Differential gene expression in multiple neurological, inflammatory and connective tissue pathways in a spontaneous model of human small vessel stroke

Abstract

Aims: Cerebral small vessel disease (SVD) causes a fifth of all strokes plus diffuse brain damage leading to cognitive decline, physical disabilities and dementia. The aetiology and pathogenesis of SVD are unknown, but largely attributed to hypertension or microatheroma.

Methods: We used the spontaneously hypertensive stroke-prone rat (SHRSP), the closest spontaneous experimental model of human SVD, and age-matched control rats kept under identical, non-salt-loaded conditions, to perform a blinded analysis of mRNA microarray, qRT-PCR and pathway analysis in two brain regions (frontal and mid-coronal) commonly affected by SVD in the SHRSP at age five, 16 and 21 weeks.

Results: We found gene expression abnormalities, with fold changes ranging from 2.5 to 59 for the 10 most differentially expressed genes, related to endothelial tight junctions (reduced), nitric oxide bioavailability (reduced), myelination (impaired), glial and microglial activity (increased), matrix proteins (impaired), vascular reactivity (impaired) and albumin (reduced), consistent with protein expression defects in the same rats. All were present at age 5 weeks thus predating blood pressure elevation. 'Neurological' and 'inflammatory' pathways were more affected than 'vascular' functional pathways.

Conclusions: This set of defects, although individually modest, when acting in combination could explain the SHRSP's susceptibility to microvascular and brain injury, compared with control rats. Similar combined, individually modest, but multiple neurovascular unit defects, could explain susceptibility to spontaneous human SVD.

Keywords: blood brain barrier; lacunar stroke; neurovascular unit; small vessel disease; stroke.

Figure 1

Heat maps were generated using hierarchical clustering of all significantly differentially expressed Neurological Genes from individual biological replicates of 5-week-old SHRSP and WKY animals. The relative expression of each gene is represented by colour intensity, where red is up and green is down-regulated in (a) 79 genes from the frontal region and (b) 48 genes identified in the mid-coronal region.

Figure 2

A network from the ‘neurological disorders’ functional group, representing interactions between differentially expressed genes in SHRSP vs. WKY rats at 5 weeks of age, generated by Ingenuity Pathway Analysis software. Genes highlighted in red are down-regulated and in green are up-regulated in SHRSP compared with WKY. Solid lines indicate direct interactions. Dotted lines indicate indirect interactions. Details of affected genes are given in Tables S1 and S2.

Figure 3

Details of genes in the ‘acute phase response’ biological pathway identified in Ingenuity Pathway Analysis, which are differentially expressed in the frontal section of SHRSP vs. WKY rats at 5 weeks of age. Genes highlighted red are down-regulated and in green are up-regulated in SHRSP compared with WKY. Solid lines indicate direct interactions. Dotted lines indicate indirect interactions.

Figure 4

Details of genes in the ‘complement’ biological pathway identified in Ingenuity Pathway Analysis, which are differentially expressed in the frontal section of SHRSP vs. WKY rats at 5 weeks of age. Genes highlighted red are down-regulated and in green are up-regulated in SHRSP compared with WKY. Solid lines indicate direct interactions. Dotted lines indicate indirect interactions.

Figure 5

Validation of significant changes in gene expression of (a) GFAP, (b) MMP14 and (c) AVP in 5-week-old rats using qRT-PCR. Bars represent the difference in fold change between WKY and SHRSP. Error bars represent the standard error of the mean. Each bar represents n = 4 rats. *P < 0.05. **P < 0.01. ***P < 0.001. (a) GFAP mRNA expression was significantly reduced in the frontal section of SHRSP. (b) MMP14 mRNA expression was significantly reduced in the frontal section of SHRSP. (c) AVP mRNA expression was significantly reduced in the mid-coronal section of SHRSP.

Figure 6

Heat maps generated using hierarchical clustering of the median expression from 5-, 16- and 21-week SHRSP and WKY downstream transcriptional targets of cAMP response element-binding protein (Creb). The relative expression of each probe is represented by colour intensity, where red is up and green is down-regulated in (a) 17 genes from the frontal (F) region and (b) 18 genes identified in the mid-coronal (M) region. Genes are listed on the right hand side.