Published online before print February 28, 2003, 10.1148/radiol.2272020490
(Radiology 2003;227:169-174.)
© RSNA, 2003
Functional-Morphologic MR Imaging with Ultrasmall Superparamagnetic Particles of Iron Oxide in Acute and Chronic Soft-Tissue Infection: Study in Rats1
Achim H. Kaim, MD,
Gernot Jundt, MD,
Thorsten Wischer, MD,
Terence O’Reilly, PhD,
Johannes Fröhlich, PhD,
Gustav K. von Schulthess, MD, PhD and
Peter R. Allegrini, PhD
1 From the Department of Diagnostic Radiology (A.H.K., T.W.) and Institute of Pathology (G.J.), University Hospital Basel, Switzerland; Novartis Pharma, Basel, Switzerland (T.O., P.R.A.); Guerbet, Zurich, Switzerland (J.F.); and the Department of Nuclear Medicine, University Hospital Zurich, Switzerland (G.K.v.S.). Received April 29, 2002; revision requested June 6; revision received July 27; accepted September 23. Supported by Fröhlich Pharma Consulting, Zurich; Novartis-Stiftung, Basel; and Freiwillige Akademische Gesellschaft, Basel. 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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PURPOSE: To investigate the use of magnetic resonance (MR) imaging enhanced with ultrasmall superparamagnetic particles of iron oxide (USPIO) to identify acute, early chronic, and late chronic abscess formation in an experimental model of soft-tissue abscess.
MATERIALS AND METHODS: Experimental soft-tissue infection in 15 rats was imaged with an MR imaging unit on days 1 and 2 (acute), days 5 and 6 (early chronic), and days 8 and 9 (late chronic) after inoculation of the infectious agent. All animals were imaged without contrast enhancement and immediately and 24 hours after USPIO administration. MR and histopathologic findings were compared. The changes in relative signal intensity (SI) and in the extent and pattern of contrast enhancement (macrophage distribution) between the animal groups were analyzed. Statistical testing was performed with Kruskal-Wallis analysis of variance and the 
2 test.
RESULTS: At 24 hours after USPIO administration, the relative SI of the abscess wall and the relative macrophage extent were 0.50 (0.33–0.73) and 1.03 (0.90–1.08), respectively, for acute infection; 0.11 (0.10–0.18) and 0.94 (0.93–1.01) for early chronic infection; and 0.53 (0.44–0.58) and 0.80 (0.77–0.83) for late chronic infection. The changes in enhancement pattern (P < .001), relative SI (P < .001), and relative macrophage extent (P < .05) with time were significant.
CONCLUSION: The macrophage distribution pattern increases the specificity of MR findings in chronic infection and allows differentiation between areas with active inflammation and areas of reparative granulation tissue.
© RSNA, 2003
Index terms: Magnetic resonance (MR), contrast media • Magnetic resonance (MR), functional imaging • Soft tissues, infection • Soft tissues, MR
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INTRODUCTION
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Inflammation has been categorized as either acute or chronic, depending on the persistence of the injury, its clinical symptoms, and the nature of the inflammatory response. The features of acute inflammation result from vascular reactions to the stimulus of a pathogen (eg, bacteria). Hallmarks include vasodilatation of precapillary arterioles, accumulation of fluid and plasma components in the interstitial space of the affected tissue, and migration of granulocytes due to loss of endothelial cell integrity in the microcirculation (1,2). These morphologic changes are accurately depicted by magnetic resonance (MR) imaging and are rarely misinterpreted. Chronic inflammation, sometimes with repeated attacks of acute inflammation, may occur as a sequel to acute inflammation, and the process continues as a low-grade chronic condition that is less intense than the acute event. Chronic infection is characterized histologically by the presence of macrophages and lymphocytes in various amounts and fibrosis (1,2).
Since clinical signs of infection are frequently masked, imaging modalities are expected to yield specific and functional information, including high-quality anatomic maps that assist with therapeutic management. Granulation tissue in septic or aseptic disease cannot be reliably differentiated from active inflammation at conventional MR imaging in chronic infection (3,4). One way to provide functional information on a cellular level is to label activated macrophages magnetically with ultrasmall superparamagnetic particles of iron oxide (USPIO) and visualize them by means of susceptibility effects on MR images. The iron particles are phagocytosed by activated macrophages and may enable the differentiation of inflammatory cells from edema and fibroblast-enriched granulation tissue in chronic infection (5). The aim of the present experimental study was to investigate the use of MR imaging enhanced with USPIO to identify acute, early chronic, and late chronic abscess formation in an experimental model of soft-tissue abscess.
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MATERIALS AND METHODS
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Animals and Abscess Model
The study was performed according to the guidelines of the U.S. National Institutes of Health and the recommendations of the committee on animal research at our institution. The protocol was fully approved by the local institutional review committee on animal care. The study included 15 female Sprague-Dawley rats (stock, IcoIbm: OFA; weight range, 250–300 g). All animals were kept in cages with standardized lighting conditions and free access to water and food.
While the rats were receiving general inhalation anesthesia (1.5% isoflurane in a 1:2 mixture of O2 to N2O), a unilateral (left-sided) deep calf muscle abscess was induced by means of intramuscular inoculation of 0.1 mL of a bacterial suspension (Staphylococcus aureus, clinical strain, 10B; Novartis Pharma, Basel, Switzerland). The inoculations were performed by A.H.K. with a 25-gauge needle. 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 assessment and controlled by plating dilutions on agar plates.
The animals were assigned to one of three groups of five animals each and examined with MR imaging at three different points, as follows: group 1, acute (days 1 and 2 [ie, 12–36 hours] after infection); group 2, early chronic (days 5 and 6 after infection); and group 3, late chronic (days 8 and 9 after infection). All animals developed an inflammatory mass in the left calf muscle within 24 hours after bacterial inoculation. No clinical signs of systemic infection were observed.
Contrast Medium
The USPIO contrast agent used in this study was AMI-227 (Sinerem; 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 imaging of the macrophages outside the liver and spleen. The r1 and r2 relaxivities (at 37° C and 0.5 T) are 22 and 80 mmol/L x sec-1 and result in high signal intensity (SI) on T1-weighted images and a decrease in SI on T2-weighted images. AMI-227 is supplied as lyophilized powder and is reconstituted with 9.7 mL of sterile 0.9% normal saline solution to yield a solution containing 20 mg of iron per milliliter. The contrast agent was intravenously administered into the tail vein with a 25-gauge needle at a dose of 180 µmol of iron per kilogram.
MR Imaging
The infected left hind leg (calf and thigh) was imaged with a 4.7-T Bruker DBX imager (Bruker Medical Systems, Karlsruhe, Germany) on days 1 and 2 (group 1), 5 and 6 (group 2), or 8 and 9 (group 3). All animals were anesthetized (1.5% isoflurane in a 1:2 mixture of O2:N2O) and imaged before, immediately after, and 24 hours after administration of the contrast agent.
The imaging protocol was designed as described by Kaim et al (5). The following imaging sequences were applied: (a) a T2-weighted (repetition time msec/echo time msec, 2,000/14–74) multisection-multiecho sequence before and immediately after USPIO administration (T2 maps were calculated from these data); and (b) a T2*-weighted (500/10, 30° flip angle) gradient-echo (GRE) sequence at all three time points (before, immediately after, and 24 hours after USPIO administration).
The imaging protocols ensured evaluation of T2* effects with USPIO and comparison with T2 patterns. Imaging immediately after USPIO administration was performed only with the GRE sequence to ensure successful intravenous USPIO administration. We chose a transverse (orthogonal to the tibia) section orientation to ensure anatomic reproducibility of image position and optimal correlation with histopathologic findings.
The spatial resolution was as follows: acquisition matrix, 256 x 192; reconstruction matrix, 256 x 256 (multisection-multiecho sequence); imaging matrix, 128 x 128 (GRE sequence); field of view, 4 cm; section thickness, 1 mm contiguous. Two signals were acquired, and the mean duration of each imaging study was 15 minutes.
Sacrifice, Histologic Examination, and Electron Microscopy
Sacrifice was performed within 4 hours after the last postcontrast imaging session. All animals were sacrificed by means of administration of inhalable pure CO2. The infected hind leg was anatomically prepared, exarticulated, and fixed with 4% formaldehyde.
The pathologic specimens were embedded in methylmethacrylate according to standard procedures. Sections 4 µm thick were cut transversely and stained with hematoxylin-eosin and chloracetatesterase reaction (a histochemical stain for granulocytes) for histologic examination. Soft-tissue infection was histologically characterized on sections stained with hematoxylin-eosin and chloracetatesterase reaction. The density of the cellular components was characterized by one pathologist according to a ranking system (+++ = high, ++ = moderate, + = low, 0 = absent). Histologic findings were compared with findings seen on nonenhanced MR images (G.J. and A.H.K.). Prussian blue staining for iron was performed to detect the presence of iron particles.
Image Analysis and Statistical Evaluation
Qualitative analysis.—Iron-stained histologic sections (Prussian blue stain) and USPIO-enhanced MR images were analyzed histomorphologically by one pathologist (G.J.) and one radiologist (A.H.K.) in consensus to correlate susceptibility effects with intracellular iron distribution. An edema pattern was defined as a pattern of areas with increased T2 values compared with noninfected muscle before contrast medium administration, and USPIO distribution was observed on GRE MR images. The iron-loaded macrophage patterns 24 hours after USPIO administration were staged in consensus by two observers (A.H.K. and T.W.), who used the following ranking system: 0, no enhancement; 1, diffuse enhancement; 2, circumscribed enhancement with ill-defined borders; and 3, circumscribed enhancement that was sharply defined. The enhancement patterns of acute, early chronic, and late chronic infection were statistically analyzed with the Craddock-Flood 2 contingency test for small sample sizes.
Quantitative analysis.—SIs were measured within regions of interest (ROIs) on T2*-weighted images obtained before and 24 hours after USPIO administration. The ROIs (0.06–0.13 cm2) were placed within the cellular infiltrate of the abscess wall around the central necrosis at identical locations over time for each animal and within unaffected muscle tissue of the ipsilateral thigh (performed by A.H.K. and G.J. in consensus). Relative T2*-weighted SI values (SI of infection/SI of unaffected muscle) were calculated for each group before and 24 hours after USPIO administration. Values were expressed as medians with interquartile ranges. The time course of the ratio between relative SI after USPIO and relative SI before USPIO was assessed with Kruskal-Wallis analysis of variance and exact testing for small sample sizes, with time as the independent variable.
So that we could compare the extent of infection on T2 maps with the macrophage distribution pattern, the areas with edema pattern were defined with ROIs on three contiguous sections on T2 maps and divided throughout the whole area of the transversely imaged extremity. The macrophage distribution pattern was delineated the same way on 24-hour postcontrast GRE images (areas with contrast enhancement were defined with ROIs on three contiguous sections on T2* maps and divided throughout the whole area of the transversely imaged extremity) with identical anatomic localization. The ratio of the area of the macrophage pattern to the area of the edema pattern (ie, the relative macrophage extent) revealed the differences in the macrophage extent and edema pattern among the three groups and was assessed over time with Kruskal-Wallis analysis of variance and exact testing for small sample sizes, with time as the independent variable. P values of less than .05 were considered to indicate a statistically significant difference.
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RESULTS
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Acute abscess formation (observed 2 days after inoculation in group 1) was characterized histologically by an area of incipient central necrosis predominantly surrounded by a dense cellular infiltrate of neutrophils and some interspersed macrophages. The contrast enhancement pattern 24 hours after USPIO administration was diffuse in four animals and circumscribed with ill-defined borders in one. The mean relative SI of the abscess wall on USPIO-enhanced GRE MR images was calculated to be 0.50 (0.33–0.73). The susceptibility effect could be histopathologically correlated to some interspersed iron-loaded macrophages seen with Perls staining. Intracellular iron deposits could not be delineated within granulocytes. The relative macrophage extent (ie, the macrophage distribution pattern relative to the edema pattern) was calculated to be 1.03 (0.90–1.08), which suggests a similar distribution of cellular infiltrates and extracellular fluid. Figure 1 shows the typical MR and histologic appearance of acute abscess formation before and after USPIO administration.
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Figure 1a. MR images and photomicrographs of corresponding histologic appearance of acute soft-tissue infection. (a) Transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained before intravenous USPIO administration shows slight hyperintensity within infected muscle. (b) Diffuse edema pattern (arrows) in infected muscle is delineated on transverse T2 map obtained before USPIO administration. (c) Enhancement pattern 24 hours after USPIO administration is diffuse, relatively faint, and inhomogeneous on transverse T2*-weighted GRE MR image (500/10, 30° flip angle). (d) Inflammatory cell infiltrate consists mainly of granulocytes (arrows). (Chloracetatesterase reaction; original magnification, x10.) (e) Only some interspersed USPIO-loaded macrophages (arrows) are present. (Prussian blue stain; original magnification, x40.)
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Figure 1b. MR images and photomicrographs of corresponding histologic appearance of acute soft-tissue infection. (a) Transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained before intravenous USPIO administration shows slight hyperintensity within infected muscle. (b) Diffuse edema pattern (arrows) in infected muscle is delineated on transverse T2 map obtained before USPIO administration. (c) Enhancement pattern 24 hours after USPIO administration is diffuse, relatively faint, and inhomogeneous on transverse T2*-weighted GRE MR image (500/10, 30° flip angle). (d) Inflammatory cell infiltrate consists mainly of granulocytes (arrows). (Chloracetatesterase reaction; original magnification, x10.) (e) Only some interspersed USPIO-loaded macrophages (arrows) are present. (Prussian blue stain; original magnification, x40.)
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Figure 1c. MR images and photomicrographs of corresponding histologic appearance of acute soft-tissue infection. (a) Transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained before intravenous USPIO administration shows slight hyperintensity within infected muscle. (b) Diffuse edema pattern (arrows) in infected muscle is delineated on transverse T2 map obtained before USPIO administration. (c) Enhancement pattern 24 hours after USPIO administration is diffuse, relatively faint, and inhomogeneous on transverse T2*-weighted GRE MR image (500/10, 30° flip angle). (d) Inflammatory cell infiltrate consists mainly of granulocytes (arrows). (Chloracetatesterase reaction; original magnification, x10.) (e) Only some interspersed USPIO-loaded macrophages (arrows) are present. (Prussian blue stain; original magnification, x40.)
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Figure 1d. MR images and photomicrographs of corresponding histologic appearance of acute soft-tissue infection. (a) Transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained before intravenous USPIO administration shows slight hyperintensity within infected muscle. (b) Diffuse edema pattern (arrows) in infected muscle is delineated on transverse T2 map obtained before USPIO administration. (c) Enhancement pattern 24 hours after USPIO administration is diffuse, relatively faint, and inhomogeneous on transverse T2*-weighted GRE MR image (500/10, 30° flip angle). (d) Inflammatory cell infiltrate consists mainly of granulocytes (arrows). (Chloracetatesterase reaction; original magnification, x10.) (e) Only some interspersed USPIO-loaded macrophages (arrows) are present. (Prussian blue stain; original magnification, x40.)
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Figure 1e. MR images and photomicrographs of corresponding histologic appearance of acute soft-tissue infection. (a) Transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained before intravenous USPIO administration shows slight hyperintensity within infected muscle. (b) Diffuse edema pattern (arrows) in infected muscle is delineated on transverse T2 map obtained before USPIO administration. (c) Enhancement pattern 24 hours after USPIO administration is diffuse, relatively faint, and inhomogeneous on transverse T2*-weighted GRE MR image (500/10, 30° flip angle). (d) Inflammatory cell infiltrate consists mainly of granulocytes (arrows). (Chloracetatesterase reaction; original magnification, x10.) (e) Only some interspersed USPIO-loaded macrophages (arrows) are present. (Prussian blue stain; original magnification, x40.)
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The early chronic phase of infection (observed 6 days after inoculation in group 2) was characterized histologically as a mixed cellular infiltrate of predominantly macrophages and granulocytes around an area of central necrosis. Interspersed fibroblasts could be detected as part of granulation tissue that delineated the abscess from the surrounding muscle. The contrast enhancement pattern 24 hours after USPIO administration was sharply defined and circumscribed in all five animals. The strong susceptibility effect could be attributed to a dense infiltration of iron-loaded macrophages seen within the abscess wall with Perls staining.
The relative SI of the abscess wall on USPIO-enhanced GRE MR images was calculated to be 0.11 (0.10–0.18). The relative macrophage extent was calculated to be 0.94 (0.93–1.01), which suggests a slight decrease in the area with macrophage distribution relative to the edema pattern. Figure 2 illustrates the typical MR and histologic appearance in early chronic abscess formation before and after USPIO administration.
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Figure 2a. MR images and photomicrograph of corresponding histologic appearance in early chronic soft-tissue infection. (a) Transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained before intravenous USPIO administration shows an inhomogeneous pattern with predominantly high SI within the area of infection. (b) High SI (arrows) on transverse T2 map obtained before USPIO administration delineates the edema pattern that defines the extent of infection. (c) Enhancement pattern seen on transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained 24 hours after USPIO administration is sharply circumscribed and shows a strong decrease in SI (*) due to susceptibility effects. An area of central necrosis with high SI is surrounded by the strongly enhancing abscess wall. (d) Cellular infiltrate is characterized by a dense infiltrate of iron-loaded macrophages (arrows). (Prussian blue stain; original magnification, x10.)
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Figure 2b. MR images and photomicrograph of corresponding histologic appearance in early chronic soft-tissue infection. (a) Transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained before intravenous USPIO administration shows an inhomogeneous pattern with predominantly high SI within the area of infection. (b) High SI (arrows) on transverse T2 map obtained before USPIO administration delineates the edema pattern that defines the extent of infection. (c) Enhancement pattern seen on transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained 24 hours after USPIO administration is sharply circumscribed and shows a strong decrease in SI (*) due to susceptibility effects. An area of central necrosis with high SI is surrounded by the strongly enhancing abscess wall. (d) Cellular infiltrate is characterized by a dense infiltrate of iron-loaded macrophages (arrows). (Prussian blue stain; original magnification, x10.)
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Figure 2c. MR images and photomicrograph of corresponding histologic appearance in early chronic soft-tissue infection. (a) Transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained before intravenous USPIO administration shows an inhomogeneous pattern with predominantly high SI within the area of infection. (b) High SI (arrows) on transverse T2 map obtained before USPIO administration delineates the edema pattern that defines the extent of infection. (c) Enhancement pattern seen on transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained 24 hours after USPIO administration is sharply circumscribed and shows a strong decrease in SI (*) due to susceptibility effects. An area of central necrosis with high SI is surrounded by the strongly enhancing abscess wall. (d) Cellular infiltrate is characterized by a dense infiltrate of iron-loaded macrophages (arrows). (Prussian blue stain; original magnification, x10.)
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Figure 2d. MR images and photomicrograph of corresponding histologic appearance in early chronic soft-tissue infection. (a) Transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained before intravenous USPIO administration shows an inhomogeneous pattern with predominantly high SI within the area of infection. (b) High SI (arrows) on transverse T2 map obtained before USPIO administration delineates the edema pattern that defines the extent of infection. (c) Enhancement pattern seen on transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained 24 hours after USPIO administration is sharply circumscribed and shows a strong decrease in SI (*) due to susceptibility effects. An area of central necrosis with high SI is surrounded by the strongly enhancing abscess wall. (d) Cellular infiltrate is characterized by a dense infiltrate of iron-loaded macrophages (arrows). (Prussian blue stain; original magnification, x10.)
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The histologic appearance of the late chronic phase of infection (observed 9 days after inoculation in group 3) was characterized by a prominent layer of macrophages, interspersed lymphocytes, and some granulocytes around the residual abscess necrosis; fibroblast-enriched granulation tissue with few inflammatory cells delineated the infection in the muscle. Perls staining revealed a clear association between increased USPIO accumulation and iron-loaded macrophages within the abscess wall, and USPIO accumulation was only faintly detectable within the fibroblast-enriched granulation tissue. The contrast enhancement pattern 24 hours after USPIO administration was graded as circumscribed with ill-defined borders in four animals and as diffuse in one. The relative SI of the abscess wall on USPIO-enhanced GRE MR images was calculated to be 0.53 (0.44–0.58), and the relative macrophage extent was 0.80 (0.77–0.83). This indicates that the macrophage distribution on GRE MR images is clearly less extended than the edema pattern on T2 maps in the late chronic stage. Figure 3 shows the typical SI patterns on MR images of late chronic abscess formation obtained before and after USPIO administration, with the corresponding histologic appearance. The Table summarizes the results of histologic analysis of the cellular infiltrates.
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Figure 3a. MR images and photomicrograph of corresponding histologic appearance of late chronic soft-tissue infection. (a) High SI is seen within infected muscle on a transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained before intravenous USPIO administration. (b) Areas of high SI (arrows) are delineated on a transverse T2 map obtained before USPIO administration. The rather nonspecific term "edema pattern" is used to describe the extent of pathomorphologic changes. (c) On a transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained 24 hours after USPIO administration, enhancement appears circumscribed in the abscess wall around the area of central necrosis (*) and is surrounded by a less intense and relatively unsharp enhancement pattern. (d) Iron-loaded macrophages (arrows) are interspersed within fibroblast-enriched granulation tissue. (Prussian blue stain; original magnification, x20.)
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Figure 3b. MR images and photomicrograph of corresponding histologic appearance of late chronic soft-tissue infection. (a) High SI is seen within infected muscle on a transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained before intravenous USPIO administration. (b) Areas of high SI (arrows) are delineated on a transverse T2 map obtained before USPIO administration. The rather nonspecific term "edema pattern" is used to describe the extent of pathomorphologic changes. (c) On a transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained 24 hours after USPIO administration, enhancement appears circumscribed in the abscess wall around the area of central necrosis (*) and is surrounded by a less intense and relatively unsharp enhancement pattern. (d) Iron-loaded macrophages (arrows) are interspersed within fibroblast-enriched granulation tissue. (Prussian blue stain; original magnification, x20.)
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Figure 3c. MR images and photomicrograph of corresponding histologic appearance of late chronic soft-tissue infection. (a) High SI is seen within infected muscle on a transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained before intravenous USPIO administration. (b) Areas of high SI (arrows) are delineated on a transverse T2 map obtained before USPIO administration. The rather nonspecific term "edema pattern" is used to describe the extent of pathomorphologic changes. (c) On a transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained 24 hours after USPIO administration, enhancement appears circumscribed in the abscess wall around the area of central necrosis (*) and is surrounded by a less intense and relatively unsharp enhancement pattern. (d) Iron-loaded macrophages (arrows) are interspersed within fibroblast-enriched granulation tissue. (Prussian blue stain; original magnification, x20.)
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Figure 3d. MR images and photomicrograph of corresponding histologic appearance of late chronic soft-tissue infection. (a) High SI is seen within infected muscle on a transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained before intravenous USPIO administration. (b) Areas of high SI (arrows) are delineated on a transverse T2 map obtained before USPIO administration. The rather nonspecific term "edema pattern" is used to describe the extent of pathomorphologic changes. (c) On a transverse T2*-weighted GRE MR image (500/10, 30° flip angle) obtained 24 hours after USPIO administration, enhancement appears circumscribed in the abscess wall around the area of central necrosis (*) and is surrounded by a less intense and relatively unsharp enhancement pattern. (d) Iron-loaded macrophages (arrows) are interspersed within fibroblast-enriched granulation tissue. (Prussian blue stain; original magnification, x20.)
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The time course of the relative SI is illustrated in Figure 4. The change with time in the ratio between relative SI after USPIO and relative SI before USPIO was highly significant (2 = 9.78, P < .001). The enhancement patterns differed significantly with time (2 = 20.4, P < .001). Figure 5 demonstrates the change over time in the macrophage extent relative to the edema pattern, which was also significant (2 = 6.02, P < .05).
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Figure 4. Graph shows that decrease in relative SI was most prominent in the early chronic stage of infection due to high density of USPIO-loaded macrophages. In acute and late chronic stages, the cellular infiltrate had only some interspersed and fewer USPIO-loaded macrophages, leading to a smaller decrease in relative SI on MR images obtained 24 hours after USPIO administration.
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Figure 5. Relative macrophage extent (ratio of area of macrophage cellular pattern to area of edema pattern) decreased to 0.8 in the late chronic stage, indicating that the distribution of USPIO-loaded macrophages was less extensive than the edema pattern. In the early chronic stage, however, the extension was very similar for both patterns.
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DISCUSSION
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The present study investigated the use of functional cellular (high-spatial-resolution) MR imaging of activated macrophages in acute, early chronic, and late chronic infection. Chemotactic cytokines and phagocytic stimuli activate inflammatory cells and lead to the so-called respiratory burst (6). Energy-dependent interrelated cellular defense mechanisms are activated; these include migration, generation and release of microbicidal agents, and phagocytosis. Their activation is associated with a large increase in glucose metabolism by way of the hexose monophosphate shunt (7). Activated macrophages are powerful phagocytes that ingest all kinds of small particulate material and digest much of it in their phagosomes (6). Because of the phagocytic response, activated macrophages can be labeled magnetically with USPIO, and their susceptibility effects can be visualized with MR imaging (8–12).
The USPIO used in this study have a longer intravascular half-life than preparations composed of large particles, making them suitable for investigation of reticuloendothelial system macrophages outside the liver, spleen, and bone marrow. USPIO consist of an iron oxide core with a form of nonstoichiometric magnetite 4.3–6.0 nm in diameter coated with low-molecular-weight dextran. The histopathologic patterns of the infection model used, which depend on the time delay from infection, were recently described (13). One time-dependent parameter is macrophage density, as reflected by the time-dependent patterns seen with USPIO-enhanced MR imaging in the present study.
Imaging of acute infection does not pose major problems in the diagnostic work-up of musculoskeletal diseases. It is characterized by vasodilatation, exudation of plasma proteins, accumulation of edematous fluid in the interstitial space, and cellular migration of predominantly neutrophils (1,2). The widely used criteria for delineation of inflammatory changes on MR images include the presence of an edema pattern on T2-weighted MR images and contrast enhancement after intravenous gadolinium chelate administration on T1-weighted MR images. Our results demonstrate that, compared with unenhanced MR imaging, USPIO-enhanced MR imaging does not offer substantial improvement in diagnostic accuracy or specificity for depicting the extent of infection in the acute stage. The extension and distribution of the edema pattern and the macrophage distribution pattern were quite similar on MR images obtained before and after USPIO administration, and USPIO-enhanced MR imaging did not provide additional information. The main reason for this was that the acute soft-tissue infections in our model were particularly characterized by dense granulocyte infiltration and the presence of only some macrophages, so intense cellular labeling could not be expected.
Inaccuracy occurs with MR imaging of chronic infection due to the low specificity of the criteria used with unenhanced and gadolinium-enhanced MR imaging (4,14). The chronic stage of infection is characterized by the presence of granulation tissue, lymphocytes, and, particularly, activated macrophages. Granulation tissue can be detected with MR imaging on T2 maps, but its presence does not enable one to fully characterize the grade and extent of active inflammation. The amount of activated macrophages, however, may be used as a parameter for defining the activity and extent of inflammation. Ingestion of the USPIO was specifically found in macrophages and visualized on MR images. The delineation of the extent of magnetically labeled activated macrophages on GRE MR images and the extent of the edema pattern on T2 maps was significantly different in late chronic infection, which may be explained by the differentiation of inflammatory cells from fibroblast-enriched granulation tissue. The increased USPIO accumulation was clearly associated with iron-loaded macrophages seen within the abscess wall with Perls staining, and USPIO accumulation was only faintly detectable within the fibroblast-enriched granulation tissue. This finding was explained by the presence of some interspersed macrophages within the granulation tissue, which led to unsharp enhancement of an otherwise circumscribed contrast pattern.
The temporal course of the relative SI from early to late chronic infection suggested the specific value of USPIO-enhanced MR imaging in defining the inflammatory activity of chronic infection. Early chronic soft-tissue abscess was histologically characterized by a dense infiltrate of macrophages in the abscess wall, which substantially decreased the SI on GRE images. The increase in SI with time may be explained by ongoing consolidation of the infection with less inflammatory activity and subsequently reduced macrophage density in the late chronic stage.
One limitation of our study is that the USPIO dose used was four times the approved clinical dose. Similar effects may be expected in humans with a decreased dose, since, for the USPIO we used, the half-life in humans is twice that in rats. The blood residence time is the principal factor that influences USPIO uptake into peripheral activated macrophages. Thus, a longer half-life increases the probability of intracellular USPIO accumulation (15). Furthermore, a stronger T2* weighting of the GRE sequence might be chosen to compensate for lower USPIO load in clinical applications, as susceptibility effects due to tissue inhomogeneities are less severe in human patients than in smaller laboratory animals at 4.7 T. Since the experimental results are very promising, further studies with clinically tolerable doses at a magnetic field strength of 1.5 T should be performed to prove the clinical usefulness of the method.
Practical application: USPIO-enhanced MR imaging provides functional-morphologic information on the cellular level in imaging of infection. Activated macrophages are powerful phagocytes and can be magnetically labeled with USPIO. The macrophage distribution on MR images characterizes inflammatory activity and extent in chronic infection and allows differentiation between areas with active inflammation and areas of reparative granulation tissue. Our results strongly suggest the need for further larger-scale studies, potentially in human subjects, to detect inflammatory activity in cases of suspected postoperative infection.
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FOOTNOTES
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Abbreviations: GRE = gradient echo,
ROI = region of interest,
SI = signal intensity,
USPIO = ultrasmall superparamagnetic particles of iron oxide
Author contributions: Guarantor of integrity of entire study, A.H.K.; study concepts, A.H.K., P.R.A., J.F.; study design, A.H.K., P.R.A.; literature research, A.H.K., J.F.; experimental studies, A.H.K., T.W., P.R.A., T.O.; data acquisition, A.H.K., P.R.A., G.J.; data analysis/interpretation, A.H.K., P.R.A., T.O., G.J.; statistical analysis, A.H.K., P.R.A.; manuscript preparation, A.H.K.; manuscript definition of intellectual content, A.H.K., P.R.A.; manuscript editing, A.H.K.; manuscript revision/review, A.H.K., P.R.A., G.K.v.S., G.J.; manuscript final version approval, all authors.
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INTRODUCTION
