Accumulation of neutrophils in brain after transient focal stroke remains controversial with some studies showing neutrophils to be deleterious, whereas others suggest neutrophils do not contribute to ischemic injury. Myeloperoxidase (MPO) has been used extensively as a marker for quantifying neutrophil accumulation, but is an indirect method and does not detect neutrophils alone. To elucidate the interaction of macrophages in the neutrophil inflammatory response, we conducted double-label immunofluorescence in brain sections at 0, 1, 2, 3, 7, and 15 days after ischemia. Each of these results was obtained from the same animal to determine correlations between neutrophil infiltration and ischemic damage. It was found that MPO activity increased up to 3 days after cerebral ischemia. Dual-staining revealed that macrophages engulf neutrophils in the brain and that this engulfment of neutrophils increased with time, with 50% of neutrophils in the brain engulfed at 3 days and approximately 85% at 15 days (N = 5, P < 0.05). Interestingly, at 7 days the amount of dual-staining was decreased to 20% (N = 5, P < 0.05). Neutrophil infiltration was positively correlated with ischemic damage in both the cortex and striatum (r 2 = 0.86 and 0.80, respectively, P < 0.01). The results of this study indicate that the MPO from neutrophils phagocytized by macrophages may continue to contribute to the overall MPO activity, and that previous assessments that have utilized this marker to measure neutrophil accumulation may have miscalculated the number of neutrophils within the ischemic territory and hence their contribution to the evolution of the infarct at later time points. Thus any biphasic infiltration of neutrophils may have been masked by the accumulation of macrophages.
NADPH oxidases contribute to brain injury, yet they may also have a role in brain repair, particularly in vascular signaling and angiogenesis. This study determined the temporal and spatial profile of NADPH oxidase subunit expression/activity concurrently with angiogenesis in the brain following transient ischemic stroke induced by prolonged constriction of the middle cerebral artery by perivascular injection of endothelin-1 in conscious Hooded Wistar rats (n = 47). VEGF mRNA expression was increased in the ipsilateral cortex and striatum between 6 h and 28 days post-stroke concurrently with a marked increase in Nox2 mRNA expression up to 7 days, and increased Nox4 mRNA expression detected between 7 and 28 days. Point counting of blood vessels using Metamorph imaging software showed increased vascular sprouting between 3 and 7 days after stroke with new vascular networks detected in the core infarct region by 14 days. Angiogenic blood vessels 3 and 7 days post-stroke were observed to co-localise with both Nox2 antibody and dihydroethidium fluorescence suggesting a role for Nox2 generated superoxide during the phase of vascular remodeling, whilst Nox4 expression was detected once new cerebral vessels had formed. These results indicate for the first time that ROS signaling through a cerebrovascular Nox2 NADPH oxidase may be important in initiating brain angiogenesis.
Background and purpose: Following transient focal stroke, rapid accumulation and activation of neutrophils in the ischaemic region is deleterious due to release of reactive oxygen species and myeloperoxidase (MPO). The purpose of this study was to examine whether AM-36, both a Na þ channel blocker and an antioxidant, afforded neuroprotection by modulating neutrophil accumulation into brain, following endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAo) in conscious rats. Experimental approach: AM-36 was administered at 3 and 24 h after ET-1-induced MCAo. Functional recovery was determined using grid-walking and cylinder tests. Image analysis of brain sections was used to determine infarct volume. The effect of AM-36 on neutrophil infiltration and their interaction with macrophages was examined in rats at 48 h following MCAo by both an MPO assay and double-label immunofluorescence. Blood brain barrier (BBB) breakdown was measured by the area stained by intravenous Evans Blue. Key results: AM-36 reduced functional deficits in both tests such that no difference existed from pre-ischaemic values at 48 h. Neutrophil infiltration, assessed by MPO activity, and infarct volume were significantly reduced following AM-36 administration by 54 and 60% respectively. Similarly, immunofluorescence revealed that AM-36 reduced neutrophil infiltration by B50% in selected brain regions, when compared to controls, and also modulated macrophage phagocytosis of neutrophils. Breakdown of the BBB was significantly reduced by 60% following AM-36 treatment. Conclusions and Implications: These findings suggest that AM-36 can directly modulate the neutrophil inflammatory response and reduce BBB breakdown following MCAo.
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