Sample preparation with sucrose cryoprotection dramatically alters Zn distribution in the rodent hippocampus, as revealed by elemental mapping.

Transition metal ions (Fe, Mn, Cu, Zn) are essential for healthy brain function, but altered concentration, distribution, or chemical form of the metal ions has been implicated in numerous brain pathologies. Currently, it is not possible to image the cellular or sub-cellular distribution of metal ions in vivo and therefore, studying brain-metal homeostasis largely relies on ex vivo in situ elemental mapping. Sample preparation methods that accurately preserve the in vivo elemental distribution are essential if one wishes to translate the knowledge of elemental distributions measured ex vivo toward increased understanding of chemical and physiological pathways of brain disease. The choice of sample preparation is particularly important for metal ions that exist in a labile or mobile form, for which the in vivo distribution could be easily distorted by inappropriate sample preparation. One of the most widely studied brain structures, the hippocampus, contains a large pool of labile and mobile Zn. Herein, we describe how sucrose cryoprotection, the gold standard method of preparing tissues for immuno-histochemistry or immuno-fluorescence, which is also often used as a sample preparation method for elemental mapping studies, drastically alters hippocampal Zn distribution. Based on the results of this study, in combination with a comparison against the strong body of published literature that has used either rapid plunge freezing of brain tissue, or sucrose cryo-protection, we strongly urge investigators in the future to cease using sucrose cryoprotection as a method of sample preparation for elemental mapping, especially if Zn is an analyte of interest.

Elemental characterisation of the pyramidal neuron layer within the rat and mouse hippocampus.

A unique combination of sensitivity, resolution, and penetration make X-ray fluorescence imaging (XFI) ideally suited to investigate trace elemental distributions in the biological context. XFI has gained widespread use as an analytical technique in the biological sciences, and in particular enables exciting new avenues of research in the field of neuroscience. In this study, elemental mapping by XFI was applied to characterise the elemental content within neuronal cell layers of hippocampal sub-regions of mice and rats. Although classical histochemical methods for metal detection exist, such approaches are typically limited to qualitative analysis. Specifically, histochemical methods are not uniformly sensitive to all chemical forms of a metal, often displaying variable sensitivity to specific "pools" or chemical forms of a metal. In addition, histochemical methods require fixation and extensive chemical treatment of samples, creating the strong likelihood for metal redistribution, leaching, or contamination. Direct quantitative elemental mapping of total elemental pools, in situ within ex vivo tissue sections, without the need for chemical fixation or addition of staining reagents is not possible with traditional histochemical methods; however, such a capability, which is provided by XFI, can offer an enormous analytical advantage. The results we report herein demonstrate the analytical advantage of XFI elemental mapping for direct, label-free metal quantification, in situ within ex vivo brain tissue sections. Specifically, we definitively characterise for the first time, the abundance of Fe within the pyramidal cell layers of the hippocampus. Localisation of Fe to this cell layer is not reproducibly achieved with classical Perls histochemical Fe stains. The ability of XFI to directly quantify neuronal elemental (P, S, Cl, K, Ca, Fe, Cu, Zn) distributions, revealed unique profiles of Fe and Zn within anatomical sub-regions of the hippocampus i.e., cornu ammonis 1, 2 or 3 (CA1, CA2 or CA3) sub-regions. Interestingly, our study reveals a unique Fe gradient across neuron populations within the non-degenerating and pathology free rat hippocampus, which curiously mirrors the pattern of region-specific vulnerability of the hippocampus that has previously been established to occur in various neurodegenerative diseases.

Antihypertensive agents do not prevent blood-brain barrier dysfunction and cognitive deficits in dietary-induced obese mice.

BACKGROUND: While vascular risk factors including Western-styled diet and obesity are reported to induce cognitive decline and increase dementia risk, recent reports consistently suggest that compromised integrity of cerebrovascular blood-brain barrier (BBB) may have an important role in neurodegeneration and cognitive deficits. A number of studies report that elevated blood pressure increases the permeability of BBB. METHODS: In this study, we investigated the effects of antihypertensive agents, candesartan or ursodeoxycholic acid (UDCA), on BBB dysfunction and cognitive decline in wild-type mice maintained on high fat and fructose (HFF) diet for 24 weeks. RESULTS: In HFF-fed mice, significantly increased body weight with elevated blood pressure, plasma insulin and glucose compared with mice fed with low-fat control chow was observed. Concomitantly, significant disruption of BBB and cognitive decline were evident in the HFF-fed obese mice. Hypertension was completely prevented by the coprovision of candesartan or UDCA in mice maintained on HFF diet, while only candesartan significantly reduced the body weight compared with HFF-fed mice. Nevertheless, BBB dysfunction and cognitive decline remained unaffected by candesartan or UDCA. CONCLUSIONS: These data conclusively indicate that modulation of blood pressure and/or body weight may not be directly associated with BBB dysfunction and cognitive deficits in Western diet-induced obese mice, and hence antihypertensive agents may not be effective in preventing BBB disruption and cognitive decline. The findings may provide important mechanistical insights to obesity-associated cognitive decline and its therapy.

Long-Term Supplementation of Microencapsulated ursodeoxycholic Acid Prevents Hypertension in a Mouse Model of Insulin Resistance.

Hypertension is a significant comorbidity associated with insulin resistance and type-2 diabetes. Limited evidence show that ursodeoxycholic acid (UDCA) has some anti-hypertensive effects. However, the potential effect of UDCA on hypertension induced by type-2 diabetic insulin resistance has not been reported. In C57Bl6 wild-type mice, insulin resistance was induced by the chronic ingestion of diet enriched in fat and fructose (HFF). HFF mice were randomized to treatment with UDCA or candersartan incorporated into the diet to achieve an ingested dose of approximately 70 mg/kg/day of UDCA or 3 mg/kg/day respectively. Systolic and diastolic blood pressure were measured with tail-cuff method. At 4 weeks of dietary treatment systolic and diastolic blood pressure were comparable in HFF and low-fat (LF) control mice. Co-administration of candesartan at 4 weeks significantly decreased systolic and diastolic blood pressure, UDCA showed no anti-hypertensive effect at 4 weeks. At 24 weeks of dietary intervention, HFF fed mice had substantially elevated systolic blood pressure compared to LF controls. The provision of UDCA substantially attenuated the dietary HFF induced increase in systolic blood pressure concomitant with significantly lower plasma angiotensin II. The anti-hypertensive effect of UDCA in HFF mice was comparable to candesartan. The data suggests that long term supplementation of UDCA effectively lowers hypertension in a dietary induced model of type-2 diabetic insulin resistance.

Biostatistical analysis of quantitative immunofluorescence microscopy images.

Semiquantitative immunofluorescence microscopy has become a key methodology in biomedical research. Typical statistical workflows are considered in the context of avoiding pseudo-replication and marginalising experimental error. However, immunofluorescence microscopy naturally generates hierarchically structured data that can be leveraged to improve statistical power and enrich biological interpretation. Herein, we describe a robust distribution fitting procedure and compare several statistical tests, outlining their potential advantages/disadvantages in the context of biological interpretation. Further, we describe tractable procedures for power analysis that incorporates the underlying distribution, sample size and number of images captured per sample. The procedures outlined have significant potential for increasing understanding of biological processes and decreasing both ethical and financial burden through experimental optimization.

Plasma concentrations of coffee polyphenols and plasma biomarkers of diabetes risk in healthy Japanese women.

Coffee consumption has been reported to reduce the risk of type 2 diabetes in experimental and epidemiological studies. This anti-diabetic effect of coffee may be attributed to its high content in polyphenols especially caffeic acid and chlorogenic acid. However, the association between plasma coffee polyphenols and diabetic risks has never been investigated in the literature. In this study, fasting plasma samples were collected from 57 generally healthy females aged 38-73 (mean 52, s.d. 8) years recruited in Himeji, Japan. The concentrations of plasma coffee polyphenols were determined by liquid chromatography coupled with mass tandem spectrometer. Diabetes biomarkers in the plasma/serum samples were analysed by a commercial diagnostic laboratory. Statistical associations were assessed using Spearman's correlation coefficients. The results showed that plasma chlorogenic acid exhibited negative associations with fasting blood glucose, glycated hemoglobin and C-reactive protein, whereas plasma total coffee polyphenol and plasma caffeic acid were weakly associated with these biomarkers. Our preliminary data support previous findings that coffee polyphenols have anti-diabetic effects but further replications with large samples of both genders are recommended.

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.

Dietary fats, cerebrovasculature integrity and Alzheimer's disease risk.

An emerging body of evidence is consistent with the hypothesis that dietary fats influence Alzheimer's disease (AD) risk, but less clear is the mechanisms by which this occurs. Alzheimer's is an inflammatory disorder, many consider in response to fibrillar formation and extracellular deposition of amyloid-beta (Abeta). Alternatively, amyloidosis could notionally be a secondary phenomenon to inflammation, because some studies suggest that cerebrovascular disturbances precede amyloid plaque formation. Hence, dietary fats may influence AD risk by either modulating Abeta metabolism, or via Abeta independent pathways. This review explores these two possibilities taking into consideration; (i) the substantial affinity of Abeta for lipids and its ordinary metabolism as an apolipoprotein; (ii) evidence that Abeta has potent vasoactive properties and (iii) studies which show that dietary fats modulate Abeta biogenesis and secretion. We discuss accumulating evidence that dietary fats significantly influence cerebrovascular integrity and as a consequence altered Abeta kinetics across the blood-brain barrier (BBB). Specifically, chronic ingestion of saturated fats or cholesterol appears to results in BBB dysfunction and exaggerated delivery from blood-to-brain of peripheral Abeta associated with lipoproteins of intestinal and hepatic origin. Interestingly, the pattern of saturated fat/cholesterol induced cerebrovascular disturbances in otherwise normal wild-type animal strains is analogous to established models of AD genetically modified to overproduce Abeta, consistent with a causal association. Saturated fats and cholesterol may exacerbate Abeta induced cerebrovascular disturbances by enhancing exposure of vessels of circulating Abeta. However, presently there is no evidence to support this contention. Rather, SFA and cholesterol appear to more broadly compromise BBB integrity with the consequence of plasma protein leakage into brain, including lipoprotein associated Abeta. The latter findings are consistent with the concept that AD is a dietary-fat induced phenotype of vascular dementia, reflecting the extraordinary entrapment of peripherally derived lipoproteins endogenously enriched in Abeta. Rather than being the initiating trigger for inflammation in AD, accumulation of extracellular lipoprotein-Abeta may be a secondary amplifier of dietary induced inflammation, or possibly, simply be consequential. Clearly, delineating the mechanisms by which dietary fats increase AD risk may be informative in developing new strategies for prevention and treatment of AD.

Chylomicron amyloid-beta in the aetiology of Alzheimer's disease.

Alzheimer's disease is characterized by inflammatory proteinaceous deposits comprised principally of the protein amyloid-beta (Abeta). Presently, the origins of cerebral amyloid deposits are controversial, though pivotal for the prevention of Alzheimer's disease. Recent evidence suggests that in blood, Abeta may serve as a regulating apoprotein of the triglyceride-rich-lipoproteins and we have found that the synthesis of Abeta in enterocytes and thereafter secretion as part of the chylomicron cascade is regulated by dietary fats. It is our contention that chronically elevated plasma levels of Abeta in response to diets rich in saturated fats may lead to disturbances within the cerebrovasculature and exaggerated blood-to-brain delivery of circulating Abeta, thereby exacerbating amyloidosis. Consistent with this hypothesis we show that enterocytic Abeta is increased concomitant with apolipoprotein B48. Furthermore, cerebral extravasation of immunoglobulin G, a surrogate marker of plasma proteins is observed in a murine model of Alzheimer's disease maintained on a saturated-fat diet and there is diminished expression of occludin within the cerebrovasculature, an endothelial tight junction protein.

Three-dimensional immunofluorescent double labelling using polyclonal antibodies derived from the same species: enterocytic colocalization of chylomicrons with Golgi apparatus.

Double immunolabelling is a useful technique to determine cellular colocalization of proteins, but is prone to false-positive staining because of cross-reactivity between antibodies. In this study, we established a simple and quick method to demonstrate the immunofluorescent double labelling with two rabbit-derived polyclonal antibodies. The principle used was to establish a dilution of primary antibody for the first protein of interest, which would only be detectable following biotin-avidin amplification. Thereafter, the second protein of interest was assessed via standard secondary antibody detection, ensuring no cross-reactivity with the first protein antibody-antigen complex. We successfully demonstrated the three-dimensional colocalization of enterocytic apolipoprotein B, an equivocal marker of intestinal lipoproteins with Golgi apparatus. Colocalization of apo B and Golgi apparatus (75.2 +/- 8.5%) is consistent with the purported mode of secretion of these macromolecules.

Impaired postprandial apolipoprotein-B48 metabolism in the obese, insulin-resistant JCR:LA-cp rat: increased atherogenicity for the metabolic syndrome.

AIM: Postprandial lipaemia is a significant contributor to the development of dyslipidaemia and cardiovascular disease, which has more recently been shown as a potential risk factor for obesity and pre-diabetes. Clinically however, the diagnosis of early insulin-resistance remains confounded due to the fact that aberrations in lipid metabolism are not often readily identified using classic indicators of hypercholesterolemia (i.e. LDL). METHODS: In this study, we assessed the metabolism of apolipoprotein-B48 (apoB48)-containing lipoproteins in an animal model of obesity and insulin-resistance, the JCR:LA-cp rat. The contribution of lipoproteins from the intestine was assessed by measuring plasma apoB48 concentration in the postprandial period following an oral fat load. Plasma apoB48 was measured by improved enhanced chemiluminescent detection and other biochemical parameters measured by established analysis. RESULTS: Fasting concentrations of plasma apoB48, postprandial apoB48 area under the curve (AUC), as well as incremental-AUC (iAUC), were all significantly greater in the obese phenotype compared to lean controls. Fasting apoB48 correlated significantly with apoB48-iAUC, triglyceride (TG)-iAUC and insulin-iAUC. In addition, there was a highly significant association with fasting insulin and the postprandial ratio of TG:apoB48, a relationship not often detected in humans during insulin-resistance. CONCLUSIONS/INTERPRETATION: We conclude that the JCR:LA-cp rat can be used as a model of postprandial lipemia to explore chylomicron metabolism during the onset and development of insulin-resistance, including the increased cardiovascular complications of the metabolic syndrome.