Effects of dietary salt on gene and protein expression in brain tissue of a model of sporadic small vessel disease

Background: The effect of salt on cerebral small vessel disease (SVD) is poorly understood. We assessed the effect of dietary salt on cerebral tissue of the stroke-prone spontaneously hypertensive rat (SHRSP) – a relevant model of sporadic SVD – at both the gene and protein level. Methods: Brains from 21-week-old SHRSP and Wistar-Kyoto rats, half additionally salt-loaded (via a 3-week regime of 1% NaCl in drinking water), were split into two hemispheres and sectioned coronally – one hemisphere for mRNA microarray and qRT-PCR, the other for immunohistochemistry using a panel of antibodies targeting components of the neurovascular unit. Results: We observed differences in gene and protein expression affecting the acute phase pathway and oxidative stress (ALB, AMBP, APOH, AHSG and LOC100129193, up-regulated in salt-loaded WKY versus WKY, >2-fold), active microglia (increased Iba-1 protein expression in salt-loaded SHRSP versus salt-loaded WKY, p<0.05), vascular structure (ACTB and CTNNB, up-regulated in salt-loaded SHRSP versus SHRSP, >3-fold; CLDN-11, VEGF and VGF down-regulated >2-fold in salt-loaded SHRSP versus SHRSP) and myelin integrity (MBP down-regulated in salt loaded WKY rats versus WKY, >2.5-fold). Changes of salt-loading were more pronounced in SHRSP and occurred without an increase in blood pressure in WKY rats. Conclusion: Salt exposure induced changes in gene and protein expression in an experimental model of SVD and its parent rat strain in multiple pathways involving components of the glio-vascular unit. Further studies in pertinent experimental models at different ages would help clarify the short- and long-term effect of dietary salt in SVD.

the results by age and brain section, then Ingenuity Pathway Analysis® (IPA) (Ingenuity Systems, http://www.ingenuity.com) analysed data using both a prespecified candidate gene approach (looking for changes in genes and pathways thought to be relevant from previous work by ourselves and others) and a genome-wide approach (to generate new hypotheses).
Significance of pathways was assessed using one-sided Fisher's exact tests.
qRT-PCR data (cycle threshold (CT) values) was analysed in Microsoft® Excel by comparing mean delta cycle threshold (dCT) values vs. the housekeeper gene using a Student's t-test.

Immunohistochemistry
Tissue preparation: Formalin-fixed tissue was sectioned into 3-mm-thick coronal slices, processed and embedded in paraffin blocks, which were then cut into 7-µm sections. Similar frontal and mid coronal sections were chosen for analysis. "Standard coordinates from a stereotactic atlas were used to identify prominent structures (e.g. the internal capsule). The frontal region encompassed a region approximately +1.8-mm bregma (containing the anterior commissure and anterior lateral ventricle) and the mid coronal captured a region at −1.72-mm bregma (including areas of the basal ganglia and internal capsule)".
Antibody staining: Antibodies assessed various components of the neurovascular unit, claudin-5, collagen IV, smooth muscle actin (SMA), collagen I, glial fibrillary acidic protein (GFAP), matrix metalloproteinase 9 (MMP9), ionized calcium-binding adaptor molecule 1 (Iba-1) and Myelin Basic Protein (MBP). We previously assessed this panel of antibodies in 5 and 16 week old WKY and SHRSP [8]. All immunohistochemical studies used the ABC immunoperoxidase method (Vector Laboratories, Peterborough, UK). Antigen heat retrieval using a pressure cooker (with slides immersed in citric acid buffer) was performed before slides were blocked in hydrogen peroxide followed by either rabbit or swine serum. 3.3′-diaminobenzidine tetrahydrochloride with a haematoxylin counterstain revealed immunoreactivity. Tris-buffered saline replaced the primary antibody in negative controls.
Data analysis: Percentage staining within a defined area of interest was generated using ImagePro™ software (version 6.2; Media Cybernetics, Bethesda, MD, USA), blinded to species and salt, in cortical, deep grey and white matter. A standard sampling protocol was developed by identifying cross sections using landmarks such as the rhinal fissure and piriform cortex (for full protocol see [8]). Each cross section was divided into cortical grey matter, white matter and deep grey matter using the freehand tool on ImagePro™. Intraobserver reliability was assessed using a randomly selected slide counted on five separate occasions and revealed less than 10% of the variance within the counts was due to the effect of day. Colour-matched pixel counts applied to the entire hemisphere and subsequently converted into percentage areas of staining as a measure of immunoreactivity using ImagePro™, once each region of interest (e.g. cortical grey) had been defined by hand.
We performed statistical analysis in Minitab using a general linear model (two-way ANOVA) followed by Tukeys test for pairwise comparisons. The effect of both salt diet and strain were analysed. Frontal cortex and mid-coronal sections were analysed separately due to significant differences in the distribution of grey and white matter, and the influence of brain territory on expression was also included. P values of p<0.05 were considered statistically significant. All data are shown as mean ± SEM.
Data taken from frontal section tissue in 21 week old SHRSP versus WKY rats fed either a normal diet or salt-loaded with 1% NaCl from the age of 18 weeks. Numbers indicate percentage staining ± the standard error of the mean from n=5 animals. NS = No significant difference.