A time course of NADPH-oxidase up-regulation and endothelial nitric oxide synthase activation in the hippocampus following neurotrauma

Abstract

Nicotinamide adenine dinucleotide phosphate oxidase (NADPH-oxidase; NOX) is a complex enzyme responsible for increased levels of reactive oxygen species (ROS), superoxide (O2.−). NOX derived O2.− is a key player in oxidative stress and inflammation mediated multiple secondary injury cascades (SIC) following traumatic brain injury (TBI). The O2.− reacts with nitric oxide (NO), produces various reactive nitrogen species (RNS), and contributes to apoptotic cell death. Following a unilateral cortical contusion, young adult rats were killed at various times post injury (1, 3, 6, 12, 24, 48, 72, and 96 h). Fresh tissue from the hippocampus was analyzed for NOX activity, and level of O2.−. In addition we evaluated the translocation of cytosolic NOX proteins (p67Phox, p47Phox and p40Phox) to the membrane, along with total NO and the activation (phosphorylation) of endothelial nitric oxide synthase (p-eNOS). Results show that both enzymes and levels of O2.− and NO have time dependent injury effects in the hippocampus. Translocation of cytosolic NOX proteins into membrane, NOX activity and O2.− were also increased in a time dependent fashion. Both, NOX activity and O2.− were increased at 6 h. Levels of p-eNOS increased within 1 h, with significant elevation of NO at 12 h post TBI. Levels of NO failed to show a significant association with p-eNOS, but did associate with O2.−. NOX up-regulation strongly associated with both the levels of O2.− and also total NO. The initial 12 hours post TBI are very important as a possible window of opportunity to interrupt SIC. It may be important to selectively target the translocation of cytosolic subunits for the modulation of NOX function.

Comprehensive Profiling of Modulation of Nitric Oxide Levels and Mitochondrial Activity in the Injured Brain: An Experimental Study Based on the Fluid Percussion Injury Model in Rats

“…These findings might thereby not account for the intracellular NO levels within different brain regions. Nevertheless, our findings were supported by studies reporting postinjury increases in NO metabolites, 16,46,47 iNOS activity 17,48,49 or iNOS expression. [50][51][52] Correspondingly, we also observed severity-independent increases in iNOS, but not nNOS mRNA expression at 4 hours in cortex and hippocampus in ipsilateral brain regions.…”

Temporal profile of M1 and M2 responses in the hippocampus following early 24 h of neurotrauma

“…Our previous studies also showed that oxidative stress in the cerebral tissue occurs as early as 3 h post TBI [4][5][6]. The superoxide producing enzyme (NADPH-oxidase), a known contributor of inflammation and oxidative-stress mediated neurodegeneration [21,64] is up-regulated within 6 h after neurotrauma [7]. The pro-inflammatory cytokine's up-regulation at 48 h [71] and significant loss of synaptic components at 96 h [6] support that "time-post-injury" is an important factor in developing therapeutic strategies after TBI.…”

“…Surgical procedure and cortical contusions were made under isoflurane anesthesia (2%) as previously described [7]. Rats were subjected to a unilateral moderate controlled cortical impact (CCI) of 2.0 mm depth at 3.5 m/s and 500 ms dwell time using the TBI 0310, a pneumatic impacting device (Precision Systems & Instrumentation, Fairfax Station, VA) with a hard stop Bimba cylinder (Bimba Manufacturing, Monee, IL).…”

“…Following a mechanical trauma, delayed pathology involves multiple secondary injury cascades/mechanisms (SIC) that include altered cell signal transduction and cytoskeletal damage, resulting in neuronal dysfunction or death. Various cellular and molecular changes play a dynamic role in these cascades including ionic imbalance [77], mitochondrial dysfunction [83], oxidative stress [4,7], excitotoxicity [15,77], and inflammation [25,51,71,76]. (See Table 1.…”

“…) TBI causes a complex series of inflammatory responses with the expression and release of several cytokine/chemokines [25,34,41,65,76] and the production of free radicals [7,16,51]. Our previous studies have demonstrated that the pro-inflammatory cytokines TNFα, IL-1β, and IL-6 [71] and oxidative stress are significantly increased in the cerebral cortex after TBI [4,5,7,71]. Together, enhanced free radicals, cytokine/chemokine expression, and excitotoxicity increase the complexity of SIC following TBI.…”