Identification of New Therapeutic Targets by Genome-Wide Analysis of Gene Expression in the Ipsilateral Cortex of Aged Rats after Stroke

<div><h3>Background</h3><p>Because most human stroke victims are elderly, studies of experimental stroke in the aged rather than the young rat model may be optimal for identifying clinically relevant cellular responses, as well for pinpointing beneficial interventions.</p> <h3>Methodology/Principal Findings</h3><p>We employed the Affymetrix platform to analyze the whole-gene transcriptome following temporary ligation of the middle cerebral artery in aged and young rats. The correspondence, heat map, and dendrogram analyses independently suggest a differential, age-group-specific behaviour of major gene clusters after stroke. Overall, the pattern of gene expression strongly suggests that the response of the aged rat brain is qualitatively rather than quantitatively different from the young, i.e. the total number of regulated genes is comparable in the two age groups, but the aged rats had great difficulty in mounting a timely response to stroke. Our study indicates that four genes related to neuropathic syndrome, stress, anxiety disorders and depression (<em>Acvr1c</em>, <em>Cort</em>, <em>Htr2b</em> and <em>Pnoc</em>) may have impaired response to stroke in aged rats. New therapeutic options in aged rats may also include <em>Calcrl</em>, <em>Cyp11b1, Prcp, Cebpa</em>, <em>Cfd, Gpnmb</em>, <em>Fcgr2b, Fcgr3a</em>, <em>Tnfrsf26, Adam 17</em> and <em>Mmp14</em>. An unexpected target is the enzyme 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 in aged rats, a key enzyme in the cholesterol synthesis pathway. Post-stroke axonal growth was compromised in both age groups.</p> <h3>Conclusion/Significance</h3><p>We suggest that a multi-stage, multimodal treatment in aged animals may be more likely to produce positive results. Such a therapeutic approach should be focused on tissue restoration but should also address other aspects of patient post-stroke therapy such as neuropathic syndrome, stress, anxiety disorders, depression, neurotransmission and blood pressure.</p> </div

Patterns of gene expression after stroke.

<p>There were several distinct patterns of gene regulation: persistently upregulated (black line), transiently upregulated, (orange line), “late-upregulated” (red line), “late-downregulated” (yellow line), transiently downregulated (blue line), and persistently downregulated (green line). Aged animals showed larger numbers than young of genes that were late-upregulated, persistently upregulated and persistently downregulated. The young rats, in contrast, had a much larger number of transiently upregulated and delayed downregulated genes. Note that this representation does not take into account the fold changes for individual genes but the relative change in gene expression at days 3 and 14 post-stroke.</p

Heatmap of genes differentially expressed between post-stroke and naïve animals.

<p>Scaled expression values of all 1,658 differentially expressed genes are shown for each group with light red being the lowest and light green the highest expression level. The depicted dendrograms cluster samples (top) and genes (left) employing average agglomeration and euclidian distance measure.</p

Correspondence analysis of differentially expressed genes and samples grouped by animal age.

<p>The left panel depicts the Eigenvalues of the correspondence analysis and shows that the major factors contributing to the variance of stroketomics analysis were stroke (52%), post-stroke time (25%) and age (12%). (Right panel): The first two sources of variability, stroke and post-stroke time formed the coordinates of the right panel. The graph shows the distribution of transcripts (black dotes) as a function of treatment (stroke) and post-stroke time. Samples from young (green) and aged (red) animals particularly differ in their post-stroke response (illustrated by ellipses that form non-parallel planes). Transcripts with characteristic expression in naive samples are encircled in black.</p

Venn diagrams for 3d and 14d post-stroke showing genes that were up- or down-regulated exclusively in old or young rats, or in both age groups.

<p>Note that at 14 days post-stroke, the differences between the age groups were more pronounced.</p

Gene expression for several new potential therapeutic targets to improve post-stroke recovery in aged animals.

<p>Legend</p><p>↔ no changes vs contralateral side.</p><p>↑ upregulated vs contralateral side.</p><p>↓ downregulated vs contralateral side.</p><p>y/3d Young, 3d post-stroke.</p><p>y/14d Young, 14d post-stroke.</p><p>A/3d Aged, 3d post-stroke.</p><p>A/14d Aged, 14d post-stroke.</p><p>Confirmation of arrays data for new stroke-related genes was done by RT-PCR. Modulation of gene/protein activity by the indicated drugs may, in combination or alone, improve post-stroke recovery in aged animals. For most of the upregulated genes there is a therapeutic option but not for downregulated genes.</p