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Grey matter atrophy measured with MRI correlates with reduced neuronal density in the experimental autoimmune encephalomyelitis model of multiple sclerosis

A. Max Hamilton^1,2,3,4, Nils D. Forkert^1,2, Ying Wu^1,2,3,4, James A. Rogers^2,3, V. Wee Yong^2,3, and Jeff F. Dunn^1,2,3,4

¹Radiology, University of Calgary, Calgary, AB, Canada, ²Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada, ³Clinical Neurosciences, University of Calgary, Calgary, AB, Canada, ⁴Experimental Imaging Center, University of Calgary, Calgary, AB, Canada

Synopsis

Atrophy is a clinical marker of neurodegeneration and progressive disability in multiple sclerosis (MS). To test neuroprotective treatments aimed at reducing atrophy, mouse models featuring atrophy are needed. We have shown the experimental autoimmune encephalomyelitis (EAE) mouse model features atrophy, though we do not know if EAE atrophy is caused by neurodegeneration, as it is in MS. We used MRI and atlas-based regional volumetrics to measure atrophy in EAE, while using immunohistochemistry to measure neurodegeneration. Atrophy measured in the cerebellum and cerebral cortex correlated with neuronal loss, suggesting we can use EAE along with MRI to test neuroprotective therapies.

Purpose

Current evidence indicates that neurodegenerative processes play an important role in multiple sclerosis (MS) alongside inflammation and demyelination¹. The accumulation of neurodegeneration, through neuron and axon loss, results in a loss of brain volume known as atrophy². Atrophy has been associated with disease progression³, as well as cognitive⁴ and physical disability⁵ making atrophy an important clinical target for treating MS. To treat atrophy in MS, there is a need to test neuroprotective therapies in mouse models that feature neuroinflammation and atrophy. One potential model is experimental autoimmune encephalomyelitis (EAE), which is a model of central nervous system (CNS) targeted autoimmunity. Previously we have shown EAE features atrophy⁶; however we do not know if atrophy is the result of accumulated neurodegeneration similar to MS. We used high resolution magnetic resonance imaging (MRI), a Bruker cryoprobe, and atlas-based regional volumetrics to measure atrophy in the brains of EAE mice. We compared these MRI measurements with histological measurements of neuronal density to determine whether MRI measurements of atrophy could be a useful marker of neurodegeneration.

Methods

Female C57BL/6 mice were induced with EAE as described.⁷ Motor disability and disease severity were assessed using a 15-point scoring system based on individual limb paralysis.⁸ Mice were imaged at 66 days post induction [n=10 EAE, 5 Naïve, 5 CFA (the last received immune stimulant but no myelin peptides)]. Imaging was conducted using a 9.4T MRI with a helium cooled Bruker cryoprobe using a FLASH sequence (TR/TE/α=2000ms/6.5ms/60o voxel=37.5-x-37.5-x-250μm3) (Figure 1a-b). Using the program Niftyreg, an averaged brain atlas⁹ was registered non-linearly to each dataset and the volume of each registered atlas brain region was determined (Figure 1c-d). Following imaging, mice were sacrificed and brains were extracted for histology. Cortex and cerebellar sections were stained with NeuN. Quantification of NeuN stained neurons was performed using stereology with the StereoInvestigator program Optical Fractionator¹⁰. Regions of interest were traced at low magnification (4x magnification, NA 0.16). Counting was performed using random counting grids (35um x 35um cortex, 10um x 10um, and cerebellum) at 40x magnification (NA 0.45). Counting sites per section ranged from 25-30 for the cortex, and 40-50 for the cerebellum. Statistics were performed using an ANOVA test with a Tukey post-hoc. Correlations between disease score and brain volume were quantified using Spearman’s test.

Results

EAE mice had smaller cerebral cortex and cerebellar volumes compared to Naïve and CFA mice (Figure 2). Both the cortex and cerebellum also showed reduced neuronal densities based on NeuN staining (Figure 3). In EAE mice, cortical and cerebellar volumes correlated with neuronal density (Figure 4) as well as cumulative long-term disease scores (Figure 5). This was not seen with peak clinical disease scores (day 14-16).

Discussion

EAE mice showed atrophy in the cortex and cerebellum, similar to previous work by our lab⁶ as well as others.¹¹ This further supports that EAE mice experience grey matter atrophy at long-term disease duration. These atrophic regions also had a reduction in neuronal density suggesting EAE mice experience atrophy as a result of neurodegeneration, rather than other issues such as weight loss or dehydration. As EAE is a model of CNS targeted inflammation, this suggests that inflammation may play a role in neurodegeneration/atrophy. The EAE model may be a good model to study how neuroinflammation is connected to neurodegenerative mechanisms in MS.

We found that volumes of atrophic regions, measured using MRI, correlated with stereological measurements of neuronal loss. Atrophy in MS is seen as the accumulation of neurodegeneration and our data suggests this appears to be the case in EAE. Atrophy also correlated with long-term disease scores, suggesting atrophy is associated with long-term permanent motor disability in EAE. This is similar to MS where atrophy is linked to disease progression³ and physical disability⁵. Previous research has shown axonal loss in the EAE spinal cord also corresponded with long-term clinical disease scores¹². It is possible that degeneration in both the spinal cord and brain is driven by similar mechanisms and combines to drive EAE disease progression.

Conclusion

EAE mice exhibit significant reductions in both volume and neuronal density of the cerebral cortex and cerebellum at long-term disease duration. Correlations between atrophy, neuronal loss, and long-term disease scores suggest that atrophy can be used as a marker of neurodegeneration, disease progression, and permanent motor disability in the EAE model. Using MRI and atlas-based volumetrics, EAE has the potential to be used for testing neuroprotective therapeutic treatments aimed at reducing neurodegeneration and atrophy in MS.

Acknowledgements

Funding for this study was provided by the AIHS CRIO team grant.

References

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6. Hamilton AM, Forkert ND, Yang R, Wu Y, Rogers J, Yong VW, Dunn JF. Grey matter atrophy measured in-vivo with 9.4T MRI in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. 25th Annual Meeting of the International Society for Magnetic Resonance in Medicine, Honolulu, USA 2017, Proceedings of the International Society for Magnetic Resonance 25: 0188, 2017.

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12. Wujek JR, Bjartmar C, Richer E, et al. Axon loss in the spinal cord determines permanent neurological disability in an animal model of multiple sclerosis. J Neuropathol Exp Neurol. 2002; 61: 23-32.

Figures

Figure 1. Atlas-based regional volumetrics. Gradient echo MRI images of an EAE mouse brain A) cerebral cortex, B) cerebellum 66 days post induction. B) EAE mouse brain C) cerebral cortex, D) cerebellum) segmented into 62 measured structures after registration to the atlas.

Figure 2. EAE mice have significantly smaller regional brain volumes at long-term disease duration. A) cerebral cortex, B) cerebellum. Animals imaged at 66 days post induction. p < 0.05*; <0.01**, <0.001***.

Figure 3. EAE mice have significantly reduced neuronal densities in atrophic regions at long-term disease duration (66 days). Neuronal nucleus staining (NeuN - red) was significantly reduced in EAE mice in the cortex (a-d) and cerebellum (e-h) compared with Naïve or CFA controls. p < 0.05*; <0.01**, <0.001***.

Figure 4. EAE mice exhibit correspondence between the volumes of atrophic regions and neuronal densities. A) cerebral cortex and B) cerebellum volume against neuronal density (percent of averaged neuronal density in naïve mice). p values determined using Spearman’s correlation test.

Figure 5. EAE mice exhibit correlations between the volumes of atrophic regions and long-term disease scores. A) cerebral cortex and B) cerebellum volume against cumulative long-term disease score (summed disease scores from day 30 to end-point). p values determined using Spearman’s correlation test.

Proc. Intl. Soc. Mag. Reson. Med. 27 (2019)

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