Hyperacute changes in blood mRNA expression profiles of rats after middle cerebral artery occlusion: Towards a stroke time signature.

Stroke evolution is a highly dynamic but variable disease which makes clinical decision making difficult. Biomarker discovery programs intended to aid clinical decision making have however largely ignored the rapidity of stroke evolution. We have used gene array technology to determine blood mRNA expression changes over the first day after stroke in rats. Blood samples were collected from 8 male spontaneously hypertensive rats at 0, 1, 2, 3, 6 and 24h post stroke induction by middle cerebral artery occlusion. RNA was extracted from whole blood stabilized in PAXgene tubes and mRNA expression was detected by oligonucleotide Affymetrix microarray. Using a pairwise comparison model, 1932 genes were identified to vary significantly over time (p≤0.5x10(-7)) within 24h after stroke. Some of the top20 most changed genes are already known to be relevant to the ischemic stroke physiopathology (e.g. Il-1R, Nos2, Prok2). Cluster analysis showed multiple stereotyped and time dependent profiles of gene expression. Direction and rate of change of expression for some profiles varied dramatically during these 24h. Profiles with potential clinical utility including hyper acute or acute transient upregulation (with expression peaking from 2 to 6h after stroke and normalisation by 24h) were identified. We found that blood gene expression varies rapidly and stereotypically after stroke in rats. Previous researchers have often missed the optimum time for biomarker measurement. Temporally overlapping profiles have the potential to provide a biological "stroke clock" able to tell the clinician how far an individual stroke has evolved.

Discovery and Longitudinal Evaluation of Candidate Biomarkers for Ischaemic Stroke by Mass Spectrometry-Based Proteomics.

Application of acute therapies such as thrombolysis for ischaemic stroke (IS) is constrained because of diagnostic uncertainty and the dynamic nature of stroke biology. To investigate changes in blood proteins after stroke and as a result of thrombolysis treatment we performed label-free quantitative proteomics on serum samples using high-resolution mass spectrometry and long high-performance liquid chromatography gradient (5 hours) combined with a 50-cm column to optimise the peptide separation. We identified (false discovery rate [FDR]: 1%) and quantified a total of 574 protein groups from a total of 92 samples from 30 patients. Ten patients were treated by thrombolysis as part of a randomised placebo-controlled trial and up to 5 samples were collected from each individual at different time points after stroke. We identified 26 proteins differently expressed by treatment group (FDR: 5%) and significant changes of expression over time for 23 proteins (FDR: 10%). Molecules such as fibrinogen and C-reactive protein showed expression profiles with a high-potential clinical utility in the acute stroke setting. Protein expression profiles vary acutely in the blood after stroke and have the potential to allow the construction of a stroke clock and to have an impact on IS treatment decision making.

Stimulation of axonal sprouting by trophic factors immobilized within the wound core.

Traumatic injury to the CNS results in peri-wound sprouting without significant axonal growth beyond the lesion edge. We have previously demonstrated that dopaminergic sprouting in the injured striatum follows an increasing gradient of BDNF and GDNF expression, with sprouting ceasing at the point of maximal factor expression. Progressively more complicated associations of sprouting fibers with increasingly activated microglia and macrophages suggest these factors are localized to the cell surface. To establish whether an increased concentration of immobilized BDNF and GDNF could stimulate axonal growth beyond the lesion edge, both factors were covalently attached to 10 microm polycarbonate microspheres. These spheres were implanted into the site of striatal injury 1 week after lesioning. A profusion of axons grew from the region of the lesion edge across the surface of the spheres. Some axons traversed the entire site of injury. Ultrastructural examination demonstrated the juxtaposition of regenerating axons to the surface of implanted spheres. CSPG immunostaining demonstrated that, in animals implanted with neurotrophin-microspheres, axonal growth was induced beyond the area of maximal CSPG reactivity. Surprisingly however, CSPG production at the wound edge was greater in control animals than those implanted with neurotrophin-microspheres. Overall, we show that axonal growth can be encouraged beyond the wound edge by an elevated concentration of immobilized trophic factors. This growth occurs despite the presence of inhibitory CSPGs at the lesion edge. Axonal growth appears to be stimulated mainly via the direct effects of neurotrophins. However, there also appears to be an indirect mechanism whereby neurotrophins reduce the synthesis of CSPG at the wound edge, making the peri-wound environment more permissive.