Mechanisms of ischaemic stroke damage

Abstract

Brain inflammation contributes to ischaemic and reperfusion injury, and thus worsens outcome after stroke. This thesis aimed to identify and investigate some of the inflammatory mechanisms that occur after cerebral ischaemia-reperfusion, to further enhance our understanding of them, and to potentially target them for future ischaemic stroke therapies. This study primarily used C57Bl6/J mice, as well as genetically modified mice. The model of focal cerebral ischaemia utilised was the intraluminal filament-induced middle cerebral artery occlusion. Real-time PCR and Western blotting were used to examine mRNA and protein expression levels, respectively, in the brain, and immunofluorescence and immunohistochemistry were used to localise proteins and cells in the brain. T lymphocytes were isolated from the blood and spleen using Dynal negative isolation kits, and T lymphocyte-generated superoxide was measured using L-012-enhanced chemiluminescence. Chapters 3 and 4 provided the first evidence that the larger infarct volume in males versus females following cerebral ischaemia is reperfusion-dependent and may be due to greater neuro-inflammation and brain infiltration of Nox2-expressing CD3+ T lymphocytes in male mice. Moreover, this gender difference was found to be dependent on Nox2 expression. The study also demonstrated for the first time that Nox2-expressing circulating CD3+ T lymphocytes produce ~15-fold more superoxide after stroke, compared to CD3+ T lymphocytes from control mice. These findings raise the possibility that therapies to reduce CD3+ T lymphocyte infiltration and/or the production of superoxide from these cells in ischaemic stroke patients who receive recombinant t-PA, might be useful for reducing reperfusion injury. Chapter 5 confirmed and extended the above findings and demonstrated for the first time that circulating Nox2-containing CD4+ and CD8+ T lymphocytes generate substantially higher levels of superoxide after cerebral ischaemia-reperfusion compared with similar T lymphocyte subsets from control mice. Chapter 6 demonstrated that the mRNA expression of various chemokines and chemokine receptors, including the potent neutrophil chemoattractants, CXCR2, CXCL1 and CXCL2, are increased in the brain after ischaemia-reperfusion. Administration of the CXCR2 antagonist, SB 225002, reduced the expression of these chemokines in the brain, as well as the infiltration of neutrophils, but did not improve outcome at 72 h after ischaemia-reperfusion. These findings suggest that the infiltration of neutrophils do not contribute to ischaemia-reperfusion injury. Chapter 7 examined for the first time the role of the endogenous calcineurin inhibitor, Down syndrome candidate region 1 (DSCR1), on outcome following cerebral ischaemia-reperfusion. We found that the over-expression of DSCR1 improves neurological outcome, and reduces infarct and oedema volume. This protection was most likely through the inhibition of calcineurin in neurons and also potentially in T lymphocytes, and the subsequent reduction in pro-inflammatory mediators. Interestingly, we also found preliminary evidence that the deficiency of DSCR1 improves neurological outcome and reduces infarct and oedema volume. DSCR1-deficiency thus may also reduce calcineurin activity, however the precise mechanisms underlying this are still unclear. Overall, this thesis presents important findings on the role of inflammation in ischaemic injury following cerebral ischaemia-reperfusion, and supports the concept that strategies targeting inflammation in combination with recombinant t-PA may be a successful stroke therapy.