Full Paper Received: 2 July 2013,

Revised: 10 January 2014,

Accepted: 19 February 2014,

Published online in Wiley Online Library: 22 April 2014

(wileyonlinelibrary.com) DOI: 10.1002/cmmi.1602

In vivo MR imaging of intercellular adhesion molecule-1 expression in an animal model of multiple sclerosis Erwin L. A. Blezera*, Lisette H. Deddensa, Gijs Kooijb, Joost Drexhageb, Susanne M. A. van der Polb, Arie Reijerkerkb, Rick M. Dijkhuizena and Helga E. de Vriesb Upregulation of intercellular adhesion molecule 1 (ICAM-1) is an early event in lesion formation in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Monitoring its expression may provide a biomarker for early disease activity and allow validation of anti-inflammatory interventions. Our objective was therefore to explore whether ICAM-1 expression can be visualized in vivo during EAE with magnetic resonance imaging (MRI) using micron-sized particles of iron oxide (MPIO), and to compare accumulation profiles of targeted and untargeted MPIO, and a gadolinium-containing agent. Targeted αICAM-1-MPIO/untargeted IgG-MPIO were injected at two model-characteristic phases of EAE (in myelin oligodendrocyte glycoprotein35–55-immunized C57BL/6 J mice), that is, at the peak of the acute phase (14 ± 1 days post-immunization) and during the chronic phase (26 ± 1 days post-immunization), followed by T2*-weighted MRI. Blood–brain barrier (BBB) permeability was measured using gadobutrol-enhanced MRI. Cerebellar microvessels were analyzed for ICAM-1 mRNA expression using quantitative PCR (qPCR). ICAM-1 and iron oxide presence was examined with immunohistochemistry (IHC). During EAE, ICAM-1 was expressed by brain endothelial cells, macrophages and T-cells as shown with qPCR and (fluorescent) IHC. EAE animals injected with αICAM-1-MPIO showed MRI hypointensities, particularly in the subarachnoid space. αICAM-1-MPIO presence did not differ between the phases of EAE and was not associated with BBB dysfunction. αICAM-1-MPIO were associated with endothelial cells or cells located at the luminal side of blood vessels. In conclusion, ICAM-1 expression can be visualized with in vivo molecular MRI during EAE, and provides an early tracer of disease activity. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: molecular MRI; experimental autoimmune encephalomyelitis; intercellular adhesion molecule; MS model; micron-sized particles of iron oxide; multiple sclerosis; MPIO

1. INTRODUCTION

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* Correspondence to: E. L. A. Blezer, Biomedical MR Imaging and Spectroscopy Group, Image Sciences Institute, University Medical Center Utrecht, Yalelaan 2, 3584 CM Utrecht, The Netherlands. E-mail: [email protected] a E. L. A. Blezer, L. H. Deddens, R. M. Dijkhuizen Biomedical MR Imaging and Spectroscopy Group, Image Sciences Institute, University Medical Center Utrecht, Yalelaan 2, 3584 CM Utrecht, The Netherlands b G. Kooij, J. Drexhage, S. M. A. van der Pol, A. Reijerkerk, H. E. Vries Department of Molecular Cell Biology and Immunology, Neuroscience Campus Amsterdam, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands

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Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by leukocyte infiltrates, leading to demyelination and axonal damage (1,2). In this process, immune activation of endothelial cells in the brain vasculature, evidenced by the enhanced expression of cell adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1), is a key event (3). Noninvasive imaging strategies that enable the in vivo detection of these markers, known to induce leukocyte diapedesis, would greatly enhance our understanding on the role of vascular inflammation in MS. More importantly, such strategies may provide excellent opportunities to monitor the occurrence of new relapses and will have a high potential to validate the efficacy of newly developed treatment regimes that are aimed to dampen neuroinflammation (4). Magnetic resonance imaging (MRI) has become the established diagnostic imaging tool for MS patients. MRI protocols entail the acquisition of non-contrast T1- and T2-weighted images and gadolinium-enhanced T1-weighted images to

visualize ongoing inflammation (5,6). These MRI techniques mainly visualize the consequences of leukocyte influx and the resulting inflammatory process, that is, edema formation, loss of tight junction integrity or myelin destruction. However, to date no techniques are available that can measure earlier stages of lesion formation, such as immune activation of endothelial cells, which may indicate or predict leukocyte influx. Recently, cellular and molecular MRI emerged as powerful tools to visualize pathogenic processes at the cellular and

E. L. A. BLEZER ET AL. molecular level (7). Novel MR probes have been developed which can be used to study events involved in the brain entry of leukocytes. For example, detection of macrophage influx into the CNS with superparamagnetic particles of iron oxide (SPIO) has been successfully accomplished in experimental autoimmune encephalomyelitis (EAE) (8,9), an animal model of MS, as well as in patients with MS (10). Micron-sized particles of iron oxide (MPIO) have been put forward as MR probes for the detection of inflammatory endothelial markers in the brain (11,12) . Using this approach, VCAM-1 expression has recently been visualized in vivo in EAE (13,14). The use of MPIO has several advantages. The iron oxide core of these relatively large particles (micrometer range) induces strong MR contrast-enhancing effects that allow detection at relatively low doses. Furthermore, blood clearance is in the order of minutes (15) and any undesirable, nonspecific blood pool contrast effect of MPIO is negligible when scans are made after 30–60 min. Finally, their large size limits passive leakage over a damaged blood–brain barrier (BBB), a phenomenon which may undermine the specificity of the detection method, as has been shown for the smaller SPIO (tens of nanometers) used for macrophage detection during EAE (9). For molecular MRI of EAE-induced neurovascular inflammation and potential leukocyte infiltration, ICAM-1 is a relevant target. Like VCAM-1, ICAM-1 is significantly expressed during early lesion formation both in EAE (16,17) and in MS (18). Both endothelial adhesion molecules are important for the transmigration of leukocytes over the brain endothelium. However, there are important differences. ICAM-1, unlike VCAM-1, can mediate the crawling of T-cells against the direction of blood flow to sites of diapedesis (19). Furthermore, ICAM-1 has been associated with T-cell diapedesis, whereas VCAM-1 is thought to predominantly mediate monocyte migration across the brain endothelium (20). The significance of ICAM-1 in the development of EAE has been revealed in ICAM-1-deficient mice (21). Moreover, in studies targeted to block ICAM-1 functionality, the development of the clinical symptoms of EAE was ameliorated (22). This central role of ICAM-1 in EAE development warrants its potential as an important imaging target. The aim of this study was therefore to explore if ICAM-1 expression can be visualized in vivo with MRI in a mouse model of MS using antibody-conjugated MPIO directed to ICAM-1. Our study shows that ICAM-1 expression can be detected during the progression of EAE, and that antibody-conjugated MPIO mainly target endothelial cells.

2. RESULTS AND DISCUSSION 2.1. Clinical Scores, Animal Weight and ICAM-1 Expression During EAE Progression

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EAE was induced in 10-week-old female C57BL/6 J mice using myelin oligodendrocyte glycoprotein (MOG 35–55) as described previously (23). The time courses of the development of neurological deficits and body weight are shown in Fig. 1(A). First neurological deficits (partially reduced tail tone; score 0.5) were detected 7 days after immunization. Animals with EAE were injected with MPIO at two characteristic phases of disease progression in this model (gray bars in Fig. 1A), that is, at the peak of the acute phase (14 ± 1 days post-immunization) and during the chronic phase of EAE (26 ± 1 days post-immunization). Quantitative PCR analyses showed that levels of ICAM-1 mRNA transcripts in isolated brain capillaries were significantly increased

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in the acute phase of EAE (Fig. 1B) compared with onset of disease and compared with complete Freund’s adjuvant (CFA) immunized control animals. In the current study, ICAM-1 mRNA levels were not specifically determined for the chronic phase of EAE. However, it has been shown that ICAM-1 remains highly expressed in the chronic phase of the disease during EAE (24). Immunohistochemical analysis (IHC) revealed that ICAM-1 expression was upregulated throughout the brain, particularly in subarachnoid and periventricular spaces, both in the acute and chronic phases of EAE (Figs. 1C and 4A and B). Together, these results indicate that in EAE, ICAM-1 expression is elevated in the vasculature already in early stages of disease, in correspondence with earlier studies in MS (18) and EAE (16,17,24). 2.2. In Vivo MRI Assessment Shows αICAM-1-MPIO Presence in Brains of Mice With EAE Animals with EAE were injected with αICAM-1-MPIO of which the physical nuclear magnetic resonance properties were characterized recently (12). MR relaxivities at 9.4 T were 0.3 ± 0.0 mM 1 s 1 for r1 and 91 ± 3 mM 1 s 1 for r2. Experiments were performed at room temperature and samples were dissolved in storage solution (150 mM NaCl with 0.002% azide). In vitro experiments on cytokine-stimulated brain endothelial cells showed that there was no significant change in T1 values after αICAM-1-MPIO uptake, whereas T2 values dramatically decreased. The current study revealed that animals injected with αICAM-1-MPIO showed abnormal hypointensities on 3D T*2-weighted gradient echo images 40 min after injection of MPIO, irrespective of the phase of EAE. These focal hypointensities were observed throughout the brain, and specifically in the subarachnoid space, and were not necessarily associated with gadobutrol leakage. These abnormalities were not observed after IgG-MPIO injection, ruling out the possibility that conjugated MPIO passively leak into the brain parenchyma. Typical MR images are shown in Fig. 2. The current study is the first to demonstrate that ICAM-1 presence can be visualized in vivo during EAE using MRI. Previously, antibody-conjugated gadolinium-containing liposomes (25) and SPIO (26) have been used to visualize ICAM-1 expression in EAE on post-mortem tissue. So far, imaging studies aimed to visualize ICAM-1 presence in vivo in EAE have been performed with ultrasound techniques, using ICAM-1 antibody-conjugated air microbubbles (27,28). These microbubbles have altered acoustic properties that make them visible using specific ultrasound techniques (29). Image resolutions of the used ultrasound technique were comparable with the present study. However, precise spatial localization of ICAM-1 in the brain could not be determined as ultrasound does not provide anatomical information. Furthermore, it is unlikely that ultrasound can be used to measure cerebral ICAM-1 presence in humans as the penetration depth of this imaging technique is limited. These disadvantages do not apply for MRI since MRI can simultaneously provide anatomical detail of tissue and is in principle not limited by any penetration depth. We here show that hypointensities were predominantly detected in cerebellum, the subarachnoid space between the temporal cortex and the midbrain, caudate putamen and cortical areas in the cerebrum. Specifically, the latter two areas showed bands of MR hypointensities suggestive of vascular ICAM-1 presence. In particular, the subarachnoid space between cortex and midbrain, an area richly fenestrated by blood vessels, showed massive αICAM-1-MPIO presence. This has also been reported

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Figure 1. Neurological score, animal weight and ICAM-1 (mRNA) expression during progression of experimental autoimmune encephalomyelitis (EAE). (A) Neurological score (○) and body weight (●) of animals with EAE. Animals were immunized with myelin oligodendrocyte glycoprotein (MOG 35–55) peptide at day 0. The time periods of injection of micron-sized particles of iron oxide (MPIO) conjugates for MRI detection at the different phases of EAE is indicated with gray bars. Data are presented as means ± SEM. (B) mRNA expression levels of intercellular adhesion molecule 1 (ICAM-1) in isolated brain endothelial cells from CFA-control and EAE mice. Expression levels of ICAM-1 are relative to CD31, which is used as a marker for brain endothelial # cells, at two different time points (day 9: before onset EAE; day 15: acute phase of EAE). * P < 0.05 vs complete Freund’s adjuvant (CFA); p < 0.05 vs acute phase of disease. (C) Immunohistochemistry showing upregulation of ICAM-1 at different phases of EAE. Sections through the cerebellum, midbrain and cerebrum are shown. ICAM-1 (brown) is particularly present in the subarachnoid and periventricular spaces, both in the acute (upper row) as in the chronic (lower row) phase of EAE.

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choroid plexus expressed ICAM-1 in both the acute and chronic phases of the disease. Only αICAM-1-MPIO-injected animals showed MPIO presence in the choroid plexus (data not shown). The bead-like hypointensities may be suggestive for αICAM-1MPIO binding to the choroid plexus. An increase in ICAM-1 expression has been reported for choroid epithelial cells during EAE (31), and it has recently been suggested that this area may serve as an alternative entry site for circulating lymphocytes to enter the CSF (32,33). 2.3. In Vivo MRI Assessment of BBB Damage Shows That αICAM-1-MPIO Presence is not Necessarily Associated With BBB Damage We explored if ICAM-1 upregulation was associated with leakage of the BBB. The integrity of the BBB was examined with the vascular contrast agent gadobutrol (34). Gadobutrol is an inert MR contrast agent and its presence in brain parenchyma is typically the result of a disrupted BBB (35). Subarachnoid and ventricular spaces showed clear gadobutrol leakage on the T1-weighted difference images. However in the brain parenchyma gadobutrol leakage was less obvious than αICAM-1-MPIO accumulation.

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using the above-mentioned ultrasound techniques (27,28), but not for the antibody-conjugated paramagnetic gadoliniumcontaining liposomes (25). VCAM-1 expression is also upregulated in this particular area, as has been shown with αVCAM-1-MPIO (14). To date, the precise involvement of this specific area in the pathogenesis of EAE remains unclear. The visualization of bands of MR hypointensities deep inside the cortical areas, corresponding to vascular ICAM-1 identified by IHC, may be of special interest as cortical pathology is nowadays increasingly recognized as an important feature in the pathogenesis of MS. The in vivo visualization of cortical pathology with conventional MRI is however limited, although advances have been made using inversion recovery-based MR techniques that suppress MR signal from cerebrospinal fluid (CSF) and white matter (30). MPIO-based MRI of ICAM-1 upregulation in the cortex may therefore offer an attractive alternative tool to study molecular mechanisms of cortical pathology in patients with MS. Seven out of eight αICAM-1 MPIO injected animals showed bead-like hypointensities within the CSF (inset Fig. 2). Only one of the IgG-MPIO-injected animals showed the presence of MPIO in the CSF, which makes it unlikely that there is passive leakage of conjugated MPIO into the CSF. IHC analyses showed that the

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Figure 2. Typical examples of MR images of animals injected with (A) αICAM-1-MPIO (neurological score 3) and (B) IgG-MPIO (neurological score 3.5) at the acute phase of disease. (A) The animal that received αICAM-1- MPIO showed focal hypointensities (black/gray arrow heads) throughout the brain, which was confirmed on the MPIO difference images. MPIO hypointensities were not necessarily associated with gadobutrol leakage (typical examples are denoted by black arrowheads), as shown on the gadobutrol difference image. Ventricular areas were occasionally positive for gadobutrol leakage, but negative for MPIO presence (denoted by white arrow heads), although small bead-like hypointensities could be observed within the ventricular CSF (gray arrowhead with asterisk). The inset on the post αICAM-1-MPIO image shows a magnification of this area. (B) No specific MPIO presence was observed after IgG-MPIO administration, although gadobutrol leakage (white arrow heads) could be observed. Similar observations were made for animals injected with MPIO during the chronic phase of EAE. Pre, 3D multigradient echo T2*-weighted image before injection of MR contrast; Post, the same after injection of MR contrast; Δ SI, difference in signal intensity as a result of MPIO/gadobutrol presence; gray arrowhead, MPIO positive/gadobutrol positive; black arrowhead, MPIO positive/gadobutrol negative; white arrowhead, MPIO negative/gadobutrol positive.

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Often, areas that showed αICAM-1-MPIO presence did not show leakage of gadobutrol (black arrowheads in Fig. 2). The opposite pattern, that is, gadobutrol leakage without αICAM-1-MPIO presence, was only observed in ventricular tissue (white arrowheads in Fig. 2). All areas in the brain parenchyma that showed gadobutrol leakage also showed αICAM-1-MPIO presence. Based on our results, we conclude that endothelial expression of ICAM-1 and loss of BBB integrity can be dissociated during

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EAE. Similar results have been obtained with the VCAM-1targeted MPIO (14), which stresses the capacity of these MPIO constructs to detect early distinct pathological events involved in MS lesion formation. As a footnote it is important to mention that in our study gadobutrol was injected after MPIO injection (approximately 1 h later). Consequently, MPIO presence may have confounded the detection of gadobutrol specifically in areas with high MPIO presence. MPIO may reduce the MR

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MOLECULAR MRI OF ICAM-1 PRESENCE IN AN MS MODEL detection sensitivity for gadobutrol as they have a strong T2(*) reducing effect. Theoretically MR signal could be abolished in MPIO overloaded areas, which would prevent the detection of subsequent gadobutrol-induced effects. However, for gadobutrol detection we acquired T 1 -weighted images with minimal T2 * -weighting (TE = 4 ms). Still, MPIO presence was occasionally observed on the pre T1-weighted gadobutrol images (data not shown) despite the short echo times used. 2.4. Quantification of MR Signal Abnormalities Induced by Contrast Agents The amount of voxels with altered signal intensity after injection of the two types of contrast agents was determined in four characteristic areas in the brain: brainstem, cerebellum, subarachnoid space and ventricular area. Only the amount of voxels with abnormal signal intensity change on the post-contrast (MPIO/ gadobutrol) images, as a result of the presence of the contrast

agent, is depicted in Fig. 3. To determine significant differences between the experimental groups, we grouped the IgG-MPIO injected animals and compared them with the grouped αICAM1-MPIO-injected animals (seven or eight animals). Results are shown in Table 1. Theoretically, when assuming a Gaussian distribution of voxel intensities, approximately 2.5% of the voxels may be considered to have an abnormally low intensity in the preMPIO images. In the pre-contrast images the amount of voxels with abnormally low values (averaged for all animals) ranged from 0.79 ± 0.63 (subarachnoid space) to 2.99 ± 1.19% (ventricular area). When results of all IgG-MPIO receiving animals were grouped, it was shown (Table 1) that the percentage of voxels with abnormally low intensity did not significantly increase in any of the regions of interest, confirming that MPIO do not cross the BBB. When all animals receiving αICAM-1-MPIO were grouped, it was shown that there was a significant increase of voxels with abnormally low intensity in all analyzed areas. The subarachnoid space (between the temporal cortex and the

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Figure 3. Amount of voxels with abnormal signal intensity after injection of MPIO (A) or gadobutrol (B). The percentage of voxels with signal intensities lower (MPIO) or higher (gadobutrol) than twice the standard deviation of the voxel intensities on the pre-injection images was determined in specific areas at different phases of disease. (A) Specifically the subarachnoid space (note different scale of y-axis) between the midbrain and temporal lobe of the cortex showed significantly increased number of voxels with abnormal signal intensity after αICAM-1-MPIO injection. (B) Voxels with abnormal signal intensity after gadobutrol injection were mainly detected in the ventricular area († p < 0.05 vs pre-injection levels). The number of animals in each group is denoted below the bars. Only animals receiving αICAM-1-MPIO were statistically evaluated to test differences between pre/post-contrast levels and acute/chronic EAE phase. All animals receiving IgG-MPIO were compared as a single group with the αICAM-1-MPIO-receiving animals, and results are shown in Table 1.

3.04 ± 0.44(3) 2.65 ± 0.42(3) 0.99 ± 0.63(5) 3.51 ± 0.87(5)

Pre

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Values represent the results from grouped animals, irrespective of the phase of disease, injected with either IgG- or αICAM-1-MPIO. Number of animals per group is indicated in parentheses in the Pre columns. *p < 0.05 vs IgG-MPIO; †p < 0.05, ††p < 0.001, vs pre-injection levels. Pre, Before the injection of contrast agent; Post, after the injection of contrast agent.

1.51 ± 0.77† 3.53 ± 2.88† 11.06 ± 6.49† 26.35 ± 13.44†† 0.68 ± 0.40(8) 1.18 ± 0.38(8) 1.37 ± 0.40(8) 1.96 ± 0.73(8) 1.34 ± 0.65 2.61 ± 1.58 21.07 ± 17.80† 30.28 ± 16.77† 1.03 ± 0.52(3) 1.46 ± 0.50(3) 1.62 ± 0.97(4) 2.28 ± 0.65(4) 5.50 ± 2.56 5.07 ± 1.45† 26.96 ± 15.68 *†† 6.13 ± 2.40† 4.91 ± 3.40 4.04 ± 1.78 3.56 ± 3.64 4.98 ± 2.88

Post

2.84 ± 0.26(7) 2.83 ± 0.41(7) 0.67 ± 0.65(8) 2.66 ± 1.28(8)



Post Pre

ICAM-1 MPIO IgG

Cerebellum Brain stem Subarachnoid space Ventricle

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Table 1. Percentage of voxels with abnormal signal intensity before and after injection of contrast agents

Pre

IgG

Post

Gadobutrol

Pre

ICAM-1

Post

E. L. A. BLEZER ET AL. midbrain) showed the highest increase (from 0.67 ± 0.65 to 29.96 ± 15.68, p < 0.001; see Table 1). The amount of voxels with abnormal signal intensity in the subarachnoid space after αICAM-1-MPIO injection was also significantly higher than after IgG-MPIO. The number of animals in the αICAM-1-MPIO-injected group was also sufficient to test if αICAM-1-MPIO presence changed during the different phases of EAE, that is during the initial peak of the disease and at the chronic phase when animals showed some regression of disease. No statistical differences were found and apparently irrespective of disease phase the extent of ICAM-1 expression, as determined by the αICAM-1-MPIO, is quite similar between the two phases of disease, when animals have clear neurological deficits, in this specific model. Gadobutrol was injected after MPIO injection. Pre-injection percentage of voxels with abnormal high signal intensity (averaged for all values) ranged between 0.79 ± 0.43 (cerebellum) and 2.07 ± 0.69% (ventricular area). When all αICAM-1- or IgG-MPIO receiving animals were grouped, it was shown that significant differences between pre- and post-gadobutrol amount of voxels with abnormal signal intensity could be observed for almost all analyzed areas (Table 1). No differences were detected between the αICAM-1-/IgG-MPIO receiving groups. Apparently, in all analyzed areas there is a detectable global leakage of the BBB when animals have neurological deficits. Importantly the detection of this leakage is not detrimentally influenced by the presence of MPIO even in the areas with the highest MPIO presence, that is, the subarachnoid space of the αICAM-1-injected animals. Both the αICAM-1- and IgG-MPIO-injected animals showed similar significantly increased levels of abnormal hyperintense voxels owing to gadobutrol presence. 2.5.

Immunohistochemistry

IHC was used to determine the cellular source of ICAM-1 expression and the localization of MPIO. Both IgG- and αICAM-1-MPIOinjected animals showed presence of MPIO in the spleen (data not shown), indicative of the aspecific uptake of conjugated MPIO by macrophages residing in the spleen (36). Similar to the MRI results, IHC showed that ICAM-1 was massively expressed on blood vessels in the subarachnoid space between the temporal cortex and the midbrain but also on other vessels throughout the brain (Figs. 1C and 4A&B) in animals with EAE. MR hypointensities of animals injected with αICAM-1-MPIO locally coincided with the presence of ICAM-1. Individual MPIOs could be histologically observed in these animals. Their presence co-localized with ICAM-1-positive vessel-like structures. MPIO were mainly associated with cells located at the luminal side of these structures. No MPIO were detected deeper in the brain parenchyma. Notably, no MPIO were detected in animals injected with IgG-MPIO, which precludes once more passive leakage of MPIO into the brain parenchyma. ICAM-1 expression was observed on endothelial cells, T-cells and monocytes/macrophages but not on B-cells and dendritic cells (Fig. 4C). These results show that endothelial cells are most likely the main target of αICAM-1-MPIO, confirming previous studies showing that ICAM-1-targeted MPIO were associated with endothelial cells in vitro (37) and in vivo (12). Our IHC findings confirm previous studies that showed that ICAM-1 is expressed on macrophages (38), microglia (39) and T-cells (21). Interestingly we have recently observed in an animal model of stroke that, in addition to endothelial cells, αICAM-1-MPIO may co-localize with CD45-positive leukocytes that are associated with blood vessels (12). It is assumable

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Figure 4. Spatial comparison of αICAM-1-MPIO binding and ICAM-1 presence determined by IHC. (A) Animal with EAE injected with αICAM-1-MPIO. Corresponding T2*-weighted 3D multigradient echo images and IHC ICAM- 1/MPIO images are displayed. IHC shows the localization of ICAM-1 (brown) on blood vessels in the subarachnoid space (upper) or cerebellum (lower). Individual MPIO, as determined with Perls’s staining (white arrowheads pointing to blue beads in the ‘+ Iron’ insets), correspond to the black arrowheads depicted in the post-contrast images, and were confined to ICAM-1-positive vessels. No MPIO were observed in the brain parenchyma, nor in animals injected with IgG-MPIO. (B) Typical examples of ICAM-1-positive blood vessels of animals injected with αICAM-1-MPIO showing MPIO presence in cells located at the luminal side of these vessels. These cells were either lining (white arrowheads) or protruded into the lumen (black arrowheads). (C) Fluorescent IHC showing that endothelial cells (glut-1), T-cells (KT3) and macrophages/monocytes (MΦ, F4/80) expressed ICAM-1 (yellow arrow heads). Dendritic cells (CD11c) and B-cells (B220) were negative for ICAM-1 expression. Green, ICAM-1 expression; red, designated cell type; yellow, both.

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3.

CONCLUSION

In conclusion, this study shows the feasibility of αICAM-1-MPIO to measure ICAM-1 expression in a rodent MS model, thereby providing opportunities for diagnosis of the early processes of MS lesion formation. For clinical application it is essential that MPIO are fully biodegradable and nontoxic which is not the case for the inert polymer coat of the MPIO used in this experimental study which most likely ends up in the reticuloendothelial system of cells located in the liver and spleen (15). Fortunately, the first biodegradable MPIO have recently been developed using US Food and Drug Administration-approved polymers (41), which opens the

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that the cells attached to the endothelial cells on the luminal side of the blood-vessel in the current study are also leukocytes. Finally ICAM-1 expression has also been shown in epithelial cells of the choroid plexus (31) and astrocytes (39). Whether αICAM-1-MPIO is able to visualize ICAM-1 expression on all of the above-mentioned ICAM-1 positive cell types during EAE is unclear. In the current study, MR images were acquired within 1 h after administration of MPIO. Given the extreme short half-life of MPIO of less than a minute (40) and the suggestion that the BBB was not permeable for MPIO, it is unlikely that the post-αICAM-1-MPIO MR images will visualize ICAM-1 presence on cells residing in the brain parenchyma.

E. L. A. BLEZER ET AL. possibility of deployment of antibody-conjugated MPIO for clinical applications. Eventually, providing clinicians with tools to visualize endothelial activity will further guide them to diagnose MS earlier, but also to tailor and measure effectiveness of treatments for specific time windows and distinct patient populations.

4. EXPERIMENTAL 4.1.

Materials

ProMag™ 1 Series, Bind-IT™ MPIO (25 mg MPIO ml 1, 26.5% ironcontent, 1 μm) were obtained from Bangs Laboratories Inc. (Fisher, IN, USA). Diaminobenzidine solution (DAB+) was obtained from Sigma-Aldrich (St Louis, MO, USA). Monoclonal antibodies against mouse ICAM-1 (αICAM-1, YN1/1.7.4) and irrelevant immunoglobulin G antibodies (IgG, RTK4530) were obtained from BioLegend (San Diego, CA, USA). Goat anti-rat IgG (H + L) was obtained from Jackson Immuno Research (Suffolk, UK), and rat-anti-mouse CD31 (clone MEC13.3) from BD Pharmingen (Breda, The Netherlands). A monoclonal antibody against mouse CD31 (αCD31) was produced from hybridoma ERMP12. Horseradish peroxidase-labeled streptavidin (HRPstrep) was obtained from DAKO (Glostrup, Denmark). ICAM-1 was obtained from eBioscience (Hatfield, UK), Glut-1 from Abcam (Camebridge, UK), and F4/80 and KT3 were harvested from various hybridoma. Hoechst 33258, goat-antirat-Alexa488, goat-anti-rabbit-Alexa488 and Streptavidin-Alexa555 were obtained from Molecular Probes (Bleiswijk, The Netherlands). Goat serum came from Jackson ImmunoResearch (West Grove, PA, USA). Primers were obtained from Ocimum Biosolutions (IJsselstein, The Netherlands). M199 was obtained from Gibco/Life Technologies (Breda, The Netherlands), fetal calf serum (FSC) from Lonza (Breda, The Netherlands) and Blendzyme II Liberase TM and bovine serum albumin (BSA) from Roche (Woerden, The Netherlands). MOG35–55 peptide (MEVG-W-YRSPFSR-V-V-HLYRNGKamide) was obtained from Cambridge Research Biochemicals (Billingham, UK), Mycobacterium tuberculosis H37Ra as well as CFA from BD Difco (Alphen aan de Rijn, The Netherlands), and pertussis toxin and dextran from Sigma-Aldrich (Zwijndrecht, The Netherlands). Euthesate was obtained from Apharmo BV (Arnhem, The Netherlands). Gadobutrol (Gd-DO3A-butriol, Gadovist®) was from Bayer Schering Pharma (Berlin, Germany). Polythene tubes (inner diameter 0.28 mm) were from Smiths Industries (Kent, UK). 4.2.

Preparation of Antibody-Functionalized MPIO

MPIO were extracted from their original buffer by magnetic separation, and resuspended in coupling buffer [50 mM 2-(Nmorpholino)ethanesulfonic acid (MES); pH 5.2]. Prior to the coupling procedure, αICAM-1 or IgG were buffer-exchanged to coupling buffer by centrifugation, resulting in a final antibody concentration of 1.0 mg ml 1. Next, MPIO and αICAM-1 or IgG were added in a 1:1 (v/v) ratio, vortexed and left to incubate for 60 min at room temperature (RT) on a roller-bench. Following incubation, antibody-MPIO were buffer-exchanged to storage solution (150 mM NaCl with 0.002% azide) by magnetic separation and stored at 4 °C. 4.3.

Induction of EAE

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All animal procedures were approved by the local (Amsterdam/ Utrecht, The Netherlands) ethical committee on animal experiments, and experiments were performed in accordance with the guidelines of the European Communities council directive.

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Twenty-five 10-week-old male C57BL/6 J mice were obtained from Harlan (Boxmeer, The Netherlands). All mice were kept under pathogen-free conditions. EAE was induced by subcutaneous inoculation of 100 μg MOG35–55 peptide in each flank, in an equal volume of CFA containing 3 mg ml 1 of heat-killed M. tuberculosis, as described before (23). Mice were subsequently intraperitoneally (i.p.) injected with 200 μl of pertussis toxin (200 ng), which was repeated 2 days post-immunization (p.i.). Control mice, used for the determination of ICAM-1 mRNA levels on cerebellar capillaries, were immunized with CFA only. All mice were examined daily for clinical signs of EAE and were scored as follows: 0, no disease; 1, limp tail; 2, hind limb weakness; 3, complete hind limb paralysis; 4, hind limb paralysis plus front limb paralysis; and 5, moribund or dead. The model is characterized by three characteristic phases. Typically EAE onset is around 9 days after immunization, with peak of disease around 14–15 days p.i., followed by partial recovery of disease. A total of four controls and eight EAE animals were used for determination of mRNA levels of ICAM-1 in cerebellar capillaries. Thirteen animals were used for MRI experiments. 4.4. Determination of ICAM-1 mRNA Levels on Isolated Brain Endothelial Cells Cerebellums were harvested at 9 (before the first clinical signs of EAE: ‘before’) and 15 days (height of the acute phase of EAE: ‘acute’) p.i. Two controls and four animals with EAE were used for both time points. Cerebellums were homogenized in M199 containing 0.2% FCS using a pair of tight and loose pestles. Cells and capillaries were pelleted at 2000 rpm for 10 min at 4 °C in a swinging bucket rotor. The pellet was re-suspended in 15% dextran in M199 and centrifuged at 2500 rpm for 25 min at 4 °C in a swinging bucket rotor. Subsequently, the microvessel pellet was resuspended in M199, enzymatically dissociated with Blendzyme II Liberase TM for 30 min at 37 °C and finally washed with M199. Each panning plate (10 cm Petri dish) was first coated with 10 ml of 50 mM Tris/HCl pH 9.5 containing 30 μl of goat antirat IgG (H + L), at 4 °C overnight. Next, the plates were washed with PBS, blocked with 0.2% BSA and incubated for at least 2 h at RT with 10 ml of 0.2% BSA containing 40 μl of rat-anti-mouse CD31 (clone MEC13.3). Finally, the plates were washed with PBS and endothelial cells were bound for 60 min at RT. After washing with PBS (8x), mRNA of panned endothelial cells was harvested. Endothelial cells were lysed using 1 ml of Trizol and stored at 80 °C. Quantitive PCR (qPCR) was used to determine the expression of ICAM-1 and CD31. Equal amounts of RNA were converted into cDNA and analyzed for mRNA presence according to the manufacturer’s protocol (Applied Biosystems, Bleiswijk, the Netherlands). The cycle threshold values were used to calculate the relative fold difference in mRNA levels. All experiments were performed using experimental duplicates. Expression levels of ICAM-1 are expressed relative to CD31. 4.5.

Experimental Design MRI

Animals were examined with MRI during two characteristic phases of disease (indicated as gray bars in Fig. 1), that is, when animal displayed maximal neurological deficits at the acute phase of EAE (14 ± 1 days p.i.), and at the chronic phase of EAE (26 ± 1 days p.i : ‘chronic’). Animals were either injected with αICAM-1- or IgG-MPIO (5 mg iron/kg). Respectively five and three animals received αICAM-1- or IgG-MPIO during the acute phase

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MOLECULAR MRI OF ICAM-1 PRESENCE IN AN MS MODEL of EAE. For the chronic phase this was respectively three and two animals. All animals were sacrificed at the end of the MR experiment by an overdose of pentobarbital (i.p., Euthesate) to harvest tissue for IHC. One hemisphere and parts of the spleen were snap-frozen with liquid N2 and stored at 80 °C. The other hemisphere was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer and stored for future experiments. The spleen was exclusively tested for MPIO presence to verify proper delivery of MPIO into animals. 4.6.

MRI Protocol

MR experiments were performed on a 9.4 T horizontal 20 cm bore MR system (Varian Inc., Palo Alto, CA, USA). For in vivo experiments, mice were anesthetized with isoflurane (3.5% induction, 1.5–2.0% maintenance) in air/O2 (2:1). The tail vein was cannulated and connected with a polythene tube (inner diameter 0.28 mm, volume approximately 100–150 μl) for contrast agent delivery during MRI. Animals were positioned in a specially designed cradle and inserted into the magnet. A birdcage volume coil (diameter 70 mm) and an inductively coupled home-built surface coil (diameter 30 mm) were used for radiofrequency transmission and signal detection, respectively. The MR protocol consisted of three phases, that is, a pre-contrast agent phase, a post-MPIO contrast agent phase, and a postgadolinium (gadobutrol) contrast agent phase. During the first phase, volume locations for 2D/3D MRI were determined from a transversal scout image. The central position of this volume was positioned directly caudal to the cerebellum. The volume included the upper part of the spinal cord, the cerebellum and the caudal part of the cerebrum. Two subsets of T2*-weighted images (3D multigradient echo; repetition time (TR)/echo time (TE) = 37/(5,10,15,20) ms; number of acquisitions (NA) = 4; flip angle 10°; 150 × 150 × 150 μm3 voxel size; acquisition time = ~20 min per set) were acquired. These two subsets were averaged and were designated as the pre-MPIO 3D image. MPIO-conjugates were injected at the end of this part of the protocol. In the post-MPIO injection phase, three subsets of the T*2-weighted 3D images were collected. The first and second 3D post-MPIO image were averaged (total acquisition time = ~40 min), and designated as the post-MPIO 3D image, which implies that MPIO were on average 20 min in circulation during the collection of the post-MPIO images. At the end of this part of the protocol, 2D T1-weighted images were acquired (gradient echo; TR/TE = 1600/4 ms, 100 × 200 μm in-plane resolution, 43 slices of 450 μm, NA = 4, flip angle 90°; experimental time = ~5 min) and gadobutrol (0.3 mM/kg) was injected to investigate BBB integrity. In the post-gadobutrol phase, two additional sets of 2D T1-weighted images were acquired. The center of k-space was acquired respectively 2.5 and 7.5 min after gadobutrol injection. The last T1-weighted image set, which was thus collected approximately 70–75 min after MPIO injection, was used to measure the extent of BBB permeability. It is likely that any unbound MPIO will be cleared from the blood at this time-point as the half-life of MPIO is less than a minute (40) as mentioned before. 4.7.

MRI Analysis

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4.8.

Immunohistochemistry

IHC was used to visualize ICAM-1, iron oxide presence and various cell types (endothelial cells, T-cells, macrophages/monocytes, B-cells dendritic cells). Cryosections of 10 μm of brain and spleen (iron oxide detection only) tissue were air-dried overnight, and acetone-fixed for 10 min. Dry, fixed sections were hydrated for 15 min in PBS containing 0.1% BSA. Next, sections were incubated with biotinylated αICAM-1 (10 μg antibody ml 1) in PBS/0.1% BSA for 1 h. Subsequently, sections were incubated for 45 min with horseradish peroxidase-labeled streptavidin (HRPstrep, prepared according to the manufacturer’s description). Diaminobenzidine solution (DAB) was used as chromogen (used according to the manufacturer’s instructions). Between incubation steps, sections

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MPIO and gadobutrol presence were quantified on the 3D images (MPIO) or 2D T1-weighted images (gadobutrol). The latter images were registered to the 3D images (see later). Signal inhomogeneity as a result of surface coil-based detection was

corrected using a nonparametric nonuniform intensity normalization procedure. Images acquired at the individual four echo times of the multiecho 3D protocol were summed to increase signal-to-noise ratio and used in the image analyses pipe-line. The averaged 3D image of each individual animal sets acquired in the pre-contrast MR phase were designated as reference image for registration procedures and delineation of regionsof-interest (ROIs). All 2D/3D MR images were rigidly registered to this reference image by maximizing mutual information using the Advanced Matters Mutual Information Metric in Elastix (42). All subsequent analyses were performed on these registered images. The change in signal intensity as a result of MR contrast agent was calculated by subtracting averaged post-MPIO 3D images from pre-MPIO 3D images, and subtracting pre-gadobutrol 2D T1-weighted images from post-gadobutrol images. Difference images were only used for qualitative evaluations, as breathinginduced movement occasionally induced voxel-level shifts, especially in caudal part of the brain. The effect of MPIO/gadobutrol presence on the signal intensity of the MR images was therefore quantified by calculating the percentage of voxels with abnormal voxel intensities in well-defined ROIs. For each of these ROIs a threshold was determined in the pre-contrast images which was defined as the mean signal intensity minus (MPIO) or plus (gadobutrol) twice the standard deviation of the signal intensity in that area. Voxel intensities on the post-contrast images below (MPIO) or above (gadobutrol) these thresholds were considered abnormal. The specific ROIs were manually outlined on the 3D reference image using well-defined hallmarks. These ROIs were outlined (see also Supporting Information, document 1) in the cerebellum, brain stem, the subarachnoid space between the temporal cortex and the midbrain, and the ventricular area near the conjunction of the lateral and third ventricle. The rationale to choose these areas was the qualitatively assessed presence of MPIO on the 3D images, combined with the knowledge that these brain regions are prone to develop abnormalities in this model (43). The ventricular area was chosen to visualize contrast leakage through the choroid plexus. One animal died during the MRI experiment after the injection of gadobutrol, and post-gadobutrol images for this specific animal were therefore excluded from further MRI analysis. Furthermore, individual MR experiments were blindly evaluated on the presence of breathing-induced movement artifacts, which could adversely affect the level of detection thresholds for the analyzed ROI. Caudal areas were especially prone to movement artifacts. Data with these artifacts were also excluded from further analyses. The resulting final numbers of αICAM-1-/IgGMPIO-injected animals per classified group are shown in Fig. 3.

E. L. A. BLEZER ET AL. were thoroughly washed with PBS. Tissue was subsequently incubated with Perls’s solution [2 M HCl and 2% ferrocyanide in Milli-Q in 1:1 (v/v) ratio] for 20 min, thoroughly washed with Milli-Q, dipped in a nuclear fast red solution and dehydrated; slides were mounted in Entallan. All steps were performed at RT. For fluorescent IHC, sections were also acetone fixed (10 min) and blocked using 10% goat serum in PBS. Sections were then incubated with primary antibodies (Glut-1 for endothelial cells, KT3 for T-cells, F4/80 for macrophages/monocytes, B220 for B-cells and CD11c for dendritic cells) at RT for 1 h and subsequently washed with PBS. Subsequently, sections were incubated with the secondary antibody (goat-anti-rat-Alexa488 for KT3, F4/80, B220 and CD11c or goat-anti-rabbit-Alexa488 for Glut-1) at RT for 1 h. All used antibodies were diluted in PBS with 1% goat serum. Sections were then washed with PBS and incubated overnight with ICAM-biotin (diluted in 0.1% BSA in PBS) at 4 °C. Finally, sections were washed with PBS and incubated with streptavidin-Alexa555 (diluted in 0.1% BSA in PBS) at RT for 1 h and after washing stained with Hoechst Dye Nuclear (DNA) staining for 1 min, washed in PBS and finally mounted in Vinol mounting medium. 4.9.

Statistical Analysis

Statistical analyses were performed using the statistical software package SigmaPlot (version 11 Germany). Analyses were performed to evaluate whether:

• •





There was a change in mRNA ICAM-1 expression in the cerebral capillaries of animals with EAE during the different phases of EAE. Data were evaluated by a one-way analysis of variance (ANOVA) for possible significant differences. There was a change in count of voxels with abnormal intensity as a result of the presence of MR contrast agents (MPIO: αICAM-1/IgG; gadobutrol) in the ROIs during the different phases of EAE. Data were evaluated for significant differences by two-way ANOVA for repeated measures. Factors were: type of contrast agent; voxels with abnormal intensity in pre/post MR image; and ROIs. Owing to the low numbers in the IgG-MPIO-injected groups, this was only evaluated for the αICAM-1-MPIO-injected animals. There was a difference between count of voxels with abnormal intensity as a result of the presence of MR contrast agents in the ROIs during the different phases of EAE, again alone for the αICAM-1-MPIO-injected animals. Data were evaluated for significant differences by two-way ANOVA. Factors were: type of contrast agent; voxels with abnormal intensity in pre/post MR image; ROIs; and phase of EAE. There were differences between the two above mentioned evaluations after grouping animals receiving the different MPIO conjugates, that is, excluding phase of disease as a factor.

When passing the ANOVA tests, data were tested using the Student–Newman–Keuls post-hoc test. A value of p < 0.05 was considered to be statistically significant. If not otherwise stated, data are presented as means ± standard deviation.

Acknowledgments

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The authors would like to thank Astrid Bovens, Hedi Hunt and Wouter Mol for their biotechnical support. We gratefully acknowledge funding from the Netherlands Organization for Scientific Research (NWO; VIDI 917.76.347), the Dutch foundation

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of Multiple Sclerosis Research (grant MS 07-615/09-689) and Top Institute Pharma (Grant T2-108).

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Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web site.

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In vivo MR imaging of intercellular adhesion molecule-1 expression in an animal model of multiple sclerosis.

Upregulation of intercellular adhesion molecule 1 (ICAM-1) is an early event in lesion formation in multiple sclerosis (MS) and experimental autoimmun...
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