Magnetic resonance imaging (MRI) allows a noninvasive assessment of treatment efficacy for multiple sclerosis (MS).^{1, 2} Diffusion basis spectrum imaging (DBSI) has successfully distinguished and quantified coexisting pathologies in MS and its animal models.^3-6 Herein, DBSI derived “axon volume”, i.e., DBSI fiber fraction multiplying ROI-based nerve volume on diffusion-weighted images, was used to serve as an outcome measure to assess therapeutic efficacy of Lenaldeker (LDK)⁷ and rapamycin (RAPA)⁸ in experimental autoimmune encephalomyelitis (EAE) mice.Animal Model: EAE was induced in 14 ten-week-old female C57BL/6 mice with MOG35-55 in complete Freund’s adjuvant emulsion. Four age- and gender-matched mice were used as a naïve control. After immunization, mice were scored daily for clinical signs using a standard 0-5 scoring system.6 Treatment Srategy: Mice were randomly assign to LDK (40mg/kg, n=4), RAPA (1mg/kg, n=5), or vehicle (DMSO in RPMI 1640, n=5) treatment. The treatment (intraperitoneal injection) started on the 4th day of the attack which was around day 12 post-immunization. Sample Preparation for Ex-Vivo MRI: All mice were euthanized and perfusion fixed at day 37 post-immunization. DBSI: An 8-mm-diameter (25 mm long) solenoid coil was used to acquire data. Ex-vivo DBSI was performed on a 4.7-T Agilent small-animal MR scanner utilizing a standard spin-echo sequence. A 99-direction diffusion scheme was employed.5 Acquisition parameters are: TR = 1 s, TE = 36 ms, Δ = 20 ms, δ = 5 ms, maximal b-value = 3,000 s/mm², slice thickness = 1.5 mm (five slices were acquired at the center of each vertebral segment from T11 to L2), FOV (field of view) = 10 × 10 mm², in-plane resolution = 78 × 78 µm² (before zero-fill).Axonal injury was detected in LDK, RAPA, and vehicle treated groups as reflected by the decreased DTI and DBSI λ∥ (Fig. 1 and 2). Unlike DTI λ⊥, significant DBSI λ⊥ increase was only seen in the vehicle spinal cords, suggesting myelin injury (Fig. 2 B and D). DBSI detected significantly increased restricted isotropic diffusion tensor fraction, as a putative biomarker for cell infiltration, in LDK, RAPA, and Vehicle groups (Fig. 3 B). Significantly increased non-restricted fraction, as a putative biomarker for edema, was shown in LDK and vehicle treated spinal cords but not in RAPA group (Fig. 3 A). DBSI-derived “axon volume”, suggested a significant axonal loss in RAPA- and vehicle-treated spinal cords (Fig. 3 C). Representative IHC staining of spinal cords exhibited mild axonal swelling in LDK group, and severe axon/myelin damages with axonal loss in RAPA and vehicle groups (Fig. 4). The time course of CS reflected that LDK and RAPA treatments could ameliorate neurological dysfunction, comparing to vehicle group (Fig. 5 A). The cumulative CS correlated well with DBSI-derived “axon volume” (Fig. 5B).DBSI quantitative assessed axonal injury and demyelination in the residual axons, in addition to the extent of axonal loss. Our data suggested that LDK and RAPA treatments lessened the severity of axon/myelin injury and inflammation. RAPA failed to prevent axonal loss. LDK holds the potential to ameliorate disease progression and recover in MOG_35-55 induced EAE.