Virginie Lam

The cerebral vasculature maintains brain function through precise blood flow regulation and its disruption is increasingly recognised as an early and functionally significant feature of Alzheimer’s disease. Understanding these pathologies requires high-resolution imaging capturing cerebral microvascular flow in vivo, particularly in rodent models enabling longitudinal investigation. However, existing modalities involve inherent trade-offs between spatial and temporal resolution, depth, sensitivity and accessibility, while anaesthetic and methodological variability limit cross-study comparability. High-resolution optical imaging, such as two-photon microscopy, provides capillary-level visualisation but is limited by imaging depth and invasiveness. Non-invasive techniques like multi-exposure speckle imaging and optical coherence tomography assess superficial cerebral blood flow dynamics, while magnetic resonance imaging and positron emission tomography offer whole-brain coverage at lower resolution and accessibility. Contrast enhanced ultrasound presents a promising balance of resolution, sensitivity, non-invasiveness and depth penetration. Recently, ultrafast ultrasound localisation ultrasound microscopy has enabled super-resolution imaging by tracking individual microbubbles to quantitatively map microvascular flow. This review critically evaluates preclinical imaging techniques for assessing cerebral microvascular flow in rodents, highlighting their methodological considerations, strengths and limitations. We provide a framework for modality selection and identify ultrafast ultrasound localisation microscopy as the modality best positioned to deliver the standardised, longitudinal and multiscale imaging approach currently lacking.

BACKGROUND: Effective treatments for neurodegenerative diseases remain elusive, underscoring the importance of preventive strategies. Probucol, a cholesterol lowering and antioxidant drug with established cardiovascular use, has shown neuroprotective effects in preclinical models of dementia by modulating peripheral lipoprotein amyloid metabolism and preserving capillary integrity. However, no large-scale human studies have examined its association with dementia risk. OBJECTIVE: To examine the association between probucol use and incident dementia in older adults. DESIGN, SETTING, AND PARTICIPANTS: This retrospective cohort study used the Japan Medical Data Centre claims database from 2014 to 2023. Adults aged 50 years or older prescribed probucol or statins were included, excluding those with prior dementia or recent drug exposure. Participants were categorized as probucol monotherapy users, statin monotherapy users, or combination users (≥2 prescriptions). Propensity score matching was used to balance baseline comorbidities. EXPOSURES: Probucol or statin therapy. MAIN OUTCOMES AND MEASURES: The primary outcome was incident all cause dementia. Secondary outcomes included Alzheimer’s disease and mixed Alzheimer’s and vascular dementia. Odds ratios (ORs) with 95% CIs were calculated using logistic regression adjusted for age and sex. RESULTS: Among 57 231 individuals (52.6% female) followed for up to 10 years (median, 3 years), 7 387 (12.9%) developed dementia. The cohort included 2 896 probucol users (5.1%) and 54 335 statin users (94.9%). Dementia incidence was higher among females (14.7%) than males (10.9%). Dementia incidence was lower in probucol users (5.6%, 162/2 896) than in statin users overall (13.2%, 8 248/62 519), with individual statins ranging from 11.4% (fluvastatin) to 16.7% (pravastatin). Probucol use was associated with a 62% lower adjusted risk of dementia compared with all statin users combined (adjusted OR, 0.38; 95% CI, 0.37-0.38). Protective associations were consistent across individual statin comparisons, with adjusted ORs ranging from 0.30 (pitavastatin) to 0.57 (fluvastatin). CONCLUSIONS AND RELEVANCE: In this large national cohort of Japanese adults, probucol use was associated with a substantially lower risk of incident dementia compared with statins. These findings provide the first large-scale human evidence linking probucol exposure with reduced dementia risk, supporting its further evaluation as a preventive therapy in prospective clinical trials. Key Points: Question: Is use of the lipid-lowering and antioxidant agent probucol associated with a reduced risk of incident dementia compared with statin therapy in older adults? Findings: In this nationwide cohort study of 57 231 Japanese adults aged 50 years and older, dementia incidence was nearly halved in probucol users (5.6%) compared with statin users overall (13.2%), corresponding to 62% lower adjusted odds of dementia. Protective associations were consistent across dementia subtypes and individual statins. Meaning: These findings suggest that probucol, a long-standing and well-tolerated cardiovascular drug, may offer a mechanistically distinct and potentially scalable strategy for dementia prevention, warranting confirmation in prospective intervention trials.

Despite its critical role in neurodevelopment and brain function, vitamin D (vit-D) homeostasis, metabolism, and kinetics within the central nervous system remain largely undetermined. Thus, it is of critical importance to establish an accurate, highly sensitive, and reproducible method to quantitate vit-D in brain tissue. Here, we present a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method and for the first time, demonstrate detection of seven major vit-D metabolites in brain tissues of C57BL/6J wild-type mice, namely 1,25(OH)₂D₃, 3-epi-1,25(OH)₂D₃, 1,25(OH)₂D₂, 25(OH)D₃, 25(OH)D₂, 24,25(OH)₂D₃, and 24,25(OH)₂D₂. Chromatographic separation was achieved on a pentaflurophenyl column with 3 mM ammonium formate water/methanol [A] and 3 mM ammonium formate methanol/isopropanol [B] mobile phase components. Detection was by positive ion electrospray tandem mass spectrometry with the EVOQ elite triple quadrupole mass spectrometer with an Advance ultra-high-performance liquid chromatograph and online extraction system. Calibration standards of each metabolite prepared in brain matrices were used to validate the detection range, precision, accuracy, and recovery. Isotopically labelled analogues, 1,25(OH)₂D₃-d₃, 25(OH)D₃-c₅, and 24,25(OH)₂D₃-d₆, served as the internal standards for the closest molecular-related metabolite in all measurements. Standards between 1 fg/mL and 10 ng/mL were injected with a resulting linear range between 0.001 and 1 ng, with an LLOD and LLOQ of 1 pg/mL and 12.5 pg/mL, respectively. The intra-/inter-day precision and accuracy for measuring brain vit-D metabolites ranged between 0.12–11.53% and 0.28–9.11%, respectively. Recovery in acetonitrile ranged between 99.09 and 106.92% for all metabolites. Collectively, the sensitivity and efficiency of our method supersedes previously reported protocols used to measure vit-D and to our knowledge, the first protocol to reveal the abundance of 25(OH)D₂, 1,25(OH)D₂, and 24,25(OH)₂D₂, in brain tissue of any species. This technique may be important in supporting the future advancement of pre-clinical research into the function of vit-D in neurophysiological and neuropsychiatric disorders, and neurodegeneration. Graphical Abstract

Ex vivo FTIR imaging reveals lipid distributions (red) alongside other spectroscopic markers in brain tissue, which can be used to provide greater biochemical insight when combined with in vivo MRI data sets collected from the same brain.

Introduction Preclinical, clinical and epidemiological studies support the hypothesis that aberrant systemic metabolism of amyloid beta (Aβ) in the peripheral circulation is causally related to the development of Alzheimer’s disease (AD). Specifically, recent studies suggest that increased plasma concentrations of lipoprotein-Aβ compromise the brain microvasculature, resulting in extravasation and retention of the lipoprotein-Aβ moiety. The latter results in an inflammatory response and neurodegeneration ensues. Probucol, a historic cholesterol-lowering drug, has been shown in murine models to suppress lipoprotein-Aβ secretion, concomitant with maintaining blood–brain-barrier function, suppressing neurovascular inflammation and supporting cognitive function. This protocol details the probucol in Alzheimer’s study, a drug intervention trial investigating if probucol has potential to attenuate cognitive decline, delay brain atrophy and reduce cerebral amyloid burden in patients with mild-to-moderate AD. Methods and analysis The study is a phase II, randomised, placebo-controlled, double-blind single-site clinical trial held in Perth, Australia. The target sample is 314 participants with mild-to-moderate AD. Participants will be recruited and randomised (1:1) to a 104-week intervention consisting of placebo induction for 2 weeks followed by 102 weeks of probucol (Lorelco) or placebo. The primary outcome is changed in cognitive performance determined via the Alzheimer’s Disease Assessment Scales-Cognitive Subscale test between baseline and 104 weeks. Secondary outcomes measures will be the change in brain structure and function, cerebral amyloid load, quality of life, and the safety and tolerability of Lorelco, after a 104week intervention. Ethics and dissemination The study has been approved by the Bellberry Limited Human Research Ethics Committee (approval number: HREC2019-11-1063; Version 4, 6 October 2021). Informed consent will be obtained from participants prior to any study procedures being performed. The investigator group will disseminate study findings through peer-reviewed publications, key conferences and local stakeholder events. Trial registration number Australian New Zealand Clinical Trials Registry (ACTRN12621000726853).

Brown adipose tissue (BAT) activation is associated with increased energy expenditure by inducing non-shivering thermogenesis. The ingestion of a milk fat globule membrane (MFGM) supplement and a high calorie diet are reported gateways into BAT activation. However, little is known about the effect of the MFGM and high calorie diets on BAT volume. To gain insight into this, mice were maintained on a high-fat (HF) or low-fat (LF) diet in conjunction with either full-cream (FC) or skim bovine dairy milk (BDM). After being maintained on their respective diets for 13 weeks, their body composition, including BAT volume, was measured using X-ray microtomography. A high calorie diet resulted in an increase in the BAT volume and mice consuming an HF diet in conjunction with FC BDM had a significantly greater BAT volume than all the other groups. Conversely, mice consuming an HF diet in addition to skim milk had a lower BAT volume compared to the HF control. The data presented suggest that the consumption of a high calorie diet in conjunction with FC BDM increases the BAT volume in wild-type mice. This study may provide valuable insight into future studies investigating BAT volume and BAT activity in relation to environmental factors, including diet.

Introduction: Preclinical, clinical and epidemiological studies support the hypothesis that aberrant systemic metabolism of amyloid-beta (Aβ) in the peripheral circulation is causally related to the development of Alzheimer’s disease (AD). Specifically, recent studies suggest that increased plasma concentrations of lipoprotein-Aβ compromises the brain microvasculature, resulting in extravasation and retention of the lipoprotein-Aβ moiety. The latter results in an inflammatory response and neurodegeneration ensues. Probucol, a historic cholesterol-lowering drug, has been shown in murine models to suppress lipoprotein-Aβ secretion, concomitant with maintaining blood-brain-barrier function and suppressing neurovascular inflammation. Probucol has also been shown to protect cognitive function in dietary-induced amyloidogenic mice. This protocol details the Probucol in Alzheimer’s Study (PIA-study), a double-blind, randomised, placebo-controlled drug intervention trial investigating if Probucol attenuates cognitive decline in patients with mild-to-moderate AD. Objectives: The primary objective of the 104-week study is to assess whether Probucol supports cognitive function and delays brain atrophy in AD patients. A secondary objective is to determine whether Probucol treatment will reduce cerebral amyloid burden. Methods & Analysis: The study is a phase II single-site, randomised, placebo-controlled, double-blind clinical trial assessing the efficacy of Probucol in AD. A total of 300 participants with mild-to-moderate AD will be recruited and randomised 1:1 (active: placebo). Cognitive function, regional volumetric changes in brain and cerebral amyloid load will be evaluated via the cognitive subscale test, AD assessment scales (ADAS-Cog), magnetic resonance imaging (MRI) and positron emission tomography (PET) scans, respectively, after a 104-week intervention. Ethics & Dissemination: The study has been approved by the Bellberry Limited Human Research Ethics Committee (Approval number: HREC2019-11-1063; Version 4, 6 ^th October 2021). The investigator group will disseminate study findings through peer-reviewed publications, key conferences and local stakeholder events. Trial registration: This trial has been registered with the Australian New Zealand Clinical Trial Registry (ACTRN12621000726853). ARTICLE SUMMARY: Strengths and limitations of this study: This is the first-in-human (FIH) randomised double-blind placebo-controlled study to assess the efficacy of Probucol in delaying cognitive decline in individuals with mild cognitive impairment (MCI) and mild-to-moderate dementia due to Alzheimer’s disease (AD). The 24-month intervention study will be the first to investigate whether treatment with Probucol will stabilise structural/functional changes in brain and if cerebral amyloid load will decrease in individuals with AD, following treatment with Probucol. Probucol is clinically used to treat cardiovascular disease with well-characterised efficacy and safety profiles, thus reducing risk of the study, and if applicable, accelerate clinical translation of the study findings.

Brown adipose tissue (BAT) activation is associated with increased energy expenditure by inducing non-shivering thermogenesis. Ingestion of a milk fat globule membrane (MFGM) supplement and a high calorie diet are reported gateways into BAT activation. However, little is known about the effect of MFGM and high calorie diets on BAT volume. To gain insight into this, mice were maintained on a high fat (HF) or low-fat (LF) diet in conjunction with either full-cream (FC) or skim bovine dairy milk (BDM). After being maintained on their respective diets for 13 weeks, body composition, including BAT volume, was measured using X-ray microtomography. A high calorie diet resulted in an increase in BAT volume and mice consuming a HF diet in conjunction with FC BDM had significantly greater BAT volume than all other groups. Conversely, mice consuming a HF diet in addition to skim milk had lower BAT volume compared to the HF control. The data presented suggests that consumption of a high calorie diet in conjunction with FC BDM increases BAT volume in wild-type mice. This study may provide valuable insight into future studies investigating BAT volume and BAT activity in relation to environmental factors including diet.

Background: The ingestion of combinatory Alcohol Mixed with Energy Drink (AMED) beverages continues to increase markedly, particularly among young adults. Some studies suggest detrimental health effects related to the combination of alcohol with energy drink formulations; however, the consumption of AMED has not been investigated in context of the cerebral microvasculature or neuroinflammation. We hypothesized that cerebral capillary integrity and glial cells are particularly vulnerable to the combination of AMED. Methods:12-week old wild-type C57BL/6J mice were orally gavaged with either vehicle (water), alcohol (vodka), an energy drink (Mother^TM), or a combination AMED, daily for five days. Thereafter, mice were sacrificed, blood alcohol concentrations were analysed and cryosections of brain specimens were subjected to confocal immunofluorescent analysis for measures of cerebral capillary integrity via immunoglobulin G (IgG), and markers of neuroinflammation, ionized-calcium-binding-adaptor-molecule 1 (Iba1) and Glial-Fibrillary-Acidic-Protein (GFAP). Proinflammatory cytokines, IL-2, IL-17A, IFN-ϒ, and anti-inflammatory cytokines, IL-4, IL-6 and IL-10, were also measured in serum. Results: Consistent with previous studies, cerebral capillary dysfunction and astroglial cell activation were markedly greater in the alcohol-only group (AO); however, the AO-induced effects were profoundly attenuated with the AMED combination. Mice maintained on AO and AMED interventions exhibited a moderate increase in microglial recruitment. There were no significant changes in pro-inflammatory nor anti-inflammatory cytokines in ED or AMED treated mice. Conclusion: This study suggests that paradoxically the acute detrimental effects of alcohol on cerebral capillary integrity and astrogliosis are counteracted with the co-provision of an ED, rich in caffeine and taurine and containing B-group vitamins.

Several lines of study suggest that peripheral metabolism of amyloid beta (Aß) is associated with risk for Alzheimer disease (AD). In blood, greater than 90% of Aß is complexed as an apolipoprotein, raising the possibility of a lipoprotein-mediated axis for AD risk. In this study, we report that genetic modification of C57BL/6J mice engineered to synthesise human Aß only in liver (hepatocyte-specific human amyloid (HSHA) strain) has marked neurodegeneration concomitant with capillary dysfunction, parenchymal extravasation of lipoprotein-Aß, and neurovascular inflammation. Moreover, the HSHA mice showed impaired performance in the passive avoidance test, suggesting impairment in hippocampal-dependent learning. Transmission electron microscopy shows marked neurovascular disruption in HSHA mice. This study provides causal evidence of a lipoprotein-Aß /capillary axis for onset and progression of a neurodegenerative process.