Aging-Related Changes in Blood-Brain Barrier Integrity and the Effect of Dietary Fat

Background: Disturbances in blood-brain barrier (BBB) integrity contribute to the onset and progression of neurodegenerative diseases including Alzheimer’s disease (AD) and vascular dementia (VaD). Aging is positively associated with AD and VaD risk, but this may reflect comorbidities or the effects of other chronic modulators of vascular function such as diet. Objective: To explore putative synergistic effects of aging with diet, in this study genetically unmanipulated mice were maintained on diets enriched in saturated fatty acids (SFA) or cholesterol and compared to mice provided with low-fat (LF) feed formula. Methods: The functional integrity of the BBB was assessed following 3, 6 and 12 months of dietary intervention commenced at 6 weeks of age, by determining the brain parenchymal extravasation of immunoglobulin G (IgG). Results: Mice maintained on the SFA- or cholesterol-enriched diet showed significant parenchymal IgG abundance following 3 months of feeding, concomitant with diminished expression of the tight junction protein occludin. LF control mice had essentially no evidence of BBB disturbances. Six months of SFA feeding exacerbated the difference in IgG abundance compared to the LF mice. At 12 months of feeding, the control LF mice also had significant parenchymal IgG that was comparable to mice fed the SFA- or cholesterol-enriched diet for 3 months. However, there may have been an adaptation to the fat-enriched diets because SFA and cholesterol did not exacerbate IgG parenchymal accumulation beyond 6 months of feeding. Conclusion: Collectively, the study suggests that diets enriched in SFA or cholesterol accelerate the onset of BBB dysfunction that otherwise occurs with aging

Long-term probucol therapy continues to suppress markers of neurovascular inflammation in a dietary induced model of cerebral capillary dysfunction

Background: Probucol has been shown to prevent cerebral capillary disturbances characterized by blood-to-brain extravasation of plasma derived proteins and neurovascular inflammation in mice maintained on western-styled diets for 12 weeks. However the effect of probucol on capillary integrity in aging models with capillary dysfunction is not known. Methods: Wild-type C57BL6 mice were randomized to a low-fat (LF); saturated-fat (SFA); or SFA + Probucol diet for up to12 months of intervention. Results: Mice fed the LF diet had substantially greater parenchymal abundance of plasma derived IgG and apo B lipoproteins at 12 months, compared to LF mice at 3 months of intervention. Markers of neurovascular inflammation were also greater at 12 months in LF fed mice compared to LF mice at 3 months. The SFA diet exacerbated the aging induced parenchymal abundance of IgG and of apo B lipoproteins and neurovascular inflammation at 12 months. The SFA effects were associated with increased production of intestinal lipoprotein amyloid-ß (Aß). The co-provision of probucol with the SFA completely abolished heightened inflammation at 12 months. Probucol attenuated SFA-induced capillary permeability but had only a modest inhibitory effect on parenchymal retention of apoB lipoproteins. The improvements in markers of inflammation and capillary integrity because of probucol correlated with enterocytic genesis of chylomicron Aß. Conclusion: In this long-term feeding study, probucol profoundly suppressed dietary SFA induced disturbances in capillary integrity but had a more modest effect on age-associated changes

Amyloid-β colocalizes with apolipoprotein B in absorptive cells of the small intestine

Background: Amyloid-β is recognized as the major constituent of senile plaque found in subjects with Alzheimer's disease. However, there is increasing evidence that in a physiological context amyloid-β may serve as regulating apolipoprotein, primarily of the triglyceride enriched lipoproteins. To consider this hypothesis further, this study utilized an in vivo immunological approach to explore in lipogenic tissue whether amyloid-β colocalizes with nascent triglyceride-rich lipoproteins. Results: In murine absorptive epithelial cells of the small intestine, amyloid-β had remarkable colocalization with chylomicrons (Manders overlap coefficient = 0.73 ± 0.03 (SEM)), the latter identified as immunoreactive apolipoprotein B. A diet enriched in saturated fats doubled the abundance of both amyloid-β and apo B and increased the overlap coefficient of the two proteins (0.87 ± 0.02). However, there was no evidence that abundance of the two proteins was interdependent within the enterocytes (Pearson's Coefficient < 0.02 ± 0.03), or in plasma (Pearson's Coefficient < 0.01). Conclusion: The findings of this study are consistent with the possibility that amyloid-β is secreted by enterocytes as an apolipoprotein component of chylomicrons. However, secretion of amyloid-β appears to be independent of chylomicron biogenesis

A Diet Enriched in Docosahexanoic Acid Exacerbates Brain Parenchymal Extravasation of Apo B Lipoproteins Induced by Chronic Ingestion of Saturated Fats

Chronic ingestion of saturated fatty acids (SFAs) was previously shown to compromise blood-brain barrier integrity, leading to brain parenchymal extravasation of apolipoprotein B (apo B) lipoproteins enriched in amyloid beta. In contrast, diets enriched in mono- or polyunsaturated (PUFA) oils had no detrimental effect. Rather, n3 and n6 oils generally confer protection via suppression of inflammation. This study investigated in wild-type mice if a PUFA diet enriched in docosahexanoic acid (DHA) restored blood-brain barrier integrity and attenuated parenchymal apo B abundance induced by chronic ingestion of SFA. Cerebrovascular leakage of apo B was quantitated utilising immunofluorescent staining. The plasma concentration of brain-derived S100β was measured as a marker of cerebrovascular inflammation. In mice fed SFA for 3 months, provision thereafter of a DHA-enriched diet exacerbated parenchymal apo B retention, concomitant with a significant increase in plasma cholesterol. In contrast, provision of a low-fat diet following chronic SFA feeding had no effect on SFA-induced parenchymal apo B. The findings suggest that in a heightened state of cerebrovascular inflammation, the provision of unsaturated fatty acids may be detrimental, possibly as a consequence of a greater susceptibility for oxidation

Restoration of dietary-fat induced blood-brain barrier dysfunction by anti-inflammatory lipid-modulating agents

Background: Several studies have identified use of non-steroidal-anti-inflammatory drugs and statins for prevention of dementia, but their efficacy in slowing progression is not well understood. Cerebrovascular disturbances are common pathological feature of Alzheimer’s disease. We previously reported chronic ingestion of saturated fatty acids (SFA) compromises blood–brain barrier (BBB) integrity resulting in cerebral extravasation of plasma proteins and inflammation. However, the SFA-induced parenchymal accumulation of plasma proteins could be prevented by co-administration of some cholesterol lowering agents. Restoration of BBB dysfunction is clinically relevant, so the purpose of this study was to explore lipid-lowering agents could reverse BBB disturbances induced by chronic ingestion of SFA’s. Methods: Wild-type mice were fed an SFA diet for 12 weeks to induce BBB dysfunction, and then randomised to receive atorvastatin, pravastatin or ibuprofen in combination with the SFA-rich diet for 2 or 8 weeks. Abundance of plasma-derived immunoglobulin-G (IgG) and amyloid-β enriched apolipoprotein (apo)-B lipoproteins within brain parenchyme were quantified utilising immunofluorescence microscopy. Results: Atorvastatin treatment for 2 and 8 weeks restored BBB integrity, indicated by a substantial reduction of IgG and apo B, particularly within the hippocampus. Pravastatin, a water-soluble statin was less effective than atorvastatin (lipid-soluble). Statin effects were independent of changes in plasma lipid homeostasis. Ibuprofen, a lipid-soluble cyclooxygenase inhibitor attenuated cerebral accumulation of IgG and apo B as effectively as atorvastatin. Our findings are consistent with the drug effects being independent of plasma lipid homeostasis. Conclusion: Our findings suggest that BBB dysfunction induced by chronic ingestion of SFA is reversible with timely introduction and sustained treatment with agents that suppress inflammatio

Synergistic effects of high fat feeding and apolipoprotein E deletion on enterocytic amyloid-beta abundance

Background: Amyloid-β (Aβ), a key protein found in amyloid plaques of subjects with Alzheimer's disease is expressed in the absorptive epithelial cells of the small intestine. Ingestion of saturated fat significantly enhances enterocytic Aβ abundance whereas fasting abolishes expression. Apolipoprotein (apo) E has been shown to directly modulate Aβ biogenesis in liver and neuronal cells but it's effect in enterocytes is not known. In addition, apo E modulates villi length, which may indirectly modulate Aβ as a consequence of differences in lipid absorption. This study compared Aβ abundance and villi length in wild-type (WT) and apo E knockout (KO) mice maintained on either a low-fat or high-fat diet. Wild-type C57BL/6J and apo E KO mice were randomised for six-months to a diet containing either 4% (w/w) unsaturated fats, or chow comprising 16% saturated fats and 1% cholesterol. Quantitative immunohistochemistry was used to assess Aβ abundance in small intestinal enterocytes. Apo E KO mice given the low-fat diet had similar enterocytic Aβ abundance compared to WT controls. Results: The saturated fat diet substantially increased enterocytic Aβ in WT and in apo E KO mice, however the effect was greater in the latter. Villi height was significantly greater in apo E KO mice than for WT controls when given the low-fat diet. However, WT mice had comparable villi length to apo E KO when fed the saturated fat and cholesterol enriched diet. There was no effect of the high-fat diet on villi length in apo E KO mice. Conclusion: The findings of this study are consistent with the notion that lipid substrate availability modulates enterocytic Aβ. Apo E may influence enterocytic lipid availability by modulating absorptive capacity

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 antioxidative 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 Suppresses Enterocytic Accumulation of Amyloid-β Induced by Saturated Fat and Cholesterol Feeding

Amyloid-β (Aβ) is secreted from lipogenic organs such as intestine and liver as an apolipoprotein of nascent triacylglycerol rich lipoproteins. Chronically elevated plasma Aβ may compromise cerebrovascular integrity and exacerbate amyloidosis—a hallmark feature of Alzheimer’s disease (AD). Probucol is a hypocholesterolemic agent that reduces amyloid burden in transgenic amyloid mice, but the mechanisms for this effect are presently unclear. In this study, the effect of Probucol on intestinal lipoprotein-Aβ homeostasis was explored. Wild-type mice were fed a control low-fat diet and enterocytic Aβ was stimulated by high-fat (HF) diet enriched in 10% (w/w) saturated fat and 1% (w/w) cholesterol for the duration of 1 month. Mice treated with Probucol had the drug incorporated into the chow at 1% (w/w). Quantitative immunofluorescence was utilised to determine intestinal apolipoprotein B (apo B) and Aβ abundance. We found apo B in both the perinuclear region of the enterocytes and the lacteals in all groups. However, HF feeding and Probucol treatment increased secretion of apo B into the lacteals without any change in net villi abundance. On the other hand, HF-induced enterocytic perinuclear Aβ was significantly attenuated by Probucol. No significant changes in Aβ were observed within the lacteals. The findings of this study support the notion that Probucol suppresses dietary fat induced stimulation of Aβ biosynthesis and attenuate availability of apo B lipoprotein-Aβ for secretion

Evaluating fibre orientation dispersion in white matter: comparison of diffusion MRI, histology and polarized light imaging

Diffusion MRI is an exquisitely sensitive probe of tissue microstructure, and is currently the only non-invasive measure of the brain’s fibre architecture. As this technique becomes more sophisticated and microstructurally informative, there is increasing value in comparing diffusion MRI with microscopic imaging in the same tissue samples. This study compared estimates of fibre orientation dispersion in white matter derived from diffusion MRI to reference measures of dispersion obtained from polarized light imaging and histology. Three post-mortem brain specimens were scanned with diffusion MRI and analyzed with a two-compartment dispersion model. The specimens were then sectioned for microscopy, including polarized light imaging estimates of fibre orientation and histological quantitative estimates of myelin and astrocytes. Dispersion estimates were correlated on region – and voxel-wise levels in the corpus callosum, the centrum semiovale and the corticospinal tract. The region-wise analysis yielded correlation coefficients of r=0.79 for the diffusion MRI and histology comparison, while r=0.60 was reported for the comparison with polarized light imaging. In the corpus callosum, we observed a pattern of higher dispersion at the midline compared to its lateral aspects. This pattern was present in all modalities and the dispersion profiles from microscopy and diffusion MRI were highly correlated. The astrocytes appeared to have minor contribution to dispersion observed with diffusion MRI. These results demonstrate that fibre orientation dispersion estimates from diffusion MRI represents the tissue architecture well. Dispersion models might be improved by more faithfully incorporating an informed mapping based on microscopy data

Nutritional and pharmacological regulation of cerebral capillary function

Alzheimer’s disease (AD) is the most common cause of dementia pathologically characterised by neurovascular inflammation, extracellular proteinaceous deposits enriched in amyloid-β (Aβ) and formation of neurofibrillar tangles. The cerebrovasculature in subjects with AD is also significantly altered and is first indicated by the progressive dysfunction of the cerebral capillary vessels. Despite evidence of an underlying vascular contribution to onset and progression of AD, most research focuses on factors regulating the more advanced pathological processes.Clinical and experimental evidence suggest vascular risk factors influence the onset and progression of AD. Several studies have demonstrated that atherosclerosis, cardiovascular disease, hypertension, dyslipidemia and insulin resistance are positively associated with AD risk. Furthermore, population studies have found that chronic ingestion of pro-inflammatory diets enriched in saturated fatty acids (SFA), trans-fatty acids and cholesterol are positive risk factors for AD and markedly exacerbate cerebral pathology in animal models. A putative mechanism for the dietary-fat/amyloidogenic pathway was demonstrated in recent animal studies where chronic consumption of diets enriched in SFA and cholesterol significantly compromised blood-brain barrier (BBB) function in wild-type (WT) mice, resulting in substantial blood-to-brain delivery of circulating Aβ and potential exacerbation of cerebral amyloid load. Significant plasma Aβ is derived from liver and small intestine, secreted into circulation associated with triglyceride-rich lipoproteins (TRLs).Dietary lipid regulation of TRL-Aβ was equivocally demonstrated in WT mice fed an SFA enriched diet, where intestinal Aβ biogenesis and secretion was enhanced. Given that humans are predominantly in an absorptive state, the cumulative cerebrovascular effects of transient exposure to dietary stimulated plasma Aβ may be AD-risk relevant. However, a dietary-lipid/Aβ axis for cerebrovascular function could also provide therapeutic opportunities to reduce AD risk.Observational studies suggest that calorie restriction and regular consumption of diets which supress inflammation may delay development and progression of AD. Recent findings have demonstrated that consumption of diets rich in monounsaturated fatty acids (MUFAs) and ω-3 polyunsaturated fatty acids (PUFAs), in particular docosahexaenoic acid (DHA), may reduce risk for AD via suppression of inflammatory pathways. Furthermore, beneficial effects of unsaturated fatty acids on prevention of cerebrovascular dysfunction have been demonstrated in vivo. The association between dietary fat intake, hypercholesterolemia and increased AD prevalence suggests the possibility that lipid-modulating agents might also delay onset and progression of AD. Some epidemiological studies have provided evidence of a lower prevalence of diagnosed AD in patients with hypercholesterolaemia treated with statins (3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors). In one clinical study, an older generation cholesterol lowering agent probucol was found to stabilise cognitive impairment in elderly AD subjects. The mechanisms for the pharmacological effects of lipid lowering agents in prevention of AD are unclear, but may include vascular benefits associated with lower plasma lipids, regulation of Aβ biogenesis and kinetics or indicative of anti-oxidant and antiinflammatory properties of the agents.At present, there is lack of effective strategies for the treatment of AD. Given that cerebral capillary disturbances is amongst the earliest indicators of disease onset, it seems a reasonable proposition to suggest that recognition of and timely intervention to restore capillary integrity would be therapeutically beneficial. This thesis explores the novel hypothesis that “dietary fat induced cerebral capillary dysfunction can be corrected by timely provision of an anti-inflammatory diet enriched in ω-3 fatty acids, or lipid lowering agents that attenuate cerebrovascular exposure to peripheral Aβ and inflammation”. The hypothesis presented is supported by a substantial literature review in Chapter 1 of this thesis.As a ‘proof of concept’ approach to the indicated hypothesis, the effects of lipid lowering agents, atorvastatin, pravastatin and probucol, for ‘prevention’ of dietary fat induced BBB dysfunction was determined (Chapter 1: article 5). Wildtype mice were chronically fed an SFA diet supplemented with atorvastatin and pravastatin. In addition, the effects of probucol on BBB integrity were determined in mice fed a cholesterol supplemented diet. All mice were maintained on their respective diets for 12 weeks. Cerebral extravasation of plasma protein immunoglobulin-G (IgG) was assessed utilising 3-dimensional (3-D) immunofluorescent microscopy. Immunoglobulin-G was used as a surrogate marker of non-specific cerebrovascular permeability. Significant BBB dysfunction and enhanced cerebral extravasation of IgG was evident in mice fed SFA and cholesterol diets. Atorvastatin, a lipid soluble HMG-CoA reductase inhibitor prevented SFA induced parenchymal extravasation of IgG at 12 weeks when incorporated into the diet. In contrast, hydrophilic pravastatin had no effect on BBB integrity. In cholesterol supplemented mice, probucol maintained BBB function and extravasation of IgG was not evident. These findings suggest that some lipid lowering agents may effectively prevent dietary fat induced BBB dysfunction and their efficacy may be dependent on solubility. The drug effects on BBB integrity were independent of significant modifications on plasma lipid homeostasis. These findings support the hypothesis that pleiotropic properties of lipid lowering agents maintain BBB integrity.Evidence of protective effect of lipid modulating agents on BBB function was concomitant with an attenuation in TRL-Aβ biogenesis is reported in Chapter 2. The article presented in this chapter investigated in vivo the putative effects of probucol on in TRL-Aβ homeostasis in absorptive epithelial cells of the small intestine in WT mice. Intestinal Aβ abundance was stimulated in mice fed a high-fat (HF) diet enriched in SFA and cholesterol for 4 weeks. Drug effects were determined in mice given low-fat (LF) and HF diets supplemented with 1% probucol. Quantitative immunofluorescent microscopy determined intestinal Aβ and apolipoprotein B (apo B) abundance. Apo B is an obligatory structural component of TRL originating from liver and absorptive epithelial cells of the small intestine. Apo B staining was detected in both the perinuclear region of the enterocytes and the lacteals in all groups. However, HF feeding and probucol treatment increased secretion of apo B into the lacteals without any change in net villi abundance. On the other hand, HF induced enterocytic perinuclear Aβ was significantly attenuated by probucol. No significant changes in Aβ were observed within the lacteals. These findings support the notion that probucol normalised the HF induced intestinal Aβ biogenesis and availability of TRL-Aβ for secretion and may confer protection against AD by reducing exposure to plasma TRL-Aβ.Chronic ingestion of SFAs compromise BBB integrity, leading to cerebral extravasation of apo B lipoproteins enriched in Aβ. In contrast, diets enriched in PUFA oils had no detrimental effect. Rather, ω-3 and ω-6 fatty acids generally confer protection via suppression of inflammation. Chapter 3 investigated in WT mice if a PUFA diet enriched in DHA restored BBB integrity and attenuated parenchymal apo B abundance induced by chronic ingestion of SFA. Cerebrovascular leakage of apo B was quantitated utilising immunofluorescent staining. The plasma concentration of brain-derived S100B was measured as a measure of brain-to blood leakage and complimentary marker of BBB function. In mice fed SFA for 12 weeks, provision thereafter of a DHA enriched diet exacerbated parenchymal apo B retention, concomitant with a significant increase in plasma cholesterol and S100B. In contrast, provision of a LF diet following chronic SFA feeding had no effect on SFA induced parenchymal apo B abundance. In the established mouse model of BBB dysfunction with a heightened state of cerebrovascular inflammation, the provision of unsaturated fatty acids may be detrimental, possibly as a consequence of a greater susceptibility for lipid peroxidation.The ‘proof of concept’ study presented in Chapter 1 (article 5) reported that the SFA induced parenchymal accumulation of plasma proteins could be prevented by co-administration of some lipid lowering agents. Restoration of BBB function is clinically relevant in a therapeutic context. Chapter 4 explored whether lipidlowering agents could reverse BBB disturbances induced by chronic SFA feeding. Wild-type mice were fed an SFA diet for 12 weeks to induce BBB dysfunction and then randomised to receive atorvastatin, pravastatin or ibuprofen in combination with the SFA-rich diet for an additional 2 or 8 weeks. Abundance of plasma-derived IgG and apo B lipoproteins within brain parenchyme were quantified utilising immunofluorescence microscopy. Atorvastatin treatment for 2 and 8 weeks restored BBB integrity, indicated by a substantial reduction of IgG and apo B, particularly within the hippocampus. Pravastatin, a water-soluble statin was less effective than atorvastatin (lipid-soluble). Statin effects were independent of changes in plasma lipid homeostasis. Ibuprofen, a lipid-soluble cyclooxygenase (COX) inhibitor attenuated cerebral accumulation of IgG and apo B as effectively as atorvastatin. These findings are consistent with the drug effects being independent of plasma lipid homeostasis.The outcomes presented in this thesis provide novel insight into the positive effects of lipid lowering agents and non-steroidal anti-inflammatory drugs (NSAIDs) on regression of SFA induced BBB dysfunction. The effects of lipid lowering statins on BBB dysfunction were similar to that of ibuprofen. In addition, statin effects were independent of significant modifications in plasma lipid homeostasis, suggesting pleiotropic anti-inflammatory anti-oxidative effects. Moreover, statins enhance endothelial nitric oxide (NO) bioavailability that is essential for regulation of cerebral perfusion and improved endothelial function. In the established mouse model of BBB dysfunction induced by SFA feeding, provision of diet enriched in ω- 3 fatty acid DHA exaggerated BBB dysfunction. At high concentrations, ω-3 fatty acids are subject to lipid peroxidation and could notionally promote oxidative stress under certain conditions. At a heightened state of inflammation, provision of ω-3 fatty acid may exacerbate BBB dysfunction. Given the importance of cerebral capillary vessels in AD pathophysiology, it is our contention that animal model studies investigating the putative role of anti-inflammatory agents on restoration of BBB integrity will be exceedingly informative for clinical research studies