kaim et al (17), have used T2- or T2*-weighted sequences for the detection of intracytoplasmatically deposited iron oxide crystals in macrophages (1,3,6–14). In addition to the T2 and T2* effects, USPIOs also have inherent T1-shortening properties (3,19,20). However, to our knowledge, in vivo imaging of macrophage activity based on T1-shortening properties of USPIO has not yet been demonstrated. This may be due to the fact that most studies dealing with in vivo MR imaging of macrophage activity were performed by using relatively high intravenous doses of USPIO. By taking into consideration the r1 and r2 relaxivity properties of USPIO, it might be expected that T1 effects would be more pronounced at lower doses, which would theoretically result in lower intracellular concentrations of USPIO within the macrophages (2,3,19).

The purposes of this study were to describe the dose-dependent signal intensity (SI) characteristics of experimentally induced soft-tissue abscesses on T1- and T2*-weighted images obtained 24 hours after intravenous administration of USPIO in a 1.5-T magnetic field and to describe the relationship between SI characteristics and the amount of USPIO uptake and macrophage iron content.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Animal Model
All experiments, including the pilot study, were conducted on 7-week-old female Sprague-Dawley rats (stock Icolbm; OFA, Fullinsdorf, Switzerland) weighing 250–300 g. All experiments were fully approved by the local institutional review committee on animal care, and the study was performed according to the guidelines of the National Institutes of Health and of the committee on animal research at University Hospital Zurich.

Similar to the experimental abscess model described by Kaim et al (21), unilateral calf muscle abscess was induced in all animals with an inoculation of 0.2 mL of a Staphylococcus aureus suspension (clinical strain 10B; Novartis Pharma, Basel, Switzerland) into the dorsal calf muscles of the left hind leg. The inoculation was performed by using a 25-gauge needle with general inhalation anesthetic (1.5% isoflurane in 1:2 mixture of O₂ and N₂O, Forene; Abbott Laboratories, Abbott Park, Ill). The day of inoculation was considered day 0. Subsequently, the animals were kept in cages with standardized conditions and free access to water and food for the next 5 days. Five days following S aureus inoculation, rats are known to develop intramuscular abscesses that manifest as palpable fluctuating masses in the calf (21).

Experimental Setup
Twenty-one animals were randomly divided into three groups: high-dose USPIO group, low-dose USPIO group, and control group. Each group consisted of seven animals. To use the concept of replacement, reduction, and refinement ("3R" concept) in performing animal experiments, as described by Russel and Burch (22), and as few animals as possible, we consulted a statistician before this study was started. When the Wilcoxon signed rank test is used for non-Gaussian variables, at least six animals have to be examined to prove an observed effect to be statistically significant, given the fact that this effect will be present in all animals. To account for the possibility that the expected effect might not be observed in all animals on the one hand, and to use the "3R" concept (22) on the other hand, three groups consisting of seven animals each were included in the study.

Each group underwent two MR imaging sessions—one on day 5 and one on day 6—after the injection of the bacterial suspension. General inhalation anesthetic (2.5% isoflurane in O₂, Forene; Abbott Laboratories) was administered in all rats for each MR imaging session.

Immediately after the second MR imaging session (day 6), the animals were sacrificed with an overdose of pentobarbital (Vetanarcol; Veterinaria, Zurich, Switzerland). The infected calf muscles of all animals were dissected and fixed in 4% formalin for histologic study.

Contrast Agent
The USPIOs used in this study were nanoparticles that consist of a core of iron oxide crystals measuring 4–6 nm in diameter and are coated with dextran (ferumoxtran, Sinerem, Guerbet, Aulnay-sous-Bois, France; or Combidex, Advanced Magnetics, Boston, Mass) and stabilized with sodium citrate. The overall mean particle diameter is approximately 20–30 nm (measured with photon correlation spectroscopy). The r1 and r2 relaxivities were 22.5–22.9 L · mmol^–1 · sec^–1 and 49.8–56.4 L · mmol^–1 · sec^–1, respectively (measured at 39.5° and 20 MHz with an NMR spectrometer [IBM-PC 20; IBM, White Plains, NY]) (23). The blood half-life of ferumoxtran in rats is approximately 2 hours at a dose of 20 µmol of iron per kilogram of body weight (hereafter, Fe/kg) (19). This relatively long half-life makes it a suitable contrast agent for the study of macrophages outside of the organs of the mononuclear phagocyte system (1,24,25). Currently, ferumoxtran is undergoing phase III clinical trials.

Ferumoxtran was administered intravenously through the tail vein on day 5 in both high- and low-dose animal groups. A dose of 150 µmol Fe/kg (corresponding to a dose of 0.45 mL/kg ferumoxtran) was administered in the high-dose USPIO group, and a dose of 50 µmol Fe/kg (corresponding to a dose of 0.15 mL/kg ferumoxtran) was administered in the low-dose USPIO group. The animals in the control group received intravenous isotonic saline solution instead of a contrast agent.

MR Imaging
All MR imaging examinations were performed with a clinically available 1.5-T MR system (Signa CV/i; GE Medical Systems, Milwaukee, Wis). The animals were placed in a dedicated wrist coil in prone position to encompass both hind legs. The signal uniformity of this coil within a selected volume of interest was verified in phantom studies prior to use in this study. All MR imaging examinations were performed in the transverse plane, that is, perpendicular to the diaphysis of the animal’s femoral bones.

The first MR imaging session was performed as the baseline study before the administration of USPIO on day 5 (precontrast MR study). The second MR imaging session was performed 24 hours after USPIO administration on day 6 (postcontrast MR study). The 24-hour delay should allow clearance of all particles from the vascular space by means of phagocytic uptake (2,19).

The following transverse sequences were performed during both MR imaging sessions: T1-weighted spin-echo (repetition time msec/echo time msec, 420/13; field of view, 10 x 5 cm; matrix, 256 x 192), T2*-weighted fast gradient-echo (GRE) (300/15; flip angle, 20°; field of view, 10 x 5 cm; matrix, 256 x 192), and short inversion time inversion-recovery (STIR) (repetition time msec/echo time msec/inversion time msec, 3000/34/150; field of view, 10 x 5 cm; matrix, 256 x 192) sequences. Section thickness was 2 mm without an intersection gap for all sequences, which resulted in a voxel size of 0.39 x 0.26 x 2 mm for all acquired data sets. After completion of this MR imaging protocol on day 5, the two doses of USPIO were administered to the tail vein of the rats in both USPIO groups. To ensure intravascular administration of the contrast agent, a transverse three-dimensional spoiled fast GRE MR sequence (12.9/4.3/34; field of view, 10 x 5 cm; matrix, 256 x 192) was performed immediately after intravenous administration of ferumoxtran. This data set was not used during further study.

Pilot Study
The aforementioned imaging protocol and the USPIO dose were evaluated in the pilot study. This pilot study was performed with three additional animals in which intramuscular abscesses were induced in the same fashion as described earlier. Each animal underwent two MR imaging sessions at the same time points and under the same conditions as described earlier. Immediately following the precontrast imaging on day 5, ferumoxtran was administered intravenously to one animal each at the following doses: 150 µmol Fe/kg, 100 µmol Fe/kg, and 50 µmol Fe/kg.

The three USPIO doses were chosen on the basis of previous study findings (12,13,15,17–19,26). Since a dose between 130 and 180 µmol Fe/kg has been shown to produce significant T2* effects in the wall of soft-tissue abscesses at 4.7 T (17,18), we chose a dose of 150 µmol Fe/kg for our study. The dose of 50 µmol Fe/kg was chosen on the basis of the recommended ferumoxtran dose of 45 µmol Fe/kg for the use in humans (12,13,15,26). The dose of 100 µmol Fe/kg was chosen to cover an intermediate-dose level.

In addition to the sequences described earlier, a T2*-weighted fast GRE sequence with varying repetition and echo times (120/20 and 1000/7, 25° flip angle, 10 x 5 cm field of view, 256 x 192 matrix) and a T1-weighted spoiled GRE acquisition in the steady state (63/1.7, 30° flip angle, 10 x 5 cm field of view, 256 x 192 matrix) MR sequence were performed in each animal. For all sequences, section thickness was 2 mm without an intersection gap.

The T2*-weighted sequence with 300/15 and a 20° flip angle was found to provide the best image quality combined with marked T2* effects at the site of the abscess walls 24 hours after the administration of USPIO (change in contrast-to-noise ratio [CNR]: 1.6 with precontrast and –6.2 with postcontrast sequence and 150 µmol Fe/kg, 0.9 with precontrast and –2.1 with postcontrast sequence and 100 µmol Fe/kg, and 1.6 with precontrast and 1.8 with postcontrast sequence and 50 µmol Fe/kg). Compared with the T1-weighted spoiled GRE acquisition in the steady state sequence, the T1-weighted spin-echo sequence was found to provide the most distinct T1 effects (change in CNR: 1.2 with precontrast and 14.9 with postcontrast sequence and 150 µmol Fe/kg, 1.3 with precontrast and 7.9 with postcontrast sequence and 100 µmol Fe/kg, 0.8 with precontrast and 5.8 with postcontrast sequence and 50 µmol Fe/kg) at the site of the abscess wall, in particular with regard to the differentiation of the different abscess components. To keep the duration of inhalation anesthesia for the animals as short as possible, only T2*-weighted fast GRE and T1-weighted spin-echo sequences were chosen for further use in the main study.

Data Analysis
Both qualitative visual analysis and quantitative SI measurements were performed. Two radiologists and one veterinary pathologist (A.M.L., D.W., E.P.) evaluated all MR studies in consensus on a commercially available workstation (Advantage Windowing; GE Medical Systems, Buc, France). All three observers were blinded to the animal groups. There were substantial delays between image acquisition and image analysis (3 months) and between image analysis and analysis of the histopathologic specimens (3 months). For image and histopathologic analyses, each animal was randomly assigned a number. This way, it was guaranteed that a potential memory bias for any reader was ruled out and that image interpretation and histopathologic analysis were performed in a blinded fashion and in randomized order.

For the USPIO groups and the control group, pre- and postcontrast MR data sets of all MR sequences were compared. On the basis of visual impression, SI within the abscess was classified as hypointense, hyperintense, or isointense in comparison with the SI of the surrounding healthy muscle.

A radiologic-histologic correlation between all MR images and the corresponding histologic slices was performed by two observers (E.P., A.M.L.), who were blinded to the animal groups. For this purpose, the harvested abscesses were cut transversely into 2–3-mm-thick slices (the same plane as the MR images). The usually required decalcification process was avoided by manually extracting each animal’s tibial and fibular bones out of the cut slices. After the slices were embedded in paraffin, Perls Prussian blue stain and Turnbull blue reaction were used to detect presence of iron at light microscopy, with magnifications from x50 to x1000 (27).

With light microscopy, the iron content of the macrophagic cytosol was assessed semiquantitatively on specimens of both iron stains by using the following grading system: low iron content, less than 30% of the individual cell cytosol filled with iron vesicles in the majority of macrophages; moderate iron content, 30%–50% of the individual cell cytosol filled with iron vesicles; and high iron content, more than 50% of the individual cell cytosol filled with iron vesicles.

Hematoxylin-eosin-saffron stain and immunolabeling specific for rat macrophages were then used to determine the quantity and distribution of macrophages within the different components of the intramuscular abscesses. For immunolabeling, 3–5-µm-thick histologic slices were cut from the paraffin blocks and immunostained with an ED-1 mouse monoclonal antibody specific for rat monocytes and macrophages (MCA 341R; Serotec, Oxford, England). Immunohistochemical reactions were performed with a common staining method by using an immunostainer (Ventana, Strasbourg, France), with the biotinylated immunoglobulin–avidin–horseradish peroxidase method (lot no. 12381; Ventana) (28,29) and prior antigen retrieval in a 0.02 mol/L boric acid solution (pH 7.0) with a pressure cooker. The slides were counterstained with hematoxylin (Gill No. 1; Sigma Diagnostics, Buchs, Switzerland).

On the basis of visual impression, the amount of macrophages was semiquantitatively analyzed by a veterinarian pathologist (E.P.) using the following four-point scale: score of 0, no macrophages; score of 1, small amount of macrophages; score of 2, moderate amount of macrophages; and score of 3, large amount of macrophages. In two abscesses in two rats (one rat of each USPIO-dose group), electron microscopy was performed by an experienced pathologist (B.S.). For this purpose, very small parts of the abscesses were fixed in buffered 2.5% glutaraldehyde. Electron microscopy was used to identify iron particles, with magnifications from x3000 to x30 000, and to determine the exact localization of iron within the intracellular or interstitial spaces.

The quantitative analysis of all MR imaging examinations was performed by two radiologists (A.M.L., D.W.) working in consensus. The SI was measured in defined regions of interest, which were set in comparable locations within the abscess center (representing the necrosis of the abscess based on histopathologic analysis) and the periphery (representing the abscess wall based on histopathologic analysis). In addition, SIs of the healthy contralateral muscle (within an area of the ipsilateral musculature presenting with the same SI characteristics as the healthy musculature on the contralateral side) and of the background noise (in the air surrounding the affected limb) were measured. The size of regions of interest varied 5–7 mm² (49–69 pixels), and CNRs were then calculated. The CNR was determined by measuring the SI in regions of interest (either within the abscess center or its periphery), subtracting the mean SI of the healthy adjacent muscle, and dividing it by the standard deviation of the noise. The relative SI change (SI_change) of the healthy contralateral muscle between precontrast (SI_pre) and postcontrast (SI_post) images was calculated by using the following equation: SI_change = (SI_post – SI_pre)/SI_pre · 100.

Statistical Analysis
Statistical analysis was performed by using software (StatView, version 5.0.1; SAS Institute, Cary, NC). The SIs and CNRs were given as means ± standard deviations. The nonparametric Wilcoxon signed rank test was used to compare the differences in CNR before and after USPIO administration for each sequence. P .05 was considered to indicate a statistically significant difference.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
On the basis of histologic and MR imaging findings, all 21 animals developed a single circumscribed intramuscular abscess within the infected left calf 5 days after the intramuscular injection of the bacterial suspension. The semiquantitative evaluation of the total amount of macrophages with use of ED-1 immunolabeling (Fig 1) revealed a large amount of ED-1–positive cells (score of 3) in all cases, which was independent of whether the animals were treated with USPIO or not. Since there were no differences in the classification of the amount of ED-1–positive cells within the abscesses, no statistical test was performed to compare the amount of ED-1–positive cells between the three animal groups.

View larger version (187K): [in this window] [in a new window] [Download PPT slide]   Figure 1a. Photomicrographs of experimentally induced soft-tissue abscesses in calves of left hind legs of rats. Immunostaining performed with monoclonal ED-1 antibodies specific for rat macrophages and monocytes. (a) Infected hind leg 24 hours after injection of 150 µmol Fe/kg. (b) Infected hind leg 24 hours after injection of 50 µmol Fe/kg. (c) Infected leg 24 hours after injection of isotonic saline (control group). In all three specimens, an abundant amount (score, 3) of stained ED-1-positive cells (arrow) can be appreciated within abscess wall (AW), especially at border to the necrotic center of the abscess (AC). Only few ED-1-positive cells (score, 1) can be detected within outer parts of necrotic center itself (arrowhead). No obvious difference in amount of macrophages is noted between the three groups. (Hematoxylin counterstain; original magnification, x100.)

View larger version (198K): [in this window] [in a new window] [Download PPT slide]   Figure 1b. Photomicrographs of experimentally induced soft-tissue abscesses in calves of left hind legs of rats. Immunostaining performed with monoclonal ED-1 antibodies specific for rat macrophages and monocytes. (a) Infected hind leg 24 hours after injection of 150 µmol Fe/kg. (b) Infected hind leg 24 hours after injection of 50 µmol Fe/kg. (c) Infected leg 24 hours after injection of isotonic saline (control group). In all three specimens, an abundant amount (score, 3) of stained ED-1-positive cells (arrow) can be appreciated within abscess wall (AW), especially at border to the necrotic center of the abscess (AC). Only few ED-1-positive cells (score, 1) can be detected within outer parts of necrotic center itself (arrowhead). No obvious difference in amount of macrophages is noted between the three groups. (Hematoxylin counterstain; original magnification, x100.)

View larger version (192K): [in this window] [in a new window] [Download PPT slide]   Figure 1c. Photomicrographs of experimentally induced soft-tissue abscesses in calves of left hind legs of rats. Immunostaining performed with monoclonal ED-1 antibodies specific for rat macrophages and monocytes. (a) Infected hind leg 24 hours after injection of 150 µmol Fe/kg. (b) Infected hind leg 24 hours after injection of 50 µmol Fe/kg. (c) Infected leg 24 hours after injection of isotonic saline (control group). In all three specimens, an abundant amount (score, 3) of stained ED-1-positive cells (arrow) can be appreciated within abscess wall (AW), especially at border to the necrotic center of the abscess (AC). Only few ED-1-positive cells (score, 1) can be detected within outer parts of necrotic center itself (arrowhead). No obvious difference in amount of macrophages is noted between the three groups. (Hematoxylin counterstain; original magnification, x100.)

View larger version (160K): [in this window] [in a new window] [Download PPT slide]   Figure 2a. Transverse precontrast STIR (3000/34/150) images in rats from (a) high-dose, (b) low-dose, and (c) control groups with experimentally induced abscesses of hind leg musculature. A comparable extent of edema pattern with hyperintense center (arrow) and layered periphery of abscess (arrowhead) are noted.

View larger version (158K): [in this window] [in a new window] [Download PPT slide]   Figure 2b. Transverse precontrast STIR (3000/34/150) images in rats from (a) high-dose, (b) low-dose, and (c) control groups with experimentally induced abscesses of hind leg musculature. A comparable extent of edema pattern with hyperintense center (arrow) and layered periphery of abscess (arrowhead) are noted.

View larger version (136K): [in this window] [in a new window] [Download PPT slide]   Figure 2c. Transverse precontrast STIR (3000/34/150) images in rats from (a) high-dose, (b) low-dose, and (c) control groups with experimentally induced abscesses of hind leg musculature. A comparable extent of edema pattern with hyperintense center (arrow) and layered periphery of abscess (arrowhead) are noted.

On postcontrast T2*-weighted images, all seven abscesses showed a slightly hyperintense circular or oval center that was surrounded by a dark rim of susceptibility effects (Fig 3). In three instances, the center of the abscess was divided by thin septa also exhibiting susceptibility effects. At histopathologic examination, the hyperintense center consisted of necrotic debris with predominantly partially fragmented granulocytes and a small amount of partly disrupted macrophages. On USPIO-enhanced T2*-weighted MR images, the rim that surrounded the abscess center could be divided to three separated layers (Fig 4): an inner hypointense rim with susceptibility effects, a middle hyperintense rim without susceptibility effects, and an outer hypointense rim with susceptibility effects. At histopathologic examination, the inner and outer rims represented dense bands of accumulated macrophages within the wall of fibrotic tissue, which surrounded the liquefied abscess center. Between these two rims with susceptibility effects, there was a more hyperintense band without susceptibility effects. At histopathologic examination in this part of the abscess, there was only a small amount of iron-positive macrophages within the fibrotic tissue surrounding the liquefied center of the abscess. Both light and electron microscopy of the individual macrophages in the high-dose group demonstrated an abundance of lysosomal iron particles in the cytoplasma of each macrophage, which gave the appearance of a fully iron-loaded macrophage. All macrophages seen at light microcopy displayed high iron content, with more than 50% of the cytosol being filled with iron vesicles (Fig 5). On the basis of light and electron microscopic findings, no iron particles were found to be extracellular within the interstitial space.

View larger version (109K): [in this window] [in a new window] [Download PPT slide]   Figure 3a. Experimentally induced soft-tissue abscess in calf of left hind leg of a rat from high-dose USPIO group. (a, b) Transverse T1-weighted spin-echo (420/13) and (c, d) T2*-weighted fast GRE (300/15, 20° flip angle) images and (e) enlarged view of d. (a) Precontrast image shows that signal of the abscess area is nearly isointense compared with that of adjacent healthy musculature; only a small area of slightly hyperintense signal (arrow) is visible within the abscess. (b) Image 24 hours after USPIO administration shows distinct T1 effects (arrow) in necrotic abscess center and in abscess periphery, with some overlying T2* effects (arrowhead) surrounding the necrotic abscess center. (c) Precontrast image shows slight hyperintense signal band (arrow) at site of soft-tissue infection within the musculature. (d) Postcontrast image shows abscess site 24 hours after USPIO injection. (e) Enlarged view of d (original magnification, x2.5) shows three layers of abscess wall; directly adjacent to necrotic hyperintense centers of the three-chambered abscess is inner hypointense layer (*), followed by relatively hyperintense middle layer (black arrow) and markedly hypointense outer layer (white arrow) of abscess wall. The hypointense wall layers correspond to higher accumulations of iron-filled macrophages at histologic examination (see Fig 4).

View larger version (116K): [in this window] [in a new window] [Download PPT slide]   Figure 3b. Experimentally induced soft-tissue abscess in calf of left hind leg of a rat from high-dose USPIO group. (a, b) Transverse T1-weighted spin-echo (420/13) and (c, d) T2*-weighted fast GRE (300/15, 20° flip angle) images and (e) enlarged view of d. (a) Precontrast image shows that signal of the abscess area is nearly isointense compared with that of adjacent healthy musculature; only a small area of slightly hyperintense signal (arrow) is visible within the abscess. (b) Image 24 hours after USPIO administration shows distinct T1 effects (arrow) in necrotic abscess center and in abscess periphery, with some overlying T2* effects (arrowhead) surrounding the necrotic abscess center. (c) Precontrast image shows slight hyperintense signal band (arrow) at site of soft-tissue infection within the musculature. (d) Postcontrast image shows abscess site 24 hours after USPIO injection. (e) Enlarged view of d (original magnification, x2.5) shows three layers of abscess wall; directly adjacent to necrotic hyperintense centers of the three-chambered abscess is inner hypointense layer (*), followed by relatively hyperintense middle layer (black arrow) and markedly hypointense outer layer (white arrow) of abscess wall. The hypointense wall layers correspond to higher accumulations of iron-filled macrophages at histologic examination (see Fig 4).

View larger version (133K): [in this window] [in a new window] [Download PPT slide]   Figure 3c. Experimentally induced soft-tissue abscess in calf of left hind leg of a rat from high-dose USPIO group. (a, b) Transverse T1-weighted spin-echo (420/13) and (c, d) T2*-weighted fast GRE (300/15, 20° flip angle) images and (e) enlarged view of d. (a) Precontrast image shows that signal of the abscess area is nearly isointense compared with that of adjacent healthy musculature; only a small area of slightly hyperintense signal (arrow) is visible within the abscess. (b) Image 24 hours after USPIO administration shows distinct T1 effects (arrow) in necrotic abscess center and in abscess periphery, with some overlying T2* effects (arrowhead) surrounding the necrotic abscess center. (c) Precontrast image shows slight hyperintense signal band (arrow) at site of soft-tissue infection within the musculature. (d) Postcontrast image shows abscess site 24 hours after USPIO injection. (e) Enlarged view of d (original magnification, x2.5) shows three layers of abscess wall; directly adjacent to necrotic hyperintense centers of the three-chambered abscess is inner hypointense layer (*), followed by relatively hyperintense middle layer (black arrow) and markedly hypointense outer layer (white arrow) of abscess wall. The hypointense wall layers correspond to higher accumulations of iron-filled macrophages at histologic examination (see Fig 4).

View larger version (130K): [in this window] [in a new window] [Download PPT slide]   Figure 3d. Experimentally induced soft-tissue abscess in calf of left hind leg of a rat from high-dose USPIO group. (a, b) Transverse T1-weighted spin-echo (420/13) and (c, d) T2*-weighted fast GRE (300/15, 20° flip angle) images and (e) enlarged view of d. (a) Precontrast image shows that signal of the abscess area is nearly isointense compared with that of adjacent healthy musculature; only a small area of slightly hyperintense signal (arrow) is visible within the abscess. (b) Image 24 hours after USPIO administration shows distinct T1 effects (arrow) in necrotic abscess center and in abscess periphery, with some overlying T2* effects (arrowhead) surrounding the necrotic abscess center. (c) Precontrast image shows slight hyperintense signal band (arrow) at site of soft-tissue infection within the musculature. (d) Postcontrast image shows abscess site 24 hours after USPIO injection. (e) Enlarged view of d (original magnification, x2.5) shows three layers of abscess wall; directly adjacent to necrotic hyperintense centers of the three-chambered abscess is inner hypointense layer (*), followed by relatively hyperintense middle layer (black arrow) and markedly hypointense outer layer (white arrow) of abscess wall. The hypointense wall layers correspond to higher accumulations of iron-filled macrophages at histologic examination (see Fig 4).

View larger version (149K): [in this window] [in a new window] [Download PPT slide]   Figure 3e. Experimentally induced soft-tissue abscess in calf of left hind leg of a rat from high-dose USPIO group. (a, b) Transverse T1-weighted spin-echo (420/13) and (c, d) T2*-weighted fast GRE (300/15, 20° flip angle) images and (e) enlarged view of d. (a) Precontrast image shows that signal of the abscess area is nearly isointense compared with that of adjacent healthy musculature; only a small area of slightly hyperintense signal (arrow) is visible within the abscess. (b) Image 24 hours after USPIO administration shows distinct T1 effects (arrow) in necrotic abscess center and in abscess periphery, with some overlying T2* effects (arrowhead) surrounding the necrotic abscess center. (c) Precontrast image shows slight hyperintense signal band (arrow) at site of soft-tissue infection within the musculature. (d) Postcontrast image shows abscess site 24 hours after USPIO injection. (e) Enlarged view of d (original magnification, x2.5) shows three layers of abscess wall; directly adjacent to necrotic hyperintense centers of the three-chambered abscess is inner hypointense layer (*), followed by relatively hyperintense middle layer (black arrow) and markedly hypointense outer layer (white arrow) of abscess wall. The hypointense wall layers correspond to higher accumulations of iron-filled macrophages at histologic examination (see Fig 4).

View larger version (176K): [in this window] [in a new window] [Download PPT slide]   Figure 4a. Photomicrographs of histopathologic findings in experimental soft-tissue infections 24 hours after USPIO injection at a dose of 150 µmol Fe/kg. (a) Inner layer of abscess wall adjacent to central necrosis (N) of abscess. Numerous iron-positive blue-stained macrophages can be detected within granulation tissue of abscess wall. Most of these macrophages have entirely blue-stained cytosol (arrow) owing to high content of intracytoplasmatic iron (>50% of cytosol filled with iron vesicles). (b) Middle layer of abscess wall. Only scarce iron-positive macrophages (arrow) can be detected within granulation tissue of abscess wall. (c) Outer layer of abscess wall adjacent to surrounding healthy musculature. Numerous iron-positive macrophages (arrow) are visible within granulation tissue of abscess wall in proximity to muscle cells (M). (Turnbull stain; original magnification, x400.)

View larger version (175K): [in this window] [in a new window] [Download PPT slide]   Figure 4b. Photomicrographs of histopathologic findings in experimental soft-tissue infections 24 hours after USPIO injection at a dose of 150 µmol Fe/kg. (a) Inner layer of abscess wall adjacent to central necrosis (N) of abscess. Numerous iron-positive blue-stained macrophages can be detected within granulation tissue of abscess wall. Most of these macrophages have entirely blue-stained cytosol (arrow) owing to high content of intracytoplasmatic iron (>50% of cytosol filled with iron vesicles). (b) Middle layer of abscess wall. Only scarce iron-positive macrophages (arrow) can be detected within granulation tissue of abscess wall. (c) Outer layer of abscess wall adjacent to surrounding healthy musculature. Numerous iron-positive macrophages (arrow) are visible within granulation tissue of abscess wall in proximity to muscle cells (M). (Turnbull stain; original magnification, x400.)

View larger version (173K): [in this window] [in a new window] [Download PPT slide]   Figure 4c. Photomicrographs of histopathologic findings in experimental soft-tissue infections 24 hours after USPIO injection at a dose of 150 µmol Fe/kg. (a) Inner layer of abscess wall adjacent to central necrosis (N) of abscess. Numerous iron-positive blue-stained macrophages can be detected within granulation tissue of abscess wall. Most of these macrophages have entirely blue-stained cytosol (arrow) owing to high content of intracytoplasmatic iron (>50% of cytosol filled with iron vesicles). (b) Middle layer of abscess wall. Only scarce iron-positive macrophages (arrow) can be detected within granulation tissue of abscess wall. (c) Outer layer of abscess wall adjacent to surrounding healthy musculature. Numerous iron-positive macrophages (arrow) are visible within granulation tissue of abscess wall in proximity to muscle cells (M). (Turnbull stain; original magnification, x400.)

View larger version (148K): [in this window] [in a new window] [Download PPT slide]   Figure 5a. Differences in iron-loading pattern of individual macrophages between high-dose (150 µmol Fe/kg) and low-dose (50 µmol Fe/kg) USPIO groups. (a) Photomicrograph of abscess wall in a rat from high-dose group demonstrates numerous iron-positive macrophages with entirely blue-stained cytosol (arrow) (>50% of cytosol filled with iron vesicles). (Turnbull stain; original magnification, x1000.) (b) Photomicrograph of abscess wall in a rat from low-dose group. Within abscess wall, only scarce iron-positive blue-stained macrophages can be detected (arrows). In contrast to a, cytosol of macrophages is not entirely stained blue, only small blue spots of intracellular deposited iron can be detected within cytosol (<30% of cytosol filled with iron vesicles). (Turnbull stain; original magnification, x1000.) (c) Electron photomicrograph shows iron-positive macrophage (arrowhead) within abscess wall of a rat from high-dose group. Within cytosol of the macrophage, numerous particles can be appreciated that are partially or totally filled with electron-dense black material, corresponding to lysosomes packed with iron (arrows). (Original magnification, x5800.) (d) Electron photomicrograph shows iron-positive macrophage (arrowhead) within abscess wall of a rat from low-dose group. In comparison to macrophage of the high-dose group, this macrophage contains few particles filled with electron-dense material (arrows), corresponding to a low amount of iron within the lysosomes. (Original magnification, x5800.)

View larger version (157K): [in this window] [in a new window] [Download PPT slide]   Figure 5b. Differences in iron-loading pattern of individual macrophages between high-dose (150 µmol Fe/kg) and low-dose (50 µmol Fe/kg) USPIO groups. (a) Photomicrograph of abscess wall in a rat from high-dose group demonstrates numerous iron-positive macrophages with entirely blue-stained cytosol (arrow) (>50% of cytosol filled with iron vesicles). (Turnbull stain; original magnification, x1000.) (b) Photomicrograph of abscess wall in a rat from low-dose group. Within abscess wall, only scarce iron-positive blue-stained macrophages can be detected (arrows). In contrast to a, cytosol of macrophages is not entirely stained blue, only small blue spots of intracellular deposited iron can be detected within cytosol (<30% of cytosol filled with iron vesicles). (Turnbull stain; original magnification, x1000.) (c) Electron photomicrograph shows iron-positive macrophage (arrowhead) within abscess wall of a rat from high-dose group. Within cytosol of the macrophage, numerous particles can be appreciated that are partially or totally filled with electron-dense black material, corresponding to lysosomes packed with iron (arrows). (Original magnification, x5800.) (d) Electron photomicrograph shows iron-positive macrophage (arrowhead) within abscess wall of a rat from low-dose group. In comparison to macrophage of the high-dose group, this macrophage contains few particles filled with electron-dense material (arrows), corresponding to a low amount of iron within the lysosomes. (Original magnification, x5800.)

View larger version (207K): [in this window] [in a new window] [Download PPT slide]   Figure 5c. Differences in iron-loading pattern of individual macrophages between high-dose (150 µmol Fe/kg) and low-dose (50 µmol Fe/kg) USPIO groups. (a) Photomicrograph of abscess wall in a rat from high-dose group demonstrates numerous iron-positive macrophages with entirely blue-stained cytosol (arrow) (>50% of cytosol filled with iron vesicles). (Turnbull stain; original magnification, x1000.) (b) Photomicrograph of abscess wall in a rat from low-dose group. Within abscess wall, only scarce iron-positive blue-stained macrophages can be detected (arrows). In contrast to a, cytosol of macrophages is not entirely stained blue, only small blue spots of intracellular deposited iron can be detected within cytosol (<30% of cytosol filled with iron vesicles). (Turnbull stain; original magnification, x1000.) (c) Electron photomicrograph shows iron-positive macrophage (arrowhead) within abscess wall of a rat from high-dose group. Within cytosol of the macrophage, numerous particles can be appreciated that are partially or totally filled with electron-dense black material, corresponding to lysosomes packed with iron (arrows). (Original magnification, x5800.) (d) Electron photomicrograph shows iron-positive macrophage (arrowhead) within abscess wall of a rat from low-dose group. In comparison to macrophage of the high-dose group, this macrophage contains few particles filled with electron-dense material (arrows), corresponding to a low amount of iron within the lysosomes. (Original magnification, x5800.)

View larger version (203K): [in this window] [in a new window] [Download PPT slide]   Figure 5d. Differences in iron-loading pattern of individual macrophages between high-dose (150 µmol Fe/kg) and low-dose (50 µmol Fe/kg) USPIO groups. (a) Photomicrograph of abscess wall in a rat from high-dose group demonstrates numerous iron-positive macrophages with entirely blue-stained cytosol (arrow) (>50% of cytosol filled with iron vesicles). (Turnbull stain; original magnification, x1000.) (b) Photomicrograph of abscess wall in a rat from low-dose group. Within abscess wall, only scarce iron-positive blue-stained macrophages can be detected (arrows). In contrast to a, cytosol of macrophages is not entirely stained blue, only small blue spots of intracellular deposited iron can be detected within cytosol (<30% of cytosol filled with iron vesicles). (Turnbull stain; original magnification, x1000.) (c) Electron photomicrograph shows iron-positive macrophage (arrowhead) within abscess wall of a rat from high-dose group. Within cytosol of the macrophage, numerous particles can be appreciated that are partially or totally filled with electron-dense black material, corresponding to lysosomes packed with iron (arrows). (Original magnification, x5800.) (d) Electron photomicrograph shows iron-positive macrophage (arrowhead) within abscess wall of a rat from low-dose group. In comparison to macrophage of the high-dose group, this macrophage contains few particles filled with electron-dense material (arrows), corresponding to a low amount of iron within the lysosomes. (Original magnification, x5800.)

Low-dose group.—When precontrast and postcontrast T2*-weighted images were compared, the abscess was only barely visible in all seven infected legs since no susceptibility effects were discernable in the location of the abscess region (Fig 6). In contrast to T2*-weighted images, on postcontrast T1-weighted images there was an increase in SI of the entire abscess without a clear differentiation between the abscess center and its periphery in all seven instances (Fig 6). At histopathologic examination, the number of iron-positive macrophages in the abscess wall was much smaller in the low-dose than in the high-dose USPIO group. Both light and electron microscopy revealed only a small amount of iron-positive lysosomes in the individual macrophages. On the basis of findings at light microscopy, the iron content of the individual macrophages was low (<30% of the cytosol filled with iron vesicles) in all instances. Thus, a completely different iron content of the individual macrophages was observed in the low-dose than in the high-dose USPIO group (Fig 5). All iron particles were visible intracytoplasmically, and no iron particles were found within the interstitial spaces at electron microscopy.

View larger version (113K): [in this window] [in a new window] [Download PPT slide]   Figure 6a. Transverse (a, b) T1-weighted (420/13) spin-echo and (c, d) T2*-weighted fast GRE (300/15, 20° flip angle) images of experimentally induced soft-tissue abscess in calf of left hind leg in a rat from low-dose (50 µmol Fe/kg) group. (a) Precontrast image shows that signal of the abscess area is nearly isointense compared with that of adjacent healthy musculature, and only a small rim of slightly hyperintense signal is visible within abscess (arrow). (b) At 24 hours after USPIO administration, entire abscess demonstrates hyperintense signal (arrow) in comparison with adjacent healthy musculature. (c) Precontrast image shows that site of soft-tissue infection (arrow) within calf muscles is slightly hyperintense. (d) At 24 hours after USPIO administration, only a slight signal hyperintensity (arrow) at the site of infection is seen, but there are no susceptibility artifacts.

View larger version (117K): [in this window] [in a new window] [Download PPT slide]   Figure 6b. Transverse (a, b) T1-weighted (420/13) spin-echo and (c, d) T2*-weighted fast GRE (300/15, 20° flip angle) images of experimentally induced soft-tissue abscess in calf of left hind leg in a rat from low-dose (50 µmol Fe/kg) group. (a) Precontrast image shows that signal of the abscess area is nearly isointense compared with that of adjacent healthy musculature, and only a small rim of slightly hyperintense signal is visible within abscess (arrow). (b) At 24 hours after USPIO administration, entire abscess demonstrates hyperintense signal (arrow) in comparison with adjacent healthy musculature. (c) Precontrast image shows that site of soft-tissue infection (arrow) within calf muscles is slightly hyperintense. (d) At 24 hours after USPIO administration, only a slight signal hyperintensity (arrow) at the site of infection is seen, but there are no susceptibility artifacts.

View larger version (123K): [in this window] [in a new window] [Download PPT slide]   Figure 6c. Transverse (a, b) T1-weighted (420/13) spin-echo and (c, d) T2*-weighted fast GRE (300/15, 20° flip angle) images of experimentally induced soft-tissue abscess in calf of left hind leg in a rat from low-dose (50 µmol Fe/kg) group. (a) Precontrast image shows that signal of the abscess area is nearly isointense compared with that of adjacent healthy musculature, and only a small rim of slightly hyperintense signal is visible within abscess (arrow). (b) At 24 hours after USPIO administration, entire abscess demonstrates hyperintense signal (arrow) in comparison with adjacent healthy musculature. (c) Precontrast image shows that site of soft-tissue infection (arrow) within calf muscles is slightly hyperintense. (d) At 24 hours after USPIO administration, only a slight signal hyperintensity (arrow) at the site of infection is seen, but there are no susceptibility artifacts.

View larger version (117K): [in this window] [in a new window] [Download PPT slide]   Figure 6d. Transverse (a, b) T1-weighted (420/13) spin-echo and (c, d) T2*-weighted fast GRE (300/15, 20° flip angle) images of experimentally induced soft-tissue abscess in calf of left hind leg in a rat from low-dose (50 µmol Fe/kg) group. (a) Precontrast image shows that signal of the abscess area is nearly isointense compared with that of adjacent healthy musculature, and only a small rim of slightly hyperintense signal is visible within abscess (arrow). (b) At 24 hours after USPIO administration, entire abscess demonstrates hyperintense signal (arrow) in comparison with adjacent healthy musculature. (c) Precontrast image shows that site of soft-tissue infection (arrow) within calf muscles is slightly hyperintense. (d) At 24 hours after USPIO administration, only a slight signal hyperintensity (arrow) at the site of infection is seen, but there are no susceptibility artifacts.

At histopathologic examination, no iron-positive macrophages could be detected within the abscess wall. Only a physiologically normal quantity of iron-containing macrophages was detected within the normal musculature surrounding the abscess; comparable amounts of these cells could be detected in the normal musculature of the USPIO-treated groups as well.

Quantitative SI Analysis
The results of the calculated CNR and the associated P values of all three animal groups are listed in the Table.

Low-dose group.—Because the abscess center and its periphery could not be differentiated on postcontrast T1- and T2*-weighted images, the SIs of the abscess center and periphery were measured at comparable locations as on STIR images. The CNR measured at the location of the abscess periphery on T1-weighted images was higher on postcontrast (mean CNR, 4.21 ± 0.90) than on precontrast (mean CNR, 1.80 ± 1.51) images. This difference also reached statistical significance (P = .03). However, there was only a very subtle decrease in CNR measured at the abscess periphery on T2*-weighted images, which did not reach statistical significance (mean precontrast CNR, 0.92 ± 0.64; mean postcontrast CNR, 0.59 ± 0.81; P = .09). When measurements were obtained within the abscess center, there was a statistically significant increase in CNR on T1-weighted images (mean precontrast CNR, 0.83 ± 0.77; mean postcontrast CNR, 7.30 ± 1.67; P = .02) but not on the corresponding location on T2*-weighted images (mean precontrast CNR, 1.73 ± 0.37; mean postcontrast CNR, 1.23 ± 0.57; P = .06).

Control group.—The changes in CNR on T1- and T2*-weighted images did not reach statistical significance in either the abscess center or its periphery.

Relative SI changes in healthy contralateral muscles.—The calculation of relative SI changes in healthy contralateral muscles revealed only minor changes for all three animal groups: On T1-weighted images, the relative SI changes in the healthy muscle were 2.98% ± 2.64 and 2.38% ± 1.77 for the high-dose and low-dose USPIO group, respectively, whereas the muscular SI changes were 2.26% ± 1.14 for the control group. On T2*-weighted images, the relative SI changes in the healthy muscle were –2.46% ± 2.21 and 1.22% ± 3.42 for the high-dose and low-dose USPIO group, respectively. For the control group, SI measurements revealed relative SI changes of 1.98% ± 1.01 on T2*-weighted images. The relative SI changes within the healthy musculature did not reach statistical significance for any of the acquired images nor for any of the animal groups (P = .06–.66).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The concept of in vivo macrophage-specific MR imaging is based on the fact that USPIO particles are small enough, after a certain vascular circulation period, to be selectively ingested by macrophages, which are located in the organs of the mononuclear phagocyte system (ie, bone marrow, lymph nodes, liver, and spleen), or by migrating macrophages, which are mobilized by an inflammatory disease process in other parts of the body (2,5,6). The ability of MR imaging to depict in vivo phagocytosis of USPIO by macrophages has been demonstrated in atherosclerotic plaques; some types of nephropathy; various diseases of the central nervous system, including experimental autoimmune encephalomyelitis and transient cerebral ischemia; allograft rejections of transplanted organs; lymph node metastasis from various carcinomas; and non-Hodgkin lymphoma (5–15).

Macrophages play a key role in inflammatory processes caused by bacterial infections. Once they are activated by the release of macrophage-activating cytokines from T lymphocytes, they migrate to the site of microbial infection. With their ability to secrete numerous hydrolytic enzymes, immune modulators like interferon and ß, as well as interleukin-1, they are heavily involved in the inflammatory response to infection. Because of their enormous phagocytic abilities, macrophages support lymphocytes in the removal of cell debris at the site of microbial infection (30–32).

The use of iron oxide particles for imaging of soft-tissue abscesses has been demonstrated in a limited number of studies in which animal models were used (16–18,33). In the study of Chan et al (33), turpentine-induced sterile muscular abscesses were examined at MR imaging by using large (mean diameter, 69.5 nm) lipid-coated iron oxide particles at doses of 25–200 µmol Fe/kg, as well as large (mean diameter, 90 nm) protein-coated colloidal magnetite particles at doses of 50 µmol Fe/kg (33). The authors of that study detected hypointense signal alterations in the abscess periphery on both T1- and T2-weighted MR images. The hypointense signal alterations were more prominent after administration of the higher doses of lipid-coated iron oxide particles. However, T1 effects were noted neither after administration of protein-coated particles nor after administration of lipid-coated particles within the abscesses. In the study by Gellissen et al (16), the short-term SI alterations within bacterially induced abscesses were dynamically studied at up to a 60-minute time delay after USPIO administration. Because the imaging protocol in the study by Gellissen et al (16) was limited to GRE fast low-angle shot sequences, the occurrence of T1 effects was not evaluated. However, by demonstrating extracellular interstitial iron deposits in proximity to the abscesses at light microscopy, transcapillary iron oxide transport was proved. In a more recent study, Kaim et al (18) have demonstrated that MR imaging at 4.7 T 24 hours after injection of USPIO is useful for identification of acute, early chronic, and late chronic abscess formation.

In continuation with the studies of Kaim et al (17,18), we have demonstrated that macrophage activity in soft-tissue infections can be reliably imaged at a clinical magnetic field strength of 1.5 T. Moreover, we have shown that by using T1- and T2*-weighted sequences, USPIO-enhanced MR imaging precisely reflects the macrophage distribution in the abscess wall and in the necrotic center when correlation with histopathologic findings is made. We were able to show a good correlation between the different layers of the abscess wall and the MR imaging pattern on T2*-weighted images when the histologic distribution of the active macrophages in the abscess wall was correlated with the MR imaging data. Another important result of our study is the fact that we did not observe any significant difference in the number of ED-1–positive cells among the three animal groups at histopathologic examination when using immunolabeling with ED-1 mouse monoclonal antibody. This result may indicate that the phagocytic-active macrophages are attracted by the infection itself rather than by the USPIO. In addition, the differences in the iron-loading pattern of the individual macrophage and the amount of iron-positive macrophages cannot be explained by significant differences in the total quantity of macrophages at the site of the abscess.

So far, authors of most studies on USPIO-enhanced macrophage MR imaging, including Kaim et al (17), have focused on T2- and T2*-weighted images for the identification of iron-loaded macrophages (6–14). A decrease of SI on T2-weighted images or evidence of T2* effects when precontrast images are correlated with postcontrast images indicates the presence of phagocytosed and strongly agglomerated iron particles in the lysosomes of macrophages (1,7,8). The SI characteristics of iron-loaded phagocytic-active macrophages on USPIO-enhanced MR images in combination with T1-weighted spin-echo images are less clear and, to our knowledge, poorly investigated so far. Although some authors (1,3,19) have used T1-weighted sequences in their imaging protocols for USPIO-enhanced MR imaging, so far there has been no systematic investigation of the T1 and T2* SI characteristics in macrophage imaging.

SI can be plotted as a function of sequence parameters and molar relaxivities. Because of the reduced r2/r1 relaxivity ratio of the USPIO ferumoxtran at lower field strengths and on the basis of the relationship between dose, particle distribution, and T1 and T2 relaxivity characteristics, it might be expected that at lower USPIO concentrations the T1 effects of the contrast agent prevail over the T2 and T2* effects (19,34). This has been demonstrated by Chambon et al (19) with in vitro experiments and in vivo perfusion experiments in rats, however, not in imaging the organ-based macrophages of the liver. The dose-dependent T1 effects have been confirmed by the results of our study. In the low-dose USPIO group (ie, 50 µmol Fe/kg), there was an increase in the SI on postcontrast T1-weighted spin-echo images compared with that on precontrast images at both the quantitative and qualitative analyses. Although there was no clear visible differentiation between the center of the abscess and its wall on the T1-weighted image, there was an SI increase in the center and in the periphery of the abscess, which indicated a T1 effect of the USPIO. Since there were no visible T2* effects within the abscess in this dose group, it may be concluded that at an intravenous dose of 50 µmol Fe/kg, T1-weighted images are more sensitive in depicting USPIO-tagged macrophages than are T2*-weighted images. In contrast, 24 hours after intravenous administration of 150 µmol Fe/kg (high-dose USPIO group), there were considerable T2* effects in the abscess wall, where most of the phagocytic-active macrophages were located.

Using light and electron microscopy, we were able to demonstrate a striking difference in the content of phagocytozed iron particles in individual macrophages between the two USPIO-dose groups. Twenty-four hours after administration of 50 µmol Fe/kg USPIO, the macrophages contained much less iron in their lysosomes (<30% of the cytosol filled with iron vesicles) than did the individual macrophages 24 hours after intravenous administration of 150 µmol Fe/kg USPIO (>50% of the cytosol filled with iron vesicles). Since immunolabeling with ED-1 rat monoclonal antibodies did not reveal any obvious difference in the number of the macrophages between the two dose groups and electron microscopic analysis did not show any residual USPIO particles within the extracellular space, we hypothesize that these differences in SI pattern are mainly due to the different iron content of the individual macrophages (and as consequence, different total amount of deposited iron) 24 hours after USPIO administration.

As in the low-dose group, we also observed T1 effects in the necrotic abscess center in the high-dose group. Since there were only a few iron-positive macrophages within the abscess center in both dose groups, we hypothesize that the total content of iron was high enough to cause T1 effects but was too low to induce T2 or T2* effects. The fact that the individual macrophages in the high-dose group contain a larger iron load than those in the low-dose group may explain the more intense T1 effect in the high-dose group than in the low-dose group.

This study had several limitations. First, this was an animal study performed with rats. Although we chose an animal model that is close to human soft-tissue infection, it is unclear whether the results of this study will be entirely applicable to humans. Second, we did not systematically investigate the dose-response effect of the USPIO on the SI characteristics by using a series of various doses of the contrast agent or a variety of different T1-weighted sequences. Third, the number of animals was limited.

In conclusion, our study has demonstrated that the distribution of phagocytic-active macrophages in an experimentally induced bacterial abscess model may be detected at 1.5-T MR imaging 24 hours after USPIO injection. The T1 and T2* SI characteristics of experimentally induced soft-tissue infections 24 hours after USPIO injection strongly depend on the intravenously administrated USPIO dose. With a low intravenous USPIO dose, T1 effects were stronger than were T2* effects, probably because of differences in the iron content of macrophages.

Practical application: The feasibility of USPIO-enhanced macrophage imaging of experimental soft-tissue infections at a clinically used magnetic field strength of 1.5 T has been shown, which might accelerate the use of USPIO for this purpose in a clinical setting. Because of the central role of macrophages in inflammatory processes, including soft-tissue infections, macrophage-specific USPIO-enhanced MR imaging may be helpful in detection of small abscesses or inflammatory processes that may not be readily depicted on STIR images. The fact that T1 and T2* effects of USPIO-enhanced MR imaging are dose dependent may influence future imaging protocols to that point that, besides the widely used T2*-weighted sequences, T1-weighted sequences should also be part of the USPIO-enhanced MR imaging protocol.

	ACKNOWLEDGMENTS

 
The authors thank D. W. Crook, MD, for his help in preparing the manuscript.

	FOOTNOTES

 
Abbreviations: CNR = contrast-to-noise ratio, GRE = gradient echo, SI = signal intensity, STIR = short inversion time inversion recovery, USPIO = ultrasmall superparamagnetic iron oxide

Author contributions: Guarantors of integrity of entire study, A.M.L., D.W.; study concepts and design, A.M.L., D.W., A.H.K.; literature research, A.M.L., J.F., J.G., A.H.K.; experimental studies, A.M.L., A.H.K.; data acquisition, A.M.L., A.H.K., K.G.; data analysis/interpretation, A.M.L., D.W., E.P., B.S.; manuscript preparation, A.M.L., D.W., E.P., B.S.; manuscript definition of intellectual content, A.M.L., D.W., A.H.K., J.F.; manuscript editing, A.M.L.; manuscript revision/review, A.M.L., D.W., A.H.K., E.P., K.G., B.S., B.M., J.G.; manuscript final version approval, D.W., A.H.K.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Weissleder R, Elizondo G, Wittenberg J, Lee AS, Josephson L, Brady TJ. Ultrasmall superparamagnetic iron oxide: an intravenous contrast agent for assessing lymph nodes with MR imaging. Radiology 1990; 175:494-498.[Abstract/Free Full Text]
2. Weissleder R, Elizondo G, Wittenberg J, Rabito CA, Bengele HH, Josephson L. Ultrasmall superparamagnetic iron oxide: characterization of a new class of contrast agents for MR imaging. Radiology 1990; 175:489-493.[Abstract/Free Full Text]
3. Weissleder R, Cheng HC, Bogdanova A, Bogdanov A, Jr. Magnetically labeled cells can be detected by MR imaging. J Magn Reson Imaging 1997; 7:258-263.[Medline]
4. Schulze E, Ferrucci JT, Jr, Poss K, Lapointe L, Bogdanova A, Weissleder R. Cellular uptake and trafficking of a prototypical magnetic iron oxide label in vitro. Invest Radiol 1995; 30:604-610.[CrossRef][Medline]
5. Ruehm SG, Corot C, Vogt P, Kolb S, Debatin JF. Magnetic resonance imaging of atherosclerotic plaque with ultrasmall superparamagnetic particles of iron oxide in hyperlipidemic rabbits. Circulation 2001; 103:415-422.[Abstract/Free Full Text]
6. Hauger O, Delalande C, Trillaud H, et al. MR imaging of intrarenal macrophage infiltration in an experimental model of nephrotic syndrome. Magn Reson Med 1999; 41:156-162.[CrossRef][Medline]
7. Hauger O, Delalande C, Deminiere C, et al. Nephrotoxic nephritis and obstructive nephropathy: evaluation with MR imaging enhanced with ultrasmall superparamagnetic iron oxide-preliminary findings in a rat model. Radiology 2000; 217:819-826.[Abstract/Free Full Text]
8. Dousset V, Ballarino L, Delalande C, et al. Comparison of ultrasmall particles of iron oxide (USPIO)-enhanced T2-weighted, conventional T2-weighted, and gadolinium-enhanced T1-weighted MR images in rats with experimental autoimmune encephalomyelitis. AJNR Am J Neuroradiol 1999; 20:223-227.[Abstract/Free Full Text]
9. Dousset V, Delalande C, Ballarino L, et al. In vivo macrophage activity imaging in the central nervous system detected by magnetic resonance. Magn Reson Med 1999; 41:329-333.[CrossRef][Medline]
10. Rausch M, Baumann D, Neubacher U, Rudin M. In-vivo visualization of phagocytotic cells in rat brains after transient ischemia by USPIO. NMR Biomed 2002; 15:278-283.[CrossRef][Medline]
11. Kanno S, Wu YJ, Lee PC, et al. Macrophage accumulation associated with rat cardiac allograft rejection detected by magnetic resonance imaging with ultrasmall superparamagnetic iron oxide particles. Circulation 2001; 104:934-938.[Abstract/Free Full Text]
12. Stets C, Brandt S, Wallis F, Buchmann J, Gilbert FJ, Heywang-Kobrunner SH. Axillary lymph node metastases: a statistical analysis of various parameters in MRI with USPIO. J Magn Reson Imaging 2002; 16:60-68.[CrossRef][Medline]
13. Sigal R, Vogl T, Casselman J, et al. Lymph node metastases from head and neck squamous cell carcinoma: MR imaging with ultrasmall superparamagnetic iron oxide particles (Sinerem MR)—results of a phase-III multicenter clinical trial. Eur Radiol 2002; 12:1104-1113.[CrossRef][Medline]
14. Daldrup-Link HE, Rummeny EJ, Ihssen B, Kienast J, Link TM. Iron-oxide-enhanced MR imaging of bone marrow in patients with non-Hodgkin’s lymphoma: differentiation between tumor infiltration and hypercellular bone marrow. Eur Radiol 2002; 12:1557-1566.[CrossRef][Medline]
15. Michel SC, Keller TM, Frohlich JM, et al. Preoperative breast cancer staging: MR imaging of the axilla with ultrasmall superparamagnetic iron oxide enhancement. Radiology 2002; 225:527-536.[Abstract/Free Full Text]
16. Gellissen J, Axmann C, Prescher A, Bohndorf K, Lodemann KP. Extra- and intracellular accumulation of ultrasmall superparamagnetic iron oxides (USPIO) in experimentally induced abscesses of the peripheral soft tissues and their effects on magnetic resonance imaging. Magn Reson Imaging 1999; 17:557-567.[CrossRef][Medline]
17. Kaim AH, Wischer T, O’Reilly T, et al. MR imaging with ultrasmall superparamagnetic iron oxide particles in experimental soft-tissue infections in rats. Radiology 2002; 225:808-814.[Abstract/Free Full Text]
18. Kaim AH, Jundt G, Wischer T, et al. Functional-morphologic MR imaging with ultrasmall superparamagnetic particles of iron oxide in acute and chronic soft-tissue infection: study in rats. Radiology 2003; 227:169-174.[Abstract/Free Full Text]
19. Chambon C, Clement O, Le Blanche A, Schouman-Claeys E, Frija G. Superparamagnetic iron oxides as positive MR contrast agents: in vitro and in vivo evidence. Magn Reson Imaging 1993; 11:509-519.[CrossRef][Medline]
20. Knollmann FD, Bock JC, Rautenberg K, Beier J, Ebert W, Felix R. Differences in predominant enhancement mechanisms of superparamagnetic iron oxide and ultrasmall superparamagnetic iron oxide for contrast-enhanced portal magnetic resonance angiography: preliminary results of an animal study original investigation. Invest Radiol 1998; 33:637-643.[CrossRef][Medline]
21. Kaim AH, Weber B, Kurrer MO, Gottschalk J, von Schulthess GK, Buck A. Autoradiographic quantification of 18F-FDG uptake in experimental soft-tissue abscesses in rats. Radiology 2002; 223:446-451.[Abstract/Free Full Text]
22. Russel WMS, Burch RL. The principles of humane experimental technique London, England: Methuen, 1959.
23. Jung CW, Jacobs P. Physical and chemical properties of superparamagnetic iron oxide MR contrast agents: ferumoxides, ferumoxtran, ferumoxsil. Magn Reson Imaging 1995; 13:661-674.[CrossRef][Medline]
24. Reimer P, Tombach B. Hepatic MRI with SPIO: detection and characterization of focal liver lesions. Eur Radiol 1998; 8:1198-1204.[CrossRef][Medline]
25. Bonnemain B. Superparamagnetic agents in magnetic resonance imaging: physicochemical characteristics and clinical applications—a review. J Drug Target 1998; 6:167-174.[Medline]
26. Dousset V, Gomez C, Petry KG, Delalande C, Caille JM. Dose and scanning delay using USPIO for central nervous system macrophage imaging. Magma 1999; 8:185-189.
27. Culling C. Handbook of histopathologic and histochemical techniques 3rd ed. London, England: Butterworths, 1974.
28. Bullock GR, Petrusz P. Techniques in immunohistochemistry New York, NY: Academic Press, 1982.
29. Bourne JA. Handbook of immunoperoxidase staining methods Santa Barbara, Calif: Immunohistochemistry Laboratory, Dako, 1984.
30. Johnston RB, Jr. Current concepts: immunology—monocytes and macrophages. N Engl J Med 1988; 318:747-752.[Medline]
31. Madri J. Inflammation and healing. In: Kissane JM, eds. Anderson’s pathology. 9th ed. Vol 1. St Louis, Mo: Mosby, 1990; 67-110.
32. Richie A, ed. Inflammation In: Boyd’s textbook of pathology. 9th ed. Vol 1. Philadelphia, Pa: Lea & Febiger, 1990; 60-82.
33. Chan TW, Eley C, Liberti P, So A, Kressel HY. Magnetic resonance imaging of abscesses using lipid-coated iron oxide particles. Invest Radiol 1992; 27:443-449.[CrossRef][Medline]
34. Josephson L, Lewis J, Jacobs P, Hahn PF, Stark DD. The effects of iron oxides on proton relaxivity. Magn Reson Imaging 1988; 6:647-653.[CrossRef][Medline]

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