- Open Access
Gadofluorine M-enhanced MRI shows involvement of circumventricular organs in neuroinflammation
© Wuerfel et al; licensee BioMed Central Ltd. 2010
- Received: 27 July 2010
- Accepted: 18 October 2010
- Published: 18 October 2010
Circumventricular organs (CVO) are cerebral areas with incomplete endothelial blood-brain barrier (BBB) and therefore regarded as "gates to the brain". During inflammation, they may exert an active role in determining immune cell recruitment into the brain.
In a longitudinal study we investigated in vivo alterations of CVO during neuroinflammation, applying Gadofluorine M- (Gf) enhanced magnetic resonance imaging (MRI) in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. SJL/J mice were monitored by Gadopentate dimeglumine- (Gd-DTPA) and Gf-enhanced MRI after adoptive transfer of proteolipid-protein-specific T cells. Mean Gf intensity ratios were calculated individually for different CVO and correlated to the clinical disease course. Subsequently, the tissue distribution of fluorescence-labeled Gf as well as the extent of cellular inflammation was assessed in corresponding histological slices.
We could show that the Gf signal intensity of the choroid plexus, the subfornicular organ and the area postrema increased significantly during experimental autoimmune encephalomyelitis, correlating with (1) disease severity and (2) the delay of disease onset after immunization. For the choroid plexus, the extent of Gf enhancement served as a diagnostic criterion to distinguish between diseased and healthy control mice with a sensitivity of 89% and a specificity of 80%. Furthermore, Gf improved the detection of lesions, being particularly sensitive to optic neuritis. In correlated histological slices, Gf initially accumulated in the extracellular matrix surrounding inflammatory foci and was subsequently incorporated by macrophages/microglia.
Gf-enhanced MRI provides a novel highly sensitive technique to study cerebral BBB alterations. We demonstrate for the first time in vivo the involvement of CVO during the development of neuroinflammation.
- Experimental Autoimmune Encephalomyelitis
- Choroid Plexus
- Area Postrema
- Experimental Autoimmune Encephalomyelitis Mouse
- Central Nervous System Lesion
The central nervous system (CNS) may no longer be considered immune privileged but rather a site of selective immune activity [1, 2]. This so-called restricted immunity is warranted by the barrier function of capillary endothelium, which channels the entry of serum proteins and immune cells from the blood to the CNS or the cerebrospinal fluid (CSF), respectively . Although the blood-brain barrier (BBB) covers most parts of the CNS, certain brain regions including the choroid plexus as well as structures that line the cavity of the third and of the fourth ventricle are devoid of a tight BBB and are in permanent contact to blood-born molecules and cells. These "exposed" areas, called circumventricular organs (CVO), are characterized by a dense capillary network with wide perivascular areas. Assumably, specialized ependymal cells, the tanycytes, act as a flexible barrier controlling the exchange of substances between CVO and the surrounding brain parenchyma as well as the CSF [3, 4]. Besides neuroendocrine functions, CVO provide an access route for immune cells into the brain and might therefore guide CNS immune surveillance. The capacity of the choroid plexus to build a bridge for immune cells trafficking from the blood circulation into the CSF and the subarachnoid space was demonstrated in physiological  as well as under inflammatory conditions . Immune activation was also reported in other CVO during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), indicating, that these unprotected CNS areas play a key role for pathological immune processes of the brain . However, until recently no reliable method had been available to survey CVO in vivo. Assuming a crucial function as "gates to the brain" for immune cells, the visualization of alterations in CVO might become of diagnostic and therapeutic value for the assessment of neuroinflammatory conditions, similar to the detection of BBB impairment by gadopentetate dimeglumine- (Gd-DTPA) enhanced MRI, the current gold standard for the evaluation of disease activity in MS. New developments in high field strength MRI and novel contrast media provide optimized means to detect lesions and localize alterations in BBB integrity, even in small rodent disease models such as murine EAE . The novel gadolinium-based contrast agent Gadofluorine M (Gf) was recently shown to facilitate the visualization of CNS lesions and cranial nerve inflammation [8–10]. Gf was originally applied for the detection of malignant lymph nodes , atherosclerotic plaques , or peripheral nerve damage .
In this study, we demonstrated for the first time that Gf-enhanced MRI represents a unique tool to in vivo visualize alterations of CVO. The magnitude of Gf accumulation in the choroid plexus and other CVO increased during active inflammation, correlated with disease activity, and could be used to differentiate between EAE animals and controls. Furthermore, Gf facilitated the detection of otherwise occult inflammatory CNS lesions.
All experiments were approved by the local animal welfare committee and conformed to the European Communities Council Directive (86/609/EEC). For adoptive transfer EAE, female naïve SJL/J mice were immunized with an emulsion containing 250 μg PLP (murine proteolipid peptide p139-151; purity >95%, Pepceuticals, Leicester, UK) in equal volumes of phosphate buffered saline (PBS) and Complete Freunds Adjuvant (CFA, Difco Laboratories, Detroit, MI, USA) and 4 mg/ml Mycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, MI, USA) . Ten days after immunization cells were extracted from draining lymph nodes and restimulated with 12.5 μg PLP/ml in cell culture medium (RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal calf serum) for 4 days at 37°C. Then 8-12 × 106 T-cell blasts in 100 μl PBS were injected intraperitoneally into syngenic recipients .
Mice were daily weighed and scored for neurological deficits as previously described : 0, unaffected; 1, tail weakness or impaired righting on attempt to roll over; 2, paraparesis; 3, paraplegia; 4, paraplegia with forelimb weakness or complete paralysis; score >4, to be sacrificed. Mice with a score of 4 received an intraperitoneal injection of 200 μl glucose 5% daily.
After induction of EAE, mice underwent cerebral MRI between day 5 and 16 post T cell transfer on a 7 Tesla rodent scanner (Pharmascan 70/16AS, Bruker BioSpin, Ettlingen, Germany), applying a 20 mm RF-Quadrature-Volume head coil. Animals received anesthesia via facemask with 1.5 - 2.0% isoflurane (Forene, Abbot, Wiesbaden, Germany) delivered in 100% O2 under constant ventilation and body temperature control (Bio Trig System, Bruker BioSpin, Ettlingen, Germany).
We acquired axial and coronal T1-weighted images (MSME; TE 10.5 ms, TR 322 ms, 0.5 mm slice thickness, matrix 256 × 256, field of view (FOV) 2.8 cm) before and after intravenous (i.v.) injection of 0.2 mmol/kg bodyweight Gd-DTPA (gadopentetate dimeglumine, Magnevist, Bayer-Schering AG, Berlin, Germany), or 0.1 mmol/kg bodyweight Gf (Gadofluorine M, kindly provided by Drs. M. Reinhardt and B. Misselwitz, Bayer-Schering AG, Berlin, Germany). Gd-DTPA is the current gold standard for the detection of BBB breakdown in the CNS, showing as hyperintensity on T1-weighted images instantly after i.v. application. With a blood half-life of 20 min it is largely excreted from the organism after 3 h . Gf is a modified amphiphilic gadolinium complex with a molecular weight of about 1.53 g/mol, also generating bright enhancement on T1-weighted images. Gf was designed by adding a perfluoroctyl chain to a gadolinium containing macrocycle. Gf interacts with hydrophobic proteins of the extracellular matrix such as collagen, proteoglycans, decorin and tenascin, and it is largely bound to serum albumin after i.v. administration . It has a plasma half-life of 15.6 h . The dose applied in this study was approved by previous MRI studies [8, 9]; it is far below the estimated lethal dose of 5 mmol/kg .
We investigated a total number of 28 mice by MRI, comprising 21 EAE and 7 control mice. Fifteen EAE mice initially received Gd-DTPA. If BBB breakdown could be detected, Gf was applied and MRI repeated after 24 h, as established previously . A subgroup of 4 EAE mice was additionally imaged to assess early kinetics 1 h and 6 h after Gf injection. Two mice were followed up until complete clinical remission in order to investigate longitudinal Gf signal decay. A further group of 6 EAE mice received Gf pre-labelled with the red fluorescent marker Cy3.5 (Gf-Cy3.5) for subsequent immunofluorescence histology. Seven naïve mice served as healthy controls. The kinetic of disease was exclusively studied in vivo owing to ethical concerns, which forced us to minimize the number of animals investigated. Histological analyses were performed after the final MR acquisitions.
Mice were perfused for post mortem analysis in the acute disease phase within 2 to 5 days after onset. Following the final MRI, brain and spinal cord were prepared for histology, as previously described . Every second slice was stained with hematoxylin and eosin (H&E) to assess inflammation. For the evaluation of Gf distribution, consecutive slices were stained with Hoechst 33258 nuclear stain (1:10000, Molecular Probes, Leiden, the Netherlands) to visualize cellular organization. We performed immunohistochemical staining against IBA-1 to identify macrophages/microglia, using the primary antibody rabbit anti-IBA-1 (1:1000, Wako Chemicals, Neuss, Germany) and goat anti-rabbit Cy2 (Amersham, Muenchen, Germany) as secondary antibody . Selected sections were examined by epifluorescence microscopy and digitally photographed (Olympus BX-51, Hamburg, Germany).
MRI data were coregistered and corrected for magnetic field inhomogeneity using MIPAV 6.1 (Center for Information Technology, National Institutes of Health, Bethesda, MD, USA). Statistical analysis was performed with GraphPad Prism 4.0c (GraphPad Software, Inc., San Diego, CA, USA). We determined the T1 lesion load of each individual EAE animal by calculating the volume of cranial Gf enhancement in T1-weighted sequences, applying a semi-automated procedure described previously . In contrast to Gf, enhancement after Gd-DTPA administration was diffuse without clearly obtainable borders and furthermore, was often masked by strong intraventricular Gd-DTPA signal due to Gd-DTPA leakage into the CSF. Accordingly, we were not able to reliably quantify the volume of Gd-DTPA-enhancing lesions. The Gf lesion volume was correlated to 1) the EAE score at the day of imaging and 2) the day of clinical EAE onset after immunization, applying Spearman's nonparametric analysis.
For the assessment of Gf enhancement in CVO, regions of interest (ROI) were placed in corresponding positions in all animals, and the mean ROI signal intensity as well as the standard deviation was calculated. We evaluated the subfornicular organ (SFO), the organum vasculosum of the lamina terminalis (OVLT), the median eminence (ME) and the area postrema (AP) in coronal slices. The most reliable ROI placement of the choroid plexus could be achieved in axial slices of the lateral ventricles. Choroid plexus examination was generally more rater demanding due to its widespread expansion inside the ventricles with high interindividual anatomic shape differences, and was more prone to partial volume contaminations compared to other areas. The mean signal intensitiy of each ROI was expressed as Gf mean intensity ratio by division with the mean signal intensity of a masseter muscle ROI for unbiased and stable comparison between individuals.
Two-tailed Mann-Whitney-U tests were applied to express differences of the Gf mean intensity ratio in CVO between naïve versus EAE animals. Correlation analyses were performed between the Gf mean intensity ratio and 1) the EAE score at the day of MRI and 2) the day of clinical disease onset using Spearman's nonparametric analysis. A receiver-operating characteristic (ROC) was used to analyze the validity of the Gf mean intensity ratio in CVO as diagnostic test to distinguish between EAE mice and controls.
Clinical EAE course and contrast-enhancing lesions (CEL)
Clinical EAE course and Gf lesion load
EAE score at Gf imaging
Gf lesion load in mm3
Day of clinical onset
Gf enhancement correlates with clinical EAE parameters
EAE score at Gf imaging
Day of clinical onset
Gf lesion volume
Gf lesion volume
0.15 to 0.80
0.30 to 0.85
0.08 to 0.79
-0.54 to 0.41
-0.74 to -0.01
-0.70 to 0.14
Contrast-enhancing lesions (CEL) exclusively detected by Gf
Total number of CEL
Number of CEL detected with Gd-DTPA and Gf
Number of CEL detected exclusively with Gf
Percentage of CEL detected exclusively with Gf
Visualization of CVO alterations by Gf-enhanced MRI
Gf enhancement in CVO correlates with disease severity
Gf enhancement of CVO differentiates EAE from control animals
Gf enhancement differentiates between EAE animals and healthy controls
Area under the ROC curve
Standard error (95% CI)
0.07 (0.77 to 1.03)
0.09 (0.64 to 0.98)
0.08 (0.68 to 1.01)
P value (two-tailed)
Sensitivity (95% CI)
89% (65 to 99%)
67% (41 to 87%)
82% (57 to 96%)
Specificity (95% CI)
80% (28 to 100%)
86% (42 to 100%)
83% (36 to 100%)
Gf accumulates in the extracellular matrix and is incorporated by monocytic cells
This study provides the first in vivo evidence for the participation of the choroid plexus and other CVO in neuroinflammation. Applying the novel MRI contrast agent Gf in high field MRI, we successfully visualized alterations in the CVO, which in part, correlated to the severity of CNS inflammation. Transmigration across the choroid plexus is a well defined entry route for leucocytes into the CNS . Already under physiological conditions, immune cells home to the choroid plexus and other CVO . Macrophages and dendritic cells, key players in CNS antigen presentation, have been localized to the choroid plexus of naïve mice [6, 24]. Recent histopathological studies stressed the role of the CVO also during inflammation, suggesting an initial recruitment of encephalitogenic T cells via the choroid plexus in EAE: Antigen presentation and subsequent reactivation of T lymphocytes is facilitated due to the presence of macrophages and microglia  and the upregulation of major histocompatibility complex antigens in these areas [7, 26]. Expression of ICAM-1 and VCAM-1, molecules that are crucial for lymphocyte adhesion and transmigration, were induced on choroid plexus epithelium  and other CVO during inflammation .
Although MRI has emerged as a powerful tool to assess disease activity in MS and EAE, alterations of CVO during inflammation have not been monitored in vivo up to date. Nevertheless, the invasion of macrophages into inflammatory plaques was demonstrated in vivo in a recent study on MS patients. This finding was partially independent from simultaneous BBB breakdown, and the route of entry is still not completely understood . We hypothesized that Gf-enhanced MRI might help to elucidate kinetics of CNS immune surveillance. Gf was originally developed as a marker for detecting lymph node metastasis in MR lymphography . Meding et al. demonstrated, that Gf binds to serum albumin and components of the extracellular matrix such as collagens, proteoglycans, fibronectin and tenascin , explaining a possible mechanism of Gf accumulation in atherosclerotic plaques [12, 28]. Furthermore, Gf is taken up by macrophages in vivo and in vitro [13, 29] and thus could highlight spots of immune activity in autoimmune neuritis, EAE and peripheral nerve degeneration [8, 9, 13, 30]. Recently, Gf was also applied to detect disease progression in an animal model of muscular dystrophy .
Here we demonstrate, that Gf accumulates weakly in the choroid plexus and other CVO involved in gating immune cell migration into the CNS in naïve mice, indicating an immunological function of those organs under physiological conditions. Interestingly, after initialization of EAE, pronounced Gf enhancement of the choroid plexus, the SFO and the AP became visible. Whereas leakage of Gd-DTPA into the CSF often prevented a reliable evaluation of the choroid plexus or periventricular brain regions in EAE mice, Gf-enhanced MRI allowed for the detailed quantitative evaluation of signal alterations in these areas. A correlation analysis in EAE mice exposed that high Gf enhancement of the SFO and the AP went along with an early disease onset and severe clinical affection. Regarding the SFO, these associations were even higher than the correlation of the same clinical parameters to the Gf lesion volume. Furthermore, we found that calculating the Gf mean intensity ratio in CVO can be applied as diagnostic tool to discriminate EAE from control animals, with a sensitivity of 89% and a specificity of 80% for the choroid plexus. In histological sections, we could confirm an accumulation of Gf in the choroid plexus stroma initially and a subsequent internalization into macrophages/microglia resident in the choroid plexus after 72 h. However, we did not detect significant alterations of the enhancement pattern in OVLT and ME during EAE. The predominance of the observed changes in periventricular and brain stem CVO might reflect a heightened regional vulnerability in developing inflammatory plaques during EAE in these areas .
Furthermore, we noted a high sensitivity of Gf to detect BBB leakage and associated parenchymal inflammation, particularly optic neuritis, resulting in a 42% increased number of contrast-enhancing lesions compared to Gd-DTPA-enhanced MRI, in line with a recent report by Bendszus et al. . In the circulation, Gf is largely bound to albumin . Brain parenchymal enhancement is due to a locally disturbed BBB and subsequent capturing of Gf molecules by protein interaction . There is no evidence for an active transport mechanism of Gf across the intact BBB. Gf accumulated with delay within CNS lesions yielding a signal intensity peak after 24 h in contrast to Gd-DTPA that immediately passed the disrupted BBB. Gf enhancement, as quantified in our study, thus reflects the accumulation during 24 h of focal BBB disruption. High binding affinity to plasma albumin and extracellular matrix proteins may explain the delayed initial CNS tissue accumulation but also the persisting presence of Gf within the brain parenchyma for up to 10 days [9, 18]. The inflammatory cascade in EAE includes a plethora of effector mechanisms such as secretion of matrix metalloproteases and other digesting enzymes by immune cells  initiating tissue degradation. Proteins liberated during this process are likely to trap Gf once it passed the disrupted BBB . The histological analysis applying fluorescent Gf likewise indicated a diffuse extracellular enrichment pattern. The clearance of Gf from the brain parenchyma after 14 days might be a result of a gradual loss of local molecule binding affinity as well as the general clearance and molecular turnover at lesion sites. We also noted a local uptake of Gf molecules by macrophages/microglia. However, we could not establish a correlation to repair mechanisms such as remyelination (data not shown).
Despite a delayed brain parenchymal enhancement Gf accumulated in CVO already 1 h after application. CVO are primarily exposed to Gf molecules due to the dense capillary network devoid of a tight BBB. Endothelial cells of the choroid plexus and other CVO express proteoglycans, fibronectin and other extracellular matrix proteins [33, 34], which may bind Gf. During CNS inflammation, such molecules are upregulated and mediate adhesion and subsequent transmigration of immune cells into the CNS [33, 35]. Thus, adhesion to extracellular matrix proteins presented on CVO endothelium might explain both, Gf enhancement in CVO of healthy control mice and increased enhancement under inflammatory conditions.
In summary, in this first in vivo MRI investigation visualizing inflammatory alterations in the CVO, Gf enhancement correlated to disease severity as well as to time of onset, indicating an active participation of the CVO in CNS inflammation. Gf enhancement in CVO was a good discriminator between healthy and EAE animals. Our results suggest that Gf enhancement could serve as sensitive marker for different hallmarks of CNS inflammation, including molecular "arming", immune cell recruitment, BBB breakdown and tissue damage. However, extensive preclinical testing is needed before a clinical application of Gf. This is eligible since Gf could not only facilitate the paraclinical diagnosis and follow-up of neuroinflammatory diseases such as MS but may also help to elucidate basic principles of CNS immunity.
We thank Susanne Mueller and Susan Pikol for assistance with animal scanning, Nancy Nowakowksi for animal care, and Martina Paetzel for correcting this manuscript as native English speaker. The authors declare no conflict of interest.
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