L-arginine and aged garlic extract for the prevention of migraine: a study protocol for a randomised, double-blind, placebo-controlled, phase-II trial (LARGE trial).

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.

Attenuation of chronic tension headache frequency and severity with daily L-arginine and aged garlic extract dietary supplementation.

A 74-year-old female subject with suboptimal management of episodic tension headache was treated with a daily dose of 1.5 g L-arginine and 1.2 g aged garlic extract (AGE). The aim of the intervention was to promote vasodilation of parenchymal cerebral blood vessels. Within 6 weeks of commencing treatment, her self-reported symptoms improved markedly and were sustained at 2 years following commencement. We propose that the putative beneficial effect of L-arginine and AGE in this patient is because of the well-established systemic vasodilatory effects of L-arginine and aged garlic extract. On the hypothesis that migraine is precipitated by cerebral microvascular constriction, we recommend a double-blind randomised controlled trial to clinically test this hypothesis in migraine patients.

The Effects of Chronic Consumption of Lipid-Rich and Delipidated Bovine Dairy Milk on Brown Adipose Tissue Volume in Wild-Type Mice.

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.

The differential effects of fatty acids on enterocytic abundance of amyloid-beta.

BACKGROUND: Consumption of a Western-styled diet enriched in saturated fatty acids (SFA) relative to polyunsaturated fatty acids is positively associated with risk for Alzheimer's disease. Whilst potential causal mechanism are unclear, there is increasing evidence that chronic ingestion of SFA enriched diets promote increase the plasma levels of lipoprotein-associated amyloid-ß (Aß). However, the effects of dietary mono- and poly-unsaturated fats (MUFA/PUFA) on nascent lipoprotein Aß abundance have not been previously reported. METHODS: Wild-type C57BL/6 J mice were maintained on low-fat control chow (LF) or diets enriched in either SFA, MUFA, or PUFA for 9 months. Enterocytic abundance of Aß was determined with quantitative immunofluorescent microscopy and plasma Aß was measured by ELISA. RESULTS: The chronic ingestion of SFA-enriched diet increased the enterocytic abundance and plasma concentration of Aß compared to LF control mice. The mice maintained on MUFA or PUFA diet showed comparable enterocytic and plasma Aß levels to the LF control mice. CONCLUSIONS: The data indicates that a diet enriched in SFA significantly increases the enterocytic Aß production and secretion into the circulation, whilst MUFA and PUFA enriched diet do not exert such effects.

Dietary fat and physiological determinants of plasma chylomicron remnant homoeostasis in normolipidaemic subjects: insight into atherogenic risk.

TAG depleted remnants of postprandial chylomicrons are a risk factor for atherosclerosis. Recent studies have demonstrated that in the fasted state, the majority of chylomicrons are small enough for transcytosis to arterial subendothelial space and accelerate atherogenesis. However, the size distribution of chylomicrons in the absorptive state is unclear. This study explored in normolipidaemic subjects the postprandial distribution of the chylomicron marker, apoB-48, in a TAG-rich lipoprotein plasma fraction (Svedberg flotation rate (Sf>400), in partially hydrolysed remnants (Sf 20-400) and in a TAG-deplete fraction (Sf<20), following ingestion of isoenergetic meals with either palm oil (PO), rice bran or coconut oil. Results from this study show that the majority of fasting chylomicrons are within the potentially pro-atherogenic Sf<20 fraction (70-75 %). Following the ingestion of test meals, chylomicronaemia was also principally distributed within the Sf<20 fraction. However, approximately 40 % of subjects demonstrated exaggerated postprandial lipaemia specifically in response to the SFA-rich PO meal, with a transient shift to more buoyant chylomicron fractions. The latter demonstrates that heterogeneity in the magnitude and duration of hyper-remnantaemia is dependent on both the nature of the meal fatty acids ingested and possible metabolic determinants that influence chylomicron metabolism. The study findings reiterate that fasting plasma TAG is a poor indicator of atherogenic chylomicron remnant homoeostasis and emphasises the merits of considering specifically, chylomicron remnant abundance and kinetics in the context of atherogenic risk. Few studies address the latter, despite the majority of life being spent in the postprandial and absorptive state.

Serum 25-hydroxyvitamin D is associated with reduced verbal episodic memory in healthy, middle-aged and older adults.

BACKGROUND: There is increasing evidence supporting an association of higher serum vitamin D concentration with better cognitive performance in older individuals. However, to date, consideration of the putative association between vitamin D and cognition has been based principally on studies investigating clinical participant samples manifesting vitamin D deficiency, particularly in older people. Moreover, relationships between vitamin D and cognition are typically not considered in the context of counter-regulatory calcium-modulating hormones or calcium homeostasis. OBJECTIVE: Serum vitamin D/bioactive (ionised) calcium/parathyroid hormone homeostasis was considered in the context of cognitive performance in healthy, middle-aged and older individuals. DESIGN: A cross-sectional sample of 179 participants between the ages of 47-84 years was recruited for this study (114 females, 65 males). Participants provided fasting blood samples for analysis of serum 25-hydroxyvitamin D levels, ionised calcium (iCa) and parathyroid hormone (PTH) and completed cognitive measures of verbal episodic learning and memory. RESULTS: Serum 25-hydroxyvitamin D concentrations were negatively associated (with and without covariates of age, gender, depression and NART scores, iCa, and PTH) with measures of verbal episodic learning and memory, in particular with trial 5 of the Rey Auditory Verbal Learning Test (RAVLT) and long-delay free recall on the RAVLT. CONCLUSION: Overall, the findings from this study suggest an association between higher vitamin D status and poorer performance on verbal episodic memory in middle-aged and older individuals with normal vitamin D-calcium-PTH homeostasis. Despite requiring replication in other participant samples, this is a potentially important finding as it indicates that it may not be beneficial from a cognitive perspective to provide vitamin D supplements in individuals with already adequate vitamin D status.

The vitamin D, ionised calcium and parathyroid hormone axis of cerebral capillary function: therapeutic considerations for vascular-based neurodegenerative disorders.

Blood-brain barrier dysfunction characterised by brain parenchymal extravasation of plasma proteins may contribute to risk of neurodegenerative disorders, however the mechanisms for increased capillary permeability are not understood. Increasing evidence suggests vitamin D confers central nervous system benefits and there is increasing demand for vitamin D supplementation. Vitamin D may influence the CNS via modulation of capillary function, however such effects may be indirect as it has a central role in maintaining calcium homeostasis, in concert with calcium regulatory hormones. This study utilised an integrated approach and investigated the effects of vitamin D supplementation, parathyroid tissue ablation (PTX), or exogenous infusion of parathyroid hormone (PTH) on cerebral capillary integrity. Parenchymal extravasation of immunoglobulin G (IgG) was used as a marker of cerebral capillary permeability. In C57BL/6J mice and Sprague Dawley rats, dietary vitamin D was associated with exaggerated abundance of IgG within cerebral cortex (CTX) and hippocampal formation (HPF). Vitamin D was also associated with increased plasma ionised calcium (iCa) and decreased PTH. A response to dose was suggested and parenchymal effects persisted for up to 24 weeks. Ablation of parathyroid glands increased CTX- and HPF-IgG abundance concomitant with a reduction in plasma iCa. With the provision of PTH, iCa levels increased, however the PTH treated animals did not show increased cerebral permeability. Vitamin D supplemented groups and rats with PTH-tissue ablation showed modestly increased parenchymal abundance of glial-fibrillary acidic protein (GFAP), a marker of astroglial activation. PTH infusion attenuated GFAP abundance. The findings suggest that vitamin D can compromise capillary integrity via a mechanism that is independent of calcium homeostasis. The effects of exogenous vitamin D supplementation on capillary function and in the context of prevention of vascular neurodegenerative conditions should be considered in the context of synergistic effects with calcium modulating hormones.

Nutraceutical agents with anti-inflammatory properties prevent dietary saturated-fat induced disturbances in blood-brain barrier function in wild-type mice.

BACKGROUND: Emerging evidence suggests that disturbances in the blood-brain barrier (BBB) may be pivotal to the pathogenesis and pathology of vascular-based neurodegenerative disorders. Studies suggest that heightened systemic and central inflammations are associated with BBB dysfunction. This study investigated the effect of the anti-inflammatory nutraceuticals garlic extract-aged (GEA), alpha lipoic acid (ALA), niacin, and nicotinamide (NA) in a murine dietary-induced model of BBB dysfunction. METHODS: C57BL/6 mice were fed a diet enriched in saturated fatty acids (SFA, 40% fat of total energy) for nine months to induce systemic inflammation and BBB disturbances. Nutraceutical treatment groups included the provision of either GEA, ALA, niacin or NA in the positive control SFA-group and in low-fat fed controls. Brain parenchymal extravasation of plasma derived immunoglobulin G (IgG) and large macromolecules (apolipoprotein (apo) B lipoproteins) measured by quantitative immunofluorescent microscopy, were used as markers of disturbed BBB integrity. Parenchymal glial fibrillar acidic protein (GFAP) and cyclooxygenase-2 (COX-2) were considered in the context of surrogate markers of neurovascular inflammation and oxidative stress. Total anti-oxidant status and glutathione reductase activity were determined in plasma. RESULTS: Brain parenchymal abundance of IgG and apoB lipoproteins was markedly exaggerated in mice maintained on the SFA diet concomitant with significantly increased GFAP and COX-2, and reduced systemic anti-oxidative status. The nutraceutical GEA, ALA, niacin, and NA completely prevented the SFA-induced disturbances of BBB and normalized the measures of neurovascular inflammation and oxidative stress. CONCLUSIONS: The anti-inflammatory nutraceutical agents GEA, ALA, niacin, or NA are potent inhibitors of dietary fat-induced disturbances of BBB induced by systemic inflammations.

Probucol prevents blood-brain barrier dysfunction in wild-type mice induced by saturated fat or cholesterol feeding.

Dysfunction of the blood-brain barrier (BBB) is an early pathological feature of vascular dementia and Alzheimer's disease (AD) and is triggered by inflammatory stimuli. Probucol is a lipid-lowering agent with potent anti-oxidant properties once commonly used for the treatment of cardiovascular disease. Probucol therapy was found to stabilize cognitive symptoms in elderly AD patients, whereas in amyloid transgenic mice probucol was shown to attenuate amyloidosis. However, the mechanisms underlying the effects of probucol have note been determined. In the present study we investigated whether probucol can prevent BBB disturbances induced by chronic ingestion of proinflammatory diets enriched with either 20% (w/w) saturated fats (SFA) or 1% (w/w) cholesterol. Mice were fed the diets for 12 weeks before they were killed and BBB integrity was measured. Mice maintained on either the SFA- or cholesterol-supplemented diets were found to have a 30- and sevenfold greater likelihood of BBB dysfunction, respectively, as determined by the parenchymal extravasation of plasma-derived immunoglobulins and endogenous lipoprotein enrichment with ß-amyloid. In contrast, mice fed the SFA- or cholesterol-enriched diets that also contained 1% (w/w) probucol showed no evidence of BBB disturbance. The parenchymal expression of glial fibrillary acidic protein, a marker of cerebrovascular inflammation, was significantly greater in mice fed the SFA-enriched diet. Plasma lipid, ß-amyloid and apolipoprotein B levels were not increased by feeding of the SFA- or cholesterol-enriched diets. However, mice fed the SFA- or cholesterol-enriched diets did exhibit increased plasma non-esterified fatty acid levels that were not reduced by probucol. The data suggest that probucol prevents disturbances of BBB induced by chronic ingestion of diets enriched in SFA or cholesterol by suppressing inflammatory pathways rather than by modulating plasma lipid homeostasis.

Differential effects of dietary fatty acids on the cerebral distribution of plasma-derived apo B lipoproteins with amyloid-beta.

Some dietary fats are a risk factor for Alzheimer's disease (AD) but the mechanisms for this association are presently unknown. In the present study we showed in wild-type mice that chronic ingestion of SFA results in blood-brain barrier (BBB) dysfunction and significant delivery into the brain of plasma proteins, including apo B lipoproteins that are endogenously enriched in amyloid-beta (Abeta). Conversely, the plasma concentration of S100B was used as a marker of brain-to-blood leakage and was found to be increased two-fold because of SFA feeding. Consistent with a deterioration in BBB integrity in SFA-fed mice was a diminished cerebrovascular expression of occludin, an endothelial tight junction protein. In contrast to SFA-fed mice, chronic ingestion of MUFA or PUFA had no detrimental effect on BBB integrity. Utilising highly sensitive three-dimensional immunomicroscopy, we also showed that the cerebral distribution and co-localisation of Abeta with apo B lipoproteins in SFA-fed mice are similar to those found in amyloid precursor protein/presenilin-1 (APP/PS1) amyloid transgenic mice, an established murine model of AD. Moreover, there was a strong positive association of plasma-derived apo B lipoproteins with cerebral Abeta deposits. Collectively, the findings of the present study provide a plausible explanation of how dietary fats may influence AD risk. Ingestion of SFA could enhance peripheral delivery to the brain of circulating lipoprotein-Abeta and exacerbate the amyloidogenic cascade.

The effect of exogenous cholesterol and lipid-modulating agents on enterocytic amyloid-beta abundance.

Dietary cholesterol may influence Alzheimer's disease risk, because it regulates the synthesis of amyloid-beta. It was recently demonstrated in enterocytes of wild-type mice that intracellular amyloid-beta expression is enhanced in response to a high-fat diet made up of SFA and cholesterol. Intestinally derived amyloid-beta may be associated with postprandial lipoproteins in response to dietary fats and could be a key regulator in chylomicron metabolism. The present study was designed to investigate the role of cholesterol in modulating amyloid-beta abundance in enterocytes. Wild-type mice were fed a low-fat diet supplemented with 2 % (w/w) cholesterol. The effects of cholesterol absorption inhibition and cholesterol biosynthesis inhibition utilising ezetimibe and atorvastatin, respectively, were also studied. Quantitative immunohistochemistry was utilised to determine enterocytic amyloid-beta homeostasis. We found that enterocytic amyloid-beta concentration was significantly attenuated in mice fed the 2 % (w/w) cholesterol diet. However, blocking cholesterol absorption reversed the cholesterol-feeding effect. Consistent with a suppressive effect of cholesterol on enterocytic amyloid-beta abundance, atorvastatin, an inhibitor of cholesterol biosynthesis, enhanced amyloid-beta. However, providing exogenous cholesterol abolished the atorvastatin-induced effect. In contrast to the suppression of enterocytic amyloid-beta by dietary cholesterol, mice fed a diet enriched in SFA had markedly greater abundance. Collectively, the findings suggest that exogenous and endogenous cholesterol reduce amyloid-beta concentration in enterocytes by suppressing production, or enhancing secretion associated with postprandial lipoproteins. Intestinally derived amyloid-beta will contribute to the pool of plasma protein and may influence cerebral amyloid homeostasis by altering the bi-directional transfer across the blood-brain barrier.