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Nox2 knockout delays infarct progression and increases vascular recovery through angiogenesis in mice following ischaemic stroke with reperfusion.

McCann SK, Dusting GJ, Roulston CL - PLoS ONE (2014)

Bottom Line: Transient cerebral ischaemia was induced via intraluminal filament occlusion and resulted in reduced infarct volumes in Nox2 KO mice at 24 h post-stroke compared to wild-type controls.The effect of Nox2 deletion on vascular damage and recovery was also examined 24 h and 72 h post-stroke using an antibody against laminin.Additionally, we show for the first time that Nox2 deletion increases re-vascularisation of the damaged brain by 72 h, which may be important in promoting endogenous brain repair mechanisms that rely on re-vascularisation.

View Article: PubMed Central - PubMed

Affiliation: Stroke Injury and Repair Team, O'Brien Institute, St Vincent's Hospital, Melbourne, Victoria, Australia; Department of Surgery, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Melbourne, Victoria, Australia.

ABSTRACT
Evidence suggests the NADPH oxidases contribute to ischaemic stroke injury and Nox2 is the most widely studied subtype in the context of stroke. There is still conjecture however regarding the benefits of inhibiting Nox2 to improve stroke outcome. The current study aimed to examine the temporal effects of genetic Nox2 deletion on neuronal loss after ischaemic stroke using knockout (KO) mice with 6, 24 and 72 hour recovery. Transient cerebral ischaemia was induced via intraluminal filament occlusion and resulted in reduced infarct volumes in Nox2 KO mice at 24 h post-stroke compared to wild-type controls. No protection was evident at either 6 h or 72 h post-stroke, with both genotypes exhibiting similar volumes of damage. Reactive oxygen species were detected using dihydroethidium and were co-localised with neurons and microglia in both genotypes using immunofluorescent double-labelling. The effect of Nox2 deletion on vascular damage and recovery was also examined 24 h and 72 h post-stroke using an antibody against laminin. Blood vessel density was decreased in the ischaemic core of both genotypes 24 h post-stroke and returned to pre-stroke levels only in Nox2 KO mice by 72 h. Overall, these results are the first to show that genetic Nox2 deletion merely delays the progression of neuronal loss after stroke but does not prevent it. Additionally, we show for the first time that Nox2 deletion increases re-vascularisation of the damaged brain by 72 h, which may be important in promoting endogenous brain repair mechanisms that rely on re-vascularisation.

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Related in: MedlinePlus

Immunofluorescent co-localisation of ROS generation with Iba1 at 24 h.Representative fluorescent micrographs of ROS-sensitive DHE, the macrophage/activated microglia antibody Iba1 and merged images from the contralateral and ipsilateral penumbral cortex of Nox2 WT (A) and Nox2 KO (B) mice at 24 h post-stroke. Scale bars  = 50 µm.
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pone-0110602-g006: Immunofluorescent co-localisation of ROS generation with Iba1 at 24 h.Representative fluorescent micrographs of ROS-sensitive DHE, the macrophage/activated microglia antibody Iba1 and merged images from the contralateral and ipsilateral penumbral cortex of Nox2 WT (A) and Nox2 KO (B) mice at 24 h post-stroke. Scale bars  = 50 µm.

Mentions: In both genotypes, Iba1 immunoreactivity increased in the ipsilateral penumbra over time. At 6 h post-stroke a low level of activation was observed in the ipsilateral cortex of both Nox2 WT and Nox2 KO mice (Fig 5). These cells were also positive for DHE fluorescence, although Iba1 co-localisation did not appear to account for the majority of DHE signal detected. Iba1-positive labelling in Nox2 WT brain increased in the ipsilateral cortex from 24 h (Fig 6A) to 72 h (Fig 7A) post-stroke, while staining in the contralateral cortex remain unchanged over time. At 72 h post-stroke Iba1-positive cells in the Nox2 KO mice appeared to be less activated (Fig 7B, inset), with cells exhibiting more ramified processes when compared to the amoeboid-like cells observed in Nox2 WT mice (Fig 7A, inset). Iba1-positive cells at all recovery times were co-labelled with DHE in both genotypes. Similar morphological changes in Iba1-positive cells were observed in the striatum at all recovery times but the degree of activation appeared to be less than that seen in the cortex (images not shown).


Nox2 knockout delays infarct progression and increases vascular recovery through angiogenesis in mice following ischaemic stroke with reperfusion.

McCann SK, Dusting GJ, Roulston CL - PLoS ONE (2014)

Immunofluorescent co-localisation of ROS generation with Iba1 at 24 h.Representative fluorescent micrographs of ROS-sensitive DHE, the macrophage/activated microglia antibody Iba1 and merged images from the contralateral and ipsilateral penumbral cortex of Nox2 WT (A) and Nox2 KO (B) mice at 24 h post-stroke. Scale bars  = 50 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222846&req=5

pone-0110602-g006: Immunofluorescent co-localisation of ROS generation with Iba1 at 24 h.Representative fluorescent micrographs of ROS-sensitive DHE, the macrophage/activated microglia antibody Iba1 and merged images from the contralateral and ipsilateral penumbral cortex of Nox2 WT (A) and Nox2 KO (B) mice at 24 h post-stroke. Scale bars  = 50 µm.
Mentions: In both genotypes, Iba1 immunoreactivity increased in the ipsilateral penumbra over time. At 6 h post-stroke a low level of activation was observed in the ipsilateral cortex of both Nox2 WT and Nox2 KO mice (Fig 5). These cells were also positive for DHE fluorescence, although Iba1 co-localisation did not appear to account for the majority of DHE signal detected. Iba1-positive labelling in Nox2 WT brain increased in the ipsilateral cortex from 24 h (Fig 6A) to 72 h (Fig 7A) post-stroke, while staining in the contralateral cortex remain unchanged over time. At 72 h post-stroke Iba1-positive cells in the Nox2 KO mice appeared to be less activated (Fig 7B, inset), with cells exhibiting more ramified processes when compared to the amoeboid-like cells observed in Nox2 WT mice (Fig 7A, inset). Iba1-positive cells at all recovery times were co-labelled with DHE in both genotypes. Similar morphological changes in Iba1-positive cells were observed in the striatum at all recovery times but the degree of activation appeared to be less than that seen in the cortex (images not shown).

Bottom Line: Transient cerebral ischaemia was induced via intraluminal filament occlusion and resulted in reduced infarct volumes in Nox2 KO mice at 24 h post-stroke compared to wild-type controls.The effect of Nox2 deletion on vascular damage and recovery was also examined 24 h and 72 h post-stroke using an antibody against laminin.Additionally, we show for the first time that Nox2 deletion increases re-vascularisation of the damaged brain by 72 h, which may be important in promoting endogenous brain repair mechanisms that rely on re-vascularisation.

View Article: PubMed Central - PubMed

Affiliation: Stroke Injury and Repair Team, O'Brien Institute, St Vincent's Hospital, Melbourne, Victoria, Australia; Department of Surgery, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Melbourne, Victoria, Australia.

ABSTRACT
Evidence suggests the NADPH oxidases contribute to ischaemic stroke injury and Nox2 is the most widely studied subtype in the context of stroke. There is still conjecture however regarding the benefits of inhibiting Nox2 to improve stroke outcome. The current study aimed to examine the temporal effects of genetic Nox2 deletion on neuronal loss after ischaemic stroke using knockout (KO) mice with 6, 24 and 72 hour recovery. Transient cerebral ischaemia was induced via intraluminal filament occlusion and resulted in reduced infarct volumes in Nox2 KO mice at 24 h post-stroke compared to wild-type controls. No protection was evident at either 6 h or 72 h post-stroke, with both genotypes exhibiting similar volumes of damage. Reactive oxygen species were detected using dihydroethidium and were co-localised with neurons and microglia in both genotypes using immunofluorescent double-labelling. The effect of Nox2 deletion on vascular damage and recovery was also examined 24 h and 72 h post-stroke using an antibody against laminin. Blood vessel density was decreased in the ischaemic core of both genotypes 24 h post-stroke and returned to pre-stroke levels only in Nox2 KO mice by 72 h. Overall, these results are the first to show that genetic Nox2 deletion merely delays the progression of neuronal loss after stroke but does not prevent it. Additionally, we show for the first time that Nox2 deletion increases re-vascularisation of the damaged brain by 72 h, which may be important in promoting endogenous brain repair mechanisms that rely on re-vascularisation.

Show MeSH
Related in: MedlinePlus