Diffusion MRI has been a promising tool to investigate white matter/axonal pathology and structure for over two decades.¹ However, diffusion-weighted imaging (DWI), and diffusion tensor imaging (DTI) are limited to quantify co-existing pathologies in CNS.² Diffusion basis spectrum imaging (DBSI) has successfully detected and distinguished co-existing pathologies in multiple sclerosis and its mouse models.^3-6 Herein, we employed DBSI to longitudinally monitor axonal degeneration after optic nerve crush (ONC) in mice. Our results demonstrated that DBSI derived fiber fraction noninvasively quantified axonal loss, in the absence of anatomically assessed atrophy, 7 days after ONC. The longitudinal progression of axon/myelin damage and axonal loss in vivo was followed by quantitative immunohistochemistry (IHC) validation to confirm in vivo MRI findingsAnimal model: 10-week-old female C57BL/6 mice (n=4) were anesthetized by intraperitoneal injection of ketamine (87 mg/kg) and xylazine (13 mg/kg). A drop of topical antibiotics was applied to both eyes. Under the binocular scope, a small incision was made at the conjunctiva by a spring scissor at the left eye. Two 45-degree-bent micro-forceps were used to hold left eyeballs and grasp the nerve at the location approximately 1-3 mm from the eyeball for 20 s. Right eye underwent the same procedure without crush as a sham control. After surgery, antibiotic gel was applied to the eye. Visual Acuity (VA): Normal VA was confirmed before baseline MRI. At day 7 post-ONC, VA was performed again.5 DBSI: A pair of 8-cm diameter volume and 1.7-cm diameter surface active-decoupled coils was used. DBSI was performed before ONC (as baseline) and at day 7 post ONC on a 4.7-T Agilent small-animal MR scanner utilizing a multiple-echo spin-echo diffusion-weighted sequence.7 A 25-direction diffusion scheme was employed.5 All images were obtained with following acquisition parameters: TR = 1.5 s, TE = 35 ms, inter-echo delay = 20.7 ms, Δ = 18 ms, δ = 6 ms, maximal b-value = 2,200 s/mm2, slice thickness = 1.0 mm, FOV (field of view) = 22.5 × 22.5 mm2, in-plane resolution = 117 × 117 µm2 (before zero-fill). Data analysis: A lab-developed DBSI code was performed on diffusion weighted MR data to estimate $$$\lambda$$$_{$$$\parallel$$$}, $$$\lambda$$$_$$$\perp$$$, and FA derived by DBSI and DTI, and DBSI specific fiber, restricted (putative cellularity) and non-restricted isotropic (putative edema) diffusion tensor fractions. The ONC eyes were blind (VA=0) and sham eyes maintained normal vision 7 days after injury (Fig.1). Axonal injury was observed in the ONC nerves revealed by the reduced $$$\lambda$$$_{$$$\parallel$$$} and FA derived by both DTI and DBSI (Fig. 2 and 3, less severe seen by DBSI) 7 days after ONC. The ONC nerves developed mild myelin damage suggested by the marginally but significantly increased DBSI $$$\lambda$$$_$$$\perp$$$ (Fig. 2 and 3), a stark contrast to DTI $$$\lambda$$$_$$$\perp$$$ (significantly increased). DBSI detected inflammation, manifested as increased cellularity and edema, in ONC nerves (Fig.4 and 5). DBSI estimated approximately 40% axon loss in the ONC nerves at day 7 post-ONC (Fig. 5E). DBSI quantitatively assessed axonal integrity without the confounding effects from surrounding pathologies. The DBSI assessed axon loss, an irreversible pathology, may prove crucial in assessing disease progression and the efficacy of therapeutic efficacy. Varying ONC severity will be pursued, in conjunction with visual function measurement and quantitative histology, to determine the threshold of axonal loss to blindness.