PURPOSE:We developed a dynamic, heavily T2-weighted (hT2w) FLAIR protocol to resolve acute Mn2+ transport kinetics, as a proxy for Ca2+, across the choroid plexus (ChP)-cerebrospinal fluid (CSF) interface, in vivo. By quantifying intercompartmental Mn2+ dynamics, and comparing these with Gd-DOTA, we sought to characterize blood-CSF-barrier (BCSFB)-mediated tracer transport, combining high in-plane resolution and 4-min temporal sampling. METHODS:hT2w-FLAIR DCE MRI was conducted in anesthetized C57BL/6 mice, receiving intravenous Mn2+ or Gd-DOTA. Semi-quantitative tracer kinetic modeling permitted regional and inter-tracer comparisons. RESULTS:Mn2+ rapidly crossed from the vasculature into the CSF, across the ChP, with similar arrival times and time-to-peak across compartments. Mn2+ delivery into the ChP exhibited a 5.7-fold steeper uptake rate (p = 0.031) and a 5.6-fold higher peak signal amplitude (p = 0.0028) than in the LV-CSF. In contrast, Gd-DOTA exhibited a 2.2-fold shorter ChP plateau duration (p = 0.034) and 2.2-fold faster ChP clearance rate (p = 0.0047), compared to Mn2+. Within the CSF, there were significant temporal delays with Gd-DOTA relative to Mn2+: a lengthened arrival (eight-fold, p = 0.045) and time-to-peak (six-fold, p = 0.024). CONCLUSION:Our hT2w-FLAIR DCE-MRI approach captured the early BCSFB transport window for Mn2+, providing distinct ChP and LV-CSF kinetics at a higher temporal resolution and with reduced CSF signal contamination compared to prior approaches. These findings highlight that MRI tracer choice strongly shapes measured BCSFB kinetics, and supports the use of Mn2+ as a tool for probing compartment-specific ChP transport.

The choroid plexus (CP), or blood-cerebrospinal fluid barrier, performs unique and diverse roles in support of brain homeostasis. Novel and non-invasive imaging biomarkers of choroid plexus physiology may be useful to further our understanding of its role in the development of pathology. Here, we introduce the concept of measuring water exchange between the choroid plexus tissue and the proximal CSF using a multiple TE fluid-suppressed (FLAIR) acquisition. By fitting the MRI signal at the choroid plexus acquired with a multi-TE FLAIR readout to a two-compartment bi-exponential model, we observed that the slow decaying T2 component of the signal had a T2 highly similar to that of cerebrospinal fluid (CSF). This finding, in turn, provides evidence that, paradoxically, this signal derives from CSF in a FLAIR image. The specific spatial co-localization of this long-T2 signal to the CP within the lateral ventricles provides evidence that this reflects the exchange of water molecules between the CP tissue and the CSF during the inversion time. A reduction in choroid plexus-CSF water exchange rate was then detected in the aged vs. young mouse brain using this method. Preliminary application of the method to the human brain at 3T, however, yielded weaker results which suggest that the method may not be able to capture this phenomenon clinically with equivalent sensitivity. Nonetheless, in pre-clinical studies, this novel MRI contrast mechanism provides a specific and quantitative signature of choroid plexus physiology and thus may represent a useful non-invasive imaging biomarker of CP dysfunction.

Introduction Vascular-cognitive-impairment and dementia (VCID) is a leading cause of disability in an increasingly ageing population. Impaired cerebral blood flow is a key characteristic of this dementia, of which few effective preclinical models are available. In this study, we aim to characterise changes in both cerebral and cardiac vascular function using MRI in the novel hyperhomocysteinemia (HHcy) induced mouse model of VCID. Methods C57BL6 mice of both sexes were subject to a HHcy inducing diet (n=10), which is known to develop cognitive impairment and cerebral microbleeds, or a control diet (n=10). After 10 weeks, mice underwent nesting, open field (OFT) and novel object recognition (NOR) behavioural testing to identify any cognitive changes. Basal cerebral blood flow (CBF) and cerebrovascular reactivity to hypercapnia in the cortex, hippocampus and midbrain were quantified using arterial spin labelling (ASL) MRI (9.4T Bruker) under medetomidine anaesthesia. As HHcy is known to increase cardiac atrial fibrillation and infarcts, cardiovascular function was additionally assessed using cine MRI with electrocardiogram-triggering (9.4T Agilent). Results At 10 weeks, no differences were identified between control and HHcy diet treated mice with nesting, OFT or NOR behavioural testing. Although no differences in basal CBF were detected, cerebrovascular reactivity to hypercapnia was significantly reduced in mice on the HHcy diet compared to those on the control diet (Figure 1, p<0.05). This occurred specifically in the cortex, and no significant difference was found in the hippocampus or midbrain. At 10 weeks, cardiovascular function was not found to be significantly affected by the HHcy inducing diet, although there was a trend towards decreased stroke volume and average mass of the left ventricle. Conclusions Although the HHcy inducing diet did not lead to detectable changes in cognitive function or basal CBF in this study, a decreased ability to respond to hypercapnia was found. This is suggestive of early disrupted cerebrovascular reactivity, a translational biomarker which could precede cognitive deficits and changes to basal perfusion. Further studies are underway to understand the mechanisms driving this impairment and characterise the potential for cerebrovascular reactivity to be used as an early biomarker of VCID.

Therapies targeting blood vessels hold promise for autosomal dominant polycystic kidney disease (ADPKD), the most common inherited disorder causing kidney failure. However, the onset and nature of kidney vascular abnormalities in ADPKD are poorly defined. Accordingly, we employed a combination of single-cell transcriptomics; three-dimensional imaging with geometric, topological and fractal analyses; and multimodal magnetic resonance imaging with arterial spin labelling to investigate aberrant microvasculature in ADPKD kidneys. Within human ADPKD kidneys with advanced cystic pathology and excretory failure, we identified a molecularly distinct blood microvascular subpopulation, characterised by impaired angiogenic signalling and metabolic dysfunction, differing from endothelial injury profiles observed in non-cystic human kidney diseases. Next, Pkd1 mutant mouse kidneys were examined postnatally, when cystic pathology is well established, but before excretory failure. An aberrant endothelial subpopulation was also detected, concurrent with reduced cortical blood perfusion. Disorganised kidney cortical microvasculature was also present in Pkd1 mutant mouse fetal kidneys when tubular dilation begins. Thus, aberrant features of cystic kidney vasculature are harmonised between human and mouse ADPKD, supporting early targeting of the vasculature as a strategy to ameliorate ADPKD progression.

The choroid plexus plays an important role in brain homeostasis, including the active secretion of cerebrospinal fluid. Its function and structure have been reported to be affected by normal ageing. However, existing measures of choroid plexus volume may be complicated by partial volume (in vivo MRI) and tissue fixation artefacts (histology). In this study, we investigate possible changes in choroid plexus volume within the lateral ventricles of aged mice utilising two structural MRI protocols explicitly designed for time-efficient, high-resolution in vivo imaging of the choroid plexus. Two MRI sequences were utilised to examine in vivo choroid plexus volume in the lateral ventricles of young (∼ 6 months) and aged (∼ 24 months) mouse brains: (1) an ultra-long echo-time T2 weighted fast-spin-echo and (2) a multi-TE T2* mapping protocol. A test-retest study was performed on a subset of the data to examine the reproducibility of choroid plexus volume estimation based on manual segmentation. A two-way ANOVA test was performed to determine possible differences in choroid plexus volume in young and aged mouse groups across the two distinct MRI protocols. Reproducibility tests showed a low test-retest variability of the manual segmentation pipeline for both MRI protocols. A statistically significant reduction of in vivo choroid plexus volume was found in the aged mouse brain. This finding is concordant with previous histological observations of a reduction in epithelial cell height with ageing across a wide range of species. We present an in vivo investigation of changes to lateral ventricle choroid plexus volume in the mouse brain utilising a manual segmentation approach based on two bespoke MRI protocols designed for time-efficient high resolution imaging of the choroid plexus. Based on these protocols, we provide evidence for a reduction in choroid plexus volume in the aged brain. This research provides insight for studies utilising MRI measurements of choroid plexus volume as a biomarker of age-related neurologic conditions as it indicates that the ageing process itself does not result in hypertrophy of the choroid plexus, but a decrease in tissue volume.

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.

Hallmarks of autosomal dominant polycystic kidney disease (ADPKD), the most common hereditary kidney anomaly, include expanding fluid-filled epithelial cysts, inflammation, and fibrosis. Despite previous work showing the potential of vascular-based therapies, renal microvascular alterations in ADPKD, and their timing, are poorly understood. Using single-cell transcriptomics of human kidney microvasculature, we identify a population of endothelial cells adjacent to cysts in ADPKD. This pericystic endothelium, distinguishable by its expression of osteopontin (SPP1), has a distinct molecular profile compared to the common endothelial cell injury signature in other kidney diseases. SPP1+ pericystic endothelium was also present in an orthologous mouse model of ADPKD before overt kidney functional decline. By interrogating geometric, topological and fractal properties from three-dimensional imaging of early ADPKD mouse kidneys, we show that pericystic endothelium associates with disorganisation and non-uniformity of the renal cortical microvasculature. Concurrently, we detected region-specific reductions in cortical blood flow within ADPKD murine kidneys using arterial spin labelling. We conclude that ADPKD kidneys contain a unique subset of endothelium manifesting with aberrant remodelling and impaired blood perfusion. Its detection, prior to renal functional decline, advocates the vasculature as a therapeutic target to modulate or preserve renal function in early ADPKD. ### Competing Interest Statement The authors have declared no competing interest.

Chronic hypertension is a major risk factor for the development of neurodegenerative disease, yet the etiology of hypertension-driven neurodegeneration remains poorly understood. Forming a unique interface between the systemic circulation and the brain, the blood-cerebrospinal fluid barrier (BCSFB) at the choroid plexus (CP) has been proposed as a key site of vulnerability to hypertension that may initiate downstream neurodegenerative processes. However, our ability to understand BCSFB’s role in pathological processes has, to date, been restricted by a lack of non-invasive functional measurement techniques. In this work, we apply a novel Blood-Cerebrospinal Fluid Barrier Arterial Spin Labeling (BCSFB-ASL) Magnetic resonance imaging (MRI) approach with the aim of detecting possible derangement of BCSFB function in the Spontaneous Hypertensive Rat (SHR) model using a non-invasive, translational technique. SHRs displayed a 36% reduction in BCSFB-mediated labeled arterial water delivery into ventricular cerebrospinal fluid (CSF), relative to normotensive controls, indicative of down-regulated choroid plexus function. This was concomitant with additional changes in brain fluid biomarkers, namely ventriculomegaly and changes in CSF composition, as measured by T1 lengthening. However, cortical cerebral blood flow (CBF) measurements, an imaging biomarker of cerebrovascular health, revealed no measurable change between the groups. Here, we provide the first demonstration of BCSFB-ASL in the rat brain, enabling non-invasive assessment of BCSFB function in healthy and hypertensive rats. Our data highlights the potential for BCSFB-ASL to serve as a sensitive early biomarker for hypertension-driven neurodegeneration, in addition to investigating the mechanisms relating hypertension to neurodegenerative outcomes.

Pharmacological MRI (phMRI) studies seek to capture changes in brain haemodynamics in response to a drug. This provides a methodological platform for the evaluation of novel therapeutics, and when applied to disease states, may provide diagnostic or mechanistic information pertaining to common brain disorders such as dementia. Changes to brain perfusion and blood-cerebrospinal fluid barrier (BCSFB) function can be probed, non-invasively, by arterial spin labelling (ASL) and blood-cerebrospinal fluid barrier arterial spin labelling (BCSFB-ASL) MRI respectively. Here, we introduce a method for simultaneous recording of pharmacological perturbation of brain perfusion and BCSFB function using interleaved echo-time ASL, applied to the anesthetized mouse brain. Using this approach, we capture an exclusive decrease in BCSFB-mediated delivery of arterial blood water to ventricular CSF, following anti-diuretic hormone, vasopressin, administration. The commonly used vasodilatory agent, CO2, induced similar increases (~21%) in both cortical perfusion and the BCSFB-ASL signal. Furthermore, we present evidence that caffeine administration triggers a marked decrease in BCSFB-mediated labelled water delivery (41%), with no significant changes in cortical perfusion. Finally, we demonstrate a marked decrease in the functional response of the BCSFB to, vasopressin, in the aged vs adult brain. Together these data, the first of such kind, highlight the value of this translational approach to capture simultaneous and differential pharmacological modulation of vessel tone at the blood brain barrier and BCSFB and how this relationship may be modified in the ageing brain.

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.