Enhanced Oxidative Stress in iNOS-Deficient Mice after Traumatic Brain Injury: Support for a Neuroprotective Role of iNOS

Abstract

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.

Postischemic hyperoxia reduces hippocampal pyruvate dehydrogenase activity

“…More importantly, the exacerbation of tyrosine nitration during early reperfusion by hyperoxic resuscitation is associated with worse neurologic outcome [15] and greater hippocampal cell death compared to that observed after normoxic resuscitation [14]. These relationships add to the body of evidence that protein tyrosine nitration contributes to a wide range of neuropathologies, including traumatic brain injury [30,31], focal ischemia [32], global ischemia [33,34], and neurodegenerative diseases [35,36].…”

“…More importantly, the exacerbation of tyrosine nitration during early reperfusion by hyperoxic resuscitation is associated with worse neurologic outcome [15] and greater hippocampal cell death compared to that observed after normoxic resuscitation [14]. These relationships add to the body of evidence that protein tyrosine nitration contributes to a wide range of neuropathologies, including traumatic brain injury [30,31], focal ischemia [32], global ischemia [33,34], and neurodegenerative diseases [35,36].The chemical basis for increased tyrosine nitration in the hippocampus after hyperoxic resuscitation is most likely linked to the stimulation of peroxynitrite formation by increased production of superoxide and (or) nitric oxide, the two substrates required for peroxynitrite formation. Whereas prolonged hyperoxia can stimulate the expression of inducible nitric oxide synthase [37], increased levels of this protein are not likely to occur within the 2-h reperfusion period used in our measurements.…”

A New Chiral Pyrrolyl α-Nitronyl Nitroxide Radical Attenuates β-Amyloid Deposition and Rescues Memory Deficits in a Mouse Model of Alzheimer Disease

“…3-Nitrotyrosine (3-NT) measurements were performed in the supernatants as described [31]. Samples and standards were incubated in microtiter wells coated with antibodies recognizing protein-bound and free 3-NT (Cell Sciences, Norwood, MA).…”

Tissue Damage and Oxidant/Antioxidant Balance

“…The levels of free oxygen radicals increase and the levels of antioxidants decrease depending on the level of consumption of antioxidants in tissues affected by traumatic brain damage. Increasing levels of free oxygen radicals due to the consumption of antioxidants cause lipid, protein and DNA oxidation [6][7][8]. An oxidant/antioxidant imbalance in favor of oxidants has also been reported in the context of brain damage caused by radiation [9].…”