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Experimental Studies |
1 From the Department of Nuclear Medicine, University Hospital Zurich, Switzerland (A.H.K., G.K.v.S.); Departments of Diagnostic Radiology (A.H.K., T.W.) and Pathology (G.J.), University Hospital Basel, Switzerland; Novartis Pharma, Basel, Switzerland (T.O., P.R.A.); and Guerbet, Zurich, Switzerland (J.F.). Received September 5, 2001; revision requested November 7; final revision received April 10, 2002; accepted May 6. Supported in part by Novartis-Stiftung (Basel, Switzerland), EMDO-Stiftung (Zurich, Switzerland), Freie Akademische Gesellschaft (Basel, Switzerland), and Fröhlich Pharma Consulting (Zurich, Switzerland). Address correspondence to A.H.K., Brachmattstrasse 6, CH-4144 Arlesheim, Switzerland (e-mail: akaim@uhbs.ch).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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MATERIALS AND METHODS: Thirteen rats with unilateral calf-muscle infection were imaged with a 4.7-T MR imager at an early chronic stage of infection (day 4 before contrast material injection, days 4–7 after injection). Eleven animals were imaged before and 3 and 24 hours after intravenous application of ultrasmall superparamagnetic iron oxide (USPIO), and eight animals were additionally imaged 48 hours and three animals 72 hours after USPIO application. Two infected rats served as controls. T1- and T2-weighted spin-echo and T2*-weighted gradient-echo sequences were applied. All animals were sacrificed, and histopathologic findings were correlated with findings on MR images. Electron microscopy was performed in two rats. For quantitative analysis, signal intensities on T2*-weighted images and T2 values on T2 maps were measured within regions of interest, and the temporal variation was analyzed by using the signed rank test.
RESULTS: Visualization of USPIO-loaded macrophages was most sensitive with a T2*-weighted sequence. USPIO distribution pattern and quantitative analysis of T2 and T2* effects 3 hours after USPIO application were significantly different (P < .05) from those at 24 and 48 hours, reflecting the dynamic transit of the particle accumulation from the intravascular to the intracellular compartment by means of macrophage phagocytosis. Local signal intensity alterations could be correlated with iron-loaded macrophages at histopathologic examination.
CONCLUSION: Activated macrophages in acute soft-tissue infection can be labeled with USPIOs and detected with MR imaging because of susceptibility effects.
© RSNA, 2002
Index terms: Animals • Contrast media, experimental studies • Iron • Magnetic resonance (MR), contrast media • Soft tissues, infection
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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The extent of infection is usually defined by the extent of edema pattern. By using this criterion, however, lesion size is frequently overestimated (8), although no histomorphologic correlation study has been performed to validate this limitation until now. An alternative criterion to define the extent of infectious tissue would be the area of cellular inflammatory infiltrates.
Cell-specific imaging is becoming an increasingly important field of MR imaging (9,10), and it represents a potential means of overcoming these limitations. Superparamagnetic iron oxide, or SPIO, particles have been used clinically for several years as contrast agents in MR imaging. They accumulate in the mononuclear phagocyte system and lead to an SI decrease on T2- or T2*-weighted images, particularly in the liver, spleen, and hematopoietic bone marrow. Development of SPIOs has been followed by the development of ultrasmall superparamagnetic iron oxide (USPIO) particles, which are long-circulating dextran-coated iron oxide nanoparticles. They are internalized predominantly by the mononuclear phagocyte system and cause shortening of R1 and R2. It has been observed that USPIOs may also be internalized by tumor cells (11,12).
Furthermore, results of clinical trials have proven the usefulness of USPIOs as a contrast agent for human MR lymphography (13,14) and characterization of hepatosplenic tumors (15,16). Experimental studies with USPIOs for the detection of macrophages and the characterization of cellular infiltration were performed in models for different disease entities of the central nervous system, such as inflammation (17), tumors (11,12,18), or ischemia (19), in bone marrow alterations (20,21) and atherosclerotic changes (22), and in a nephropathy model (23). Results of in vitro experiments with USPIO-loaded macrophages (24) showed that alteration of SI on T1-, T2-, and T2*-weighted MR images were mainly correlated with different intra- or extracellular particle concentrations.
Since macrophages are activated during infection and migrate to the site of microbiotic invasion (25), we developed the hypothesis that USPIO-loaded macrophages might be detectable with MR imaging in soft-tissue infection. The aim of this study was to investigate the feasibility of macrophage MR imaging in rats by using an experimental soft-tissue infection model.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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Bacterial suspensions were prepared by washing the organisms once with 0.9% saline, resuspending them in phosphate-buffered saline, and diluting them to 1 x 109 colony-forming units per milliliter. Bacterial concentration was measured by means of optical density and controlled by means of plating dilutions on agar plates.
All animals developed inflammatory masses in the left calf muscle within 24 hours after bacterial inoculation. A circumscribed infection was palpable on days 4–7. No systemic infection occurred. Two groups were defined: (a) intramuscular infection with USPIO-enhanced MR imaging (n = 11) and (b) intramuscular infection without USPIO-enhanced MR imaging (control group; n = 2).
The study design required two measurements on day 4, which limited the total number of animals for this experiment. Only two animals could be considered controls for documentation of no similar spontaneous effects that occurred after USPIO application in the remaining 11 rats.
Contrast Medium
The USPIO contrast agent used in this study was AMI-227 (Sinerem; Laboratory Guerbet, Roissy, France). The nanoparticles consist of a nucleus of iron oxide crystals coated with dextran. The mean diameter of the particles is 35 nm; the blood half-life in rats is about 2–3 hours at a dose of 40 µmol of iron per kilogram. The relatively long half-life renders the particles suitable for examining macrophages outside the liver and spleen. The R1 and R2 (37°C, 0.5 T) are 22 and 80 mmol/L · sec-1, respectively, and they provide high SI on T1-weighted images and low SI on T2-weighted images. AMI-227 is supplied as a lyophilized powder and reconstituted with 9.7 mL of sterile 0.9% saline solution to yield a solution containing 20 mg of iron per milliliter. The contrast agent was administered intravenously by means of the animals’ tail vein at a dose of 130 µmol of iron per kilogram by using a 25-gauge needle.
MR Imaging
Imaging of the infected left hind leg (calf and thigh) was performed with a 4.7-T Bruker DBX imager (Bruker Medical Systems, Karlsruhe, Germany) at an early chronic stage of infection (day 4 before USPIO application, days 4–7 after USPIO application). Eleven anesthetized animals (1.5% isofluran in a 1:2 mixture of O2/N2O) were imaged before and 3 and 24 hours after intravenous USPIO application, and eight animals were additionally imaged 48 hours and three animals 72 hours after USPIO application.
The following imaging sequences were applied: (a) T1-weighted spin echo 350/13 (repetition time msec/echo time msec), (b) T2-weighted multisection multiecho spin echo 2,000/14–74, and (c) T2*-weighted gradient echo (GRE) 500/10 with a 30° flip angle. T2 maps were calculated from multisection multiecho data.
The imaging protocols ensured evaluation of T1, T2, and T2* effects with USPIO enhancement. Section orientation was transverse (orthogonal to the tibia) to ensure anatomic reproducibility of image position and to obtain optimal correlation with histopathologic findings.
The spatial resolution parameters were as follows: acquisition matrix of 256 x 192 and reconstruction matrix of 256 x 256 for the multisection multiecho spin-echo sequence, imaging matrix of 128 x 128 for the GRE sequence, field of view of 4 cm, and section thickness of 1 mm contiguous. Two signals were acquired, and the mean duration of each imaging session was 25 minutes.
Sacrifice
Sacrifice was performed within 4 hours following the last imaging session after USPIO application. All animals were sacrificed by means of inhalation of pure CO2. The infected hind leg was anatomically prepared, exarticulated, and fixed with formaldehyde (2%).
Histologic Findings and Electron Microscopy
The pathologic specimens were cut transversely and stained with hematoxylin-eosin for histologic examination. Perls Prussian blue stain was used to detect the presence of iron particles. Two animals without USPIO application (group 2) served as controls.
To identify iron particles and determine the location of iron within the intracellular or interstitial space, electron microscopy was performed with magnification from x4,000 to x12,500. Small portions of the abscess wall of two rats (24 and 48 hours after USPIO application) were sampled and prefixed with 2% glutaraldehyde. After the samples were washed with potassium phosphate buffer, half of the samples were postfixed with 1% OsO4, whereas the other half were not osmicated to avoid any precipitate, which may be misinterpreted as iron particles. The samples were dehydrated in a graded concentration of ethanol and embedded in mixtures of Epon (Fluka, Buchs, Switzerland) and finally in pure Epon. The samples were placed into gelatin capsules filled with pure Epon and polymerized over night. Thin sections were cut and collected on pallodion copper grids and stained with uranyl acetate (1 hour) and lead citrate (2 minutes).
Image Analysis and Statistical Evaluation
Histomorphologic analysis of iron-stained histologic samples (Perls Prussian blue stain) and MR imaging were performed to correlate susceptibility effects with intra- or extracellular iron distribution by one pathologist (G.J.) and one radiologist (A.H.K.) in consensus. Areas with increased T2 values compared with those of noninfected muscle were defined according to edema pattern, which was assessed on T2 maps prior to USPIO application, and USPIO distribution was defined on GRE images. Analysis of the patterns at the different time points was performed in consensus by two observers (A.H.K., T.W.), and the pattens were compared by using the parameters (a) less or more extended and (b) similar or dissimilar.
For quantitative analysis, SIs on T2*-weighted GRE images and T2 values on T2 maps were measured within regions of interest before USPIO application, as well as on all images obtained 3, 24, 48, and 72 hours after USPIO administration. The regions of interest (3–4 mm2) were placed within the infection (abscess wall or intermuscular fascia) at identical locations over time per animal and within the unaffected muscle of the ipsilateral thigh. Relative SI (SI of the infected muscle divided by SI of the unaffected muscle) on T2*-weighted GRE images and T2 values on T2 maps were calculated over time. Values were expressed as median values with interquartile ranges. The temporal variation of T2 values and relative SIs (on T2*-weighted GRE images) was statistically analyzed by using the signed rank test. The data from 3 hours versus 24 hours after USPIO application and from 24 hours versus 48 hours were compared. Statistical significance was assigned if P was less than .05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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Quantitative measurements of relative SI on T2*-weighted GRE images (Fig 2) confirmed the visual impression that the T2* effect was most prominent 3 hours after USPIO application because of the high amount of intravascular USPIO (relative SI: median, 0.11; interquartile range, 0.08–0.20) and that a significant decrease occurred 24 hours after USPIO application (relative SI: median, 0.47; interquartile range, 0.38–0.58; P < .05), which reflects the intracellular iron accumulation within macrophages. Only a slight decrease of T2* shortening could be delineated at 48 hours (relative SI: median, 0.67; interquartile range, 0.64–0.77; P > .05) and 72 hours (relative SI: median, 0.79; interquartile range, 0.69–0.84) after USPIO application compared with that at 24 hours. This probably reflected gradual emigration of iron-loaded macrophages from the infectious site.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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When exposed to particular stimuli, phagocytes undergo an activation, which is known as "respiratory burst," with an increase of glucose metabolism (28). Energy-dependent, interrelated cellular defense mechanisms are activated that include migration, generation, release of microbicidal agents, and phagocytosis. Activated macrophages are powerful phagocytes that ingest all kinds of small particulate material and digest much of it in their phagosomes (26,27). With an experimental bacterial soft-tissue model in the early chronic stage, which was histologically characterized by granulocytes, macrophages, and interspersed fibroblasts, results of the present study demonstrate that USPIOs are specifically phagocytosed by activated macrophages and can be detected with MR imaging.
Histologic findings demonstrated that USPIOs were not ingested by granulocytes. The mechanism by which cellular uptake occurs is receptor-mediated endocytosis for macrophages (12). Since endocytotic activity needs high metabolic activity, USPIO enhancement patterns might reflect not only engulfment of particles or cellular mobilization but also the increased metabolic activity of phagocytosing cells. The USPIO particles used in the present study have a prolonged intravascular half-life compared with that of SPIO preparations composed of large particles that render them suitable for investigation of the reticuloendothelial system macrophages outside the liver, spleen, and bone marrow. They consist of an iron oxide core with a form of nonstoichiometric magnetite 43°–60° in diameter, coated with low-molecular-weight dextran. It is generally agreed that the main factors influencing the distribution of these typical subcolloidal systems after intravenous administration are the mean particle diameter (35 nm), the citrate-dependent negative surface charge, and the dextran-dependant hydrophilic coating, which reduces opsonization and thus the immediate cellular uptake of the nanoparticles (12,29).
Size and surface properties are the determining factors for physiologic distribution. It is well known that larger particles like SPIO with a mean diameter of 150 nm are quickly phagocytosed, mainly by means of hepatic Kupffer cells and the mononuclear phagocyte system. The human blood half-life is much shorter, with 10–15 minutes compared with 2–3 hours for USPIOs in rats and up to 36 hours in humans with high intraindividual heterogeneity. The blood residence time is the principal factor that influences USPIO uptake into peripheral activated macrophages, and a longer half-life increases the probability of intracellular USPIO accumulation (30).
USPIO-loaded macrophages are characterized by T1, T2, and T2* shortening effects because of their magnetic properties. Depending on the intra- and extracellular particle concentration (24) and the external magnetic field, the effect on SI varies considerably. The T1 shortening effect with time could not be analyzed clearly in our experimental model, since the T2 effect of the edema pattern predominated even the T1-weighted spin-echo MR images at 4.7 T and did not allow discrimination from particle-related T1 shortening. Particles internalized into cells exerted strong R2* effects, resulting in SI loss on T2*-weighted GRE images and reduction of T2 at any time after USPIO application.
The influence on imaging contrast was most prominent on T2*-weighted GRE images, whereas contrast attributes on T2-weighted images were less evident. The prolonged T2 values of the edema pattern in comparison to those of muscle tissue were opposed to the USPIO-related T2 effects, which may explain the indistinguishable T2 of uninfected muscle from the infected area 24 and 48 hours after USPIO infusion. The first measurement, performed 3 hours after intravenous USPIO administration, reflects combined intra- and extracellular particle distribution that exhibited substantial SI loss on T2*-weighted GRE images and shortening of T2 because of infection-related hyperemia with high intravascular particle concentration. Twenty-four hours after USPIO administration, all particles had accumulated in macrophages, as seen at histologic analysis. Thus, SI pattern alterations are caused by macrophages and reveal their distribution. The significant increase of T2 values and SI on T2*-weighted GRE images obtained 24 hours after USPIO application in comparison to the measurement performed 3 hours after application is explained by the overall decreased particle concentration. The intravascular USPIOs had disappeared, and SI alterations were due to intralysosomal intracellular iron particles.
Results of a previous electron microscopy study (10), combined with our results, confirmed that the particles are in rat macrophage phagolysosomes at this time point. Moreover, this resulted in a different USPIO accumulation pattern 3 hours after intravenous USPIO injection, in comparison to the cellular distribution patterns 24, 48, and 72 hours after intravenous USPIO administration, which were similar over time. The edema patterns on T2-weighted images also seemed different and more extended than those at 24 hours after USPIO application, suggesting incongruent distribution of exudation-related interstitial fluid and macrophages.
The statistical analysis in our study was limited because of the small number of animals, which was defined by the experimental design. Although we demonstrated statistical significance of time-dependent effects on T2*-weighted GRE images and T2 maps 3 and 24 hours after USPIO application, the small sample size may be responsible for lack of significance between data obtained at later measurement points.
The site of USPIO loading of macrophages is still hypothetical and may take place in monocytes and/or macrophages of the bloodstream before the cells reach the infected area. Another hypothesis is that the particles may escape into the extravascular space because of the damage to the endothelium and subsequent increase of vessel permeability in the infected area and are then ingested by macrophages. Since our histopathologic findings did not show any extracellular iron deposits, we favor the theory of bloodstream labeling with migration of USPIO-loaded macrophages to the infected area. The slight increase of T2 and SI on T2*-weighted GRE images after 48 and 72 hours compared with that at previous time points may be explained by a reduction in the number of USPIO-loaded macrophages over time, since many of them leave the site of infection and go back into a vessel and are removed by the flowing blood or the lymph (26,27).
One major limitation of former macrophage MR imaging studies in inflammatory diseases (9,17,19,22) was the intravenous doses, which exceeded the tolerable clinical dose (45 µmol/kg) five- to 20-fold. We used a dose increased by approximately threefold, which resulted in excellent detection of USPIO-loaded macrophages in soft-tissue infection. This may be a result of (a) the macrophage density and (b) increased phagocytotic activity of activated macrophages. Although, to our knowledge, a dose-finding study has not been performed yet, it seems possible to detect the same effects with a clinically acceptable dose in humans, since the blood half-life of USPIO particles in humans is at least twice that in rats (30).
Despite the relatively few animals used in this study, our results demonstrate the feasibility of macrophage MR imaging in soft-tissue infection at 4.7 T. Further experiments at a clinically available magnetic field strength of 1.5 T with clinical doses have to be performed to prove the usefulness of the method in a routine clinical setting. The potential value of therapeutic monitoring of infection to discriminate septic lesions from other causes of edema formation on T2-weighted MR images warrants further investigation prior to routine application in humans.
Practical application: The phagocytic response to a stimulus allows us to label macrophages magnetically by means of USPIO particles and to visualize them because of susceptibility effects at MR imaging. Ingestion of USPIO particles was specifically found in macrophages, which will potentially enable differentiation of inflammatory cells from fibroblast-enriched granulation tissue and define inflammatory activity in chronic inflammation. USPIO-enhanced MR imaging provides functional information on a cellular level, but the specific value of USPIO-enhanced MR imaging in chronic inflammation has to be validated in further experimental and clinical studies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Author contributions: Guarantor of integrity of entire study, A.H.K.; study concepts, G.K.v.S., A.H.K., J.F.; study design, A.H.K., P.R.A.; literature research, T.W., A.H.K.; experimental studies, T.O., A.H.K., T.W., P.R.A.; data acquisition, P.R.A., A.H.K., T.O.; data analysis/interpretation, A.H.K., P.R.A., G.J.; statistical analysis, P.R.A., A.H.K.; manuscript preparation, A.H.K.; manuscript definition of intellectual content, A.H.K., P.R.A.; manuscript editing, J.F., G.K.v.S., A.H.K., P.R.A.; manuscript revision/review, G.K.v.S., A.H.K.; manuscript final version approval, A.H.K., P.R.A.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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= relative SI (SI of infected muscle divided by SI of noninfected muscle) on T2*-weighted GRE images before and after USPIO application over time (median values with interquartile range). 
= control values (without USPIO application). The T2* effect was most prominent 3 hours after USPIO application because of the high level of intravascular USPIO and because a significant increase of T2* occurred 24 hours after USPIO application, reflecting the intracellular iron accumulation within macrophages. Only a slight increase of T2* could be delineated at 48 and 72 hours after USPIO application compared with that at 24 hours.
) muscle before and after USPIO application over time. 
