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Diffusion Basis Spectrum Imaging (DBSI): Successful in vivo Use to Assess Axonal Protection in Mouse Optic Neuritis treated with Fingolimod
Tsen-Hsuan (Abby) Lin1, Rui-Meng Yang1,2, Jie Zhan 1,3, Chunyu Song4, Peng Sun1, Michael Wallendorf5, Anne H Cross6,7, and Sheng-Kwei Song1,4,7

1Radiology, Washington University School of Medicine, St Louis, MO, United States, 2Radiology, Guangzhou Medical University, Guangzhou, China, 3Radiology, The First Affiliated Hospital of Nanchang University, Nanchang, China, 4Biomedical Engineering, Washington Univerisyt is St. Louis, St Louis, MO, United States, 5Biostatistics, Washington University School of Medicine, St Louis, MO, United States, 6Neurology, Washington University School of Medicine, St Louis, MO, United States, 7Hope Center for Neurological Disorders, Washington University School of Medicine, St Louis, MO, United States

Synopsis

We previously introduced diffusion basis spectrum imaging (DBSI) to noninvasively assess coexisting pathologies in central nervous system (CNS) tissues.Previously, we showed in multiple sclerosis (MS) spinal cord specimens and mice with experimental autoimmune encephalomyelitis (EAE), the main animal model of MS, that DBSI reflected coexisting white matter pathologies in CNS. Fingolimod, a disease-modifying treatment approved for relapsing MS, is thought to preserve axons. In the current study, we employed longitudinal DBSI to noninvasively assess fingolimod treatment efficacy in EAE mouse optic nerve, followed by histological validation.

Introduction

Previously, we introduced diffusion basis spectrum imaging (DBSI)1 and used it to successfully and noninvasively assess coexisting pathologies in multiple sclerosis (MS) spinal cord specimens2 and experimental autoimmune encephalomyelitis (EAE)3 mice, the main animal model of MS. Our prior data supported the ability of DBSI to reflect central nervous system (CNS) pathology. Moreover, we previously employed DBSI and post-DBSI histology to document an early axonal loss, which is correlated with irreversible visual impairment, at the onset of optic neuritis in EAE-affected mice.3 Fingolimod, an approved disease-modifying treatment for relapsing MS, is believed to preserve axons in relapsing MS.4 In the current study, we employed longitudinal DBSI to noninvasively assess the efficacy of fingolimod in optic nerves of EAE-affected mice, with concurrent optic neuritis (ON), followed by histological validation.

Materials and Methods

Animal model: EAE was induced with an emulsion of MOG35-55 peptide in incomplete Freund's adjuvant in 17 female C57BL/6 mice. After immunization, daily clinical score (CS) and body weight were assessed. Visual Acuity (VA) & treatment strategy: VA was assessed daily after immunization. When VA ≤ 0.25 cycle/degree (defining onset ON)3, EAE mice were treated with saline (n=9) or fingolimod (n=8) in parallel by oral gavage for three months beginning at onset of ON (Fig. 1A). DBSI: A pair of active-decoupled volume and surface coils were used. A 25-direction diffusion-weighted imaging (DWI) was performed at baseline (naïve, before immunization), one, two, and three months after treatment on a 4.7-T Agilent small-animal MR scanner utilizing a multiple-echo spin-echo diffusion-weighted sequence.5 All images were obtained with the 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, with the same FOV = 22.5 × 22.5 mm2, slice thickness = 0.8 mm, and matrix size = 192 × 192 (before zero-filling). Data analysis: A lab-developed DBSI code was performed on DWI data to estimate DBSI derived λǁ, λ┴, restricted (putative cellularity) and non-restricted isotropic (putative edema) diffusion tensor fractions. Histology: mice were perfusion-fixed immediately after the last in vivo MR measurements for immunohistochemistry (IHC) staining of SMI-312, MBP, SMI-31, and DAPI. Statistics: For all the box plots, degrees of freedom were adjusted with Kenward-Rogers method. The associations of histology data with DBSI metrics were analyzed by mixed random effects regression with correlation calculated as the mean of Pearson correlations on left and right sides.

Results

Worse VA was seen in the saline (placebo) group (Fig. 1B), indicating more severe optic nerve damage than in the fingolimod-treated group. Using DBSI, less non-restricted and restricted isotropic fractions were seen in optic nerves of the fingolimod group than saline group (Fig. 2), indicating anti-inflammatory effects of fingolimod. Axonal volume (Fig. 3 D) was derived by multiplying fiber fraction (Fig. 3 B) by nerve volume (Fig. 3 C). Significantly reduced DBSI-derived axonal volume was seen in some saline-treated optic nerves but not in fingolimod-treated group (Fig. 3 D), suggesting axonal preservation with fingolimod. At months 2 and 3, increased λ⊥ was seen using DBSI in saline-treated optic nerves, suggesting more demyelination in the saline-treated than fingolimod-treated group (Fig. 4). Regarding fingolimod-treated group, significant decreased λǁ and increased λ⊥ were only shown one month after treatment, suggesting axonal prevention (Fig. 4). Comparing to fingolimod-treated optic nerves, the IHC results revealed axonal loss (reduced SMI-312 intensity, Fig.5 A) demyelination (reduced MBP intensity, Fig.5 C), axonal swelling (Fig. 5, white arrows), axonal injury (reduced SMI-31 intensity, Fig.5 E) in saline-treated optic nerves and cell infiltration (increased DAPI intensity, Fig.5 G). The correlations of DBSI metrics and corresponding IHC markers (Fig.5 I - L) suggested that DBSI metrics correctly reflected coexisting pathologies of ON.

Conclusions

Our results indicate that optic nerves of EAE-affected mice treated three months with fingolimod developed relatively mild damage compared to saline-treated mice. This results was shown by DBSI and confirmed with pathological examination. This ‘proof-of-concept’ study demonstrates that it is feasible to use DBSI as a noninvasive imaging biomarker to assess the effect of a drug (in this case fingolimod) on underlying anthologies. DBSI could potentially be used as an outcome measure in human clinical studies to monitor disease progression and validate therapeutic efficacy.

Acknowledgements

This work was supported in part by NIH R01-NS047592, P01-NS059560, U01-EY025500, National Multiple Sclerosis Society (NMSS) RG 4549A4/1, RG-1507-05315 and Department of Defense Idea Award W81XWH-12-1-0457.

References

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

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

3. Lin TH, Chiang CW, Perez-Torres CJ, et al. Diffusion MRI quantifies early axonal loss in the presence of nerve swelling. Journal of neuroinflammation 2017;14:78.

4. Brinkmann V, Billich A, Baumruker T, et al. Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis. Nature reviews Drug discovery 2010;9:883-897.

5. Tu TW, Budde MD, Xie M, et al. Phase-aligned multiple spin-echo averaging: a simple way to improve signal-to-noise ratio of in vivo mouse spinal cord diffusion tensor image. Magnetic resonance imaging 2014;32:1335-1343.

Figures

Figure 1 The course of daily treatment started from the onset of optic neuritis for three months (A). DBSI was performed at baseline (before immunization), one, two, and three months after immunization. Visual acuity was lower in saline-treated mice (B), suggesting poor vision and more severe optic nerve injury.

Figure 2 DBSI-derived non-restricted isotropic (putative edema marker) and restricted isotropic (putative cellularity marker) diffusion tensor fraction maps from representative mice (A) reflected findings supporting of more severe inflammation in saline-treated optic nerves. Optic nerves of fingolimod-treated mice showed significantly lower non-restricted (all time-points, B) and restricted fraction (month 3, B), suggesting anti-inflammatory fingolimod effects.

Figure 3 DBSI-derived fiber fraction reflects total signal from anisotropic diffusion tensor components, suggesting axonal fiber density (A). Decreased axonal density was shown in saline-treated optic nerves but not in fingolimod-treated optic nerves (B), suggesting axonal prevention with fingolimod treatment. To remove the confounding effects from inflammation, DBSI axon volume (D) was derived by fiber fraction (B) multiplying ROI-derived nerve volume (C) in each individual optic nerve. Comparing to saline group, fingolimod-treated optic nerves showed significant higher axon volume (D), suggesting axonal prevention with fingolimod treatment.

Figure 4 DBSI-derived axial (λǁ) and radial (λ⊥) diffusivity maps (A) were derived from anisotropic diffusion tensor components and reflected axonal injury and demyelination, respectively. Comparing to fingolimod-treated optic nerves, mild but not significant DBSI λǁ decrease (B). Meanwhile, significant higher DBSI λ⊥ were seen in saline-treated optic nerves two and three months after treatment indicated demyelination (B). Regarding fingolimod-treated group, significant decreased λǁ and increased λ⊥ were only shown one month after treatment, suggesting axonal prevention.

Figure 5 Representative 100x immunohistochemical staining images of total neurofilaments (SMI-312, injured and intact axons), myelin basic protein (MBP, myelin), phosphorylated neurofilament (SMI-31, intact axons), and 4’, 6-dianidino-2-phenylindole (DAPI, nuclei) from saline- and fingolimod-treated optic nerves at three months after treatment. Saline-treated optic nerves showed more severe damages (A, C, E, G). The correlations of SMI-312 volume, MBP and SMI-31 area fractions, and DAPI counts with DBSI-derived axon volume (I), DBSI λ⊥ (J), DBSI λǁ (K), DBSI restricted isotropic fraction (L) suggested that DBSI was able to reflect the severity of axonal loss, demyelination, axonal injury, and cell infiltration, respectively.


Proc. Intl. Soc. Mag. Reson. Med. 26 (2018)
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