Axon Loss as an Outcome Measure for Assessing Therapeutic Efficacy
Tsen-Hsuan Lin1, Mitchell Hallman1,2, Mattew F. Cusick3, Jane E. Libbey3, Peng Sun1, Yong Wang1,4,5,6, Robert S. Fujinami3, and Sheng-Kwei Song1,5,6

1Radiology, Washington University School of Medicine, St. Louis, MO, United States, 2Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States, 3Pathology, University of Utah School of Medicine, Salt Lake City, UT, United States, 4Obstertic and Gynecology, Washington University School of Medicine, St. Louis, MO, United States, 5The Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, United States, 6Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, United States


Diffusion basis spectrum imaging (DBSI) has successfully distinguished co-existing pathologies in CNS, such as MS. The utility of DBSI derived “axon volume” has not been explored previously. In this study, we demonstrated the use of axon loss, reflecting irreversible tissue damage, as an outcome measure for assessing therapeutic efficacy in a mouse model of multiple sclerosis.


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.

Methods and Materials

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 MOG35-55 induced EAE.


Supported in part by NIH R01-NS047592, P01-NS059560, U01-EY025500, and NMSS RG 5258-A-5.


1. Lovblad KO, Anzalone N, Dorfler A, et al. MR imaging in multiple sclerosis: review and recommendations for current practice. AJNR American journal of neuroradiology 2010;31:983-989.

2. Ge Y. Multiple sclerosis: the role of MR imaging. AJNR American journal of neuroradiology 2006;27:1165-1176.

3. Wang Y, Sun P, Wang Q, et al. Differentiation and quantification of inflammation, demyelination and axon injury or loss in multiple sclerosis. Brain 2015;138:1223-1238.

4. Wang Y, Wang Q, Haldar JP, et al. Quantification of increased cellularity during inflammatory demyelination. Brain : a journal of neurology 2011;134:3590-3601.

5. Chiang CW, Wang Y, Sun P, et al. Quantifying white matter tract diffusion parameters in the presence of increased extra-fiber cellularity and vasogenic edema. Neuroimage 2014;101:310-319. 6. Wang X, Cusick MF, Wang Y, et al. Diffusion basis spectrum imaging detects and distinguishes coexisting subclinical inflammation, demyelination and axonal injury in experimental autoimmune encephalomyelitis mice. Nmr Biomed 2014;27:843-852.

7. Cusick MF, Libbey JE, Trede NS, Eckels DD, Fujinami RS. Human T cell expansion and experimental autoimmune encephalomyelitis inhibited by Lenaldekar, a small molecule discovered in a zebrafish screen. Journal of neuroimmunology 2012;244:35-44.

8. Esposito M, Ruffini F, Bellone M, et al. Rapamycin inhibits relapsing experimental autoimmune encephalomyelitis by both effector and regulatory T cells modulation. Journal of neuroimmunology 2010;220:52-63.


Representative DTI and DBSI metric maps overlaid on gray-scale diffusion-weighted images (DWI) from each group.

The exaggerated DTI index change reflected the confounding effects of co-existing pathologies. LDK- and RAPA-treated spinal cords still developed significant axonal injury (A) and mild myelin damage (B). * indicates p < 0.05

Significant inflammation, seen as the increased non-restricted (A) or restricted fraction (B), was observed in LDK- and RAPA-treated spinal cords. Significantly reduced DBSI derived “axon volume”, i.e., DBSI fiber fraction multiplying ROI-based nerve volume on diffusion-weighted images, in RAPA group (C) suggested that RAPA failed to prevent axonal loss in this EAE mouse model. * indicates p < 0.05

Representative double immunohistochemistry (IHC) staining for each group. The RAPA- and vehicle-treated spinal cords exhibited less positive SMI312 and MBP staining, suggesting relatively more severe axonal injury and loss.

The time course of clinical score (CS) for each group of animals was shown (A). The cumulative CS (from day 0 – 37 post-immunization) correlated with DBSI derived “axon volume” suggesting the less severe axon loss and accumulative CS in LDK-treated mice.

Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)