Radiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published online before print May 5, 2008, 10.1148/radiol.2481071260
This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2481071260v1
248/1/114    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bierry, G.
Right arrow Articles by Kremer, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bierry, G.
Right arrow Articles by Kremer, S.
Related Collections
Right arrowRelated Article
(Radiology 2008;248:114-123.)
© RSNA, 2008

Experimental Studies

Macrophage Activity in Infected Areas of an Experimental Vertebral Osteomyelitis Model: USPIO-enhanced MR Imaging—Feasibility Study1

Guillaume Bierry, MD, François Jehl, MD, PhD, Nelly Boehm, MD, PhD, Philippe Robert, PhD, Gilles Prévost, MD, PhD, Jean-Louis Dietemann, MD, Hubert Desal, MD, and Stéphane Kremer, MD, PhD

1 From the Departments of Radiology 2 (G.B., J.L.D., S.K.) and Bacteriology (F.J., G.P.), University Hospital of Strasbourg, Strasbourg, France; EA 3432 (G.B., F.J., G.P., J.L.D., S.K.), Institute of Histology, Faculty of Medicine (N.B.), and INSERM U666 (N.B.), University Louis Pasteur, Strasbourg, France; Guerbet Research, Aulnay-sous-Bois, France (P.R.); and Department of Neuroradiology, University Hospital of Nantes, Nantes, France (H.D.). Received July 19, 2007; revision requested September 20; revision received December 2; accepted January 28, 2008; final version accepted January 31. Address correspondence to G.B., Department of Radiology 2, University Hospital, Hautepierre Hospital, Avenue Molière, 67098 Strasbourg, France (e-mail: guillaume.bierry{at}chru-strasbourg.fr).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCE IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 
Purpose: To prospectively evaluate ultrasmall superparamagnetic iron oxide (USPIO) magnetic resonance (MR) imaging for the depiction of macrophages in infected areas of an experimental rabbit vertebral osteomyelitis model.

Materials and Methods: Lumbar vertebral osteomyelitis was induced in 10 rabbits with intradiscal injection of bacteria in a vertebral disk (test level) versus saline injection in another disk (control level). After a mean interval of 12 days, rabbits were imaged prior to and 24 hours after administration of USPIO. The MR imaging protocol included T1-weighted spin-echo, T2-weighted fast spin-echo, and T2*-weighted gradient-echo sequences. MR findings were compared with histologic findings (macrophage immunostaining and Perls Prussian blue staining). A Wilcoxon signed rank test was used to compare signal-to-noise ratio (SNR) results before and after USPIO administration.

Results: T1-weighted MR images of infected vertebral test levels obtained 24 hours after USPIO administration showed a significant increase in SNR (P = .005), whereas T2- and T2*-weighted images showed no significant changes in SNR (P = .14 and P = .87, respectively). Histologic examination results of infected areas demonstrated complete replacement of hematopoietic bone marrow by macrophage infiltration. Perls Prussian blue staining showed that some macrophages were iron loaded. T1- (P = .02), T2- (P = .04), and T2*-weighted (P = .04) images of control vertebrae showed a significant decrease in SNR. Histologic examination results confirmed the persistence of normal hematopoietic bone marrow without macrophage infiltration, which was reflected by more intensive Perls Prussian blue staining compared with that in infected areas.

Conclusion: MR imaging can depict USPIO-loaded macrophage infiltration present in infected areas in an experimental rabbit model of vertebral osteomyelitis.

© RSNA, 2008


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCE IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 
Magnetic resonance (MR) imaging is the modality of choice for the detection of vertebral osteomyelitis because of its ability to demonstrate pathologic bone marrow changes (1,2). Bone edema and contrast material enhancement, which reflect hyperemia and increased endothelial permeability, are generally observed. The presence of these imaging patterns associated with clinical and/or laboratory signs of infection suggests a diagnosis of vertebral osteomyelitis. These MR imaging criteria have a high sensitivity but poor specificity, because they are observed in infection, as well as degenerative diseases, osteonecrosis, and tumors, and are unreliable in posttraumatic or postoperative settings (3,4). Gadolinium-enhanced MR images may show persistent and even increased enhancement of involved areas after resolution of infection because of the presence of granulation tissue (1,5).

Because activated macrophages are reliable indicators of infected tissues, their presence may therefore allow more accurate evaluation of infected bone marrow (6,7). Activated macrophages can be labeled with ultrasmall superparamagnetic iron oxide (USPIO) contrast agents to visualize the localization of macrophages at MR imaging. USPIO contrast agents can be considered to be an in vivo marker of macrophage activity (8–11). They are progressively taken up by the mononuclear phagocytic system, particularly in the liver, spleen, and hematopoietic bone marrow cells (12). Because of their prolonged intravascular half-life, USPIO particles can be taken up by macrophages outside of the reticuloendothelial system, such as macrophages in infected tissues (13). Because of their ability to shorten T1, T2, and T2*, USPIO particles induce signal changes in tissues containing macrophages. At high concentrations, USPIO particles induce signal loss and susceptibility artifacts on T2- and T2*-weighted images, respectively, while a T1 effect prevails at low concentrations (14).

Experimental studies with USPIO-enhanced MR imaging for the detection of macrophages and the characterization of cellular infiltration have been successfully performed in models of various diseases, such as inflammation, tumors, and ischemia (6,10,13–24). USPIO-loaded macrophages present in infected areas of vertebral osteomyelitis should therefore be detected at MR imaging. Thus, the purpose of our study was to prospectively evaluate USPIO MR imaging for the depiction of macrophages in infected areas of an experimental rabbit vertebral osteomyelitis model.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCE IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 
The USPIO agent (Sinerem) was provided by Guerbet Laboratories (Roissy, France). One author (P.R.) is currently an employee of Guerbet. The other authors had control of data inclusion and manuscript preparation that might present a conflict of interest for the industry-employed author.

Study Design
Ten adult male rabbits (New Zealand White; Grimaud Freres Selection, Roussay, France) (weight, 2.9 kg ± 0.3 [standard deviation]) were included in this study, which was approved by the University Hospital of Strasbourg’s institutional review committee on animal care. Vertebral osteomyelitis was induced with intradiscal bacterial injection. Two MR imaging examinations were performed, one before and one after the administration of USPIO contrast agent. During bacterial inoculation and imaging examinations, animals were anesthetized by using intravenous infusion of 1% propofol (Fresenius Kabi, Sèvres, France) (20 mL of a 10-µmol/mL solution) mixed with midazolam (Merck, Lyon, France) (10 mL of a 1-µmol/mL solution) through a peripheral ear vein catheter at a rate of 10–15 mL/hr, depending on the animal's reactivity.

Osteomyelitis Model
The animal's lumbar region was shaved 24 hours before the procedure, and the skin was cleansed with povidone solution. With fluoroscopic guidance and a posterolateral approach, 22-gauge spinal needles (22 gauge x 3 inches, BD, Franklin Lakes, NJ) were placed percutaneously by one author (G.B.) in the L3-4 and L5-6 intervertebral disks. L5-6 disks were used as test levels and were injected with 0.2 mL of a 5–15.103 colony-forming units per milliliter bacterial suspension (Staphylococcus aureus, Newman strain, NTCC 8178; University Hospital of Strasbourg). Each animal was inoculated with 103–3.103 colony-forming units. Bacterial concentrations were controlled by placing dilutions on agar plates. L3-4 disks were injected with 0.2 mL of saline solution and were used as control levels. After injection procedures, animals were kept in cages and were given free access to food and water. Rabbits were weighed each day by one author (G.B.). The first MR imaging sequences were performed as soon as an animal showed substantial weight loss (about 10% of baseline value).

MR Imaging
MR imaging examinations of rabbit spines were performed with a 1.5-T unit (Avanto; Siemens Medical System, Erlangen, Germany). Two phased-array carotid coils were placed on the skin along the lumbar spine and were coupled with a 12-channel head coil. The body coil was used for radiofrequency transmission, and the combination of carotid coils and head coil was used for reception. MR imaging was performed in the sagittal plane of the vertebrae.

The first MR imaging examination was performed to confirm the presence of vertebral infection and to provide a baseline study before USPIO administration. The MR imaging protocol included four sequences as follows: T1-weighted spin echo (repetition time msec/echo time msec, 588/12; number of signals acquired, four; section thickness, 1 mm; field of view, 200 mm; matrix, 256 x 256), frequency selective fat-suppression T1-weighted spin echo (588/12; number of signals acquired, four; section thickness, 1 mm; field of view, 200 mm; matrix, 256 x 256), T2-weighted spin echo (2000/81; number of signals acquired, three; section thickness, 1.3 mm; field of view, 200 mm; matrix, 256 x 256), and T2*-weighted gradient echo (60/24; flip angle, 25°; number of signals acquired, one; section thickness, 1 mm; field of view, 200 mm; matrix, 256 x 256). The field of view was chosen to allow an examination of the entire spine, including the control and test levels, and to improve the signal-to-noise ratio (SNR). In-plane resolution was 0.8 x 0.8 mm. T1-weighted MR images were obtained to demonstrate signs of vertebral infection, while fat-suppression T1-weighted MR images were used as baseline images prior to USPIO administration.

On completion of the first MR imaging examination, an USPIO contrast agent (Sinerem), with particles of a mean diameter of 35 nm, was administered intravenously (G.B.) through an ear vein at a dose of 45 µmol of iron per kilogram of body weight in a single injection. This dose, usually recommended for human applications, has been shown to be efficient in inducing T1 signal positive enhancement in a rat model of soft-tissue infection (14,25,26).

The second MR imaging examination was performed 24 hours after USPIO administration. This interval allowed clearance of iron particles from the blood circulation and complete phagocytosis by macrophages outside the reticuloendothelial system (27,28). The MR imaging protocol included three sequences as follows: fat-suppression T1-, T2-, and T2*-weighted. The T1-weighted MR imaging sequence was performed only at the first examination to demonstrate vertebral low signal intensity, which indicates the presence of osteomyelitis. It was not used as a baseline for USPIO evaluation.

The mean time between bacterial inoculation and the first MR imaging session was 12 days (range, 10–15 days).

MR Data Analysis
Qualitative and quantitative analyses of signal intensity were performed. MR images were evaluated by using Digital Imaging and Communications in Medicine viewer software (OsiriX; The OsiriX Foundation, Geneva, Switzerland) (29).

Qualitative analysis was performed by two radiologists working in consensus (G.B. and S.K., with 4 and 10 years of experience, respectively, in spinal imaging). Vertebral signal intensity changes before USPIO administration in control and test levels were evaluated on T1-, T2-, and T2*-weighted MR images in each rabbit according to the following three-point scale: a score of 0 indicated no signal intensity changes; a score of 1, slight signal intensity changes; and a score of 2, distinct signal intensity changes (30). Vertebral signal intensity changes in control and test levels between pre- and postcontrast fat-suppression T1-, T2-, and T2*-weighted MR images were evaluated according to the following three-point scales: On fat-suppression T1-weighted MR images, T1 effects were scored as 0 for no signal enhancement, 1 for presence of slight enhancement, and 2 for presence of distinct enhancement. On T2-weighted MR images, T2 effects were scored as 0 for no signal loss, 1 for slight signal loss, and 2 for distinct signal loss. On T2*-weighted MR images, susceptibility effects were scored as 0 for no effects, 1 for slight effects, and 2 for distinct effects.

Quantitative analysis was performed by the same two radiologists working in consensus. Three regions of interest with an area of 2–6 mm2 were placed on vertebral areas previously found to be abnormal on unenhanced T1-, T2-, and T2*-weighted images, at both test and control levels. Signal intensities were measured at the corresponding sites on unenhanced fat-suppression T1-, USPIO-enhanced fat-suppression T1-, unenhanced T2-, USPIO-enhanced T2-, unenhanced T2*-, and USPIO-enhanced T2*-weighted images. The mean value of the three regions of interest was then calculated. Three regions of interest were also placed within the air adjacent to the vertebrae to determine background noise. SNRs of normal (control) and pathologic (test) vertebrae were calculated by dividing the mean signal intensity of these sites by the standard deviation of the background noise. The relative SNR changes were then calculated as follows: SNR change = [(SNRpost – SNRpre)/SNRpre] · 100, where SNRpre and SNRpost correspond to the SNR of pre- and postcontrast images, respectively (30).

Histologic Examination
Animals were sacrificed with intravenous injection of 10 µmol/kg pentobarbital (Abbott, Rungis, France) within 2 hours after completion of the second MR imaging examination. Spines were harvested, and bacteriologic samples of disks and vertebrae were obtained from test and control levels. The L3-4 and L5-6 blocks were fixed in buffered formaldehyde solution and were decalcified and embedded in paraffin, and 5-µm slices were cut in the sagittal axis.

Hematoxylin-eosin staining was performed to assess modifications of the infected vertebrae. Immunochemical analyses were performed (research technician) with a primary monoclonal mouse antirabbit antimacrophage antibody (RAM 11, M0633; Dako, Trappes, France) associated with a secondary polyclonal goat antimouse antibody (E0433; Dako) to identify the site and quantity of macrophages.

Perls Prussian blue stain was used to detect the presence of iron at light microscopy. Iron-stained histologic samples and MR images were analyzed together by a pathologist (N.B., 25 years of experience in human pathology) to compare MR signal intensity changes with iron distribution.

Statistical Analysis
Statistical analysis was performed with software (Statistica, version 6; Statsoft, Tulsa, Okla). A Wilcoxon signed rank test for small sample size was used to assess the difference in SNR calculations of infected and control vertebrae on fat-suppression T1-, T2-, and T2*-weighted MR images before and after USPIO administration. A P value of .05 or less was considered to indicate a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ADVANCE IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 
Bacteriologic examination results showed that all 10 vertebrae injected with bacterial suspension had S aureus infection. No infection was in any of the control intervertebral disk spaces.

Vertebral MR Signal Intensity Changes
Osteomyelitis was identified in all vertebrae at the infected levels at MR imaging. Infected vertebral areas were clearly characterized and involved the part of vertebral bodies adjacent to the inoculated disk. On T1-weighted MR images, signal intensity changes were scored as 2 in all animals. On T2-weighted MR images, signal intensity changes were scored as 1 in one animal and as 2 in nine animals. On T2*-weighted MR images, signal intensity changes were scored as 1 in one animal and as 2 in nine animals (Table 1). No changes were observed in any of the control vertebrae (Fig 1, Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1. Scores of MR Signal Intensity Changes in Infected and Control Vertebrae before USPIO Administration

 

Figure 1
View larger version (99K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1: Sagittal T1-weighted spin-echo MR image (588/12) shows changes in test (infected) vertebra with an area of decreased signal intensity (arrow), while control vertebra remained unchanged (arrowhead).

 
USPIO Enhancement at MR Imaging
Twenty-four hours after USPIO administration, fat-suppression T1-weighted images showed distinct signal enhancement (score of 2) in 100% of the infected vertebrae. Signal enhancement was identified in vertebral areas with an abnormal appearance prior to USPIO administration (Table 2, Fig 2). USPIO-enhanced T2- and T2*-weighted images did not show signal loss or susceptibility effects, respectively, in any of the infected vertebral areas (Table 2, Fig 3).


View this table:
[in this window]
[in a new window]

  		Table 2. Scores of MR Signal Intensity Changes in Infected and Control Vertebrae 24 Hours after USPIO Administration

 

Figure 2A
View larger version (101K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2a: Sagittal fat-suppression T1-weighted spin-echo MR images (588/12) demonstrate T1 effects at MR imaging of macrophage activity in bacterial osteomyelitis. (a) Precontrast MR image shows no substantial signal intensity changes in test (infected) or control vertebrae. (b) Postcontrast MR image (24 hours after USPIO administration) shows increased signal intensity of infected vertebral area (arrow) and decreased signal intensity of control vertebra (arrowhead).

 

Figure 2B
View larger version (116K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2b: Sagittal fat-suppression T1-weighted spin-echo MR images (588/12) demonstrate T1 effects at MR imaging of macrophage activity in bacterial osteomyelitis. (a) Precontrast MR image shows no substantial signal intensity changes in test (infected) or control vertebrae. (b) Postcontrast MR image (24 hours after USPIO administration) shows increased signal intensity of infected vertebral area (arrow) and decreased signal intensity of control vertebra (arrowhead).

 

Figure 3A
View larger version (112K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3a: Sagittal T2*-weighted gradient-echo MR images (60/24; flip angle, 25°) show a difference in signal intensity changes between infected and control vertebrae before and after USPIO administration. (a) Precontrast MR image shows only a slight signal enhancement of infected vertebra (arrow), while control vertebra has a normal signal intensity (arrowhead). (b) Postcontrast MR image (24 hours after USPIO administration) shows susceptibility effects with decreased signal intensity of control vertebra (arrowhead) and no changes in infected area (arrow).

 

Figure 3B
View larger version (109K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3b: Sagittal T2*-weighted gradient-echo MR images (60/24; flip angle, 25°) show a difference in signal intensity changes between infected and control vertebrae before and after USPIO administration. (a) Precontrast MR image shows only a slight signal enhancement of infected vertebra (arrow), while control vertebra has a normal signal intensity (arrowhead). (b) Postcontrast MR image (24 hours after USPIO administration) shows susceptibility effects with decreased signal intensity of control vertebra (arrowhead) and no changes in infected area (arrow).

 
Quantitative analysis confirmed the visual evaluation, because a statistically significant (P = .005) relative mean SNR increase of 113% was observed between unenhanced and USPIO-enhanced fat-suppression T1-weighted images in the infected areas (Fig 4, Table 3). Quantitative analysis demonstrated the absence of any statistically significant changes in SNR on either T2- (–13%, P = .14) or T2*-weighted MR images (–10%, P = .87) (Fig 4, Table 3).


Figure 4
View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4: Graph of SNR of test (infected) vertebrae on fat-suppression T1-, T2-, and T2*-weighted MR images before and 24 hours after USPIO administration. A significant increase of SNR is present on fat-suppression T1-weighted images, while no significant changes are observed on T2- and T2*-weighted images. P values were determined with Wilcoxon signed rank test. Error bars = standard deviation of the mean. n.s. = no significant difference (P > .05).

 

View this table:
[in this window]
[in a new window]

  		Table 3. SNRs of Infected and Control Vertebrae before and 24 Hours after USPIO Administration

 
Distinct signal loss (score of 2) was observed in control vertebrae on fat-suppression T1-, T2-, and T2*-weighted images (Figs 2, 3). Quantitative analysis showed a statistically significant decrease in the relative SNR of vertebrae on T1- (–22%, P = .02), T2- (–35%; P = .04), and T2*-weighted images (–43%; P = .04) (Fig 5, Table 3).


Figure 5
View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5: Graph of SNR of control vertebrae on fat-suppression T1-, T2-, and T2*-weighted MR images before and 24 hours after USPIO administration. A significant decrease of SNR is present on fat-suppression T1-, T2-, and T2*-weighted images. P values were determined by using Wilcoxon signed rank test. Error bars = standard deviation of the mean.

 
Histologic Findings
Histologic examination results confirmed MR findings by showing replacement of normal hematopoietic bone marrow of vertebral bodies by an inflammatory tissue with marked cellular infiltrate and suppuration in all infected vertebrae at hematoxylin-eosin staining (Fig 6). The infectious changes were more intense in the vertebral area adjacent to the inoculated disk. Immunohistochemical analysis demonstrated intense staining inside infected vertebrae, revealing the massive presence of macrophages (Fig 7). Perls Prussian blue staining showed that few macrophages in these areas were iron loaded, and no extracellular iron deposits were observed (Fig 8). Iron-loaded macrophages that showed a positive finding at Perls Prussian blue staining were found at histologic examination in the vertebral areas that showed increased signal intensity on USPIO-enhanced fat-suppression T1-weighted images.


Figure 6A
View larger version (135K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6a: Histologic examination results in an infected vertebra. (a) Photomicrograph of infected vertebral endplate adjacent to test intervertebral disk shows replacement of normal bone marrow by intense inflammation (arrow). (Original magnification, x10; hematoxylin-eosin stain.) (b) Photomicrograph of infected vertebra. The part of the vertebra situated away from discal site of bacterial inoculation contains normal bone marrow (arrow), while the part adjacent to site of inoculation shows intense infectious changes with a marked cellular infiltrate (arrowhead). (Original magnification, x20; hematoxylin-eosin stain.)

 

Figure 6B
View larger version (145K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6b: Histologic examination results in an infected vertebra. (a) Photomicrograph of infected vertebral endplate adjacent to test intervertebral disk shows replacement of normal bone marrow by intense inflammation (arrow). (Original magnification, x10; hematoxylin-eosin stain.) (b) Photomicrograph of infected vertebra. The part of the vertebra situated away from discal site of bacterial inoculation contains normal bone marrow (arrow), while the part adjacent to site of inoculation shows intense infectious changes with a marked cellular infiltrate (arrowhead). (Original magnification, x20; hematoxylin-eosin stain.)

 

Figure 7
View larger version (110K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 7: Photomicrograph of immunohistochemical analysis shows specific antimacrophage antibodies demonstrating intense staining of the cytoplasm of macrophages, which reflects massive infiltration of infected vertebral areas (arrow). (Original magnification, x100.)

 

Figure 8A
View larger version (120K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 8a: Photomicrographs obtained in infected and control bone marrow 24 hours after USPIO administration. (a) In infected areas, staining is reduced and limited to some cells that correspond morphologically to macrophages with iron uptake after USPIO administration (arrow). (b) In control areas, numerous blue iron-positive cells are present, which reflects intense USPIO uptake by hematopoietic bone marrow (arrow). (Perls Prussian blue stain; original magnification, x200.)

 

Figure 8B
View larger version (112K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 8b: Photomicrographs obtained in infected and control bone marrow 24 hours after USPIO administration. (a) In infected areas, staining is reduced and limited to some cells that correspond morphologically to macrophages with iron uptake after USPIO administration (arrow). (b) In control areas, numerous blue iron-positive cells are present, which reflects intense USPIO uptake by hematopoietic bone marrow (arrow). (Perls Prussian blue stain; original magnification, x200.)

 
No pathologic changes were observed in the control vertebrae. Immunohistochemical staining showed the presence of only a few macrophages compared with that in infected areas. Bone marrow of control vertebrae presented intense Perls Prussian blue staining, revealing intense uptake of iron particles, and no extracellular iron deposits were observed (Fig 8).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ADVANCE IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 
The classic MR imaging pattern of vertebral infection is low signal intensity on T1-weighted images, high signal intensity on T2-weighted images, and enhancement after administration of a gadolinium-based contrast agent. Tissue enhancement induced by using extracellular gadolinium-based contrast agents merely indicates increased capillary permeability of the inflamed tissue with accumulation of contrast agent in the extracellular space. This technique does not reflect tissue- or cell-specific labeling by the contrast agent. This technique is not specific of infection as tissue enhancement can also be seen in inflammation, degenerative disease, or trauma. Gadolinium-based enhancement observed in infected vertebrae is seen during the acute inflammatory phase and persists to varying degrees for several weeks or months. Loss of gadolinium-based enhancement is considered to be a sign of healing. However, it may persist for a long time, and it is unknown whether this indicates persistent sterile inflammation or recrudescence of the infective process (31).

Specific targeting of phagocytes, especially macrophages, by using MR imaging with USPIO contrast agents is now considered to be a reliable method to identify infected tissues, especially soft-tissue infection (6). In acute osteomyelitis, local massive recruitment of phagocytes (neutrophils and macrophages) helps to contain proliferation of microorganisms by generating lytic enzymes (32). This process transforms normal bone into an inflammatory tissue composed of microorganisms, phagocytes, neutrophils, and thrombosed vessels (17). At the opposite end of the spectrum, granulation tissue associated with sterile lesions or noninfectious inflammation has only a small percentage of macrophages (33).

In contrast with extracellular gadolinium-based contrast agents, iron oxide particles are cell-specific contrast agents. After intravenous administration, superparamagnetic iron oxide particles are almost entirely ingested by cells of the reticuloendothelial system of the bone marrow, liver, and spleen (28,34). A small amount is taken up by macrophages and other phagocytic cells outside of the reticuloendothelial system (34). USPIO particles (mean particle diameter, 18–30 nm) are particularly suitable for in vivo macrophage-specific MR imaging. After intravenous injection, USPIO particles are taken up by macrophages and other phagocytic cells involved in the inflammatory process (10,15,35,36). The results of our study indicate that USPIO contrast agents are phagocytosed by macrophages present in infected areas of an experimental model of bacterial vertebral osteomyelitis.

After 24 hours, most of the injected USPIO particles are phagocytosed by the reticuloendothelial system and are mainly taken up by bone marrow phagocytes. The bone marrow uptake of iron oxide particles is sufficient enough to induce loss of MR signal intensity (12). In the presence of pathologic replacement of normal bone marrow, this signal loss is reduced or disappears, which allows better depiction of pathologic areas of bone marrow. This phenomenon has been reported after bone marrow irradiation, bone tumor, and inflammation models in which normal bone marrow showed signal loss while pathologic areas remained unchanged (18,37,38).

In our model, infected areas showed no signal loss on T2- and T2*-weighted MR images, while normal vertebrae showed significant signal loss. The visual impressions were confirmed by quantitative analysis that showed no statistically significant change of signal intensity in infected areas on T2- and T2*-weighted images due to the fact that normal hematopoietic bone marrow had completely disappeared and was replaced by inflammatory tissue. Perls Prussian blue staining demonstrated that the presence of iron in these areas was strictly limited to macrophages, and the quantity of USPIO was not sufficient enough to induce T2 and T2* effects. In contrast, fat-suppression T1-weighted MR images showed intense signal enhancement of infected areas after administration of USPIO. Histologic examination results confirmed the intracellular presence of iron particles in some macrophages in these enhancing areas. In contrast, normal bone marrow in noninfected areas demonstrated significant signal loss on fat-suppression T1-weighted images. This difference in behavior was correlated with the different quantities of iron detected by using Perls Prussian blue stain in infected and control vertebrae. In infected vertebrae, only some macrophages were iron loaded, while in control vertebrae, normal bone marrow was intensely stained, which reflected intense uptake of iron particles. The presence of this type of signal enhancement on T1-weighted MR images has been previously reported in studies with a similar dose of USPIO (14,27). The decreased quantity of iron present in macrophages in infected areas may generate T1 effects but was insufficient to induce T2 or T2* effects. At low concentrations, the T1 effects of this type of contrast agent prevail over T2 and T2* effects because of their relaxation characteristics.

Several limitations of our study have to be addressed. First, the study was performed in a small number of animals, and obtained results cannot be directly extrapolated to humans. Statistical power may be increased by using a higher number of animals. Second, we used a small dose of USPIO (45 µmol of iron per kilogram) to obtain T1 effects (14). At this dose, we observed T1 effects, but no differences on T2 and T2* effects were noted. Third, immunohistochemical analysis demonstrated an intense macrophagic infiltration, but only some of the macrophages in the infection site were iron loaded. This difference between the iron load and the number of macrophages has already been noted in other study results and needs further investigation. Additionally, it can be suggested that T1 effects may have disappeared if a larger number of iron-loaded macrophages were present at the infection site. The last limitation of our study was that we only evaluated one MR examination 24 hours after USPIO administration. Different timing and earlier examinations (ie, after 3 or 6 hours) may have resulted in different signal modifications. Some authors (39) have demonstrated that, by using small superparamagnetic iron oxide particles, modifications of MR signal of bone marrow can be observed as early as 3 hours after intravenous administration. Despite different blood half-life and subsequent different absorption rate by reticuloendothelial system cells of small superparamagnetic iron oxide particles and USPIO particles, earlier MR examinations with USPIO could have provided additional information about the kinetics and the amplitude of enhancement.

In conclusion, our study results demonstrate that USPIO-enhanced MR imaging can be used to label macrophages inside infected vertebrae, as different signal intensity changes were observed on fat-suppression T1-weighted MR images in infected and healthy vertebrae 24 hours after administration of a clinical dose of USPIO. The presence of iron-loaded macrophages induces a specific T1 effect in infected areas. As bone marrow is no longer present, T1 signal changes are only related to the iron contained in macrophages. In contrast, T2- and T2*-weighted MR images are not specific of macrophage infiltration and only reflect replacement of normal bone marrow (37).

Practical applications: The specific signal enhancement observed on USPIO-enhanced fat-suppression T1-weighted images allows identification of infected areas and should facilitate distinction between infection and sterile vertebral inflammation. Moreover, monitoring of T1 signal changes during antibiotic treatment could also be useful to identify the absence of macrophages, reflecting healing.


   ADVANCE IN KNOWLEDGE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCE IN KNOWLEDGE
IMPLICATION FOR PATIENT CARE
 References
 


   IMPLICATION FOR PATIENT CARE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCE IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
References
 


   ACKNOWLEDGMENTS
 
We thank Myriam Bertrand, PhD, from the Strasbourg University Mathematical and Statistical Department for her support in the statistical analysis.


   FOOTNOTES
 

Abbreviations: SNR = signal-to-noise ratio • USPIO = ultrasmall superparamagnetic iron oxide

See also Science to Practice in this issue.

Author contributions: Guarantor of integrity of entire study, G.B.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, G.B., F.J., N.B., P.R., J.L.D., H.D., S.K.; experimental studies, G.B., F.J., N.B., P.R., G.P., J.L.D., S.K.; statistical analysis, G.B., S.K.; and manuscript editing, all authors

See Materials and Methods for pertinent discloses.


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCE IN KNOWLEDGE
 IMPLICATION FOR PATIENT CARE
 References
 

  1. Tali ET. Spinal infections. Eur J Radiol 2004;50:120–133.[CrossRef][Medline]
  2. Jevtic V. Vertebral infection. Eur Radiol 2004;14(suppl 3):E43–E52.[CrossRef][Medline]
  3. Ledermann HP, Kaim A, Bongartz G, Steinbrich W. Pitfalls and limitations of magnetic resonance imaging in chronic posttraumatic osteomyelitis. Eur Radiol 2000;10:1815–1823.[CrossRef][Medline]
  4. Kaim A, Ledermann HP, Bongartz G, Messmer P, Muller-Brand J, Steinbrich W. Chronic post-traumatic osteomyelitis of the lower extremity: comparison of magnetic resonance imaging and combined bone scintigraphy/immunoscintigraphy with radiolabelled monoclonal antigranulocyte antibodies. Skeletal Radiol 2000;29:378–386.[CrossRef][Medline]
  5. Gillams AR, Chaddha B, Carter AP. MR appearances of the temporal evolution and resolution of infectious spondylitis. AJR Am J Roentgenol 1996;166:903–907.[Abstract/Free Full Text]
  6. 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]
  7. Johnston RB Jr. Current concepts: immunology—monocytes and macrophages. N Engl J Med 1988;318:747–752.[Medline]
  8. Bellin MF, Roy C, Kinkel K, et al. Lymph node metastases: safety and effectiveness of MR imaging with ultrasmall superparamagnetic iron oxide particles—initial clinical experience. Radiology 1998;207:799–808.[Abstract/Free Full Text]
  9. Corot C, Petry KG, Trivedi R, et al. Macrophage imaging in central nervous system and in carotid atherosclerotic plaque using ultrasmall superparamagnetic iron oxide in magnetic resonance imaging. Invest Radiol 2004;39:619–625.[CrossRef][Medline]
  10. 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]
  11. Pannu HK, Wang KP, Borman TL, Bluemke DA. MR imaging of mediastinal lymph nodes: evaluation using a superparamagnetic contrast agent. J Magn Reson Imaging 2000;12:899–904.[CrossRef][Medline]
  12. Seneterre E, Weissleder R, Jaramillo D, et al. Bone marrow: ultrasmall superparamagnetic iron oxide for MR imaging. Radiology 1991;179:529–533.[Abstract/Free Full Text]
  13. 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]
  14. Lutz AM, Weishaupt D, Persohn E, et al. Imaging of macrophages in soft-tissue infection in rats: relationship between ultrasmall superparamagnetic iron oxide dose and MR signal characteristics. Radiology 2005;234:765–775.[Abstract/Free Full Text]
  15. 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]
  16. Bush CH, Mladinich CR, Montgomery WJ. Evaluation of an ultrasmall superparamagnetic iron oxide in MRI in a bone tumor model in rabbits. J Magn Reson Imaging 1997;7:579–584.[Medline]
  17. Dardzinski BJ, Schmithorst VJ, Holland SK, et al. MR imaging of murine arthritis using ultrasmall superparamagnetic iron oxide particles. Magn Reson Imaging 2001;19:1209–1216.[CrossRef][Medline]
  18. Simon GH, Raatschen HJ, Wendland MF, et al. Ultrasmall superparamagnetic iron-oxide-enhanced MR imaging of normal bone marrow in rodents: original research. Acad Radiol 2005;12:1190–1197.[CrossRef][Medline]
  19. 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]
  20. Simon GH, von Vopelius-Feldt J, Fu Y, et al. Ultrasmall supraparamagnetic iron oxide-enhanced magnetic resonance imaging of antigen-induced arthritis: a comparative study between SHU 555 C, ferumoxtran-10, and ferumoxytol. Invest Radiol 2006;41:45–51.[CrossRef][Medline]
  21. Moore A, Weissleder R, Bogdanov A Jr. Uptake of dextran-coated monocrystalline iron oxides in tumor cells and macrophages. J Magn Reson Imaging 1997;7:1140–1145.[Medline]
  22. Zimmer C, Weissleder R, Poss K, Bogdanova A, Wright SC Jr, Enochs WS. MR imaging of phagocytosis in experimental gliomas. Radiology 1995;197:533–538.[Abstract/Free Full Text]
  23. Enochs WS, Harsh G, Hochberg F, Weissleder R. Improved delineation of human brain tumors on MR images using a long-circulating, superparamagnetic iron oxide agent. J Magn Reson Imaging 1999;9:228–232.[CrossRef][Medline]
  24. Rausch M, Sauter A, Frohlich J, Neubacher U, Radu EW, Rudin M. Dynamic patterns of USPIO enhancement can be observed in macrophages after ischemic brain damage. Magn Reson Med 2001;46:1018–1022.[CrossRef][Medline]
  25. 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]
  26. 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]
  27. 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]
  28. 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]
  29. Rosset A, Spadola L, Ratib O. OsiriX: an open-source software for navigating in multidimensional DICOM images. J Digit Imaging 2004;17:205–216.[CrossRef][Medline]
  30. Lutz AM, Seemayer C, Corot C, et al. Detection of synovial macrophages in an experimental rabbit model of antigen-induced arthritis: ultrasmall superparamagnetic iron oxide-enhanced MR imaging. Radiology 2004;233:149–157.[Abstract/Free Full Text]
  31. Ledermann HP, Schweitzer ME, Morrison WB, Carrino JA. MR imaging findings in spinal infections: rules or myths? Radiology 2003;228:506–514.[Abstract/Free Full Text]
  32. Gillespie WJ, Allardyce RA. Mechanisms of bone degradation in infection: a review of current hypotheses. Orthopedics 1990;13:407–410.[Medline]
  33. Modic MT, Steinberg PM, Ross JS, Masaryk TJ, Carter JR. Degenerative disk disease: assessment of changes in vertebral body marrow with MR imaging. Radiology 1988;166:193–199.[Abstract/Free Full Text]
  34. Weissleder R, Stark DD, Engelstad BL, et al. Superparamagnetic iron oxide: pharmacokinetics and toxicity. AJR Am J Roentgenol 1989;152:167–173.[Abstract/Free Full Text]
  35. 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]
  36. 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]
  37. Daldrup HE, Link TM, Blasius S, et al. Monitoring radiation-induced changes in bone marrow histopathology with ultra-small superparamagnetic iron oxide (USPIO)-enhanced MRI. J Magn Reson Imaging 1999;9:643–652.[CrossRef][Medline]
  38. Daldrup-Link HE, Rudelius M, Oostendorp RA, et al. Targeting of hematopoietic progenitor cells with MR contrast agents. Radiology 2003;228:760–767.[Abstract/Free Full Text]
  39. Fukuda Y, Ando K, Ishikura R, et al. Superparamagnetic iron oxide (SPIO) MRI contrast agent for bone marrow imaging: differentiating bone metastasis and osteomyelitis. Magn Reson Med Sci 2006;5:191–196.[CrossRef][Medline]

Related Article

Can USPIO-enhanced Spinal MR Imaging Help Distinguish Acute Infectious Osteomyelitis from Chronic Infectious and Inflammatory Processes?
Jonathan B. Kruskal
Radiology 2008 248: 1-3. [Full Text] [PDF]




This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2481071260v1
248/1/114    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bierry, G.
Right arrow Articles by Kremer, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bierry, G.
Right arrow Articles by Kremer, S.
Related Collections
Right arrowRelated Article

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
RADIOLOGY RADIOGRAPHICS RSNA JOURNALS ONLINE