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NADPH oxidase and angiogenesis following endothelin-1 induced stroke in rats: role for nox2 in brain repair.

Taylor CJ, Weston RM, Dusting GJ, Roulston CL - Brain Sci (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Stroke Injury and Repair Team, O'Brien Institute, 42 Fitzroy St, Fitzroy, Melbourne, Victoria 3065, Australia. cj.taylor@unimelb.edu.au.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Angiogenesis and lesion pathology following ET-1 induced stroke. Hematoxylin and Eosin (H & E) stained images of the infarcted cortex between 6 h and 28 days post-stroke (A–E) were used to identify areas of interest for vWF, NeuN and OX42 histological analysis (as marked by frames). Merged immunofluorescent images of NeuN labeled neurons (red) and vWF labeled blood vessels (green) throughout the stroke affected cortex 6 h to 28 days after ET-1 induced stroke (F–J). Increased angiogenesis occurs in areas where there is greatest neuronal loss. Merged immunofluorescent images of vWF labelled blood vessels (red) with markers for microglia (OX42, green) (K–O); and astrocytes (GFAP, green) (P–T), 3, 7, 14 and 28 days post-stroke. Peak microglia activation occurs between 3 and 7 days whilst activated astrocytes are observed 3 days after stroke surrounding the damaged territory with intense staining detected by 14 days (S) that infiltrates into the core by 28 days (T). Scale bar = 100 μM.
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brainsci-03-00294-f003: Angiogenesis and lesion pathology following ET-1 induced stroke. Hematoxylin and Eosin (H & E) stained images of the infarcted cortex between 6 h and 28 days post-stroke (A–E) were used to identify areas of interest for vWF, NeuN and OX42 histological analysis (as marked by frames). Merged immunofluorescent images of NeuN labeled neurons (red) and vWF labeled blood vessels (green) throughout the stroke affected cortex 6 h to 28 days after ET-1 induced stroke (F–J). Increased angiogenesis occurs in areas where there is greatest neuronal loss. Merged immunofluorescent images of vWF labelled blood vessels (red) with markers for microglia (OX42, green) (K–O); and astrocytes (GFAP, green) (P–T), 3, 7, 14 and 28 days post-stroke. Peak microglia activation occurs between 3 and 7 days whilst activated astrocytes are observed 3 days after stroke surrounding the damaged territory with intense staining detected by 14 days (S) that infiltrates into the core by 28 days (T). Scale bar = 100 μM.

Mentions: Immunohistochemical co-localisation of neuronal marker NeuN and vWF revealed increased staining of new blood vessels in regions where there was greatest neuronal loss throughout the stroke affected brain. Increased blood vessels numbers in regions of the damaged cortex between 7 and 28 days post-stroke were associated with sustained neuronal loss over this time (Figure 3A–E). Analysis of inflammatory cell reactivity during the 28 day recovery period showed increased microglia/macrophage activation, evidenced by increased immunoreactivity to OX-42, across the infarcted cortex and striatum between 6 h and 7 days after stroke in regions also associated with increased vWF staining (Figure 3F–H). This effect appeared to subside by 14 and 28 days particularly in regions where peak angiogenesis was clearly evident (Figure 3I,J). Increased reactivity to the astrocytic marker GFAP occurred between 6 h and 3 days after stroke in the border zone to the infarct (Figure 3K,L), with intense staining detected by 14 days which extended into the core infarct region after this time. Although the temporal pattern for GFAP and vWF staining was similar, greatest levels of vWF staining occurred within the core infarct whereas increased GFAP immunoreactivity was mostly localised to the border zone (Figure 3N,O).


NADPH oxidase and angiogenesis following endothelin-1 induced stroke in rats: role for nox2 in brain repair.

Taylor CJ, Weston RM, Dusting GJ, Roulston CL - Brain Sci (2013)

Angiogenesis and lesion pathology following ET-1 induced stroke. Hematoxylin and Eosin (H & E) stained images of the infarcted cortex between 6 h and 28 days post-stroke (A–E) were used to identify areas of interest for vWF, NeuN and OX42 histological analysis (as marked by frames). Merged immunofluorescent images of NeuN labeled neurons (red) and vWF labeled blood vessels (green) throughout the stroke affected cortex 6 h to 28 days after ET-1 induced stroke (F–J). Increased angiogenesis occurs in areas where there is greatest neuronal loss. Merged immunofluorescent images of vWF labelled blood vessels (red) with markers for microglia (OX42, green) (K–O); and astrocytes (GFAP, green) (P–T), 3, 7, 14 and 28 days post-stroke. Peak microglia activation occurs between 3 and 7 days whilst activated astrocytes are observed 3 days after stroke surrounding the damaged territory with intense staining detected by 14 days (S) that infiltrates into the core by 28 days (T). Scale bar = 100 μM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061826&req=5

brainsci-03-00294-f003: Angiogenesis and lesion pathology following ET-1 induced stroke. Hematoxylin and Eosin (H & E) stained images of the infarcted cortex between 6 h and 28 days post-stroke (A–E) were used to identify areas of interest for vWF, NeuN and OX42 histological analysis (as marked by frames). Merged immunofluorescent images of NeuN labeled neurons (red) and vWF labeled blood vessels (green) throughout the stroke affected cortex 6 h to 28 days after ET-1 induced stroke (F–J). Increased angiogenesis occurs in areas where there is greatest neuronal loss. Merged immunofluorescent images of vWF labelled blood vessels (red) with markers for microglia (OX42, green) (K–O); and astrocytes (GFAP, green) (P–T), 3, 7, 14 and 28 days post-stroke. Peak microglia activation occurs between 3 and 7 days whilst activated astrocytes are observed 3 days after stroke surrounding the damaged territory with intense staining detected by 14 days (S) that infiltrates into the core by 28 days (T). Scale bar = 100 μM.
Mentions: Immunohistochemical co-localisation of neuronal marker NeuN and vWF revealed increased staining of new blood vessels in regions where there was greatest neuronal loss throughout the stroke affected brain. Increased blood vessels numbers in regions of the damaged cortex between 7 and 28 days post-stroke were associated with sustained neuronal loss over this time (Figure 3A–E). Analysis of inflammatory cell reactivity during the 28 day recovery period showed increased microglia/macrophage activation, evidenced by increased immunoreactivity to OX-42, across the infarcted cortex and striatum between 6 h and 7 days after stroke in regions also associated with increased vWF staining (Figure 3F–H). This effect appeared to subside by 14 and 28 days particularly in regions where peak angiogenesis was clearly evident (Figure 3I,J). Increased reactivity to the astrocytic marker GFAP occurred between 6 h and 3 days after stroke in the border zone to the infarct (Figure 3K,L), with intense staining detected by 14 days which extended into the core infarct region after this time. Although the temporal pattern for GFAP and vWF staining was similar, greatest levels of vWF staining occurred within the core infarct whereas increased GFAP immunoreactivity was mostly localised to the border zone (Figure 3N,O).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Stroke Injury and Repair Team, O'Brien Institute, 42 Fitzroy St, Fitzroy, Melbourne, Victoria 3065, Australia. cj.taylor@unimelb.edu.au.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus