Resting-state functional magnetic resonance imaging reveals functional connectivity alteration in the experimental autoimmune encephalomyelitis model of multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune degenerative disease targeting white matter in the central nervous system. The most common animal model that mimics MS is experimental autoimmune encephalomyelitis (EAE) and it plays a crucial role in pharmacological research, from the identification of a therapeutic target to the in vivo validation of efficacy. Magnetic resonance imaging (MRI) is largely used to detect MS lesions, and resting-state functional MRI (rsfMRI) to investigate alterations in the brain functional connectivity (FC). MRI was mainly used in EAE studies to detect lesions in the spinal cord and brain. The current longitudinal MRI study aims to validate rsfMRI as a biomarker of the disease progression in the myelin oligodendrocyte glycoprotein 35-55 induced EAE animal model of MS. MR images were acquired 14, 25, and 50 days postimmunization. Seed-based analysis was used to investigate the whole-brain FC with some predefined areas, such as the thalamic regions, cerebellum, motor and somatosensory cortex. When compared with the control group, the EAE group exhibited a slightly altered FC and a decreasing trend in the total number of activated voxels along the disease progression. The most interesting result regards the whole-brain FC with the cerebellum. A hyperconnectivity behavior was found at an early phase and a significant reduced connectivity at a late phase. Moreover, we found a negative correlation between the total number of activated voxels during the late phase and the cumulative disease index. The results obtained provide a clinically relevant experimental platform that may be pivotal for the elucidation of the key mechanisms of accumulation of irreversible disability, as well as the development of innovative therapies for MS. Moreover, the negative correlation between the disease severity and the size of the activated area suggests a possible research pathway to follow for the resolution of the clinico-radiological paradox.

Cortical matrix remodeling as a hallmark of relapsing-remitting neuroinflammation in MR elastography and quantitative MRI.

Multiple sclerosis (MS) is a chronic neuroinflammatory disease that involves both white and gray matter. Although gray matter damage is a major contributor to disability in MS patients, conventional clinical magnetic resonance imaging (MRI) fails to accurately detect gray matter pathology and establish a clear correlation with clinical symptoms. Using magnetic resonance elastography (MRE), we previously reported global brain softening in MS and experimental autoimmune encephalomyelitis (EAE). However, it needs to be established if changes of the spatiotemporal patterns of brain tissue mechanics constitute a marker of neuroinflammation. Here, we use advanced multifrequency MRE with tomoelastography postprocessing to investigate longitudinal and regional inflammation-induced tissue changes in EAE and in a small group of MS patients. Surprisingly, we found reversible softening in synchrony with the EAE disease course predominantly in the cortex of the mouse brain. This cortical softening was associated neither with a shift of tissue water compartments as quantified by T2-mapping and diffusion-weighted MRI, nor with leukocyte infiltration as seen by histopathology. Instead, cortical softening correlated with transient structural remodeling of perineuronal nets (PNNs), which involved abnormal chondroitin sulfate expression and microgliosis. These mechanisms also appear to be critical in humans with MS, where tomoelastography for the first time demonstrated marked cortical softening. Taken together, our study shows that neuroinflammation (i) critically affects the integrity of PNNs in cortical brain tissue, in a reversible process that correlates with disease disability in EAE, (ii) reduces the mechanical integrity of brain tissue rather than leading to water accumulation, and (iii) shows similar spatial patterns in humans and mice. These results raise the prospect of leveraging MRE and quantitative MRI for MS staging and monitoring treatment in affected patients.

Intestinal MAdCAM-1 imaging as biomarker for prognostic in murine models of multiple sclerosis.

INTRODUCTION: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system. Recent evidence suggests that lymphocyte trafficking in the intestines could play a key role in its etiology. Nevertheless, it is not clear how intestinal tissue is involved in the disease onset nor its evolution. In the present study, we aimed to evaluate intestinal inflammation dynamic throughout the disease course and its potential impact on disease progression. METHODS: We used tissue immunophenotyping (immunohistofluorescence and flow cytometry) and a recently described molecular magnetic resonance imaging (MRI) method targeting mucosal addressin cell adhesion molecule-1 (MAdCAM-1) to assess intestinal inflammation in vivo in two distinct animal models of MS (Experimental Autoimmune Encephalomyelitis - EAE) at several time points of disease progression. RESULTS: We report a positive correlation between disease severity and MAdCAM-1 MRI signal in two EAE models. Moreover, high MAdCAM-1 MRI signal during the asymptomatic phase is associated with a delayed disease onset in progressive EAE and to a lower risk of conversion to a secondary-progressive form in relapsing-remitting EAE. During disease evolution, in line with a bi-directional immune communication between the gut and the central nervous system, we observed a decrease in T-CD4+ and B lymphocytes in the ileum concomitantly with their increase in the spinal cord. CONCLUSION: Altogether, these data unveil a crosstalk between intestinal and central inflammation in EAE and support the use of molecular MRI of intestinal MAdCAM-1 as a new biomarker for prognostic in MS patients.

Multiparametric magnetic resonance imaging for detection of pathological changes in the central nervous system of a mouse model of multiple sclerosis in vivo.

Multiple sclerosis (MS) is an autoimmune disease involving demyelination and axonal damage in the central nervous system (CNS). In this study, we investigated pathological changes in the lumbar spinal cord of C57BL/6 mice induced with progressive experimental autoimmune encephalomyelitis (EAE) disease using 9.4-T magnetic resonance imaging (MRI). Multiparametric MRI measurements including MR spectroscopy, diffusion tensor imaging (DTI) and volumetric analyses were applied to detect metabolic changes in the CNS of EAE mice. Compared with healthy mice, EAE mice showed a significant reduction in N-acetyl aspartate and increases in choline, glycine, taurine and lactate. DTI revealed a significant reduction in fractional anisotropy and axial diffusivity and an increase in radial diffusivity in the lumbar spinal cord white matter (WM), while in the grey matter (GM), fractional anisotropy increased. High-resolution structural imaging also revealed lumbar spinal cord WM hypertrophy and GM atrophy. Importantly, these MRI changes were strongly correlated with EAE disease scoring and pathological changes in the lumbar (L2-L6), particularly WM demyelination lesions and aggregation of immune cells (microglia/macrophages and astrocytes) in this region. This study identified changes in MRI biomarker signatures that can be useful for evaluating the efficacy of novel drugs using EAE models in vivo.

Non invasive in vivo monitoring of dimethyl fumarate treatment in EAE by assessing the glucose metabolism in secondary lymphoid organs.

[18 F]FDG-PET/CT is a high sensitive functional diagnostic imaging modality to monitor tumor but also immune cell activation by determination of the glucose metabolism. Our results show that the anti-inflammatory effects of immunotherapeutics like DMF can be assessed non invasively in vivo during Th1/Th17 cell-mediated encephalomyelitis (EAE) by [18 F]FDG-PET/CT imaging of the draining lymph nodes.

CEST MRI and MALDI imaging reveal metabolic alterations in the cervical lymph nodes of EAE mice.

BACKGROUND: Multiple sclerosis (MS) is a neurodegenerative disease, wherein aberrant immune cells target myelin-ensheathed nerves. Conventional magnetic resonance imaging (MRI) can be performed to monitor damage to the central nervous system that results from previous inflammation; however, these imaging biomarkers are not necessarily indicative of active, progressive stages of the disease. The immune cells responsible for MS are first activated and sensitized to myelin in lymph nodes (LNs). Here, we present a new strategy for monitoring active disease activity in MS, chemical exchange saturation transfer (CEST) MRI of LNs. METHODS AND RESULTS: We studied the potential utility of conventional (T2-weighted) and CEST MRI to monitor changes in these LNs during disease progression in an experimental autoimmune encephalomyelitis (EAE) model. We found CEST signal changes corresponded temporally with disease activity. CEST signals at the 3.2 ppm frequency during the active stage of EAE correlated significantly with the cellular (flow cytometry) and metabolic (mass spectrometry imaging) composition of the LNs, as well as immune cell infiltration into brain and spinal cord tissue. Correlating primary metabolites as identified by matrix-assisted laser desorption/ionization (MALDI) imaging included alanine, lactate, leucine, malate, and phenylalanine. CONCLUSIONS: Taken together, we demonstrate the utility of CEST MRI signal changes in superficial cervical LNs as a complementary imaging biomarker for monitoring disease activity in MS. CEST MRI biomarkers corresponded to disease activity, correlated with immune activation (surface markers, antigen-stimulated proliferation), and correlated with LN metabolite levels.

Fibrin-targeting molecular MRI in inflammatory CNS disorders.

BACKGROUND: Fibrin deposition is a fundamental pathophysiological event in the inflammatory component of various CNS disorders, such as multiple sclerosis (MS) and Alzheimer's disease. Beyond its traditional role in coagulation, fibrin elicits immunoinflammatory changes with oxidative stress response and activation of CNS-resident/peripheral immune cells contributing to CNS injury. PURPOSE: To investigate if CNS fibrin deposition can be determined using molecular MRI, and to assess its capacity as a non-invasive imaging biomarker that corresponds to inflammatory response and barrier impairment. MATERIALS AND METHODS: Specificity and efficacy of a peptide-conjugated Gd-based molecular MRI probe (EP2104-R) to visualise and quantify CNS fibrin deposition were evaluated. Probe efficacy to specifically target CNS fibrin deposition in murine adoptive-transfer experimental autoimmune encephalomyelitis (EAE), a pre-clinical model for MS (n = 12), was assessed. Findings were validated using immunohistochemistry and laser ablation inductively coupled plasma mass spectrometry. Deposition of fibrin in neuroinflammatory conditions was investigated and its diagnostic capacity for disease staging and monitoring as well as quantification of immunoinflammatory response was determined. Results were compared using t-tests (two groups) or one-way ANOVA with multiple comparisons test. Linear regression was used to model the relationship between variables. RESULTS: For the first time (to our knowledge), CNS fibrin deposition was visualised and quantified in vivo using molecular imaging. Signal enhancement was apparent in EAE lesions even 12-h after administration of EP2104-R due to targeted binding (M ± SD, 1.07 ± 0.10 (baseline) vs. 0.73 ± 0.09 (EP2104-R), p = .008), which could be inhibited with an MRI-silent analogue (M ± SD, 0.60 ± 0.14 (EP2104-R) vs. 0.96 ± 0.13 (EP2104-La), p = .006). CNS fibrin deposition corresponded to immunoinflammatory activity (R2 = 0.85, p < .001) and disability (R2 = 0.81, p < .001) in a model for MS, which suggests a clinical role for staging and monitoring. Additionally, EP2104-R showed substantially higher SNR (M ± SD, 6.6 ± 1 (EP2104-R) vs. 2.7 ± 0.4 (gadobutrol), p = .004) than clinically used contrast media, which increases sensitivity for lesion detection. CONCLUSIONS: Molecular imaging of CNS fibrin deposition provides an imaging biomarker for inflammatory CNS pathology, which corresponds to pathophysiological ECM remodelling and disease activity, and yields high signal-to-noise ratio, which can improve diagnostic neuroimaging across several neurological diseases with variable degrees of barrier impairment.

In vivo imaging of CNS microglial activation/macrophage infiltration with combined [18F]DPA-714-PET and SPIO-MRI in a mouse model of relapsing remitting experimental autoimmune encephalomyelitis.

PURPOSE: To evaluate the feasibility and sensitivity of multimodality PET/CT and MRI imaging for non-invasive characterization of brain microglial/macrophage activation occurring during the acute phase in a mouse model of relapsing remitting multiple sclerosis (RR-MS) using [18F]DPA-714, a selective radioligand for the 18-kDa translocator protein (TSPO), superparamagnetic iron oxide particles (SPIO), and ex vivo immunohistochemistry. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced in female SJL/J mice by immunization with PLP139-151. Seven symptomatic EAE mice and five controls underwent both PET/CT and MRI studies between 11 and 14 days post-immunization. SPIO was injected i.v. in the same animals immediately after [18F]DPA-714 and MRI acquisition was performed after 24 h. Regional brain volumes were defined according to a mouse brain atlas on co-registered PET and SPIO-MRI images. [18F]DPA-714 standardized uptake value (SUV) ratios (SUVR), with unaffected neocortex as reference, and SPIO fractional volumes (SPIO-Vol) were generated. Both SUVR and SPIO-Vol values were correlated with the clinical score (CS) and among them. Five EAE and four control mice underwent immunohistochemical analysis with the aim of identifying activated microglia/macrophage and TSPO expressions. RESULTS: SUVR and SPIO-Vol values were significantly increased in EAE compared with controls in the hippocampus (p < 0.01; p < 0.02, respectively), thalamus (p < 0.02; p < 0.05, respectively), and cerebellum and brainstem (p < 0.02), while only SPIO-Vol was significantly increased in the caudate/putamen (p < 0.05). Both SUVR and SPIO-Vol values were positively significantly correlated with CS and among them in the same regions. TSPO/Iba1 and F4/80/Prussian blue staining immunohistochemistry suggests that increased activated microglia/macrophages underlay TSPO expression and SPIO uptake in symptomatic EAE mice. CONCLUSIONS: These preliminary results suggest that both activated microglia and infiltrated macrophages are present in vulnerable brain regions during the acute phase of PLP-EAE and contribute to disease severity. Both [18F]DPA-714-PET and SPIO-MRI appear suitable modalities for preclinical study of neuroinflammation in MS mice models.

Diffusion Kurtosis Imaging maps neural damage in the EAE model of multiple sclerosis.

Diffusion kurtosis imaging (DKI) is an imaging modality that yields novel disease biomarkers and in combination with nervous tissue modeling, provides access to microstructural parameters. Recently, DKI and subsequent estimation of microstructural model parameters has been used for assessment of tissue changes in neurodegenerative diseases and associated animal models. In this study, mouse spinal cords from the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS) were investigated for the first time using DKI in combination with biophysical modeling to study the relationship between microstructural metrics and degree of animal dysfunction. Thirteen spinal cords were extracted from animals with varied grades of disability and scanned in a high-field MRI scanner along with five control specimen. Diffusion weighted data were acquired together with high resolution T2* images. Diffusion data were fit to estimate diffusion and kurtosis tensors and white matter modeling parameters, which were all used for subsequent statistical analysis using a linear mixed effects model. T2* images were used to delineate focal demyelination/inflammation. Our results reveal a strong relationship between disability and measured microstructural parameters in normal appearing white matter and gray matter. Relationships between disability and mean of the kurtosis tensor, radial kurtosis, radial diffusivity were similar to what has been found in other hypomyelinating MS models, and in patients. However, the changes in biophysical modeling parameters and in particular in extra-axonal axial diffusivity were clearly different from previous studies employing other animal models of MS. In conclusion, our data suggest that DKI and microstructural modeling can provide a unique contrast capable of detecting EAE-specific changes correlating with clinical disability.

Monitoring diffuse injury during disease progression in experimental autoimmune encephalomyelitis with on resonance variable delay multiple pulse (onVDMP) CEST MRI.

Multiple sclerosis (MS) is an autoimmune disorder that targets myelin proteins and results in extensive damage in the central nervous system in the form of focal lesions as well as diffuse molecular changes. Lesions are currently detected using T1-weighted, T2-weighted, and gadolinium-enhanced magnetic resonance imaging (MRI); however, monitoring such lesions has been shown to be a poor predictor of disease progression. Chemical exchange saturation transfer (CEST) MRI is sensitive to many of the biomolecules in the central nervous system altered in MS that cannot be detected using conventional MRI. We monitored disease progression in an experimental autoimmune encephalomyelitis (EAE) model of MS using on resonance variable delay multiple pulse (onVDMP) CEST MRI. Alterations in onVDMP signal were observed in regions responsible for hindlimb function throughout the central nervous system. Histological analysis revealed glial activation in areas highlighted in onVDMP CEST MRI. onVDMP signal changes in the 3rd ventricle preceded paralysis onset that could not be observed with conventional MRI techniques. Hence, the onVDMP CEST MRI signal has potential as a novel imaging biomarker and predictor of disease progression in MS.

Comparison of different optomotor response readouts for visual testing in experimental autoimmune encephalomyelitis-optic neuritis.

Optomotor response is increasingly used in preclinical research for evaluating the visual function in rodents. However, the most suitable measuring protocol for specific scientific questions is not always established. We aimed to determine the optimal parameters for visual function analysis in experimental autoimmune encephalomyelitis optic neuritis (EAEON), an animal model for multiple sclerosis. Contrast sensitivity as well as spatial frequency both had a low variance and a good test-retest reliability. Also, both parameters were able to differentiate between the EAEON and the control group. Correlations with the retinal degeneration, assessed by optical coherence tomography, the infiltration of immune cells, and the clinical disability score revealed that spatial frequency was superior to contrast sensitivity analysis. We therefore conclude that spatial frequency testing is better suited as visual acuity assessment in C57Bl/6 J EAEON mice. Furthermore, contrast sensitivity measurements are more time consuming, possibly leading to more stress for the animals.

PET imaging of P2X7R in the experimental autoimmune encephalomyelitis model of multiple sclerosis using [11C]SMW139.

BACKGROUND: Non-invasive imaging of the activation status of microglia and the ability to identify a pro- or anti-inflammatory environment can provide valuable insights not only into pathogenesis of neuro-inflammatory and neurodegenerative diseases but also the monitoring of the efficacy of immunomodulatory therapies. P2X7R is highly expressed on pro-inflammatory microglia and [11C]SMW139, a specific P2X7R tracer for positron emission tomography imaging, showed good pharmacokinetics, stability, and brain permeability in vivo. Our objective was to evaluate the potential of [11C]SMW139 for PET imaging of neuroinflammation in vivo in the experimental autoimmune encephalomyelitis (EAE) model. METHODS: We induced EAE in Lewis rats by immunization with MBP 69-88 in complete Freund's adjuvant (CFA). We determined the affinity of [11C]SMW139 to human and rat P2X7R using saturation binding assay. Using this tracer, PET imaging was performed at the peak of disease and in the recovery phase. In vivo blocking experiments were conducted to validate the specific brain uptake of the tracer. Immunohistochemistry staining and autoradiography were performed to evaluate the level of neuroinflammation and validate the specific binding of [11C]SMW139. RESULTS: [11C]SMW139 showed good affinity for the rat P2X7R with a Kd of 20.6 ± 1.7 nM. The uptake of [11C]SMW139 was significantly higher in EAE animals at the peak of disease compared to the recovery phase but not in CFA control animals. The amplitude of increase of [11C]SMW139 uptake showed significant positive correlation with clinical scores mainly in the spinal cord (Pearson = 0.75, Spearman = 0.76; p < 0.0001). Treating EAE animals with P2X7R antagonist JNJ-47965567 blocked the uptake of [11C]SMW139 in the spinal cord, cerebellum, and brain stem, demonstrating specific accumulation of the tracer. P-glycoprotein blocking with tariquidar (30 mg/kg) did not affect tracer penetration in the brain showing that [11C]SMW139 is not a Pgp substrate. CONCLUSION: Our data shows that [11C]SMW139 is a promising PET tracer for imaging neuroinflammation and evaluating the dynamics of pro-inflammatory microglia in the brain. This can provide crucial insights into the role of microglia in disease progression and enables the development of novel treatment strategies aimed at modulating the immune response in order to promote neuroprotection.

Development of a CD19 PET tracer for detecting B cells in a mouse model of multiple sclerosis.

BACKGROUND: B cells play a central role in multiple sclerosis (MS) through production of injurious antibodies, secretion of pro-inflammatory cytokines, and antigen presentation. The therapeutic success of monoclonal antibodies (mAbs) targeting B cells in some but not all individuals suffering from MS highlights the need for a method to stratify patients and monitor response to treatments in real-time. Herein, we describe the development of the first CD19 positron emission tomography (PET) tracer, and its evaluation in a rodent model of MS, experimental autoimmune encephalomyelitis (EAE). METHODS: Female C57BL/6 J mice were induced with EAE through immunization with myelin oligodendrocyte glycoprotein (MOG1-125). PET imaging of naïve and EAE mice was performed 19 h after administration of [64Cu]CD19-mAb. Thereafter, radioactivity in organs of interest was determined by gamma counting, followed by ex vivo autoradiography of central nervous system (CNS) tissues. Anti-CD45R (B220) immunostaining of brain tissue from EAE and naïve mice was also conducted. RESULTS: Radiolabelling of DOTA-conjugated CD19-mAb with 64Cu was achieved with a radiochemical purity of 99% and molar activity of 2 GBq/µmol. Quantitation of CD19 PET images revealed significantly higher tracer binding in whole brain of EAE compared to naïve mice (2.02 ± 0.092 vs. 1.68 ± 0.06 percentage of injected dose per gram, % ID/g, p = 0.0173). PET findings were confirmed by ex vivo gamma counting of perfused brain tissue (0.22 ± 0.020 vs. 0.12 ± 0.003 % ID/g, p = 0.0010). Moreover, ex vivo autoradiography of brain sections corresponded with PET imaging results and the spatial distribution of B cells observed in B220 immunohistochemistry-providing further evidence that [64Cu]CD19-mAb enables visualization of B cell infiltration into the CNS of EAE mice. CONCLUSION: CD19-PET imaging can be used to detect elevated levels of B cells in the CNS of EAE mice, and has the potential to impact the way we study, monitor, and treat clinical MS.

The spectrum of spinal cord lesions in a primate model of multiple sclerosis.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) in the common marmoset is a nonhuman primate model of multiple sclerosis (MS) that shares numerous clinical, radiological, and pathological features with MS. Among the clinical features are motor and sensory deficits that are highly suggestive of spinal cord (SC) damage. OBJECTIVE: To characterize the extent and nature of SC damage in symptomatic marmosets with EAE using a combined magnetic resonance imaging (MRI) and histopathology approach. MATERIALS AND METHODS: SC tissues from five animals were scanned using 7 T MRI to collect high-resolution ex vivo images. Lesions were segmented and classified based on shape, size, and distribution along the SC. Tissues were processed for histopathological characterization (myelin and microglia/macrophages). Statistical analysis, using linear mixed-effects models, evaluated the association between MRI and histopathology. RESULTS: Marmosets with EAE displayed two types of SC lesions: focal and subpial lesions. Both lesion types were heterogeneous in size and configuration and corresponded to areas of marked demyelination with high density of inflammatory cells. Inside the lesions, the MRI signal was significantly correlated with myelin content (p < 0.001). CONCLUSIONS: Our findings underscore the relevance of this nonhuman primate EAE model for better understanding mechanisms of MS lesion formation in the SC.

Axonal damage in spinal cord is associated with gray matter atrophy in sensorimotor cortex in experimental autoimmune encephalomyelitis.

BACKGROUND: Gray matter (GM) atrophy in brain is one of the best predictors of long-term disability in multiple sclerosis (MS), and recent findings have revealed that localized GM atrophy is associated with clinical disabilities. GM atrophy associated with each disability mapped to a distinct brain region, revealing a disability-specific atlas (DSA) of GM loss. OBJECTIVE: To uncover the mechanisms underlying the development of localized GM atrophy. METHODS: We used voxel-based morphometry (VBM) to evaluate localized GM atrophy and Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) to evaluate specific pathologies in mice with experimental autoimmune encephalomyelitis (EAE). RESULTS: We observed extensive GM atrophy throughout the cerebral cortex, with additional foci in the thalamus and caudoputamen, in mice with EAE compared to normal controls. Next, we generated pathology-specific atlases (PSAs), voxelwise mappings of the correlation between specific pathologies and localized GM atrophy. Interestingly, axonal damage (end-bulbs and ovoids) in the spinal cord strongly correlated with GM atrophy in the sensorimotor cortex of the brain. CONCLUSION: The combination of VBM with CLARITY in EAE can localize GM atrophy in brain that is associated with a specific pathology in spinal cord, revealing a PSA of GM loss.

Assessing the anterior visual pathway in optic neuritis: recent experimental and clinical aspects.

PURPOSE OF REVIEW: Multiple sclerosis (MS) and related autoimmune disorders of the central nervous system such as neuromyelitis optica spectrum disorders (NMOSD) are characterized by chronic disability resulting from autoimmune neuroinflammation, with demyelination, astrocyte damage, impaired axonal transmission and neuroaxonal loss. Novel therapeutics stopping or reversing the progression of disability are still urgently warranted. This review addresses research on optic neuritis in preclinical experimental models and their translation to clinical trials. RECENT FINDINGS: Optic neuritis can be used as paradigm for an MS relapse which can serve to evaluate the efficacy of novel therapeutics in clinical trials with a reasonable duration and cohort size. The advantage is the linear structure of the visual pathway allowing the assessment of visual function and retinal structure as highly sensitive outcome parameters. Experimental autoimmune encephalomyelitis is an inducible, inflammatory and demyelinating central nervous system disease extensively used as animal model of MS. Optic neuritis is part of the clinicopathological manifestations in a number of experimental autoimmune encephalomyelitis models. These have gained increasing interest for studies evaluating neuroprotective and/or remyelinating substances as longitudinal, visual and retinal readouts have become available. SUMMARY: Translation of preclinical experiments, evaluating neuroprotective or remyelinating therapeutics to clinical studies is challenging. In-vivo readouts like optical coherence tomography, offers the possibility to transfer experimental study designs to clinical optic neuritis trials.

Myeloperoxidase Molecular MRI Reveals Synergistic Combination Therapy in Murine Experimental Autoimmune Neuroinflammation.

Background Despite advances in immunomodulatory agents, most current therapies for multiple sclerosis target lymphocytes or lymphocytic function. However, therapy response may be less than optimal due to demyelination and axonal damage caused by myeloid cells. Purpose To determine if myeloperoxidase (MPO) molecular MRI can evaluate whether combination therapy targeting both lymphoid and myeloid inflammation can improve autoimmune neuroinflammation compared with either drug alone, even at suboptimal doses. Materials and Methods Four groups of 94 female mice (8-10 weeks old) were induced with experimental autoimmune encephalomyelitis (EAE) from August 2, 2016, to March 30, 2018, and divided into saline control (n = 22), 4-aminobenzoic acid hydrazide (ABAH) therapy group (n = 19), glatiramer acetate (GA) therapy group (n = 22), and combination therapy group (n = 31). Mice were administered suboptimal doses of ABAH, an irreversible inhibitor of MPO; GA, a first-line multiple sclerosis drug; both ABAH and GA; or saline (control). Mice were imaged with bis-5-hydroxytryptamide-diethylenetriaminepentaacetate gadolinium (hereafter, MPO-Gd) MRI. One-way analysis of variance, two-way analysis of variance, Kurskal-Wallis, and log-rank tests were used. P < .05 was considered to indicate statistical significance. Results The combination-treated group showed delayed disease onset (day 11.3 vs day 9.8 for ABAH, day 10.4 for GA, day 9.9 for control; P < .05) and reduced disease severity (clinical score during the acute exacerbation period of 1.8 vs 3.8 for ABAH, 3.1 for GA, 3.9 for control; P < .05). The combination-treated group demonstrated fewer MPO-positive lesions (30.2 vs 73.7 for ABAH, 64.8 for GA, 67.2 for control; P < .05), smaller MPO-positive lesion volume (16.7 mm3 vs 65.2 mm3 for ABAH, 69.9 mm3 for GA, 66.0 mm3 for control; P < .05), and lower intensity of MPO-Gd lesion activation ratio (0.7 vs 1.9 for ABAH, 3.2 for GA, 2.3 for control; P < .05). Reduced disease severity in the combination group was confirmed at histopathologic analysis, where MPO expression (1779 vs 2673 for ABAH, 2898 for GA; P < .05) and demyelination (5.3% vs 9.0% for ABAH, 10.6% for GA; P < .05) were ameliorated. Conclusion Myeloperoxidase molecular MRI can track the treatment response from immunomodulatory drugs even if the drug does not directly target myeloperoxidase, and establishes that combination therapy targeting both myeloid and lymphocytic inflammation is effective for murine autoimmune neuroinflammation, even at suboptimal doses. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Walczak in this issue.

Folate receptor-targeted positron emission tomography of experimental autoimmune encephalomyelitis in rats.

BACKGROUND: Folate receptor-ß (FR-ß) is a cell surface receptor that is significantly upregulated on activated macrophages during inflammation and provides a potential target for folate-based therapeutic and diagnostic agents. FR-ß expression in central nervous system inflammation remains relatively unexplored. Therefore, we used focally induced acute and chronic phases of experimental autoimmune encephalomyelitis (EAE) to study patterns of FR-ß expression and evaluated its potential as an in vivo imaging target. METHODS: Focal EAE was induced in rats using heat-killed Bacillus Calmette-Guérin followed by activation with complete Freund's adjuvant supplemented with Mycobacterium tuberculosis. The rats were assessed with magnetic resonance imaging and positron emission tomography/computed tomography (PET/CT) at acute (14 days) and chronic (90 days) phases of inflammation. The animals were finally sacrificed for ex vivo autoradiography of their brains. PET studies were performed using FR-ß-targeting aluminum [18F]fluoride-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid conjugated folate ([18F]AlF-NOTA-folate, 18F-FOL) and 18 kDa translocator protein (TSPO)-targeting N-acetyl-N-(2-[11C]methoxybenzyl)-2-phenoxy-5-pyridinamine (11C-PBR28). Post-mortem immunohistochemistry was performed using anti-FR-ß, anti-cluster of differentiation 68 (anti-CD68), anti-inducible nitric oxide synthase (anti-iNOS), and anti-mannose receptor C-type 1 (anti-MRC-1) antibodies. The specificity of 18F-FOL binding was verified using in vitro brain sections with folate glucosamine used as a blocking agent. RESULTS: Immunohistochemical evaluation of focal EAE lesions demonstrated anti-FR-ß positive cells at the lesion border in both acute and chronic phases of inflammation. We found that anti-FR-ß correlated with anti-CD68 and anti-MRC-1 immunohistochemistry; for MRC-1, the correlation was most prominent in the chronic phase of inflammation. Both 18F-FOL and 11C-PBR28 radiotracers bound to the EAE lesions. Autoradiography studies verified that this binding took place in areas of anti-FR-ß positivity. A blocking assay using folate glucosamine further verified the tracer's specificity. In the chronic phase of EAE, the lesion-to-background ratio of 18F-FOL was significantly higher than that of 11C-PBR28 (P = 0.016). CONCLUSION: Our EAE results imply that FR-ß may be a useful target for in vivo imaging of multiple sclerosis-related immunopathology. FR-ß-targeted PET imaging with 18F-FOL may facilitate the monitoring of lesion development and complement the information obtained from TSPO imaging by bringing more specificity to the PET imaging armamentarium for neuroinflammation.

Anti-MOG autoantibodies pathogenicity in children and macaques demyelinating diseases.

BACKGROUND: Autoantibodies against myelin oligodendrocyte glycoprotein (anti-MOG-Abs) occur in a majority of children with acquired demyelinating syndromes (ADS) and physiopathology is still under investigation. As cynomolgus macaques immunized with rhMOG, all develop an experimental autoimmune encephalomyelitis (EAE), we assessed relatedness between anti-MOG-Abs associated diseases in both species. METHODS: The study includes 27 children followed for ADS and nine macaques with rhMOG-induced EAE. MRI lesions, cytokines in blood, and CSF at onset of ADS or EAE, as well as histopathological features of brain lesions were compared. RESULTS: Twelve children with anti-MOG-Abs ADS (ADS MOG+) and nine macaques with EAE, presented increased IL-6 and G-CSF in the CSF, whereas no such signature was found in 15 ADS MOG-. Furthermore, IgG and C1q were associated to myelin and phagocytic cells in brains with EAE (n = 8) and in biopsies of ADS MOG+ (n = 2) but not ADS MOG- children (n = 1). Macaque brains also revealed prephagocytic lesions with IgG and C1q depositions but no leukocyte infiltration. CONCLUSIONS: Children with ADS MOG+ and macaques with EAE induced with rhMOG, present a similar cytokine signature in the CSF and a comparable aspect of brain lesions indicating analogous pathophysiological processes. In EAE, prephagocytic lesions points at IgG as an initial effector of myelin attack. These results support the pertinence of modeling ADS MOG+ in non-human primates to apprehend the natural development of anti-MOG-associated disease, find markers of evolution, and above all explore the efficacy of targeted therapies to test primate-restricted molecules.

CEST MRI with distribution-based analysis for assessment of early stage disease activity in a mouse model of multiple sclerosis: An initial study.

Imaging biomarkers that can detect pathological changes at an early stage of multiple sclerosis (MS) may allow earlier therapeutic intervention with an improved outcome. Using a mouse model of MS, termed as experimental autoimmune encephalomyelitis (EAE), we performed chemical exchange saturation transfer (CEST) MRI at a very early stage before symptom onset (6 days post-induction) for assessment of changes in tissues that appear "normal" with conventional MRI. The collected CEST Z-spectra signals (Ssat /S0 ) were analyzed using a histogram-guided method to determine the contributions from various offset frequencies. Histogram analysis showed that EAE mice exhibit a more heterogeneous distribution with lower peak heights in the hindbrain compared with naïve mice at saturation offsets of 1 and 2 ppm. At these two offsets, both the mean Ssat /S0 and the mean MTRasym values in the cerebellum and brain stem are significantly different between EAE and naïve mice (P < 0.05). Immunofluorescent staining validated the presence of neuroinflammation, with IBA1-positive cells detected throughout the hindbrain including the cerebellum and brain stem. Follow-up MRI at the symptom onset (score = 1.5-2.5, 13 days post-induction) confirmed gadolinium-enhanced periventricular lesions. CEST Z-spectra signals also changed by this time. The proposed three-level histogram-oriented analysis is simple to execute and robust for detecting subtle changes in Z-spectra signals, which does not require a priori knowledge of damage locations or contributing offset components. CEST MRI signals at 1 and 2 ppm were sensitive to the subtle pathological changes at an early stage in EAE mice, and have potential as novel imaging biomarkers complementary to functional and physiological MRI measures.