Neuroprotection from retinal ischemia/reperfusion injury by NOX2 NADPH oxidase deletion

Harumasa Yokota, Subhadra Priyadarshini Narayanan, Wenbo Zhang, Hua Liu, Modesto Antonio Rojas, Zhimin Xu, Tahira Lemtalsi, Taiji Nagaoka, Akitoshi Yoshida, Steven E. Brooks, Robert William Caldwell, Ruth B Caldwell

Research output: Contribution to journalArticle

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Abstract

Purpose. The aim of this study was to determine whether NOX2, one of the homologs of NADPH oxidase, plays a role in neuronal cell death during retinal ischemia. Methods. Ischemia reperfusion (I/R) injury was generated in C57/BL6 and NOX2 -/- mice by increasing the intraocular pressure (IOP) to 110 mm Hg for 40 minutes followed by reperfusion. Quantitative PCR and Western blot analysis were performed to measure NOX2 expression. Reactive oxygen species (ROS) formation was assessed by dihydroethidium imaging of superoxide formation and Western blot analysis for tyrosine nitration. TUNEL assay was performed to determine cell death at 3 days after I/R. Survival of neurons within the ganglion cell layer (GCL) was assessed at 7 days after I/R by confocal morphometric imaging of retinal wholemounts immunostained with NeuN antibody. Activation of mitogen-activated protein kinases and nuclear factor κB (NF-κβ) was measured by Western blot analysis. Results. NOX2 mRNA and protein and ROS were significantly increased in wild-type I/R retinas. This effect was associated with a 60% decrease in the number of GCL neurons and a 10-fold increase in TUNEL-positive cells compared with the fellow sham control eyes. Phosphorylation of ERK and NF-κB was significantly increased in wild-type I/R retinas. Each of these effects was markedly attenuated in the NOX2 -/- retina (P < 0.01). Conclusions. These data demonstrate that the deletion of NOX2 can reduce I/R-induced cell death and preserve retinal GCL neurons after I/R injury. The neuronal cell injury caused by I/R is associated with the activation of ERK and NF-κB signaling mechanisms.

Original languageEnglish (US)
Pages (from-to)8123-8131
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume52
Issue number11
DOIs
StatePublished - Oct 1 2011

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NADPH Oxidase
Reperfusion Injury
Reperfusion
Ischemia
Retina
Cell Death
Western Blotting
In Situ Nick-End Labeling
Neurons
Ganglia
Reactive Oxygen Species
Retinal Ganglion Cells
Mitogen-Activated Protein Kinases
Intraocular Pressure
Superoxides
Tyrosine
Cell Count
Phosphorylation
Neuroprotection
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Neuroprotection from retinal ischemia/reperfusion injury by NOX2 NADPH oxidase deletion. / Yokota, Harumasa; Narayanan, Subhadra Priyadarshini; Zhang, Wenbo; Liu, Hua; Rojas, Modesto Antonio; Xu, Zhimin; Lemtalsi, Tahira; Nagaoka, Taiji; Yoshida, Akitoshi; Brooks, Steven E.; Caldwell, Robert William; Caldwell, Ruth B.

In: Investigative Ophthalmology and Visual Science, Vol. 52, No. 11, 01.10.2011, p. 8123-8131.

Research output: Contribution to journalArticle

Yokota, H, Narayanan, SP, Zhang, W, Liu, H, Rojas, MA, Xu, Z, Lemtalsi, T, Nagaoka, T, Yoshida, A, Brooks, SE, Caldwell, RW & Caldwell, RB 2011, 'Neuroprotection from retinal ischemia/reperfusion injury by NOX2 NADPH oxidase deletion', Investigative Ophthalmology and Visual Science, vol. 52, no. 11, pp. 8123-8131. https://doi.org/10.1167/iovs.11-8318
Yokota, Harumasa ; Narayanan, Subhadra Priyadarshini ; Zhang, Wenbo ; Liu, Hua ; Rojas, Modesto Antonio ; Xu, Zhimin ; Lemtalsi, Tahira ; Nagaoka, Taiji ; Yoshida, Akitoshi ; Brooks, Steven E. ; Caldwell, Robert William ; Caldwell, Ruth B. / Neuroprotection from retinal ischemia/reperfusion injury by NOX2 NADPH oxidase deletion. In: Investigative Ophthalmology and Visual Science. 2011 ; Vol. 52, No. 11. pp. 8123-8131.
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abstract = "Purpose. The aim of this study was to determine whether NOX2, one of the homologs of NADPH oxidase, plays a role in neuronal cell death during retinal ischemia. Methods. Ischemia reperfusion (I/R) injury was generated in C57/BL6 and NOX2 -/- mice by increasing the intraocular pressure (IOP) to 110 mm Hg for 40 minutes followed by reperfusion. Quantitative PCR and Western blot analysis were performed to measure NOX2 expression. Reactive oxygen species (ROS) formation was assessed by dihydroethidium imaging of superoxide formation and Western blot analysis for tyrosine nitration. TUNEL assay was performed to determine cell death at 3 days after I/R. Survival of neurons within the ganglion cell layer (GCL) was assessed at 7 days after I/R by confocal morphometric imaging of retinal wholemounts immunostained with NeuN antibody. Activation of mitogen-activated protein kinases and nuclear factor κB (NF-κβ) was measured by Western blot analysis. Results. NOX2 mRNA and protein and ROS were significantly increased in wild-type I/R retinas. This effect was associated with a 60{\%} decrease in the number of GCL neurons and a 10-fold increase in TUNEL-positive cells compared with the fellow sham control eyes. Phosphorylation of ERK and NF-κB was significantly increased in wild-type I/R retinas. Each of these effects was markedly attenuated in the NOX2 -/- retina (P < 0.01). Conclusions. These data demonstrate that the deletion of NOX2 can reduce I/R-induced cell death and preserve retinal GCL neurons after I/R injury. The neuronal cell injury caused by I/R is associated with the activation of ERK and NF-κB signaling mechanisms.",
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AU - Yokota, Harumasa

AU - Narayanan, Subhadra Priyadarshini

AU - Zhang, Wenbo

AU - Liu, Hua

AU - Rojas, Modesto Antonio

AU - Xu, Zhimin

AU - Lemtalsi, Tahira

AU - Nagaoka, Taiji

AU - Yoshida, Akitoshi

AU - Brooks, Steven E.

AU - Caldwell, Robert William

AU - Caldwell, Ruth B

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N2 - Purpose. The aim of this study was to determine whether NOX2, one of the homologs of NADPH oxidase, plays a role in neuronal cell death during retinal ischemia. Methods. Ischemia reperfusion (I/R) injury was generated in C57/BL6 and NOX2 -/- mice by increasing the intraocular pressure (IOP) to 110 mm Hg for 40 minutes followed by reperfusion. Quantitative PCR and Western blot analysis were performed to measure NOX2 expression. Reactive oxygen species (ROS) formation was assessed by dihydroethidium imaging of superoxide formation and Western blot analysis for tyrosine nitration. TUNEL assay was performed to determine cell death at 3 days after I/R. Survival of neurons within the ganglion cell layer (GCL) was assessed at 7 days after I/R by confocal morphometric imaging of retinal wholemounts immunostained with NeuN antibody. Activation of mitogen-activated protein kinases and nuclear factor κB (NF-κβ) was measured by Western blot analysis. Results. NOX2 mRNA and protein and ROS were significantly increased in wild-type I/R retinas. This effect was associated with a 60% decrease in the number of GCL neurons and a 10-fold increase in TUNEL-positive cells compared with the fellow sham control eyes. Phosphorylation of ERK and NF-κB was significantly increased in wild-type I/R retinas. Each of these effects was markedly attenuated in the NOX2 -/- retina (P < 0.01). Conclusions. These data demonstrate that the deletion of NOX2 can reduce I/R-induced cell death and preserve retinal GCL neurons after I/R injury. The neuronal cell injury caused by I/R is associated with the activation of ERK and NF-κB signaling mechanisms.

AB - Purpose. The aim of this study was to determine whether NOX2, one of the homologs of NADPH oxidase, plays a role in neuronal cell death during retinal ischemia. Methods. Ischemia reperfusion (I/R) injury was generated in C57/BL6 and NOX2 -/- mice by increasing the intraocular pressure (IOP) to 110 mm Hg for 40 minutes followed by reperfusion. Quantitative PCR and Western blot analysis were performed to measure NOX2 expression. Reactive oxygen species (ROS) formation was assessed by dihydroethidium imaging of superoxide formation and Western blot analysis for tyrosine nitration. TUNEL assay was performed to determine cell death at 3 days after I/R. Survival of neurons within the ganglion cell layer (GCL) was assessed at 7 days after I/R by confocal morphometric imaging of retinal wholemounts immunostained with NeuN antibody. Activation of mitogen-activated protein kinases and nuclear factor κB (NF-κβ) was measured by Western blot analysis. Results. NOX2 mRNA and protein and ROS were significantly increased in wild-type I/R retinas. This effect was associated with a 60% decrease in the number of GCL neurons and a 10-fold increase in TUNEL-positive cells compared with the fellow sham control eyes. Phosphorylation of ERK and NF-κB was significantly increased in wild-type I/R retinas. Each of these effects was markedly attenuated in the NOX2 -/- retina (P < 0.01). Conclusions. These data demonstrate that the deletion of NOX2 can reduce I/R-induced cell death and preserve retinal GCL neurons after I/R injury. The neuronal cell injury caused by I/R is associated with the activation of ERK and NF-κB signaling mechanisms.

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