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Unveiling the Association between Gray Matter Atrophy and Impaired Blood Flow in EAE Mouse Model of MS with 9.4T MRI

Mada Hashem^1,2,3,4, A. Max Hamilton^1,2,3,4, Manoj Mishra^2,4, V. Wee Yong^2,4, and Jeff F. Dunn^1,2,3,4
¹Department of Radiology, University of Calgary, Calgary, AB, Canada, ²Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada, ³Experimental Imaging Centre, University of Calgary, Calgary, AB, Canada, ⁴Cumming School of Medicine, University of Calgary, Calgary, AB, Canada

Synopsis

Keywords: Multiple Sclerosis, Brain, arterial spin labeling, high field MRI, Atrophy, Perfusion

Motivation: The underlying mechanisms of Multiple Sclerosis (MS) remain unclear, and treatments are lacking. In MS, cerebral atrophy, and impaired cerebral blood flow (CBF), are both aspects of GM pathology.

Goal(s): We aim to assess the relationship between atrophy and CBF in MS, and their changes with disease duration and severity.

Approach: We applied non-invasive ASL-MRI and Atlas-based volumetrics to measure CBF and atrophy in the EAE mouse model of MS, over disease course.

Results: EAE mice showed reduced CBF during peak and long-term disease but atrophy just during long-term disease. Long-term clinical disability and atrophy were correlated with CBF.

Impact: Reduced CBF may relate to pathology in MS, including progression and atrophy. Future studies combining ASL-MRI and atlas-based volumetrics may be useful for investigating the processes underlying neurodegeneration in MS.

INTRODUCTION

Multiple Sclerosis (MS) is characterized by inflammation and progressive neurodegeneration culminating in atrophy^1-6. Atrophy has become an important clinical marker of disease progression due to its association with declining neurological function in MS^7-15. However, the underlying mechanisms of neurodegeneration and atrophy are not clear, and treatments are lacking. One mechanism of interest is cerebral perfusion, measured via cerebral blood flow (CBF). CBF plays an integral role in MS pathophysiology^16-22, and has promise as a non-invasive clinical measurement of gray matter (GM) pathology, either as a promoter or a marker of atrophy. To assess the relationship between atrophy and CBF in MS, we use the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, as it experiences GM neurodegeneration and atrophy at long-term disease duration²³. The EAE model also experiences hypoxia during peak inflammation^24-26, which could result in reduced blood supply. The aim of this study is to determine how CBF changes with disease duration in the cortex of EAE mice, and whether there is an association between atrophy and CBF. This study will demonstrate whether Arterial Spin Labeling (ASL) MRI could be a non-invasive method of assessing GM pathology.

METHODS

Forty-three C57BL/6J female mice were separated into naïve (n=10), CFA/PTX control (n=10) and EAE (n=23) groups. Naïve mice are controls without interventions. CFA/PTX mice were injected with complete Freund’s adjuvant (CFA) and pertussis toxin (PTX). EAE mice were injected with MOG35-55 peptide emulsified in CFA and PTX. Mice were imaged at “Peak” clinical disease severity and “long-term” disease (14-16 days and 40 days post EAE induction, respectively). Imaging was performed for all groups at the same time points. During imaging, the mice were spontaneously ventilated with a gas mixture of 70% N2/30% O2 and 2% isoflurane. Normal physiological temperature and respiration rate were maintained at 36.5°C and 60 breaths/min. Imaging was conducted using a 9.4T Bruker MRI with an Avance console (Bruker Biospin GmbH, Germany), paravision 5.1, and a 35mm quadrature volume coil. For single-slice perfusion imaging, axial slices were acquired around the bregma with a CASL-HASTE sequence. To account for magnetization transfer, four images consisting of two control and two tagged images were acquired with the following parameters: matrix size=128x128; FOV=30mmx30mm; TR=3000ms; TE=2.66ms; number of averages=16; RARE factor=36; voxel size=0.23mmx0.23mmx1mm; acquisition time=8 minutes. Following this, a T1 map was acquired in the same location using a rapid acquisition with refocused echoes variable repetition time (RARE-VTR) sequence with the following parameters: matrix=128x128; FOV=30mmx30mm; TR=100, 500, 1000, 3000, 7500ms; TE=10ms; voxel size=0.23mmx0.23mmx1mm; acquisition time=6 minutes. The total acquisition time for each perfusion image was 14 minutes. CBF was calculated on a voxel-by-voxel basis from an ROI in the cortex²⁷. A subset of mice at long-term (n=10 naïve, 10 CFA, 10 EAE) were also imaged using anatomical MRI for atlas-based volumetric analysis. These images were collected using a T2-weighted rapid acquisition with refocusing echoes (RARE) sequence where: matrix size=256x256; FOV=20mmx20mm; slice number=30; TR=4500ms; TE=32ms; number of averages=3; RARE factor=36; voxel size=78µm×78µm×500µm. Using the Niftyreg²⁸ software, an averaged brain atlas29 was registered to individual images, and used for volumetric analysis.

RESULTS

At peak clinical disease, EAE and CFA/PTX mice experienced reduced cortical perfusion compared to naïve controls. At long-term disease, CBF was restored to normal levels in CFA/PTX but not in EAE mice. Leukocyte congestion was found in the cortical microvasculature of EAE at peak clinical disease, and CFA/PTX mice as well. At long-term disease, EAE mice had significantly smaller cortical volume compared to naïve and CFA/PTX controls, while there was no evidence of atrophy at peak clinical disease. Cortical CBF correlated with cortical brain volume and clinical disability at long-term disease.

DISCUSSION

At peak clinical disease, reduced CBF could be partially related to inflammation, as a similar reduction was found in the CFA/PTX mouse model of inflammation. Perivascular inflammation is prominent in MS and may disrupt the microvasculature and CBF regulation^30-35. Inflammation within the vessels may also disrupt blood flow by directly occluding vessels^36,37. At long-term disease, reduced CBF in EAE mice relates to atrophy. It is possible that prolonged reduced CBF plays a role in atrophy by promoting hypoxia and therefore cell death. Alternatively, reduced CBF could be the direct result of GM volume loss, which in turn leads to a decrease in metabolic demand, and therefore lower CBF^38,39.

CONCLUSION

This study identifies atrophy and reduced CBF in the EAE model. It also supports the use of ASL-MRI and atlas-based volumetrics in future studies to investigate the processes that promote neurodegeneration and atrophy in MS.

Acknowledgements

This work was supported by the Sciences and Engineering Research Council (NSERC) RGPIN/05225Discovery grant and CIHR project 173416.

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Figures

Reduced cortical cerebral blood flow in the EAE model. A) Perfusion maps for Naïve, CFA/PTX, and EAE groups at peak clinical disease (d14-16) and long-term disease duration (d40+). There was restricted CBF in the cortex of CFA/PTX and EAE mice at peak clinical disease and in the EAE mice at long-term disease duration. B) Cortical CBF quantification at peak clinical disease. C) Cortical CBF quantification at long-term disease. CBF was compared between groups using an analysis of variance (ANOVA) with a Tukey post-hoc. n=10 Naïve, 10 CFA/PTX, 23 EAE. * = p < 0.05; ** = p <0.01 *** = p <0.001.

Cortical atrophy in EAE mice at long-term disease duration. A) Cerebral cortex volume (frontal lobe + parieto-temporal lobe + occipital lobe + entorhinal cortex) in Naïve, CFA/PTX, and EAE mice at peak clinical disease (d14-16). B) Cerebral cortex volume in Naïve, CFA/PTX, and EAE mice at long-term disease (d40+). All volumes are adjusted based on weight using an analysis of covariance (ANCOVA). n = 10 Naïve, 10 CFA, 10 EAE. * = p < 0.05; ** = p <0.01 *** = p <0.001.

Cortical cerebral blood flow (CBF) is correlated with cerebral cortex volume, and long-term disease scores. A) Correlation of CBF with cerebral cortex volume (frontal lobe + parieto-temporal lobe + occipital lobe + entorhinal cortex). Regression was controlled for body weight. n=10. B) Correlation of CBF with cumulative long-term disease score. Long-term disease scores were summed from days 30-40 to determine the total long-term burden of disease. Linear regression test was conducted to determine significance.

Proc. Intl. Soc. Mag. Reson. Med. 32 (2024)

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DOI: https://doi.org/10.58530/2024/2961