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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

Nox4 immunohistochemistry in the ipsilateral cortex 28 days after stroke. Immunofluorescent images of rabbit monoclonal Nox4 antibody (Epitomics) in the contralateral (A) and ipsilateral (B) (Stroke affected) cortex 28 days post-stroke. Adjacent immunofluorescent images of control rabbit IgG serum in the contralateral (C) and ipsilateral (D) cortex reveal a high degree of non-specific immunoreactivity to blood vessels and neurons alike. Scale bar = 100 μm.
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brainsci-03-00294-f008: Nox4 immunohistochemistry in the ipsilateral cortex 28 days after stroke. Immunofluorescent images of rabbit monoclonal Nox4 antibody (Epitomics) in the contralateral (A) and ipsilateral (B) (Stroke affected) cortex 28 days post-stroke. Adjacent immunofluorescent images of control rabbit IgG serum in the contralateral (C) and ipsilateral (D) cortex reveal a high degree of non-specific immunoreactivity to blood vessels and neurons alike. Scale bar = 100 μm.

Mentions: Immunoreactivity to a Nox4 primary antibody was also attempted. Adjacent sections were fixed in 4% PFA for 10 min prior to pre-block for 1 h in DAKO universal Blocking solution. Tissue sections were then washed in phosphate buffered saline (2 × 5 min PBS, 0.1 M, pH 7.4) and transferred for overnight incubation at 4 °C with rabbit monoclonal Nox4 antibody (1:200; Epitomics, Burlingame, CA, USA) in a mixture of PBS (0.1 M, pH 7.4) containing 2% NGS and 0.3% Triton-X. Sections were again washed (2 × 5 min PBS) and transferred for incubation with the secondary antibody Alexa 488 goat anti-rabbit (1:500) containing 2% NGS and 0.3% triton-X for 2.5 h at room temperature. Tissue sections were then washed in PBS (0.1 M, pH 7.4; 2 × 5min) and cover-slipped using fluorescent mounting medium (Dako Cytomation, Carpinteria, CA, USA). Analogous experiments were also conducted with rabbit IgG control serum in place of the primary antibody. Resulting sections were examined with a fluorescence microscope as described above. Unfortunately all attempts to localise Nox4 immunoreactivity using a specific Nox4 antibody were unsuccessful with sections incubated in the presence of the primary antibody revealing a similar pattern of distribution as sections exposed to IgG control serum in the absence of the primary antibody (Figure 8).


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)

Nox4 immunohistochemistry in the ipsilateral cortex 28 days after stroke. Immunofluorescent images of rabbit monoclonal Nox4 antibody (Epitomics) in the contralateral (A) and ipsilateral (B) (Stroke affected) cortex 28 days post-stroke. Adjacent immunofluorescent images of control rabbit IgG serum in the contralateral (C) and ipsilateral (D) cortex reveal a high degree of non-specific immunoreactivity to blood vessels and neurons alike. 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-f008: Nox4 immunohistochemistry in the ipsilateral cortex 28 days after stroke. Immunofluorescent images of rabbit monoclonal Nox4 antibody (Epitomics) in the contralateral (A) and ipsilateral (B) (Stroke affected) cortex 28 days post-stroke. Adjacent immunofluorescent images of control rabbit IgG serum in the contralateral (C) and ipsilateral (D) cortex reveal a high degree of non-specific immunoreactivity to blood vessels and neurons alike. Scale bar = 100 μm.
Mentions: Immunoreactivity to a Nox4 primary antibody was also attempted. Adjacent sections were fixed in 4% PFA for 10 min prior to pre-block for 1 h in DAKO universal Blocking solution. Tissue sections were then washed in phosphate buffered saline (2 × 5 min PBS, 0.1 M, pH 7.4) and transferred for overnight incubation at 4 °C with rabbit monoclonal Nox4 antibody (1:200; Epitomics, Burlingame, CA, USA) in a mixture of PBS (0.1 M, pH 7.4) containing 2% NGS and 0.3% Triton-X. Sections were again washed (2 × 5 min PBS) and transferred for incubation with the secondary antibody Alexa 488 goat anti-rabbit (1:500) containing 2% NGS and 0.3% triton-X for 2.5 h at room temperature. Tissue sections were then washed in PBS (0.1 M, pH 7.4; 2 × 5min) and cover-slipped using fluorescent mounting medium (Dako Cytomation, Carpinteria, CA, USA). Analogous experiments were also conducted with rabbit IgG control serum in place of the primary antibody. Resulting sections were examined with a fluorescence microscope as described above. Unfortunately all attempts to localise Nox4 immunoreactivity using a specific Nox4 antibody were unsuccessful with sections incubated in the presence of the primary antibody revealing a similar pattern of distribution as sections exposed to IgG control serum in the absence of the primary antibody (Figure 8).

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