Enhanced oxidative stress in iNOS-deficient mice after traumatic brain injury: support for a neuroprotective role of iNOS.
Studies in experimental traumatic brain injury (TBI) suggest both deleterious and protective effects of inducible nitric oxide synthase (iNOS). Early after injury, iNOS may be detrimental via formation of peroxynitrite and iNOS inhibitors are protective. In contrast, we reported impaired long-term functional outcome after TBI in iNOS knockout (ko) versus wild-type (wt) mice. To elucidate potential neuroprotective and neurotoxic mechanisms for iNOS, we studied nitric oxide formation by electron paramagnetic resonance (EPR) spectroscopy using diethyldithiocarbamate-iron (DETC-Fe) as a spin trap and markers of nitrosative (S-nitrosothiol (RSNO, Fluorescent assay); nitrotyrosine (3NT, ELISA)) and oxidative stress (ascorbate, HPLC) at 72 h after controlled cortical impact (CCI) in iNOS ko and wt and in uninjured iNOS ko and wt mice. 3NT immunostaining with macrophage and myeloperoxidase (MPO) dual labeling was also assessed in brain sections. Brain DETC-Fe-NO low-temperature EPR signal intensity was approximately 2-fold greater in wt versus iNOS ko at 72 h after CCI. Ascorbate levels decreased in injured hemisphere in wt and iNOS ko versus uninjured -this decrease was more pronounced in iNOS ko. In wt mice, RSNO and 3NT levels were increased after CCI versus uninjured (50% and 400%, respectively, P < 0.05). RSNO levels were not increased in iNOS ko after CCI. Nitrotyrosine levels increased after CCI in wt and ko versus respective uninjured -this increase was more pronounced in wt (2.34 +/- 0.95 versus 1.27 +/- 0.49 pmol/mg protein, P < 0.05). Increased 3NT immunoreactivity was detected in wt versus iNOS ko at 72 h after CCI, and colocalized with macrophage marker and MPO. Our data support a role for iNOS-derived NO as an endogenous antioxidant after CCI. iNOS also contributes protein nitrosylation and nitration. Colocalization of 3NT with macrophages and MPO suggests generation of nitrating agents by macrophages and/or phagocytosis of nitrated proteins.
Bayir
Hülya
H
Safar Center for Resuscitation Research, University of Pittsburgh Medical Center, Pennslyvania 15260, USA.
Kagan
Valerian E
VE
Borisenko
Grigory G
GG
Tyurina
Yulia Y
YY
Janesko
Keri L
KL
Vagni
Vincent A
VA
Billiar
Timothy R
TR
Williams
Deborah L
DL
Kochanek
Patrick M
PM
D000818:Animals; D001921:Brain / Q000201:enzymology / Q000473:pathology; D001930:Brain Injuries / Q000378:metabolism* / Q000473:pathology; D004578:Electron Spin Resonance Spectroscopy; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D018384:Oxidative Stress / Q000502:physiology*; D009195:Peroxidase / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D009195:Peroxidase; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse