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

Measure of Neuroinflammation.

<p>Neuroinflammation was assessed by measuring the voxel intensity of cerebral glial fibrillary acidic protein (GFAP) expression in cerebral cortex (A) and hippocampal formation (B) in Sprague-Dawley rats and C57BL/6J mice fed 80,000 IU vitamin D (VD) per kilogram of diet and their respective controls, expressed as voxels per volume unit. Cerebral GFAP expression in cortex (C) and hippocampal formation (D) in parathyroid gland ablated (PTX) rats and parathyroid hormone (PTH) infused rats with their controls are also shown. Statistical difference represented by different letters (p<0.05; n = 4–10; one way ANOVA followed by Tukey’s post hoc test). Data is shown as mean ± SEM. Representative 2-dimensional immunofluorescent micrographs of neuroinflammatory GFAP, in extended focus, are shown in the last frame. Each intervention (vitamin D enrichment; PTX; PTH-infusion) and their respective controls are indicated. GFAP is shown in yellow and nuclei in blue. Scale bar indicates 100μm.</p

Cerebral Capillary Integrity.

<p>Cerebral capillary vessel integrity was assessed using 3-dimensional semi-quantitative fluorescent immunomicroscopy by measurement of parenchymal extravasation of plasma macromolecule IgG in the cerebral cortex (cortex) and hippocampal formation (hippocampus) of wild-type rats (A) and mice (B) supplemented with units of dietary vitamin D (VD) as indicated and measured in voxels per volume unit. The bar graphs illustrate a strong dosage effect of vitamin D concentration and abundance of cerebral IgG in the cortex and hippocampal formation in both species; statistical significance is denoted by different letters (p<0.05; n = 8; one way ANOVA followed by Tukey’s post hoc analysis). The bottom graphs demonstrate duration-of-treatment effects in both rodent species fed either control (open square/dotted lines) or diets enriched with 80, 000 IU VD/kg (black circles/solid lines) for 6, 12 and 24 weeks. Different letters show p<0.05 for duration effects within each group (n = 8) whilst an asterisk show statistically significant different dietary effects between control group and its’ relevant treatment group at each time point (p<0.05; n = 8; one-way ANOVA followed by Tukey’s post hoc analysis). Data is shown as mean ± SEM. Representative 2-dimensional extended-focus fluorescent immunomicrographs (magnification x 200) of cerebral IgG distribution in animals fed control and 80, 000 IU VD/kg diets are shown in the last frame; IgG is shown in green and DAPI nuclei counterstaining is shown in blue. Scale bar represents 100μm.</p

Blood biochemistry analyses.

<p>Blood biomarkers were measured in wild-type rats and mice supplemented with different doses of dietary vitamin D (VD) to assess calcemic status. Blood Ionised Calcium (B), serum Total Calcium (C) and Serum Phosphate were measured using either an iSTAT point-of-care analyser or commercially available colorimetric kits. Data is shown as mean ± SEM. Statistical significance is denoted by different letters (p<0.05; n = 8; one-way ANOVA followed by Tukey’s post hoc test).</p

Correlation Table of Cerebral Capillary Permeability with markers of Vitamin D-Calcium-Parathyroid hormone homeostasis.

<p>Correlation coefficients of cerebral capillary permeability (IgG) in both cerebral cortex (CTX) and hippocampal formation (HPF) with vitamin D, blood ionised calcium, serum total calcium, parathyroid hormone and serum phosphate for all vitamin D supplemented experimental groups (rats and mice), parathyroid gland ablated (PTX) and parathyroid hormone-infused (PTH-infusion) groups are represented in this table. (**p<0.01; *p<0.05; n = 4–10; Pearson’s analysis).</p><p>Correlation Table of Cerebral Capillary Permeability with markers of Vitamin D-Calcium-Parathyroid hormone homeostasis.</p

Duration-of-treatment effects of exogenous vitamin D on calcemic status.

<p>The duration-of-treatment effects of dietary vitamin D on blood ionised calcium, serum total calcium and serum phosphate in either control animals (open square/dotted lines) or animals fed 80, 000 IU vitamin D/kg (black circles/solid lines) at 6, 12 and 24 weeks of age are shown. Column (A) represents data derived from Sprague-Dawley rats and column (B) of C57BL/6J mice. p<0.05 for duration effects within each group is represented by different letters (n = 8; one-way ANOVA). * p<0.05 compared to relevant control at each corresponding time point (dietary effects; n = 8; one-way ANOVA). Data is shown as mean ± SEM.</p

Measures of Parathyroid Hormone (PTH) in vitamin D-supplemented rats.

<p>After 12 weeks of intervention, circulating serum concentrations of intact parathyroid hormone (PTH) were determined via commercially available ELISA kits in each of the dietary vitamin D (VD) groups. Data shown as mean ± SEM is indicated in the top graph. Different letters denote statistical significance between groups (p<0.05; n = 8; one-way ANOVA followed by Tukey’s post hoc analysis). Duration-of-treatment effects in each representative group are shown in the bottom frame for either 6, 12 or 24 weeks (control, shown as open squares/dotted line or 80,000IU VD/kg, shown as black circles/solid black line). Different letters are indicative of p<0.05 for duration effects within each group (n = 8; one-way ANOVA) whereas * show p<0.05 compared to respective controls (n = 8; one-way ANOVA). All data is shown as mean ± SEM.</p