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MRI Assessment of Sex Differences in APOEε4 Knock-in in Rat Brains1Biomedical Engineering, University of Arizona, Tucson, AZ, United States, 2Department of Clinical Therapeutics, USC School of Pharmacy, University of Southern California, Los Angeles, CA, United States, 3Center for Innovation in Brain Science, University of Arizona, Tucson, AZ, United States, 4Department of Pharmacology, University of Arizona, Tucson, AZ, United States
Synopsis
Using a humanized APOEε4 gene knock-in ex-vivo rat brain model, the individual and combined impact of sex and APOEε4 genotype on white matter microstructure was measured using high T2-weighted and diffusion weighted MRI. Total brain volumes showed a significant sexual dimorphism in WT as well as APOEε4 animals, with the females having significantly lower volumes. Both volumetric and diffusion MRI measures were able to show trends of sexual dimorphism as well as genotype effect. These findings were supported with metabolomic data suggesting reduction in glucose utilization and possible shift to fatty acids derived from white matter catabolism as a fuel source.
Introduction
Diffusion magnetic resonance imaging (dMRI) can be used to help characterize neurological degenerative processes, such as Alzheimer’s disease (AD). Carriers of the ε4 allele of the apolipoprotein E gene (APOEε4) have been shown to undergo faster rates of cognitive decline from Mild Cognitive Impairment (MCI) to AD compared to carriers with the ε3 allele. Using a humanized APOEε4 gene knock-in rat model, the individual and combined impact of sex and APOEε4 genotype on white matter microstructure was measured using dMRI. Findings were also compared with cortical metabolomics.Methods
Wildtype (WT), and with a humanized APOEε4 knock-in, male and female rats (Sprague Dawley, n=8, 2 per group) were used in this study. At 16 months of age, metabolomic studies were conducted to investigate sex and APOEε4 genotype effects. Subsequently, rats were injected with ketamine and xylazine (i.p.), and transcardially perfused with Trump’s fixative. Rats were then decapitated and intact skulls containing the fixed brains underwent MRI on a 7T Bruker Biospec MRI scanner. Anatomical 3D T2-weighted RARE images were collected with TR/TE=1500/10ms, RARE factor of 8, and 100µm isotropic resolution. In addition, three sets of dMRI were collected using 8-shot echo planar imaging with 32 directions and a diffusion weighting of b=1000s/mm2, and 4 b=0 images. In plane resolution was 200µm and slice thickness was 600µm. Three contiguous datasets, shifted by 200µm in the slice direction were collected such that superresolution reconstruction produced dMRI datasets with 200µm isotropic resolution.
Image Analysis- High-resolution anatomical MRI images were semi-automatically brain extracted using MRIcron and Mango programs and bias field-corrected using N4 implemented in ANTs. The data was further analyzed by registering a T2-weighted reference image and atlas with 115 regions of interest (ROIs)1 to each animal using the SyN algorithm in ANTs. Volumes of specific regions of the brain, inclusive of white matter and grey matter areas, were compared across the 4 groups (male and female; WT and APOEε4) using pair-wise t-tests. Raw, low-resolution, dMRI images were motion and eddy-current corrected using FSL’s eddy-correct2 and denoised using a diffusion-matched principal component analysis technique3. Subsequently, the three low resolution datasets were reconstructed using in-house super-resolution reconstruction software, written in Julia, to generate 200µm isotropic dMRI data (Figure 1). The brain was then semi-automatically extracted from non-brain tissue and bias field corrected and two SyN registrations were performed in ANTs to create a labeled atlas in individual diffusion space (Figure 2). The high-resolution dMRI data were then fit to the diffusion tensor imaging (DTI) model using weighted linear least squares4. From the DTI fit, fractional anisotropy (FA), and mean diffusivity (MD) were calculated on a voxel-by-voxel basis using MRTrix and directionally encoded color maps were generated (Figure 3). Parameter maps were analyzed by registering the labeled rat atlas to each individual fixed rat brain dMRI data, and then comparing the mean value of the top quartile of FA in white matter ROIs.
Results
Total brain volumes showed a significant sexual dimorphism in WT as well as APOEε4 animals, with the females having significantly lower volumes. The effect of genotype on total brain volume was not significant, but APOEε4 animals trended to have larger total brain volume. Grey matter regions – such as, the neocortex and hippocampus proper trended to be larger in APOEε4 males whereas white matter areas- such as anterior commissure, fimbria of the hippocampus and hippocampal commissure trended to be larger in the APOEε4 females. Microstructural analysis revealed that there is a sex difference in FA in males and females, in which APOEε4 females trended to have lower FA values. Cortical metabolomics analyses suggested a reduction in glucose utilization as evidenced by a reduction in fumarate and malate (metabolites of the TCA cycle) and reduced fatty acid oxidation in APOEε4 females with respect to the APOEε4 male, possibly indicating an energy deprived state.Discussion
Both volumetric and dMRI measures were able to show trends of sexual dimorphism as well as genotype effect. These findings were supported with metabolomic data suggesting reduction in glucose utilization and possible shift to fatty acids derived from white matter catabolism as a fuel source5. These findings are pending ultrastructural imaging analyses, i.e. electron microscopy, to corroborate the microstructural assessment.Acknowledgements
This work was supported by NIA P01AG026572, Alzheimer’s Association SAGA-17-419459 and Arizona Alzheimer’s Consortium to RDB.References
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3. Chen NK, Chang HC, Bilgin A, Bernstein A, Trouard TP. A diffusion-matched principal component analysis (DM-PCA) based two-channel denoising procedure for high-resolution diffusion-weighted MRI. PLoS One. 2018;13(4):e0195952.
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5. Klosinski LP, Yao J, Yin F. White Matter Lipids as a Ketogenic Fuel Supply in Aging Female Brain: Implications for Alzheimer's Disease EBioMedicine. 2015; 2(12): 1888–1904.
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