Hydrogen and N-acetyl-L-cysteine rescue oxidative stress-induced angiogenesis in a mouse corneal alkali-burn model.
OBJECTIVE
To investigate the role of reactive oxygen species (ROS) as the prime initiators of the angiogenic response after alkali injury of the cornea and observe the effects of antioxidants in preventing angiogenesis.


METHODS
The corneal epithelia of SOD-1-deficient mice or wild-type (WT) mice were removed after application of 0.15 N NaOH to establish the animal model of alkali burn. ROS production was semiquantitatively measured by dihydroethidium (DHE) fluorescence. Angiogenesis was visualized by CD31 immunohistochemistry. The effects of the specific NF-κB inhibitor DHMEQ, the antioxidant N-acetyl-L-cysteine (NAC), and hydrogen (H2) solution were observed.


RESULTS
ROS production in the cornea was enhanced immediately after alkali injury, as shown by increased DHE fluorescence (P<0.01). NF-κB activation and the upregulation of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) were significantly enhanced (P<0.01), leading to a significantly larger area of angiogenesis. Angiogenesis in SOD-1-/- mice corneas were significantly higher in WT mice (P<0.01), confirming the role of ROS. Pretreatment with the specific NF-κB inhibitor DHMEQ or the antioxidant NAC significantly reduced corneal angiogenesis by downregulating the NF-κB pathway (P<0.01) in both WT and SOD-1-/- mice. Furthermore, we showed that irrigation of the cornea with hydrogen (H2) solution significantly reduced angiogenesis after alkali-burn injury (P<0.01).


CONCLUSIONS
Immediate antioxidant therapy with H2-enriched irrigation solution is a new potent treatment of angiogenesis in cornea to prevent blindness caused by alkali burn.
Kubota
Miyuki
M
Department of Ophthalmology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan.
Shimmura
Shigeto
S
Kubota
Shunsuke
S
Miyashita
Hideyuki
H
Kato
Naoko
N
Noda
Kousuke
K
Ozawa
Yoko
Y
Usui
Tomohiko
T
Ishida
Susumu
S
Umezawa
Kazuo
K
Kurihara
Toshihide
T
Tsubota
Kazuo
K
D000111:Acetylcysteine / Q000008:administration & dosage / Q000627:therapeutic use*; D000818:Animals; D000975:Antioxidants / Q000008:administration & dosage / Q000627:therapeutic use*; D001549:Benzamides / Q000494:pharmacology; D001766:Blindness / Q000517:prevention & control; D002057:Burns, Chemical / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D018932:Chemokine CCL2 / Q000378:metabolism; D016510:Corneal Neovascularization / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D003512:Cyclohexanones / Q000494:pharmacology; D017666:Deuterium Oxide / Q000008:administration & dosage / Q000627:therapeutic use*; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005126:Eye Burns / Q000139:chemically induced*; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012972:Sodium Hydroxide / Q000633:toxicity; D013482:Superoxide Dismutase / Q000235:genetics; D000072105:Superoxide Dismutase-1; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D000975:Antioxidants; D001549:Benzamides; C491768:Ccl2 protein, mouse; D018932:Chemokine CCL2; D003512:Cyclohexanones; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D042461:Vascular Endothelial Growth Factor A; C464444:dehydroxymethylepoxyquinomicin; C467480:vascular endothelial growth factor A, mouse; D012972:Sodium Hydroxide; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D017666:Deuterium Oxide; D000111:Acetylcysteine