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.

Propagation and aggregation of prion proteins, such as tau and α-synuclein (α-syn), are key pathological features of neurodegenerative diseases. Extracellular clearance pathways, such as the glymphatic system, might play a crucial role in the removal of these toxic proteins from the brain. Primarily active during sleep, this system relies on aquaporin-4 (AQP4) water channel expression and polarization to astrocytic endfeet, facilitating interstitial solute clearance. Glymphatic dysfunction has recently been implicated in Parkinson's disease, but the precise mechanisms underlying the pathogenic effect of this dysfunction remain unclear. This includes how impaired glymphatic function influences α-syn propagation dynamics, in addition to the role of propagating α-syn itself on glymphatic function. In this study, we used a mouse model of α-syn propagation to elucidate the impact of α-syn aggregation on glymphatic function by measuring CSF-interstitial fluid exchange and assessing AQP4 and associated endfoot complex proteins in the brain over time and across different regions. Our results show that direct injection of α-syn preformed fibrils leads to local reduced expression of the AQP4 endfoot complex, but propagation of α-syn pathology induces an enhancement of glymphatic function, suggesting compensatory upregulation in response to increasing α-syn aggregate load. To determine the influence of glymphatic dysfunction on α-syn propagation dynamics, we then took a pharmacological approach to inhibit glymphatic function in this model. Acute glymphatic inhibition significantly reduced brain-to-CSF clearance of misfolded α-syn, and chronic treatment exacerbated α-syn pathology, cerebral atrophy and motor behavioural deficits in mice. Together, our findings show that α-syn clearance and propagation are modulated by glymphatic function. Moreover, they suggest that AQP4 complex dysregulation might contribute to glymphatic impairment associated with Parkinson's disease, supporting further mechanistic investigation of this protein complex in the disease.

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.

Glioblastoma (GBM) is the most common and aggressive brain tumour with starkresistance to available therapies, leading to relapse and a median survival of<15 months. A key cause of therapy resistance is diffuse infiltration oftumour cells into brain regions surrounding the tumour, which presents a majorclinical challenge as existing imaging techniques offer limited detection of theresectable margin. Here, we use diffusion weighted imaging (DWI) and apply themultiple echo time neurite orientation dispersion and density imaging(MTE-NODDI) model as a tool to detect tumour cells in the hard-to-distinguishmargin. We used the G144 patient-derived xenograft model, with characteristicinvasion along white matter tracts, in combination with MTE-NODDI. Tumourdevelopment was monitored, and magnetic resonance imaging (MRI) data wereacquired over a 4-week period, starting at 4 weeks after stereotactic injectionof tumour cells. MTE-NODDI demonstrated sensitivity to the developing tumour inthe invading margin, and changes in measured parameters were apparent from 6weeks after injection. In comparison to standard DWI, MTE-NODDI showed increasedsensitivity to the tumour-associated changes in the margin. Furthermore,extraneurite volume fraction (fen ) and neuritedensity index (NDI) measured from MTE-NODDI correlated with immunohistologicalmeasurement of tumour cells. These findings suggest that MTE-NODDI maynon-invasively detect infiltrating cells and tumour-induced pathology in marginregions without T2 or DWI changes in a patient-derived mouse model of GBM.MTE-NODDI is clinically translatable and could be a powerful tool forneurosurgeons to maximise surgical resection, resulting in better survivaloutcomes for patients with GBM.

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.

Propagation and aggregation of prion proteins, such as tau and α-synuclein (αSyn), are key pathological features of neurodegenerative diseases. Extracellular clearance pathways, such as the glymphatic system, may play a crucial role in the removal of these toxic proteins from the brain. Primarily active during sleep, this system relies on aquaporin-4 (AQP4) water channel expression and polarisation to astrocytic endfeet, facilitating interstitial solute clearance. Glymphatic dysfunction has recently been implicated in Parkinson’s disease, however the precise mechanisms underlying the pathogenic effect of this dysfunction remain unclear. This includes how impaired glymphatic function influences αSyn propagation dynamics, and the role of propagating αSyn itself on glymphatic function. In this study, we used a mouse model of αSyn propagation to elucidate the impact of αSyn aggregation on glymphatic function, by measuring CSF-ISF exchange and assessing AQP4 and associated endfoot complex proteins in the brain over time and across different regions. Our results show that direct injection of αSyn pre-formed fibrils leads to reduced expression of the AQP4 endfoot complex, but propagation of endogenous αSyn induces an enhancement of glymphatic function suggesting compensatory upregulation in response to increasing endogenous αSyn load. To determine the influence of glymphatic dysfunction on αSyn propagation dynamics, we then employed a pharmacological approach to inhibit glymphatic function in this model. Acute glymphatic inhibition significantly reduced brain to CSF αSyn clearance, and chronic treatment exacerbated αSyn pathology, neurodegeneration, and motor behavioural deficits in mice. Together our findings show that αSyn clearance and propagation are modulated by glymphatic function and suggest that AQP4 complex dysregulation may contribute to glymphatic impairment associated with Parkinson’s diseases. Summary for the non-scientific community The glymphatic system clears brain waste during sleep. Lopes et al. show that α-synuclein, a protein linked to Parkinson’s, is cleared by this system. Using a mouse model of the disease, they suggest that aquaporin-4 water channels may impair glymphatic function, contributing to α-synuclein buildup in patients’ brains. ### Competing Interest Statement The authors have declared no competing interest.

Perivascular spaces mediate a complex interaction between cerebrospinal fluid and brain tissue that may be an important pathway for solute waste clearance. Their structural or functional derangement may contribute to the development of age-related neurogenerative conditions. Here, we employed a non-invasive low b-value diffusion-weighted ECG-gated MRI method to capture perivascular fluid movement around the middle cerebral artery of the anaesthetised rat brain. Using this method, we show that such MRI estimates of perivascular fluid movement directionality are highly sensitive to the cardiac cycle. We then show that these measures of fluid movement directionality are decreased in the angiotensin-II pharmacological model of acute hypertension, with an associated dampening of vessel pulsatility. This translational MRI method may, therefore, be useful to monitor derangement of perivascular fluid movement associated with cardiovascular pathologies, such as hypertension, in order to further our understanding of perivascular function in neurology.

Early in Alzheimer's disease (AD), pericytes constrict capillaries, increasing their hydraulic resistance and trapping of immune cells and, thus, decreasing cerebral blood flow (CBF). Therapeutic approaches to attenuate pericyte-mediated constriction in AD are lacking. Here, using in vivo two-photon imaging with laser Doppler and speckle flowmetry and magnetic resonance imaging, we show that Ca2+ entry via L-type voltage-gated calcium channels (CaVs) controls the contractile tone of pericytes. In AD model mice, we identifed pericytes throughout the capillary bed as key drivers of an immune reactive oxygen species (ROS)-evoked and pericyte intracellular calcium concentration ([Ca2+]i)-mediated decrease in microvascular flow. Blocking CaVs with nimodipine early in disease progression improved CBF, reduced leukocyte stalling at pericyte somata and attenuated brain hypoxia. Amyloid beta (A beta)-evoked pericyte contraction in human cortical tissue was also greatly reduced by CaV block. Lowering pericyte [Ca2+]i early in AD may, thus, offer a therapeutic strategy to enhance brain energy supply and possibly cognitive function in AD.

Abstract Background The aggregation and spread of misfolded amyloid structured proteins, such as tau and α-synuclein, are key pathological features associated with neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. These proteins possess a prion-like property, enabling their transmission from cell to cell leading to propagation throughout the central and peripheral nervous systems. While the mechanisms underlying their intracellular spread are still being elucidated, targeting the extracellular space has emerged as a potential therapeutic approach. The glymphatic system, a brain-wide pathway responsible for clearing extracellular metabolic waste from the central nervous system, has gained attention as a promising target for removing these toxic proteins. Methods In this study, we investigated the impact of long-term modulation of glymphatic function on tau aggregation and spread by chronically treating a mouse model of tau propagation with a pharmacological inhibitor of AQP4, TGN-020. Thy1-hTau.P301S mice were intracerebrally inoculated with tau into the hippocampus and overlying cortex, and subsequently treated with TGN-020 (3 doses/week, 50 mg/kg TGN-020, i.p.) for 10-weeks. During this time, animal memory was studied using cognitive behavioural tasks, and structural MR images were acquired of the brain in vivo prior to brain extraction for immunohistochemical characterisation. Results Our findings demonstrate increased tau aggregation in the brain and transhemispheric propagation in the hippocampus following the inhibition of glymphatic clearance. Moreover, disruption of the glymphatic system aggravated recognition memory in tau inoculated mice and exacerbated regional changes in brain volume detected in the model. When initiation of drug treatment was delayed for several weeks post-inoculation, the alterations were attenuated. Conclusions These results indicate that by modulating AQP4 function and, consequently, glymphatic clearance, it is possible to modify the propagation and pathological impact of tau in the brain, particularly during the initial stages of the disease. These findings highlight the critical role of the glymphatic system in preserving healthy brain homeostasis and offer valuable insights into the therapeutic implications of targeting this system for managing neurodegenerative diseases characterized by protein aggregation and spread.

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.

A detailed study into the synthesis and functional properties of layered rare-earth hydroxides (LRHs) is reported. It is possible to obtain precise control of particle size, and combining Tb and Gd in the LRH allows multi-modal imaging.

Neurofluids is a term introduced to define all fluids in the brain and spine such as blood, cerebrospinal fluid, and interstitial fluid. Neuroscientists in the past millennium have steadily identified the several different fluid environments in the brain and spine that interact in a synchronized harmonious manner to assure a healthy microenvironment required for optimal neuroglial function. Neuroanatomists and biochemists have provided an incredible wealth of evidence revealing the anatomy of perivascular spaces, meninges and glia and their role in drainage of neuronal waste products. Human studies have been limited due to the restricted availability of noninvasive imaging modalities that can provide a high spatiotemporal depiction of the brain neurofluids. Therefore, animal studies have been key in advancing our knowledge of the temporal and spatial dynamics of fluids, for example, by injecting tracers with different molecular weights. Such studies have sparked interest to identify possible disruptions to neurofluids dynamics in human diseases such as small vessel disease, cerebral amyloid angiopathy, and dementia. However, key differences between rodent and human physiology should be considered when extrapolating these findings to understand the human brain. An increasing armamentarium of noninvasive MRI techniques is being built to identify markers of altered drainage pathways. During the three-day workshop organized by the International Society of Magnetic Resonance in Medicine that was held in Rome in September 2022, several of these concepts were discussed by a distinguished international faculty to lay the basis of what is known and where we still lack evidence. We envision that in the next decade, MRI will allow imaging of the physiology of neurofluid dynamics and drainage pathways in the human brain to identify true pathological processes underlying disease and to discover new avenues for early diagnoses and treatments including drug delivery.Evidence level: 1Technical Efficacy: Stage 3

Introduction Approximately 30,000 patients experience ruptured aneurysms annually. Rupture is fatal in 50% of cases, and among those who survive, 66% suffer permanent neurological morbidity. Compliant balloons are commonly used to facilitate coil embolization of wide-necked ruptured aneurysms, however there is an increased risk of ischemia due to compromise of parent artery blood flow during embolization. Additionally, balloon inflation/deflation cycles can cause blood vessel trauma. The proposed balloon-stent device is a temporary adjunctive device composed of a self-expandable nitinol mesh structure that covers the aneurysm neck preventing intra-aneurysmal device protrusion. Unlike a balloon inflation, this design also allows blood to perfuse through the device and parent artery, thereby reducing the risk of ischemia. Materials and Methods The prototype balloon-stent is composed of a fine mesh with 250µm pores (figure 1a) up to 4x smaller area than current flow diverters. Quantification of the prototype's radial force, flow disruption effects, and ease of delivery/retrieval have been measured by the Bioengineering Devices Lab (BDL) at Northern Arizona University (NAU) using a hybrid DMA-rheometer (HR2, TA Instruments) and an advanced flow system. Radial force measurements from the DMA-rheometer were compared to those of control devices (Scepter-C and LVIS Jr.-Microvention). A sophisticated physiologically-relevant benchtop flow system was used to quantify flow disruption effects via the pressure drop measurements in 3D-printed parent vessels, across prototype and control devices. The flow system includes a programmable pulsatile pump system, mechanically relevant 3D-printed models, pressure transducers, and a blood analog fluid. Results The balloon-stent prototypes and control devices were delivered and retrieved from 3D-printed aneurysm models under fluoroscopic imaging (figure 1b). The prototype and stent devices exhibited minimal pressure drop (Fractional Pressure ratio (FPR) >0.95), with prior work showing FPR >0.75 minimizes downstream ischemic risk. The radial force/length of the balloon-stent was ~10 times lower than a self-expanding LVIS-Jr stent and ~80 times lower than a Scepter-C balloon. Conclusion The proposed device is a highly flexible, retrievable, temporary adjunctive medical device for aneurysm treatment. This device provides a smooth protective surface that effectively seals the aneurysm neck during adjunctive treatment. This device can potentially reduce embolic device complications, such as coil protrusion, resulting in a more stable and consistent embolic device placements without the need for temporary balloon protection. Further testing is underway to increase balloon-stent device radial force to maximize aneurysm neck sealing and minimize vessel trauma. Disclosures O. Asgari: 5; C; Northern Arizona University. J. Wells: 5; C; Northern Arizona University, Aneuvas Technologies, Inc. C. Fisher: 5; C; Northern Arizona University. T. Becker: 2; C; United Biologics. 4; C; Aneuvas Technologies, Inc.. 5; C; Northern Arizona University. A. Ducruet: 2; C; Medtronic, Penumbra, Oculus, Stryker, Balt, Koswire. 4; C; Aneuvas Technologies, inc.. 5; C; Barrow Neurological Institute.

During hypoxia, increases in cerebral blood flow maintain brain oxygen delivery. Here, we describe a mecha-nism of brain oxygen sensing that mediates the dilation of intraparenchymal cerebral blood vessels in response to reductions in oxygen supply. In vitro and in vivo experiments conducted in rodent models show that during hypoxia, cortical astrocytes produce the potent vasodilator nitric oxide (NO) via nitrite reduction in mitochon-dria. Inhibition of mitochondrial respiration mimics, but also occludes, the effect of hypoxia on NO production in astrocytes. Astrocytes display high expression of the molybdenum-cofactor-containing mitochondrial enzyme sulfite oxidase, which can catalyze nitrite reduction in hypoxia. Replacement of molybdenum with tungsten or knockdown of sulfite oxidase expression in astrocytes blocks hypoxia-induced NO production by these glial cells and reduces the cerebrovascular response to hypoxia. These data identify astrocyte mitochondria as brain oxygen sensors that regulate cerebral blood flow during hypoxia via release of nitric oxide.

Neurovascular coupling is a fundamental brain mechanism that regulates local cerebral blood flow (CBF) in response to changes in neuronal activity. Functional imaging techniques are commonly used to record these changes in CBF as a proxy of neuronal activity to study the human brain. However, the mechanisms of neurovascular coupling remain incompletely understood. Here we show in experimental animal models (laboratory rats and mice) that the neuronal activity-dependent increases in local CBF in the somatosensory cortex are prevented by saturation of the CO 2 -sensitive vasodilatory brain mechanism with surplus of exogenous CO 2 or disruption of brain CO 2 /HCO 3 − transport by genetic knockdown of electrogenic sodium-bicarbonate cotransporter 1 (NBCe1) expression in astrocytes. A systematic review of the literature data shows that CO 2 and increased neuronal activity recruit the same vasodilatory signaling pathways. These results and analysis suggest that CO 2 mediates signaling between neurons and the cerebral vasculature to regulate brain blood flow in accord with changes in the neuronal activity.

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.