Combined intervention strategies in a model of transient cerebral ischaemia

Rachel Shirley, Emily Ord, Christopher McCabe, I. Mhairi Macrae, Andrew H. Baker, Lorraine M. Work. University of Glasgow, Glasgow, United Kingdom.

Stroke is the 3rd leading cause of death in the UK and the major cause of long-term disability. Research has identified excitotoxicity and ion imbalance, oxidative stress and cell death mediated by necrosis and apoptosis as contributing pathways. A peripheral zone adjacent to the ischaemic core, of mild to moderate oligaemia, is referred to as the penumbra and in the acute period after the stroke contains potentially salvageable tissue. We have visualised the penumbra in Wistar Kyoto (WKY) and spontaneously hypertensive stroke prone rats (SHRSP) using magnetic resonance imaging (MRI) following permanent middle cerebral artery occlusion (MCAO) and shown the area of salvageable penumbra to be time dependent, decreasing with increasing time from MCAO. Therefore, key to improving outcome following experimental stroke is intervention at an early stage in the pathways central to this stage of lesion progression. Our research attention is focussing on combined drug and gene-therapy targeting antioxidant and anti-apoptotic pathways and assessment of the contribution of these pathways in lesion development using a model of transient MCAO (tMCAO) in the SHRSP. Integral to pro-apoptotic signalling cascades is the enzyme c-jun N-terminal kinase (JNK). Through gene expression studies using Taqman™ q-PCR we have found levels of the neuronal specific gene, jnk3, to be markedly and selectively up-regulated within penumbral tissue 2 hours after permanent MCAO whilst jnk1 and jnk2 remain unchanged (ΔCt vs GAPDH 4.6 ± 0.1 in sham vs 4.0 ± 0.3 in MCAO, n=3 per group, *p<0.05; cycle threshold (Ct) = number of cycles required for fluorescent signal to cross predetermined threshold. The Ct levels are inversely proportional to the amount of target in the sample therefore lower Ct level = greater amount of target). Short hairpin RNA (shRNA) directed against the jnk3 gene has been incorporated into a lentivirus to silence the neuronal-specific enzyme JNK3. In vitro determination of the specificity of this construct has been assessed in B50 rat neuronal cells subjected to hypoxia/reperfusion using a hypoxic chamber (1% O2, 5% CO2, balance N2). JNK3 gene and phosphorylated protein levels increased with increasing exposure to hypoxia up to 9 hrs when levels began to decline. Increasing concentration of shRNA jnk3 lentivirus infection into B50 cells prior to hypoxia/reperfusion insult blocked the increase in jnk3 gene expression levels compared to a control, scrambled shRNA lentivirus. While hypoxia resulted in a 1.9 fold increase in jnk3 gene expression levels prior infection with jnk3 shRNA expressing lentivirus resulted in a 14% (multiplicity of infection, MOI, = 10 virus particles, vp, / cell) or 94% (MOI = 50 vp / cell) reduction in jnk3 gene expression levels. Levels of jnk1 and jnk2 gene expression were unaffected by jnk3 shRNA expressing lentivirus. We have engineered a selective shRNA expressing lentivirus to counter the increased expression of the neuronal specific jnk3 pro-apoptotic gene following cerebral ischaemia.