John Charles Louis Mamo

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.

Background and aims Chronically elevated levels of circulating lipoprotein-amyloid-β (Aβ) are implicated in the disruption of the blood-brain barrier and the initiation of a neurodegenerative cascade leading to Alzheimer’s disease (AD). Type 2 diabetes is associated with BBB dysfunction, dyslipidaemia, and an increased risk of AD. However, alterations in triglyceride-rich lipoproteins (TRL)-Aβ homeostasis and its downstream effects on the BBB in a diabetes context remain explored. This study aimed to 1) investigate, in a preclinical model of diabetes, the hypothesis that diabetes-induced impairments in TRL-Aβ metabolism might compromise BBB integrity and exacerbate cognitive function and behavioural changes, and 2) assess the efficacy of interventions that improve TRL catabolism, including probucol, HA1, and ApoC3 siRNA, to prevent disease progression by lowering circulating TRL-Aβ levels. Methods Five-week-old db/db mice underwent a 9- or 23-weeks dietary interventions with probucol, a probucol prodrug HA1, or a standard diet with four-weekly injections of ApoC3 siRNA. Db/+ mice served as negative controls for each treatment duration. Blood levels of Aβ and ApoB were measured using ELISA. Immunofluorescence imaging was used to quantify enterocytic levels of Aβ and ApoB, and assess changes in neurovascular integrity (IgG, PDGFRβ, ZO1, occludin), neuroinflammation (GFAP, Iba1), and cerebral oxidative stress (8OHdG). Results Our results indicate that diabetes increased the abundance of plasma amyloid, specifically Aβ42, which correlated with enterocytic abundance, suggesting exaggerated postprandial excretion. Disrupted plasma amyloid homeostasis was associated with BBB breakdown, including diminished barrier function, and the loss of pericytes and astrocytes. Provision of the probucol analogue, HA1, normalised plasma and enterocytic amyloidemia concomitant with the preservation of the neurovascular junction. Treatment with ApoC3 siRNA attenuated plasma Aβ42 and modestly reduced neurovascular inflammation. Conclusion The findings further support the hypothesis that aberrant peripheral metabolism of lipoprotein-Aβ is associated with microvascular corruption and the development of anxiety-like behaviour. HA1 is more effective than probucol or ApoC3 siRNA in positively modulating lipoprotein-amyloid homeostasis in db/db mice and maintaining central capillary integrity. Highlights Enterocytic and plasma lipoprotein-Aβ levels are increased in diabetic db/db mice. Elevated plasma lipoprotein-Aβ levels correlate with BBB breakdown and anxiety-like behavior. HA1 lowers enterocytic and plasma Aβ levels, protects BBB, alleviates oxidative stress and anxiety. ApoC3 siRNA lowers plasma lipoprotein-Aβ, mitigates neuroinflammation and anxiety.

Migraine is one of the world’s most debilitating disorders, and it has recently been shown that changes in the retina can be a potential biomarker for the disease. These changes can be detected by optical coherence tomography (OCT), which measures retinal thickness, and optical coherence tomography angiography (OCTA), which measures vessel density. We searched the databases Google Scholar, ProQuest, Scopus, and Web of Science for studies in English using OCT and OCTA in migraineurs, using the search terms “optical coherence tomography,” “OCT,” “optical coherence tomography angiography,” “OCTA” and “migraine.” We found 73 primary studies, 11 reviews, and 8 meta-analyses pertaining to OCT and OCTA findings in migraineurs. They showed that migraineurs had reduced retinal thickness (via OCT), retinal vessel density, and greater foveal avascular zone area (via OCTA) than controls. OCTA changes reflect a perfusion compromise occurring in migraineurs as opposed to in healthy controls. OCT and OCTA deficits were worse in migraine-with-aura and chronic migraine than in migraine-without-aura and episodic migraine. Certain areas of the eye, such as the fovea, may be more vulnerable to these perfusion changes than other parts. Direct comparison between study findings is difficult because of the heterogeneity between the studies in terms of both methodology and analysis. Moreover, as almost all case–control studies were cross-sectional, more longitudinal cohort studies are needed to determine cause and effect between migraine pathophysiology and OCT/OCTA findings. Current evidence suggests both OCT and OCTA may serve as retinal markers for migraineurs, and further research in this field will hopefully enable us to better understand the vascular changes associated with migraine, perhaps also providing a new diagnostic and therapeutic biomarker.

Background: Migraine is a common and distressing neurological condition characterised by recurrent throbbing headaches, nausea and heightened sensitivity to light and sound. Accumulating evidence suggests that cerebral arteries dilate during migraine, causing distal microvessels to constrict, which could activate nociceptors and cause onset of headache pain. If so, preventing or attenuating chronic microvascular constriction, and promoting a dilatory phenotype, may reduce frequency and/or severity of migraines. The primary aim of the L-Arginine and Aged Garlic Extract (LARGE) trial is to investigate whether oral treatment with dietary nutraceuticals, L-arginine and aged garlic extract (AGE), both systemic vasodilatory agents, will alleviate migraine frequency, duration and severity in adults with chronic frequent episodic migraines. Methods: The study is a randomised double-blind placebo-controlled phase-II single-site clinical trial conducted in Perth, Australia. The target sample is to recruit 240 participants diagnosed with chronic frequent episodic migraines between 18 and 80 years of age. Participants will be randomised to one of four treatment groups for 14 weeks (placebo induction for 2 weeks, followed by 12 weeks on one of the respective treatment arms): placebo, L-arginine, AGE, or a combination of L-arginine and AGE. The doses of L-arginine and AGE are 1.5 g and 1 g daily, respectively. The primary outcome is to assess migraine response using change in migraine frequency and intensity between baseline and 12 weeks. Secondary outcomes include the impact of L-arginine and/or AGE on photosensitivity, retinal vessel changes, and blood biomarker concentrations of vascular tone, following a 12-week intervention. Discussion: The protocol describes the oral administration of 2 nutraceutical-based interventions as possible prophylactic treatments for chronic frequent episodic migraines, with potential for direct clinical translation of outcomes. Potential limitations of the study include the fixed-dose design of each treatment arm and that in vivo neuroimaging methods, such as magnetic resonance imaging (MRI), will not be conducted to determine putative cerebro-vasodilatory changes to coincide with the outcome measures. Dose-response studies may be indicated. Trial registration: The trial was retrospectively registered with the Australian New Zealand Clinical Trials Registry ACTRN12621001476820 (Universal Trial Number: U1111-1268-1117) on 04/08/2021. This is protocol version 1, submitted on 25/11/2022.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder pathologically characterized by brain parenchymal abundance of amyloid-beta (Aβ) and the accumulation of lipofuscin material that is rich in neutral lipids. However, the mechanisms for aetiology of AD are presently not established. There is increasing evidence that metabolism of lipoprotein-Aβ in blood is associated with AD risk, via a microvascular axis that features breakdown of the blood-brain barrier, extravasation of lipoprotein-Aβ to brain parenchyme and thereafter heightened inflammation. A peripheral lipoprotein-Aβ/capillary axis for AD reconciles alternate hypotheses for a vascular, or amyloid origin of disease, with amyloidosis being probably consequential. Dietary fats may markedly influence the plasma abundance of lipoprotein-Aβ and by extension AD risk. Similarly, apolipoprotein E (Apo E) serves as the primary ligand by which lipoproteins are cleared from plasma via high-affinity receptors, for binding to extracellular matrices and thereafter for uptake of lipoprotein-Aβ via resident inflammatory cells. The epsilon APOE ε4 isoform, a major risk factor for AD, is associated with delayed catabolism of lipoproteins and by extension may increase AD risk due to increased exposure to circulating lipoprotein-Aβ and microvascular corruption.

There is increasing evidence of a positive association of type 2 diabetes with Alzheimer’s disease (AD), the most prevalent form of dementia. Suggested pathways include cerebral vascular dysfunction; central insulin resistance, or exaggerated brain abundance of potentially cytotoxic amyloid-β (Aβ), a hallmark feature of AD. However, contemporary studies find that Aβ is secreted in the periphery by lipogenic organs and secreted as nascent triglyceride-rich lipoproteins (TRL’s). Pre-clinical models show that exaggerated abundance in blood of TRL-Aβ compromises blood-brain barrier (BBB) integrity, resulting in extravasation of the TRL-Aβ moiety to brain parenchyme, neurovascular inflammation and neuronal degeneration concomitant with cognitive decline. Inhibiting secretion of TRL-Aβ by peripheral lipogenic organs attenuates the early-AD phenotype indicated in animal models, consistent with causality. Poorly controlled type 2 diabetes commonly features hypertriglyceridemia because of exaggerated TRL secretion and reduced rates of catabolism. Alzheimer’s in diabetes may therefore be a consequence of heightened abundance in blood of lipoprotein-Aβ and accelerated breakdown of the BBB. This review reconciles the prevailing dogma of amyloid associated cytotoxicity as a primary risk factor in late-onset AD, with substantial evidence of a microvascular axis for dementia-in-diabetes. Consideration of potentially relevant pharmacotherapies to treat insulin resistance, dyslipidaemia and by extension plasma amyloidemia in type 2 diabetes are discussed.

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

Endometriosis is a complex and potentially debilitating condition that has major impact on quality of life. There is emerging evidence that biological compounds found in garlic (Allium sativum) may be effective for attenuating endometrial pain. Suggested mechanisms for efficacy include modulation of inflammation and potent antioxidant effects. Aged-garlic-extract (AGE) is a centuries old process describing ethanolic extracts of garlic bulbs for 12–20 months. The AGE formulation realised contains a complex array of stabilised biologics with significant immunomodulatory effects relevant to inflammatory conditions. This perspective article puts forward a hypothesis that AGE should be considered as a prophylactic to manage endometrial pain.

Introduction Migraine is a common and distressing neurological condition characterised by recurrent throbbing headaches, nausea and heightened sensitivity to light and sound. Accumulating evidence suggests that cerebral arteries dilate during migraine, causing distal microvessels to constrict, which could activate nociceptors and cause onset of headache pain. If so, preventing or attenuating chronic microvascular constriction, and promoting a dilatory phenotype, may reduce frequency and/or severity of migraines. The primary aim of the L-Arginine and Aged Garlic Extract (LARGE) trial is to investigate whether oral treatment with dietary nutraceuticals, L-arginine and aged garlic extract (AGE), both systemic vasodilatory agents, will alleviate migraine frequency, duration and severity in adults with chronic frequent episodic migraines. Methods and analysis The study is a randomised double-blind placebo-controlled phase-II single-site clinical trial conducted in Perth, Australia. The target sample is to recruit 240 participants diagnosed with chronic frequent episodic migraines between 18 and 80 years of age. Participants will be randomised to one of four treatment groups for 14 weeks (placebo induction for 2 weeks, followed by 12 weeks on one of the respective treatment arms): placebo, L-arginine, AGE, or a combination of L-arginine and AGE. The doses of L-arginine and AGE are 1.5 g and 1 g daily, respectively. The primary outcome is to assess migraine response using change in migraine frequency and intensity between baseline and 12 weeks. Secondary outcomes include the impact of L-arginine and/or AGE on photosensitivity, retinal vessel changes, and blood biomarker concentrations of vascular tone, following a 12-week intervention. Ethics and dissemination The Curtin University Human Research Ethics Committee (HREC) has approved this study (Approval number: HRE2020-0466; Version 4; 16^th August 2021). Written consent will be obtained from all participants prior to commencing their participation in the trial. The results of the study will be disseminated in peer-reviewed publications and presented at key national and international conferences and local stakeholder events. Registration details The trial is registered with the Australian New Zealand Clinical Trials Registry ACTRN12621001476820 (Universal Trial Number: U1111-1268-1117). Strengths and limitations of the study This is the first in-human randomised double-blind placebo-controlled phase-II clinical trial examining the efficacy, safety and tolerability of L-arginine and AGE, in preventing chronic frequent episodic migraines by assessing participant-reported pain-related outcomes, and changes in photosensitivity and retinal vessels. The double-blinded nature of the study, and the placebo run-in for 2 weeks at the beginning of the study, are strengths in trial methodology. The protocol describes the oral administration of 2 nutraceutical-based interventions as possible prophylactic treatments for chronic frequent episodic migraines, with potential for direct clinical translation of outcomes. Potential limitations of the study include the fixed-dose design of each treatment arm and that in vivo neuroimaging methods, such as magnetic resonance imaging (MRI), will not be conducted to determine putative cerebro-vasodilatory changes to coincide with the outcome measures. Dose-response studies may be indicated.