Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system that leads to pathologic changes in the brain. Blood brain barrier (BBB) dysregulation and transendothelial migration of activated leukocytes are among the earliest cerebrovascular abnormalities seen in MS and parallel the release of inflammatory cytokines [1]. However, the precise mechanisms underlying BBB breakdown have not been completely elucidated. MRI, a widely used non-invasive tool for diagnosing and characterizing MS pathology, is capable of quantifying BBB permeability. Gadolinium (Gd), an intravascular contrast agent, can be injected into the circulatory system and only extravasates into the brain parenchyma in regions of BBB compromise. Subsequently, T1-weighted (T1W) MRI can be used to visualize contrast agent uptake. In this study, we used MRI and lysine fixable dextran tracers to characterize the spatial and temporal profile of BBB permeability in the non-obese diabetic experimental allergic encephalomyelitis (NOD-EAE) mouse model of secondary progressive MS (SPMS). Together, the NOD-EAE model and MRI offer an invaluable toolbox to not only quantify BBB breakdown in disease contexts and test the efficacy of potential therapeutic interventions, but also to validate therapeutic benefit for translational purposes in the clinic.10-week-old female NOD/ShlTJ mice were immunized with an emulsion of MOG_35-55 peptide (150 µg/mouse) in complete Freund’s adjuvant containing 0.6 mg Mycobacterium tuberculosis delivered by subcutaneous injection. Bordetella pertussis toxin was administered via intraperitoneal (IP) injection on Day 0 and Day 2 at 150 ng/animal in 200 µL of PBS. Following EAE induction, the mice were monitored and scored daily for paralytic symptoms. MRI was performed using a 7T-Bruker scanner and 50-mm volume coil at 35 (n=4), 50 (n=5), and 80 (n=3) days post-induction. Naïve NOD mice served as the control group (n=3). At each time-point, T1W images were acquired pre-injection and from 3 minutes up to 1 hour after IP injection of Gd (0.5 mmol/kg) using RARE sequence (TR/TE=800/8.5 ms, 10 coronal slices, 0.75 mm thickness, matrix size=256×256, FOV=25 mm). The mice were then injected with 1 mg dextran tracer (biotin, 10000 mw) via cardiac injection to assess the BBB functionality using histology. For data analysis, 1-hour post-Gd injection was chosen to compare contrast uptake in the brain for all the time-points due to longer retention of contrast especially at the later disease stages. Regions of interest (ROI) were drawn around the contrast-enhanced regions within the brain parenchyma (excluding the pituitary gland) in all slices that exhibited contrast uptake relative to the corresponding pre-injection slice. The percent change in signal-to-noise-ratio (SNR) of the ROI relative to pre-contrast and the approximate volume of contrast extravasation (product of ROI area and slice thickness) was calculated for each animal at the respective time-points.The mean disease scores followed the appropriate clinical course for the NOD-EAE model of SPMS (Fig. 1). The NOD-EAE mice displayed varying degrees of Gd uptake into the brain parenchyma at different time-points after disease induction indicating a compromise of the BBB (Fig. 2). Previous works have characterized brain lesions in the NOD-EAE model at single time-points in late disease [2,3]. However, BBB permeability across disease progression has yet to be investigated. We quantified the extent of Gd uptake into the brain parenchyma extending from the corpus callosum to the cerebellum. One-way ANOVA demonstrated a significant difference in the volume of extravasation (p<0.001) and percent change in SNR at 1-hour post-Gd injection (p<0.05) between the mice at different days post-induction (Fig. 3). Naïve mice showed minimal Gd uptake primarily around the ventricles. Larger volumes of Gd extravasation were observed with disease progression (Fig. 3A), suggesting either a significant increase in area of BBB breakdown or an enhanced ability for Gd to diffuse from sites of BBB compromise through the brain parenchyma. SNR changes at the 1-hour time-point showed significant increase in signal intensities at days 50 and 80 compared to naïve (Fig. 3B). This clearly demonstrates that a greater concentration of Gd extravasated into the brain parenchyma, suggesting an increase in either paracellular or transcellular transport. Following MRI, the dextran tracer was easily visualized using immunohistochemistry (Fig. 4). The NOD-EAE mice clearly exhibited tracer extravasation into the brain parenchyma indicating a compromised BBB.The NOD-EAE mouse model showed an increase in extravasation volume of the contrast agent across the BBB with progressing disease after EAE induction which correlates with the clinical score pattern. SNR change in late disease stage clearly indicates a difference in the transport mechanism across the BBB. The quantifications presented herein can be used to potentially test the effect of therapeutic agents on BBB breakdown.