Development of magnetic resonance diffusion weighted imaging at 16.4T to assess neurological degeneration in a mouse model of multiple sclerosis: high-resolution ex-vivo diffusion weighted MRI revealed complex MS pathology in the somatosensory and motor cortex in a mild relapsing remitting EAE mouse model

Abstract

Multiple sclerosis (MS) is a disease of the central nervous system, which is characterized by neuroinflammation and demyelination that mainly affects the white matter (WM) structures. MS is one of the major causes of neurological disability worldwide, with patient symptoms vary according to the disease stage and treatment outcomes (Trapp & Nave 2008). The major hallmarks of MS include neurodegeneration, demyelination, axonal damage and nerve injury (Lassmann, H. 2013). Recently, diffuse pathological changes have been documented in the gray matter (GM) and cortical areas in patients with MS, as well as in certain animal models (Calabrese, M. et al. 2011; Pirko et al. 2009). Various rodent models of MS have been developed to investigate specific aspects of this disease and explore the underlying mechanisms. Experimental autoimmune encephalomyelitis (EAE) is a classical animal model for MS, which is characterized by the development of CNS neuroinflammation that leads to demyelination and neurodegeneration. EAE disease severity in mice varies according to the mouse strain and the immunization protocols utilized (Lassmann, H. 2008; Robinson et al. 2014). In order to assess sensory deficits in EAE-mice, the immunization protocol was optimized (in the laboratory of Prof. M. Smith, the School of Pharmacy, UQ) to produce mild relapsing remitting MS clinical symptoms with partial recovery between relapses (Khan, Woodruff & Smith 2014; Peiris et al. 2007). Using this EAE model, we assessed the correlations between clinical scores and pain behaviour with changes in the motor and sensory areas of the brain by magnetic resonance imaging (MRI). The aim of this PhD project is to develop MRI diffusion weighted imaging (DWI) at 16.4 T to detect sensory and motor deficits in a mild EAE model of relapsing and remitting MS. In rodent MRI, a high magnetic field is desirable feature, because it provides a high spatial resolution and a high signal-to-noise (SNR) ratio (Moldrich et al. 2010). Spin echo high-spatial and high-angular resolution diffusion-weighted imaging (HARDI) has been used for many ex vivo mouse brain studies using at 16.4T. Ex vivo imaging has some disadvantage as the diffusion parameters could be affected uniformly by tissue fixation (Zhang et al. 2012). On the other hand, in vivo HARDI-DWI provides potential benefits in term of tracking longitudinal pathological changes during the disease cycle. However, in vivo ultra-high field MRI has several physical challenges, including limited acquisition time, motion, magnetic susceptibility, lengthening of T1 and shortening of T2 relaxation times. In vivo HARDI acquisition using spin-echo DWI would result in long experiment time and increased sensitivity to motion (Aggarwal, Zhang & Mori 2012; Wu, D et al. 2014). The first part of this project involved the optimization of two-dimensional (2D) HARDI echo planar imaging (EPI) sequence for in vivo MS imaging at 16.4T. This sequence was optimized to overcome the technical challenges above, including the incorporation of echo-train length segmentation and partial Fourier acceleration in the phase direction. High-quality HARDI data was acquired at b=3000 s/mm2, 64 diffusion-encoding directions, 125 x 150μm2 in-plane resolution, 0.6 mm slice thickness, within a 2h acquisition time. The analyses of manually drawn regions of interest (ROIs) of DTI parameters (fractional anisotropy (FA), axial and radial diffusivity (AD) and mean diffusivity (MD) of the major WM structures, however, did not reveal significant changes between controls and MS animals. To study the MS disease model in more detail and to increase the possibility to detect subtle pathological changes unobserved in in vivo imaging, subsequent studies were performed with 3D-ex vivo MRI at 16.4T. Ex vivo imaging was done using 100μm3 isotropic spin echo DWI with 30 directions at b = 3000 s/mm2. Comparison of DTI parametric maps of manually drawn ROIs in the major WM structures did not reveal significant changes between EAE, sham and control subjects in acute and chronic stages of the MS disease. However, whole brain analysis using VBM (Voxel Based Morphometry) revealed significant reductions of FA in chronic EAE-mice compared to naive control animals in important gray matter (GM) brain areas including the primary and secondary motor areas, primary somatosensory area, anterior cingulate and rostral CA1 hippocampal regions, and a small portion of WM external capsule. These FA changes were not detected in the MS animals of the acute phase. Also, using ROI based analysis; there were significant increase of the T2 relaxation time in these areas exhibiting reduction in FA. Black and Gold II histology confirmed the presence of extensive demyelination in these gray matter and cortical areas. Our findings suggest that there is a correlation between histological changes in the brain motor and sensory areas, all which are consistent with the reductions of FA observed in these regions. The ability to detect mild EAE pathology in GM somatosensory cortex using MRI is highly important, providing better insight into neuropathological changes during the remitting-relapsing disease course.