Abstract Background Choroid plexus (CP) or blood-cerebrospinal fluid-barrier (BCSFB) is a unique functional tissue which lines the brain’s fluid-filled ventricles, with a crucial role in CSF production and clearance. BCSFB dysfunction is thought to contribute to toxic protein build-up in neurodegenerative disorders, including Alzheimer’s disease (AD). However, the dynamics of this process remain unknown, mainly due to the paucity of in-vivo methods for assessing CP function. Methods We harness recent developments in Arterial Spin Labelling MRI to measure water delivery across the BCSFB as a proxy for CP function, as well as cerebral blood flow (CBF), at different stages of AD in the widely used triple transgenic mouse model (3xTg), with ages between 8 and 32 weeks. We further compared the MRI results with Y-maze behaviour testing, and histologically validated the expected pathological changes, which recapitulate both amyloid and tau deposition. Results Total BCSFB-mediated water delivery is significantly higher in 3xTg mice (> 50%) from 8 weeks (preclinical stage), an increase which is not explained by differences in ventricular volumes, while tissue parameters such as CBF and T1 are not different between groups at all ages. Behaviour differences between the groups were observed starting at 20 weeks, especially in terms of locomotion, with 3xTg animals showing a significantly smaller number of arm entries in the Y-maze. Conclusions Our work strongly suggests the involvement of CP in the early stages of AD, before the onset of symptoms and behavioural changes, providing a potential biomarker of pathology.

INTRODUCTION Choroid plexus (CP) dysfunction is thought to contribute to toxic protein build-up in neurodegenerative disorders, including Alzheimer’s disease (AD). However, the dynamics of this process remain unknown, mainly due to the paucity of in-vivo methods capable of assessing CP function.METHODS Here, we harness recent developments in Arterial Spin Labelling MRI to measure water delivery across the blood cerebrospinal fluid barrier (BCSFB) as a proxy for CP function, as well as cerebral blood flow (CBF), at different stages of AD progression in the widely used triple transgenic mouse model (3Tg), which recapitulates aspects of disease pathology.RESULTS Total BCSFB-mediated water delivery is significantly higher in 3Tg mice (>50%) from 8 weeks (preclinical stage), while tissue parameters such as CBF and T1 are not different between groups at all ages.DISCUSSION Our work shows changes in BCSFB function in the early stages of AD, providing a novel biomarker of pathology.### Competing Interest StatementThe authors have declared no competing interest.

Alzheimer’s disease induces early morphological tissue changes which precede the onset of symptoms and can be important targets for neuroimaging. In this work we employ Soma and Neurite Density Imaging (SANDI), a biophysical modelling approach for diffusion MRI, to investigate tissue microstructure alterations in the 3xTg AD mouse model and compare it with the state-of-the-art Diffusion Kurtosis Imaging (DKI).

Cross modal validation of micro-architectural features derived from diffusion MRI with contrast from other imaging modalities is critical for developing reliable imaging biomarkers. Here, we map apparent soma and neurite densities via the SANDI methodology in the in-vivo mouse brain at 9.4T and compared the derived metrics with the contrast from the Allen Mouse Brain Atlas which reflects cell body density in the brain. Our findings suggest that soma signal fraction correlates well with atlas intensities in different parts of the brain.

Temporal Diffusion Ratio (TDR) is a recently proposed dMRI technique (Dell'Acqua et al., proc. ISMRM 2019) which provides contrast between areas with restricted diffusion and areas either without restricted diffusion or with length scales too small for characterisation. Hence, it has a potential for informing on pore sizes, in particular the presence of large axon diameters or other cellular structures. TDR employs the signal from two dMRI acquisitions obtained with the same, large, b-value but with different diffusion gradient waveforms. TDR is advantageous as it employs standard acquisition sequences, does not make any assumptions on the underlying tissue structure and does not require any model fitting, avoiding issues related to model degeneracy. This work for the first time introduces and optimises the TDR method in simulation for a range of different tissues and scanner constraints and validates it in a pre-clinical demonstration. We consider both substrates containing cylinders and spherical structures, representing cell soma in tissue. Our results show that contrasting an acquisition with short gradient duration, short diffusion time and high gradient strength with an acquisition with long gradient duration, long diffusion time and low gradient strength, maximises the TDR contrast for a wide range of pore configurations. Additionally, in the presence of Rician noise, computing TDR from a subset (50% or fewer) of the acquired diffusion gradients rather than the entire shell as proposed originally further improves the contrast. In the last part of the work the results are demonstrated experimentally on rat spinal cord. In line with simulations, the experimental data shows that optimised TDR improves the contrast compared to non-optimised TDR. Furthermore, we find a strong correlation between TDR and histology measurements of axon diameter. In conclusion, we find that TDR has great potential and is a very promising alternative (or potentially complement) to model-based approaches for informing on pore sizes and restricted diffusion in general.

Diffusion MRI (dMRI) provides unique insights into the neural tissue milieu by probing interactions between diffusing molecules and tissue microstructure. Most dMRI techniques focus on white matter (WM) tissues, nevertheless, interest in gray matter characterizations is growing. The Soma and Neurite Density MRI (SANDI) methodology harnesses a model incorporating water diffusion in spherical objects (assumed to be associated with cell bodies) and in impermeable "sticks" (assumed to represent neurites), which potentially enables the characterization of cellular and neurite densities. Recognising the importance of rodents in animal models of development, aging, plasticity, and disease, we here employ SANDI for in-vivo preclinical imaging and provide a first validation of the methodology by comparing SANDI metrics with cellular density reflected by the Allen mouse brain atlas. SANDI was implemented on a 9.4T scanner equipped with a cryogenic coil, and in-vivo experiments were carried out on N = 6 mice. Pixelwise, ROI-based, and atlas comparisons were performed, magnitude vs. real-valued analyses were compared, and shorter acquisitions with reduced the number of b-value shells were investigated. Our findings reveal good reproducibility of the SANDI parameters, including the sphere and stick fractions, as well as sphere size (CoV < 7%, 12% and 3%, respectively). Additionally, we find a very good rank correlation between SANDI-driven sphere fraction and Allen mouse brain atlas contrast that represents cellular density. We conclude that SANDI is a viable preclinical MRI technique that can greatly contribute to research on brain tissue microstructure.

Diffusion MRI (dMRI) provides unique insights into the neural tissue milieu by probing interaction of diffusing molecules and tissue microstructure. Most dMRI techniques focus on white matter tissues (WM) due to the relatively simpler modelling of diffusion in the more organized tracts; however, interest is growing in gray matter characterisations. The Soma and Neurite Density MRI (SANDI) methodology harnesses a model incorporating water diffusion in spherical objects (assumed to be associated with cell bodies) and in impermeable “sticks” (representing neurites), which potentially enables the characterisation of cellular and neurite densities. Recognising the importance of rodents in animal models of development, aging, plasticity, and disease, we here sought to develop SANDI for preclinical imaging and provide a validation of the methodology by comparing its metrics with the Allen mouse brain atlas. SANDI was implemented on a 9.4T scanner equipped with a cryogenic coil, and experiments were carried out on N=6 mice. Pixelwise, ROI-based, and atlas comparisons were performed, and results were also compared to more standard Diffusion Kurtosis MRI (DKI) metrics. We further investigated effects of different pre-processing pipelines, specifically the comparison of magnitude and real-valued data, as well as different acceleration factors. Our findings reveal excellent reproducibility of the SANDI parameters, including the sphere and stick fraction as well as sphere size. More strikingly, we find a very good rank correlation between SANDI-driven soma fraction and Allen brain atlas contrast (which represents the cellular density in the mouse brain). Although some DKI parameters (FA, MD) correlated with some SANDI parameters in some ROIs, they did not correlate nearly as well as SANDI parameters with the Allen atlas, suggesting a much more specific nature of the SANDI parameters. We conclude that SANDI is a viable preclinical MRI technique that can greatly contribute to research on brain tissue microstructure.### Competing Interest StatementThe authors have declared no competing interest.

Dementia is a devastating age-related disorder. Its therapy would largely benefit from the identification of susceptible subjects at early, prodromal stages of the disease. To search for such prognostic markers of cognitive impairment, we studied spatial navigation in male BALBc vs. B6N mice in combination with in vivo magnetic resonance spectroscopy (1H-MRS). BALBc mice consistently showed higher escape latencies than B6N mice, both in the Water Cross Maze (WCM) and the Morris water maze (MWM). These performance deficits coincided with higher levels of myo-inositol (mIns) in the dorsal hippocampus before and after training. Subsequent biochemical analyses of hippocampal specimens by capillary immunodetection and liquid chromatography mass spectrometry-based (LC/MS) metabolomics revealed a higher abundance of glial markers (IBA-1, S100B, and GFAP) as well as distinct alterations in metabolites including a decrease in vitamins (pantothenic acid and nicotinamide), neurotransmitters (acetylcholine), their metabolites (glutamine), and acetyl-L-carnitine. Supplementation of low abundant acetyl-L-carnitine via the drinking water, however, failed to revert the behavioral deficits shown by BALBc mice. Based on our data we suggest (i) BALBc mice as an animal model and (ii) hippocampal mIns levels as a prognostic marker of mild cognitive impairment (MCI), due to (iii) local changes in microglia and astrocyte activity, which may (iv) result in decreased concentrations of promnesic molecules.

Alzheimer’s disease (AD) is characterised by many pathophysiological changes, such as the accumulation of amyloid-β. The clearing of detrimental agents, including amyloid-β proteins, from brain tissue is linked to the function of choroid plexus (CP) or blood-cerebrospinal fluid barrier (BCSFB). This study investigates for the first time BCSFB function using non-invasive ASL-based methods in a mouse model of AD. Significantly higher values of total BCSFB-mediated water delivery in AD mice relative to controls were observed as early as 8 weeks of age, and a possible (though currently non-significant) correlation with behavioural tests was identified.