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.

Non-Invasive Evaluation of Retinal Vascular Alterations in a Mouse Model of Optic Neuritis Using Laser Speckle Flowgraphy and Optical Coherence Tomography Angiography.

Optic neuritis, a characteristic feature of multiple sclerosis (MS), involves the inflammation of the optic nerve and the degeneration of retinal ganglion cells (RGCs). Although previous studies suggest that retinal blood flow alterations occur during optic neuritis, the precise location, the degree of impairment, and the underlying mechanisms remain unclear. In this study, we utilized two emerging non-invasive imaging techniques, laser speckle flowgraphy (LSFG) and optical coherence tomography angiography (OCTA), to investigate retinal vascular changes in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). We associated these changes with leukostasis, RGC injury, and the overall progression of EAE. LSFG imaging revealed a progressive reduction in retinal blood flow velocity and increased vascular resistance near the optic nerve head in the EAE model, indicating impaired ocular blood flow. OCTA imaging demonstrated significant decreases in vessel density, number of junctions, and total vessel length in the intermediate and deep capillary plexus of the EAE mice. Furthermore, our analysis of leukostasis revealed a significant increase in adherent leukocytes in the retinal vasculature of the EAE mice, suggesting the occurrence of vascular inflammation in the early development of EAE pathology. The abovechanges preceded or were accompanied by the characteristic hallmarks of optic neuritis, such as RGC loss and reduced visual acuity. Overall, our study sheds light on the intricate relationship between retinal vascular alterations and the progression of optic neuritis as well as MS clinical score. It also highlights the potential for the development of image-based biomarkers for the diagnosis and monitoring of optic neuritis as well as MS, particularly in response to emerging treatments.

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.

Contribution of Intravital Neuroimaging to Study Animal Models of Multiple Sclerosis.

Multiple sclerosis (MS) is a complex and long-lasting neurodegenerative disease of the central nervous system (CNS), characterized by the loss of myelin within the white matter and cortical fibers, axonopathy, and inflammatory responses leading to consequent sensory-motor and cognitive deficits of patients. While complete resolution of the disease is not yet a reality, partial tissue repair has been observed in patients which offers hope for therapeutic strategies. To address the molecular and cellular events of the pathomechanisms, a variety of animal models have been developed to investigate distinct aspects of MS disease. Recent advances of multiscale intravital imaging facilitated the direct in vivo analysis of MS in the animal models with perspective of clinical transfer to patients. This review gives an overview of MS animal models, focusing on the current imaging modalities at the microscopic and macroscopic levels and emphasizing the importance of multimodal approaches to improve our understanding of the disease and minimize the use of animals.

High B-value diffusion tensor imaging for early detection of hippocampal microstructural alteration in a mouse model of multiple sclerosis.

Several studies have highlighted the value of diffusion tensor imaging (DTI) with strong diffusion weighting to reveal white matter microstructural lesions, but data in gray matter (GM) remains scarce. Herein, the effects of b-values combined with different numbers of diffusion-encoding directions (NDIRs) on DTI metrics to capture the normal hippocampal microstructure and its early alterations were investigated in a mouse model of multiple sclerosis (experimental autoimmune encephalomyelitis [EAE]). Two initial DTI datasets (B2700-43Dir acquired with b = 2700 s.mm-2 and NDIR = 43; B1000-22Dir acquired with b = 1000 s.mm-2 and NDIR = 22) were collected from 18 normal and 18 EAE mice at 4.7 T. Three additional datasets (B2700-22Dir, B2700-12Dir and B1000-12Dir) were extracted from the initial datasets. In healthy mice, we found a significant influence of b-values and NDIR on all DTI metrics. Confronting unsupervised hippocampal layers classification to the true anatomical classification highlighted the remarkable discrimination of the molecular layer with B2700-43Dir compared with the other datasets. Only DTI from the B2700 datasets captured the dendritic loss occurring in the molecular layer of EAE mice. Our findings stress the needs for both high b-values and sufficient NDIR to achieve a GM DTI with more biologically meaningful correlations, though DTI-metrics should be interpreted with caution in these settings.

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.

Characterization of Radioiodinated Diaryl Oxadiazole Derivatives as SPECT Probes for Detection of Myelin in Multiple Sclerosis.

Multiple sclerosis (MS) is an intractable disease of the central nervous system that results from destruction of the myelin sheath. Direct measurement of de- and remyelination is required for monitoring the disease stage of MS, but no useful method has been established. In this study, we characterized four diaryl oxadiazole derivatives as novel myelin-imaging probes for single photon emission computed tomography (SPECT). All the diaryl oxadiazole derivatives penetrated the blood-brain barrier in normal mice. Among them, the highest ratio of radioactivity accumulation in the white matter (myelin-rich region) against the gray matter (myelin-deficient region) was observed at 60 min postinjection of [125I]1,3,4-PODP-DM in ex vivo autoradiography using normal mice. In the blocking study with ex vivo autoradiography, the radioactivity accumulation of [125I]1,3,4-PODP-DM in the white matter markedly reduced. [125I]1,3,4-PODP-DM detected demyelination in the ex vivo autoradiographic images of not only the spinal cord of the experimental autoimmune encephalomyelitis mice but also the brain after lysophosphatidylcholine (LPC) injection. In addition, [123I]1,3,4-PODP-DM could image LPC-induced demyelination in the mouse brain with SPECT. These results suggest that [123I]1,3,4-PODP-DM may be a potential SPECT probe for imaging myelin in MS.

Low Frequency Ultrasound With Injection of NMO-IgG and Complement Produces Lesions Different From Experimental Autoimmune Encephalomyelitis Mice.

Neuromyelitis optica spectrum disorder (NMOSD), a relapsing autoimmune disease of the central nervous system, mainly targets the optic nerve and spinal cord. To date, all attempts at the establishment of NMOSD animal models have been based on neuromyelitis optica immunoglobulin G antibody (NMO-IgG) and mimic the disease in part. To solve this problem, we developed a rodent model by opening the blood-brain barrier (BBB) with low frequency ultrasound, followed by injection of NMO-IgG from NMOSD patients and complement to mice suffering pre-existing neuroinflammation produced by experimental autoimmune encephalomyelitis (EAE). In this study, we showed that ultrasound with NMO-IgG and complement caused marked inflammation and demyelination of both spinal cords and optic nerves compared to blank control group, as well as glial fibrillary acidic protein (GFAP) and aquaporin-4 (AQP4) loss of spinal cords and optic nerves compared to EAE mice and EAE mice with only BBB opening. In addition, magnetic resonance imaging (MRI) revealed optic neuritis with spinal cord lesions. We further demonstrated eye segregation defects in the dorsal lateral geniculate nucleus (dLGN) of these NMOSD mice.

Semi-Automated Live Tracking of Microglial Activation in CX3CR1GFP Mice During Experimental Autoimmune Encephalomyelitis by Confocal Scanning Laser Ophthalmoscopy.

Confocal scanning laser ophthalmoscopy (cSLO) is a non-invasive technique for real-time imaging of the retina. We developed a step-by-step protocol for the semi-automatic evaluation of myeloid cells in cSLO images from CX3CR1GFP mice, expressing green fluorescent protein (GFP) under control of the endogenous CX3C chemokine receptor 1 locus. We identified cSLO parameters allowing us to distinguish animals with experimental autoimmune encephalomyelitis (EAE) from sham-treated/naïve animals. Especially cell count (CC) and the total microglial area (SuA) turned out to be reliable parameters. Comparing the cSLO results with clinical parameters, we found significant correlations between the clinical EAE score and the SuA and of the inner retinal layer thickness, measured by optical coherence tomography, with the CC as well as the SuA. As a final step, we performed immunohistochemistry to confirm that the GFP-expressing cells visualized by the cSLO are Iba1 positive and validated the step-by-step protocol against manual counting. We present a semi-automatic step-by-step protocol with a balance between fast data evaluation and adequate accuracy, which is optimized by the option to manually adapt the contrast threshold. This protocol may be useful for numerous research questions on the role of microglial polarization in models of inflammatory and degenerating CNS diseases involving the retina.

Susceptibility weighted imaging detects prominent veins that precede or coincide with maximal motor disability in a model of multiple sclerosis: A pilot study.

BACKGROUND: Susceptibility weighted imaging (SWI) has detected veins in the center of white matter lesions and alterations in veins themselves in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). However, the relationship between SWI-detected venous alterations and disease progression is unclear. The objective of this study was to assess alterations in the lumbar spinal cord veins in EAE mice over the disease course using serial SWI. METHODS: EAE mice (n = 8) underwent imaging for SWI using a 9.4T Bruker Avance console at baseline, 7 days (pre-motor dysfunction), 12 days (typical motor dysfunction onset), and 16-18 days (typical peak disease) post-immunization. Naïve controls were imaged alongside EAE mice (n = 3). SWI hypointensities were counted by two subjects and compared between time points. RESULTS: SWI hypointensities appeared before motor dysfunction onset in most EAE mice. The ratio of SWI hypointensities to baseline was highly variable for EAE mice (0.45-6.75) while less so for controls (0.80-1.31). The time point for the maximum number of SWI hypointensities always preceded or coincided with maximum motor disability. CONCLUSION: Venous alterations are detected before the onset of motor disability in some EAE mice using SWI which may relate to inflammation and/or tissue hypoxia.

ELTD1 as a biomarker for multiple sclerosis: Pre-clinical molecular-targeted studies in a mouse experimental autoimmune encephalomyelitis model.

Multiple sclerosis (MS) and glioblastoma (GBM) are two distinct diseases that affect the central nervous system (CNS). However, perturbation in CNS vasculature are hallmarks of both diseases. ELTD1 (epidermal growth factor, latrophilin, and 7 transmembrane domain containing protein 1 on chromosome 1) is associated with vascular development, and has been linked with tumor angiogenesis. In glioblastomas, we detected over-expression of ELTD1, and found that an antibody targeting ELTD1 could increase animal survival and decrease tumor volumes in a xenograft GBM model. RNA-seq analysis of the preclinical data in the model for GBM identified that some of the molecular pathways affected by the anti-ELTD1 antibody therapy are also found to be associated with MS. In this study, we used molecular-targeted (mt) MR imaging and immunohistochemistry to assess ELTD1 levels in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Specifically, we found that ELTD1 is readily detected in the brains of mice with EAE and is predominantly found in the corpus callosum. In addition, we found that the blood-brain barrier (BBB) was compromised in the brains of EAE mice using contrast-enhanced MRI (CE-MRI), as well as altered relative cerebral blood flow (rCBF) in the brains and cervical spinal cords of these mice using perfusion imaging, compared to controls. These findings indicate that ELTD1 may be a promising biomarker for CNS-inflammation in MS.

Subcutaneous anti-CD20 antibody treatment delays gray matter atrophy in human myelin oligodendrocyte glycoprotein-induced EAE mice.

BACKGROUND: The human myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (huMOG-EAE) model, generates B-cell driven demyelination in mice, making it a suitable multiple sclerosis model to study B cell depletion. OBJECTIVES: We investigated the effect of subcutaneous anti-CD20 antibody treatment on huMOG-EAE gray matter (GM) pathology. METHODS: C57Bl/6, 8-week old mice were immunized with 200 huMOG1-125 and treated with 50 µg/mouse of anti-CD20 antibody (n = 16) or isotype control (n = 16). Serial brain volumetric 9.4 T MRI scans was performed at baseline, 1 and 5 wkPI. Disease severity was measured by clinical disability score (CDS) and performance on rotarod test. RESULTS: Anti-CD20 antibody significantly reduced brain volume loss compared with the isotype control across all timepoints longitudinally in the basal ganglia (p = 0.01), isocortex (p = 0.025) and thalamus (p = 0.023). The CDS was reduced significantly with anti-CD20 antibody vs. the isotype control at 3 (p = 0.003) and 4 (p = 0.03) wkPI, while a trend was observed at 5 (p = 0.057) and 6 (p = 0.086) wkPI. Performance on rotarod was also improved significantly at 3 (p = 0.007) and 5 (p = 0.01) wkPI compared with the isotype control. At cellular level, anti-CD20 therapy suppressed the percentage of proliferative nuclear antigen positive microglia in huMOG-EAE isocortex (p = 0.016). Flow cytometry confirmed that anti-CD20 antibody strongly depleted the CD19-expressing B cell fraction in peripheral blood mononuclear cells, reducing it from 39.7% measured in isotype control to 1.59% in anti-CD20 treated mice (p < 0.001). CONCLUSIONS: Anti-CD20 antibody treatment delayed brain tissue neurodegeneration in GM, and showed clinical benefit on measures of disease severity in huMOG-EAE mice.

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.

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.

[Met5]-enkephalin preserves diffusion metrics in EAE mice.

Multiple sclerosis is a chronic progressive neurological disorder that has few distinctive biomarkers associated with disease progression or response to therapy. This research investigated whether non-invasive imaging correlated with animal behavior and morphological indicators of disease in response to serum levels of [Met5]-enkephalin. Using the experimental autoimmune encephalomyelitis (EAE) model, adult female C57BL/6 J mice were randomized to receive daily injections of 0.1 mg/kg naltrexone (NTX) (= low dose naltrexone, LDN), 10 mg/kg Opioid Growth Factor (OGF) (chemically termed [Met5]-enkephalin) or saline beginning at the time of disease induction. Daily composite behavior scores were recorded over a 30-day period based on tail tone, gait, righting reflex, and limb strength. Prior to disease onset (day 7), and at peak disease (day 18), mice were imaged and tissues (blood and spinal cord) collected at day 30 for serum analyses of OGF and morphology. Serum OGF levels of EAE mice treated with saline were significantly reduced from baseline and from normal mice. Longitudinal cohort data demonstrated an increase in fractional anisotropy in all cohorts by day 18. There was a significant decrease in radial diffusivity in the saline group seen at day 18 whereas the axial diffusivity was not altered amongst treatment groups. Treatment with OGF or LDN resulted in mean diffusivity rates that were comparable to baseline (normal) levels at days 7 and 18. Luxol fast blue staining of the lumbar spinal cords demonstrated a 16 % reduction in myelin staining in saline treated EAE animals when compared to OGF and LDN treated EAE mice. Immunohistochemistry with Olig2 (pan-oligodendrocyte marker) and myelin basic protein (MBP) revealed that OGF and LDN treatment restored the area (%) of MBP and number of oligodendrocytes to that of normal spinal cord (∼75 %). Saline treated EAE mice had more demyelination and fewer oligodendrocytes than normal mice. Collectively, these data suggest that a panel of biomarkers including imaging, serum biomarker levels, and behavior correlate with progression of disease, and may begin to validate use of specific non-invasive markers for MS.

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.

Experimental mouse model of NMOSD produced by facilitated brain delivery of NMO-IgG by microbubble-enhanced low-frequency ultrasound in experimental allergic encephalomyelitis mice.

Although optic neuritis and myelitis are the core clinical characteristics of neuromyelitis optica spectrum disorders (NMOSD), appropriate animal models of NMOSD with myelitis and optic neuritis are lacking. we developed a mouse model of NMOSD by intravenously injecting 100 µg neuromyelitis optica immunoglobulin G antibody (NMO-IgG) and complement into experimental allergic encephalomyelitis (EAE) mice after reversible blood-brain barrier (BBB) opening by microbubble-enhanced low-frequency ultrasound (MELFUS). Animals were assessed by histopathology. We found noticeable inflammation and demyelination concomitant with the loss of aquaporin-4 (AQP4) and glial fibrillary acidic protein (GFAP) expression in the spinal cord, brain and optic nerve, as well as human IgG and C9neo deposition. Thus, with the help of MELFUS, we established an NMOSD mouse model with the core lesions of NMOSD by applying a considerably lower dose of human NMO-IgG, which may help identify the pathogenesis and facilitate the development of other neuroimmune disease models in the future.

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.