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Genomic Tools
Introduction
Although an enormous amount of work has been performed trying to understand the molecular events associated with stroke in animal models, much less has been done assessing the molecular biology of stroke in humans. A number of human studies have examined clotting pathways and some inflammatory pathways; most of the studies have focused on candidate molecules or pathways that are thought to be important in human stroke or hemorrhage. We took a different approach by examining all of the messenger RNA (mRNA) expressed in the blood of patients with strokes, transient ischemic attacks (TIAs), and brain hemorrhage. Since all of the mRNAs are known, this approach will capture all of the molecules and pathways in blood associated with the stroke, TIA, hemorrhage, or other cerebrovascular event.
In humans, the only viable avenue at present is to study the blood. However, blood is a particularly relevant organ for stroke since ischemic stroke due to cardioembolism, large-vessel atherosclerosis, and lacunar disease involves clotting, which includes blood proteins, blood platelets, and blood leukocytes . Evidence for clotting as being the primary event for all causes of stroke includes the fact that tissue plasminogen activator (tPA) is effective for all ischemic strokes including those due to cardioembolism, large-vessel disease, and lacunar disease. Thus, by examining the molecular biology of peripheral blood leukocytes and platelets using our genomics approach, one can examine factors that potentially promote or prevent clotting and thus play key roles in ischemic stroke and in brain hemorrhage. In addition, peripheral blood leukocytes and platelets interact with brain endothelium and atherosclerotic plaques, which changes their gene expression and function. By assessing the whole genome of blood leukocytes and platelets, we are able to assess the biology of stroke and intracerebral hemorrhage.
Ischemic Stroke Versus Controls and Intracerebral Hemorrhage
We took blood from animals 24 h after they had experimental ischemic strokes, brain hemorrhage, status epilepticus, hypoxia, and hypoglycemia . The RNA was isolated and processed on whole genome microarrays. Although no single gene could distinguish these conditions, groups of genes we termed “gene expression profiles” did distinguish ischemic stroke from intracerebral hemorrhage. Indeed, there was a unique expression profile for each condition in peripheral blood .
Human studies demonstrated similar findings. There were unique gene expression profiles in whole blood of patients who had ischemic strokes compared with matched controls without strokes . Gene expression changed within 3 h of a stroke with hundreds of genes with altered expression by 24 h after a stroke . These results were confirmed in a larger follow-up study that showed that a panel of less than 20 genes could distinguish strokes from controls with greater than 85% accuracy . Studies using RNAseq to measure alternative splicing of mRNA transcripts in blood showed that there are specific spliced gene profiles that can distinguish patients with ischemic strokes from patients with intracerebral hemorrhages and control patients .
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