Whole-Brain Ex-Vivo Imaging of Demyelination in the Cuprizone Mouse with mcDESPOT and DTI
Tobias C Wood1, Camilla Simmons1, Joel Torres1, Flavio Dell' Acqua1, Anthony Vernon2, Samuel A Hurley3, Steve CR Williams1, and Diana Cash1

1Neuroimaging, IoPPN, King's College London, London, United Kingdom, 2Basic and Clinical Neuroscience, IoPPN, King's College London, London, United Kingdom, 3FMRIB Centre, University of Oxford, Oxford, United Kingdom

### Synopsis

We demonstrate the feasibility of full-brain high-resolution ex-vivo imaging and analysis of demyelination in the Cuprizone mouse model using multi-component DESPOT and DTI. We found evidence of demyelination in the Cerebellum as well as the Corpus Callosum.

### Introduction

The Cuprizone mouse1 is an established, repeatable model of demyelination that has been used to test many different MR methods that claim sensitivity to myelination state2,3,4. Multi-component DESPOT (mcDESPOT5) is a fast method for imaging the Myelin Water Fraction (MWF), but to date has seen only a limited number of validation studies6,7. It is hence a prime candidate for use with the Cuprizone mouse, and is interesting as most previous MR studies have been restricted to a limited number of slices and Regions-of-Interest for analysis2,3,4, whereas mcDESPOT is a 3D method inherently capable of high-resolution full-brain imaging. Finally, a recent attempt to quantify the MWF in the Cuprizone mouse via Multi-Echo T2 failed even in healthy controls2.

We imaged ex-vivo Cuprizone mice using mcDESPOT and looked for differences in T1, T2 and MWF across the whole brain using non-parametric statistics. We also acquired Diffusion Tensor Imaging (DTI) for comparison, particularly the Radial Diffusivity (RD) parameter which is often interpreted as a measure of myelin “integrity”, and then performed histology to confirm any findings.

### Methods

8 control mice were fed powdered standard mouse diet while the food for another 8 mice was supplemented with 0.2% Cuprizone for 5 weeks. Mice were then sacrificed and transcardially perfused with 4% Paraformaldehyde (PFA). The heads were removed and immersed in PFA for 2 days before rehydration in Phosphate Buffered Saline for a minimum of 30 days. 2 controls and 2 Cuprizone heads at a time were then immersed in perfluorinated fluid (Galden, Solvay) to minimise susceptibility artefacts and scanned simultaneously. with a structural 3D Fast Spin-Echo, a mcDESPOT protocol with B1 correction consisting of SPGR, bSSFP & AFI7 scans, and a DTI scan. All scans had a 28.8mm FoV, other parameters were as follows. 3D Fast-Spin Echo: 256x256x256 matrix, TE/TR=40/3000 ms, scan-time 3 hours 25 minutes. SPGR: 192x192x192 matrix, TE/TR=5.1/20ms, flip-angles 4,5,6,8,10,12,16,20,24,26,28,30°, scan-time 2 hours 27 minutes. bSSFP: 192x192x192 matrix, TE/TR 3/6ms, flip-angles 8,9,10,12,15,20,30,40,50,55,60,65°, scan-time 2 hours 56 minutes. AFI: 96x96x96 matrix, TE/TR=4.3/20ms, flip-angle 55°, scan-time 40 minutes. Segmented EPI for DTI: 192x128 matrix, 40x0.5mm slices, TE/TR=43/4000ms, 4 segments, 30 diffusion directions with b=2000 & 4 b=0, scan-time 3 hours 7 minutes.

The T1, T2 & MWF maps were calculated using in-house open-source C++ software (github.com/spinicist/QUIT). DTI maps including RD were calculated using dtifit from FSL. A study template was built from the 3D FSE images using ANTs8, which was then registered to the Dorr mouse template9. All parameter maps were then transformed into the standard space. Non-parametric two-group T-tests were used to compare controls and Cuprizone mice using FSL randomise with the Threshold Free Cluster-Enhancement10,11.

After scanning the brains were extracted and cryosectioned at 20µm and sections collected serially onto slides with 240µm between each section. Slides were then stained with Luxol Fast Blue for visualisation of Myelin content and with markers of microglia (Iba1) for neuroinflammation.

### Results

Figure 1 shows example slices through an MWF map produced by mcDESPOT in a control and Cuprizone mouse. Demyelination in the Corpus Callosum (CC) and in the Arbor Vitae of the Cerebellum is clearly visible and was confirmed in the histological sections. Figure 2 shows regions of increase in T1, T2, & RD and decrease in MWF between control and Cuprizone mouse thresholded at corrected $p < 0.05$. There is significant overlap but also some differences in these maps. In general, the T1 changes are the most focal and T2 most diffuse. RD shows increases in some different regions to the DESPOT measurements. Figure 3 is a rendering of the regions of decreased MWF, emphasising that Cuprizone causes widespread changes in the mouse brain that are not limited to the CC.

### Discussion and Conclusion

mcDESPOT successfully measured the MWF in the Cuprizone mouse. Decreases are clearly visible in the CC but also in other regions including the Cerebellum. This demonstrates the power of quantitative, full-brain, high-resolution MRI and analysis methods compared to previous studies where only a limited number of ROIs have been used. Effects such as inflammation are often co-present with demyelination, and the slight differences in maps between T1, T2, MWF & RD suggest that although these are all sensitive to demyelination, they may not be specific.

### Acknowledgements

No acknowledgement found.

### References

1. Torkildsen et al. The Cuprizone Model For Demyelination Acta Neurologica Scandinavica (2008) 117:72--76 doi:10.1111/j.1600-0404.2008.01036.x

2. Thiessen et al. Quantitative Mri And Ultrastructural Examination Of The Cuprizone Mouse Model Of Demyelination NMR in Biomedicine (2013) 26:1562--1581 doi:10.1002/nbm.2992

3. Merkler et al. Multicontrast MRI Of Remyelination In The Central Nervous System NMR in Biomedicine (2005) 18:395--403 doi:10.1002/nbm.972

4. Fjær et al. Deep Gray Matter Demyelination Detected By Magnetization Transfer Ratio In The Cuprizone Model PLoS ONE (2013) 8:e84162 doi:10.1371/journal.pone.0084162

5. Deoni, Matthews, and Kolind One Component? Two Components? Three? The Effect Of Including A Nonexchanging “Free” Water Component In Multicomponent Driven Equilibrium Single Pulse Observation Of T1 And T2 Magnetic Resonance in Medicine (2013) 70:147--154 doi:10.1002/mrm.24429

6. Hurley et al. Multicomponent Relaxometry (mcDESPOT) In The Shaking Pup Model Of Dysmyelination ISMRM (2010) 18:0088

7. McDowell et al. Myelin And More: mcDESPOT Applied To Post Mortem Multiple Sclerosis Spinal Cord ISMRM (2015) 23:3277

8. Avants et al. The Optimal Template Effect In Hippocampus Studies Of Diseased Populations NeuroImage (2010) doi:http://dx.doi.org/10.1016/j.neuroimage.2009.09.062

9. Dorr et al. High Resolution Three-Dimensional Brain Atlas Using An Average Magnetic Resonance Image Of 40 Adult C57Bl/6J Mice NeuroImage (2008) 42:60 - 69 doi:http://dx.doi.org/10.1016/j.neuroimage.2008.03.037

10. Smith and Nichols. Threshold-Free Cluster Enhancement: Addressing Problems Of Smoothing, Threshold Dependence And Localisation In Cluster Inference NeuroImage (2009) 44:83 - 98 doi:http://dx.doi.org/10.1016/j.neuroimage.2008.03.061

11. Winkler et al. Permutation Inference For The General Linear Model NeuroImage (2014) 92:381 - 397 doi:http://dx.doi.org/10.1016/j.neuroimage.2014.01.060

### Figures

An axial (top) and coronal (bottom) slice through the mcDESPOT MWF map in template space of a control (left) and Cuprizone (right) mouse. Demyelination is obvious in the Corpus Callosum and Cerebellum (red arrows).

Regions of significant increase in T1,T2 & RD, and decrease in MWF, thresholded at corrected p < 0.05.

Regions of MWF decrease in the Cuprizone mouse (red), thresholded at corrected p < 0.05 and overlaid on the study template. Changes are not limited to the Corpus Callosum but include the Cerebellum.

Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
1317