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Original Research |
1 Department of Radiology, Bundang CHA General Hospital, College of Medicine,
Pochon CHA University, Kyonggi-do 463-712, Korea.
2 Department of Radiology, Asan Medical Center, University of Ulsan College of
Medicine, 388-1 Poongnap-dong, Songpa-gu, Seoul 138-736, Korea.
3 Department of Pathology, Bundang CHA General Hospital, College of Medicine,
Pochon CHA University, Kyonggi-do 463-712, Korea.
4 Department of Radiology, College of Medicine, Ewha Womans University, Seoul
158-710, Korea.
Received August 30, 2006;
accepted after revision April 23, 2007.
Address correspondence to S. H. Lee
(shlee{at}amc.seoul.kr).
Abstract
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MATERIALS AND METHODS. Nine rats with infectious arthritis of the knee or soft-tissue infection were imaged on an MRI unit on days 4-6 after IV injection of a bacterial suspension. All animals were imaged on a T2-weighted fast spin-echo sequence before and 24 hours after administration of SPIO. The nine rats were classified into two groups according to the dose of SPIO. We calculated the relative signal-to-noise ratio (SNR) change and compared the relative SNR change with the histologic findings. We analyzed iron-loaded cells and the intracellular uptake of iron particles according to the dose of SPIO.
RESULTS. The SNR value decreased in proportion to the increase in the number of iron-laden macrophages or fibroblasts in the wall of the soft-tissue abscess (p < 0.01). The intracellular uptake of iron particles was shown in fibroblasts as well as in macrophages, and their uptake in the fibroblasts was greater than that in the macrophages (p < 0.05). There was no statistically significant difference in the intracellular uptake of iron particles according to the dose of SPIO (p >0.1).
CONCLUSION. SPIO-enhanced MRI can be useful in evaluating infectious disease of the joint or soft tissue and is influenced by the uptake of iron particles in fibroblasts as well as macrophages.
Keywords: infectious disease • MRI • musculoskeletal infection • superparamagnetic iron oxide (SPIO)
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Histologically, acute inflammation shows vasodilatation of precapillary arterioles, accumulation of fluid or plasma components in the interstitial spaces of the affected tissue, and migration of granulocytes caused by the loss of endothelial cell integrity resulting from vascular reactions to the stimulus of a pathogen such as bacteria [7, 8]. However, chronic infection shows the infiltration of varying amounts of macrophages, lymphocytes, and fibrosis [7, 8].
Recently, the importance of cell-specific MRI has become increasingly recognized. MRI with superparamagnetic iron oxide (SPIO) particles accumulated in the mononuclear phagocyte system, has been performed in liver and spleen imaging. Because SPIO particles are phagocytosed by activated macrophages, we assumed that MRI with SPIO might reveal the distribution of macrophages in infectious disease and thereby reveal the activity of infection and the extent of a chronic infection. Subsequently, we would be able to differentiate between active inflammation and reparative granulation tissue. The purpose of this study was to evaluate the usefulness of MRI with SPIO in experimental models of infectious disease and to analyze the intracellular uptake of SPIO.
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General anesthesia was induced by means of intramuscular injection of a mixture of 50 mg/kg of ketamine and 5 mg/kg of xylazine hydrochloride (Rompun, Bayer HealthCare) into the buttocks. To induce infectious arthritis or a soft-tissue infection, 0.5 mL of a bacterial suspension of Staphylococcus aureus (2 x 109 colony-forming units per milliliter) was administered by means of IV injection through tail veins of all rats. The injections were performed with a 24-gauge peripheral IV catheter.
Infectious arthritis of the knee or soft-tissue infection was palpable on days 4-6 after IV injection of the bacterial suspension. This study included rats having no clinical signs of systemic infection at the time.
Contrast Medium
The SPIO contrast agent used in this study was Resovist (ferucarbotran,
Schering); 1 mL of Resovist solution contains 540 mg of ferucarbotran,
corresponding to 28 mg of iron. The mean diameter of the particles is
approximately 62 nm [9].
MRI
Two MRI sessions were performed for all animals. The first session was
performed as the base-line study before administration of SPIO (unenhanced MR
study). The second MRI session was performed 24 hours after the SPIO
administration (contrast-enhanced MR study).
MRI was performed on a 1.5-T Intera scanner (Philips Medical Systems) after palpation of the infectious arthritis of the knee or of the soft-tissue infection, on days 4-6 after IV injection of the bacterial suspension. In all animals, each MRI session was performed with the animal under general anesthesia. All animals were placed in the prone position with the knees centrally located in a microscopy coil; they were then imaged with a T2-weighted fast spin-echo sequence. The acquisition parameters were as follows: TR range/TE, 1,820-2,000/80; field of view, 6 x 6 cm; matrix, 256 x 256. MRI was performed in the sagittal plane. The section thickness was 1.5 mm without an intersection gap, and there were three excitations. The acquisition time was a mean of 2 minutes 47 seconds.
The SPIO contrast agent was administered IV through a tail vein using a 24-gauge peripheral IV catheter immediately after completing the unenhanced imaging session. The nine rats were classified into two groups according to the dose of SPIO—that is, six in the low- to intermediate-dose group (3-60 µmol Fe/300 g body weight) and three in the high-dose group (350 µmol Fe/300 g body weight). The SPIO contrast agent was administered as slowly as possible and was administered with 1 mL of normal saline. Contrast-enhanced T2-weighted fast spin-echo images were obtained using parameters identical to those for the unenhanced MR images.
Histologic Examination and Analysis
The rats were sacrificed within 30 minutes after completing the
contrast-enhanced imaging session. All animals were sacrificed by means of IV
injection of a lethal dose of a mixture of ketamine and Rompun.
The pathologic specimens were obtained from the infected synovium and soft-tissue abscess and were fixed in 4% formalin. These specimens were then cut into sagittal slices according to the imaging planes for histologic staining. After embedding of the selected slices in paraffin, H and E staining was used for histologic examination. Prussian blue staining and CD68 staining were also performed to detect the presence of iron particles and macrophages. On light microscopy with a magnification of 400, the numbers of iron-loaded cells, macrophages, and fibroblasts were counted in five areas, which included the largest amounts of them. The number of fibroblasts was counted on the section stained with H and E.
To analyze iron-loaded cells, we compared their shape and location between the sections stained with Prussian blue and comparable sections stained with H and E or CD68. We could see iron-loaded cells shaped like fibroblasts or macrophages on the sections stained with Prussian blue and confirm that they were fibroblasts or macrophages on comparable sections stained with H and E or CD68. We counted the number of iron-loaded cells according to fibroblasts or macrophages and analyzed which cells were most prevalent between the two types.
To analyze the intracellular uptake of iron particles in each cell according to the dose of SPIO, we compared the number of iron-loaded macrophages in the low to intermediate-dose group with the number in the high-dose group. We also compared the number of iron-loaded fibroblasts in both groups.
Image Analysis and Statistical Evaluation
Signal intensity (SI) was measured within the 2-4 mm2 regions of
interest (ROIs), which were placed in comparable locations within the infected
synovium or in the soft-tissue abscess wall on both unenhanced and
contrast-enhanced T2-weighted images. The signal-to-noise (SNR) values were
then calculated using the following equation:
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where SIROI and SIbackground are SIs of the ROI and
the background and SDbackground is the standard deviation of the
background noise. To evaluate the SNR change between the unenhanced and
contrast-enhanced images, the relative SNR changes were quantified using the
following equation:
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where SNRPRE and SNRPOST are the SNRs of the unenhanced and contrast-enhanced images.
We analyzed the correlation between the relative SNR change and the total number of iron-loaded cells on sections stained with Prussian blue. In addition, in the soft-tissue abscess wall, we analyzed the following: the correlation between the SNR change and the total number of iron-loaded cells on sections stained with Prussian blue, the correlation between the SNR change and the number of macrophages on sections stained with CD68, and the correlation between the SNR change and the number of fibroblasts on sections stained with H and E.
Statistical Analysis
Statistical analysis was performed using SPSS statistical package, version
11.0. The Spearman's correlation coefficient (r) was used for each
correlation. The Mann-Whitney test was used to compare the number of
iron-loaded cells according to their kind and to compare the number of
iron-loaded macrophages or iron-loaded fibroblasts according to the dose of
SPIO. Statistical significance was set at p < 0.05.
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Of the three rats given a high dose of SPIO, in one we were able to measure SI and perform histologic examination both in the infected synovium and in the soft-tissue abscess wall. Measurement in the other two rats could be obtained only in the soft-tissue abscess wall. Therefore, the cases that allowed for the measurement of SI and histologic examination of the infected synovium and of the soft-tissue abscess wall were 1 and 3, respectively (Table 1). All soft-tissue abscesses were located in calf muscles.
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There was intracellular uptake of iron particles in the fibroblasts and in the macrophages (Table 2 and Fig. 4A, 4B, 4C). The mean percentages of the number of iron-loaded macrophages and iron-loaded fibroblasts to the total number of iron-loaded cells were 21.28% ± 38.73% and 78.72% ± 38.74%, respectively. The intracellular uptake of iron particles in fibroblasts was more than that in macrophages (p < 0.05).
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Iron oxide particle-based MR contrast agents are SPIO with a mean diameter of 60-150 nm and ultrasmall superparamagnetic iron oxide (USPIO) with a mean diameter of 20-50 nm. These contrast agents are phagocytosed by the reticuloendothelial system (RES). However, size and surface properties of these contrast agents are the determining factors for their physiologic distribution. It is well known that larger particles, such as SPIO, are quickly phagocytosed, mainly by means of hepatic Kupffer cells and the mononuclear phagocyte system of the spleen. Therefore, the human blood half-life is much shorter—that is, by 10-15 minutes compared with that of USPIO.
On the other hand, a smaller number of USPIO particles, because of their smaller size, are entrapped by the mononuclear phagocytic system of the liver and the spleen. The blood half-life of USPIO is 2-3 hours in rats and up to 36 hours in humans. Therefore, SPIO has been used clinically as a contrast agent for MRI of the liver and spleen. Because USPIO has a longer blood half-life, it is suitable for use in investigating lymph nodes and bone marrow [13]. Once particles such as SPIO or USPIO are injected into the bloodstream, they are rapidly coated by components of the circulation, such as plasma proteins. This process is known as opsonization and is critical in recognizing the injected particles [14].
Normally, opsonization renders the particles recognizable by the body's major defense system, the RES [15, 16]. Therefore, more than 80% of the IV-injected SPIO particles are quickly ingested by the RES of the liver and spleen [17]. The remaining 20% of the particles are ingested in bone marrow or other sites of the human body. The particles remaining in the bloodstream may then escape into the extravascular spaces because of the damage to the endothelium and the subsequent increase of vessel permeability in the infected area and may then be ingested by phagocytes such as macrophages.
Because the hydrodynamic diameter of Resovist is 62 nm, which is the smallest of SPIO and only slightly larger than USPIO [9], the possibility of being taken into the RES system beyond the liver and spleen is high compared with that of other SPIOs. Resovist is clinically available in Europe and Asia and showed more efficient uptake into the cells than USPIO in an in vitro study [18]. Therefore, we were concerned regarding the possibility of macrophage imaging in inflammatory or infectious lesions using Resovist.
In MRI with SPIO or USPIO, the vastly different susceptibility between superparamagnetic particles and the surrounding tissue creates immense field gradients that cause an irreversible loss of phase coherence. The main effect is shortening of the T2 relaxation time and, to a lesser degree, of the T1 relaxation time [19, 20].
MRI with USPIO shows an uptake of iron particles by activated macrophages in acute soft-tissue infection in rats and a decrease of the T2 value because of the susceptibility effect [21]. Our study using SPIO, not USPIO, showed that the SNR value on T2-weighted images after SPIO administration decreased in proportion to the increase of the total number of iron-loaded cells in the infected synovium of the knee and in the soft-tissue abscess wall, similar to the results in previous reports using USPIO. The increased numbers of fibroblasts and macrophages were seen in the soft-tissue abscess wall, and fibroblasts were more prominent in our study than in the study of Kaim et al. [22], in which 5-6 days after the onset of infection, macrophages were more common than fibroblasts in the abscess wall and the prominent fibroblasts appeared in the course of chronic infection. The injection method in our study was different from the method of Kaim et al. IV injection was used in our study, and intramuscular injection was used by Kaim et al. Therefore, the concentration of bacteria in the infection area might be different in our study than in the study by Kaim et al. The different methods of causing infection might lead to a different ratio of macrophages and fibroblasts in the abscess wall. Kaim et al. insisted that the distribution pattern of the macrophages increases the specificity of the MRI findings in chronic infection and therefore allows differentiation between areas of active inflammation and reparative granulation tissue. However, in our study, because fibroblasts also showed a large amount of uptake of iron oxides, the low signal intensity in the abscess wall alone could not differentiate between acute and chronic infection.
Human fibroblasts have been used in studies of the intracellular uptake of nanoparticles [23, 24]. Dextran-coated iron oxide nanoparticles used as MR contrast agents are taken into fibroblasts via fluid-phase endocytosis [25]. The SPIO used in our study has a mean diameter of 62 nm. A small number of SPIO particles, avoiding ingestion in the RES of the liver and spleen, might escape into the extravascular space in the infected area and be ingested by phagocytosis of macrophages or into fibroblasts via fluid-phase endocytosis.
The usefulness of MRI with SPIO or USPIO in antigen-induced arthritis has recently been reported [26, 27]. Active macrophages within the arthritic synovium can ingest USPIO. Therefore, MRI with USPIO allows visualization of the decrease in SNR value on T2-weighted images and helps to evaluate the synovial activity [26]. In our study, all six cases of infected synovium revealed features of joint effusion on unenhanced T2-weighted images. However, after SPIO administration, there was little SNR change in the infected synovium, and no iron-loaded cells were seen on sections stained with Prussian blue. Lutz et al. [26] performed MRI 9-29 days after intraarticular injection of an antigen suspension into the knee joints of rabbits to construct an experimental model of chronic inflammatory arthritis, such as rheumatoid arthritis.
On the other hand, our experimental model caused infectious arthritis 4-6 days after IV injection of a bacterial suspension. Therefore, unlike the many macrophages that were visualized in the study of Lutz et al. [26], the synovium in our study revealed only a small number of macrophages. Although infectious arthritis seen 4-6 days after IV injection of a bacterial suspension showed effusion on unenhanced T2-weighted images, SPIO-enhanced T2-weighted images showed little SNR change because of the minimal inflammatory histologic change. However, soft-tissue abscesses showed extensive macrophage and fibroblast infiltration into the walls. Therefore, there were more inflammatory changes in soft-tissue abscesses than in joint infection at the acute stage of infection in our study. After a week of infection, swelling of the affected knee joint had usually improved on serial inspection of our study rats. Therefore, there was no concern regarding evaluation of the infectious arthritis in our study.
The dose of SPIO is 8-16 µmol Fe/kg for MRI of the liver or perfusion in vivo [9]. However, our study intended to show the intracellular uptake of SPIO particles escaping ingestion in the RES of the liver and spleen as much as possible in the infected soft tissue. Therefore, our study used a dose of SPIO similar to or slightly higher than that used for MRI of the liver (low- to intermediate-dose group). For evaluation of the intracellular uptake of SPIO particles into macrophages or fibroblasts as much as possible in the infected soft tissue, we also used a higher dose of SPIO than that used for MRI of the liver (high-dose group). There was no statistically significant difference in the intracellular uptake of SPIO particles into the macrophages or fibroblasts between both groups.
This study had several limitations. First, the number of rats we used was limited. Second, we did not perform the fibroblast-specific stain. However, on sections stained with Prussian blue, we could see iron particles loaded into cells showing a shape like fibroblasts. These cells were shown as fibroblasts on the sections of the same area stained with H and E. Therefore, the identification of fibroblasts was not difficult in our study. Third, cases of infectious arthritis showed minimal inflammatory change of the synovium. Therefore, SPIO-enhanced MRI findings of infectious arthritis with severe synovial hypertrophy could not be revealed. However, referring to previous literature reports [26], we assumed that the intracellular uptake of SPIO is less in the synovium with minimal inflammation and increases with severe synovial hypertrophy. Studies of MRI with SPIO for acute and chronic infectious arthritis must continue in the future.
In conclusion, the intracellular uptake of SPIO and the change of SNR values were correlated in the infected synovium and in the soft-tissue abscess wall. Therefore, SPIO-enhanced MRI can be useful in the evaluation of musculoskeletal infection and is influenced by the uptake of iron particles in fibroblasts and macrophages.
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