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 (p67^Phox, p47^Phox and p40^Phox) 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.

Introduction

Traumatic brain injury (TBI) is associated with costly health problems and mortality in the population of all age. Millions of individuals suffer TBI each year resulting in long term disabilities, with many requiring hospitalization [1]. A majority of TBI survivors have chronic neurobehavioral problems including cognitive deficits. Considering that the hippocampus is an important brain region involved in learning and memory, it is important to investigate its involvement after TBI. Following a contusion or concussion, multiple secondary injury cascades/mechanisms (SIC) initiate delayed pathology that includes cytoskeletal damage and altered cell signal transduction [2,3] resulting in neuronal dysfunction, and/or death. Various cellular and molecular changes play a key role in these cascades and include ionic imbalance [4], mitochondrial dysfunction [5-8], excitotoxicity [9], oxidative stress [10-12], and inflammation [13-16].

Oxidative stress, the imbalance between the level of free radicals and antioxidants, consists an increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and decreased antioxidant defense. These ROS/RNS have been linked with the pathogenesis after TBI [10-12,17]. High content of poly-unsaturated fatty acids in the brain and reduced levels of antioxidants after neurotrauma [11,12,15] makes it more vulnerable to free radical attacks. Increased ROS/RNS causes oxidative/nitrosative damage [18] in lipids, proteins, and nucleic acids [11,12,19] are believed to initiate neurodegenerative processes in the brain [20,21].

Several studies have shown that NADPH-oxidase (NOX) is up-regulated following TBI [22-25] and coincides with the excessive production of superoxide (O₂^.−) [26-31]. Increased ROS in the brain following TBI appears to be linked to NOX up-regulation. Increased O₂^.− can intensify oxidative stress, participate in the inflammation [28], and increase further ROS/RNS production [14,26,32,33]. Elevated ROS/RNS can cause mitochondrial dysfunction and ultimately neuronal degeneration [34-36]. The ROS producing enzyme, NOX, is expressed in the various different types of cells, i.e., phagocytic cells, endothelial cells, vascular smooth muscles, and fibroblasts [37,38]. A growing body of evidence suggests that the NOX is expressed in microglia [39-43] and neurons [44,45], and plays a key role in CNS pathophysiology [46-49]. NOX is a multi-subunit enzyme composed of cytosolic (p-67^Phox, p-47^Phox, and p-40^Phox) and membrane (p-91^Phox, and p-22^Phox) [50] subunits coupled with the GTPase protein Rac1 [51-53]. NOX is up-regulated with the translocation/interaction of its cytosolic proteins to the membrane subunit [29,31,50,54] and subsequently participates in various pathophysiological mechanisms [48,50,54-58]. Recent studies have demonstrated time dependent changes in NOX function after TBI [25]. NOX derived ROS after brain injury has been linked to the neuronal death and its inhibition has been shown to be neuroprotective [22,24,59,60]. Direct or indirect inhibition of NOX function appears to be neuroprotective after brain injury regardless if the inhibitor/modulator is administered pre- or post-injury [23-25,40].

A form of RNS, nitric oxide (NO), production is also increased following TBI [2,61-64] and participates in neurodegenerative cascades [2,27,65]. NO can be derived from different NO-synthase among which is endothelial NOS (eNOS) [66]. Although, expression of eNOS has been shown to be increased after TBI [61,62], it is unclear whether eNOS up-regulation following head trauma contributes to oxidative stress via increasing NO. Several studies have implicated eNOS in the variety of physiological functions [67-69], and it’s up-regulation might be beneficial after TBI [61,63]. The activation of eNOS by phosphorylation (p-eNOS) has been linked to several cell signaling processes [64,70]. The bioavailability of eNOS derived NO depends on the rate of reaction with O₂⁻ (NO + O₂^.− → ONOO⁻) that may be linked to NOX up-regulation. Most researchers adhere to the idea that modulation of NOX function can suppress ROS/RNS formation [22-24,30,59,60] and reduces pathology after head trauma [22,24,60].

Characterizing the time course of early changes in mechanisms responsible for both ROS/RNS, will help determine the window of opportunity for maximizing a more favorable outcome following TBI. This study evaluated possible time-dependent changes in NOX activity with including translocation of their cytosolic subunits and production of O₂^.−. The analysis also focused on the time course of NO production and its correlation with eNOS activation.

Materials and Methods

Chemicals

Mouse monoclonal anti β-actin (sc-47778), p-47^Phox (sc-17845), p-40^Phox (sc-48376), p-eNOS (sc-136519), GAPDH (sc-365062), rabbit polyclonal anti p-67^Phox (sc-15342), and Na+/K+-ATPase (sc-28800) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alkaline phosphatase conjugated anti-mouse and anti-rabbit secondary antibodies, and all other chemicals/reagents were purchased from Sigma (St Louis, MO, USA). Materials, and chemicals used in electrophoresis were purchased from Bio-Rad (Hercules, CA, USA) unless stated otherwise.

Animals and surgical procedures

Adult male Sprague-Dawley rats (250-275g) (Harlan Laboratories, Indianapolis, IN) were used in this study. Animal protocol and procedures were approved by the Institutional Animal Care and Use Committee of the University of Kentucky. Animals were housed 2/cage on a 12 h light/dark cycle and provided access to food and water ad libitum. Rats were subjected to a moderate unilateral controlled cortical impact (CCI) utilizing an electronic controlled pneumatic impact device (ECPI) (TBI0310, Precision System & Instrumentation, Fairfax Station, VA) as previously described [10,12]. Each animal was anesthetized with 2% isofluorane and placed in a Kopf stereotaxic frame (Tujunga, CA) with the incisor bar set at −5. Body temperature of each rat were monitored and maintained at 36 °C with a heating pad. Following a midline incision and retraction of the skin, a 6-mm-diameter craniotomy was made approximately midway between bregma and lamda with a Michele hand trephine (Miltex, NY). The skull disk was removed without disturbing the dura membrane. The open brain was injured using the ECPI; as a 5-mm-diameter cortex compressed for a depth of 2.0 mm at 3.5 m/s. All the surgical procedure was completed in 15-20 min. Animals were divided into nine groups (n=6/group) and allowed to survive for 1, 3, 6, 12, 24, 48, 72 and 96 h post TBI for biochemical analysis. Sham animals (n=2/group) received the craniotomy without TBI and killed at 6, 12, and 48h. Since our previous studies failed to demonstrate a significant change in oxidative stress following sham injury [10,12], tissue from all three sham groups were combined for analysis.

Tissue processing

Animals were euthanized with Fatal-Plus (Med-Vet International, Mettawa, IL) and hippocampi isolated from both the ipsilateral and contralateral hemispheres. Tissue samples were immediately placed in dry ice and then stored at −80°C until used for analysis. Tissue was homogenized (10%; w/v) on ice in 0.1M, PBS (pH 7.4) containing protease inhibitor cocktail (Millipore Inc.) and centrifuged at 1000g for 10 min/4°C to remove the cell debris pellet (P1). Collected supernatants were further centrifuged at 15,000g for 10 min/4°C and then both the pellet-2 (P2) and post mitochondrial supernatant (PMS) was collected. PMS of all samples were used for enzymatic and non-enzymatic biochemical analyses. These assays were completed in 96-well plates and analyzed with a SpectraMax® micro-plate reader (Molecular Devices, Sunnyvale, CA). Total protein concentration was determined using the Pierce BCA method (Sigma, St. Louis, MO).

Estimation of NOX activity and its derived superoxide (O₂^.−)

NOX activity and it’s derived O₂^.− was quantified as previously described using lucigenin-enhanced chemiluminescence [50]. Reaction mixture containing HEPES buffer (pH 7.4), protease inhibitor cocktail, L-NAME (1 mmol/L), triethylenetetramine (1.0 mmol/L), SDS (100 μmol/L) lucigenin (20 μmol/L), and 20 μl PMS were aliquoted in 96-well plates and luminescence values were recorded. Blank values were subtracted from sample readings and chemiluminescence/mg protein of each sample was calculated as percent change versus sham operates. Chemiluminescence was also monitored in the presence of diphenyliodonium and quinacrine (100 μM and 1.0 mM), oxypurinol (100 μM), rotenone (100 μM), or indomethacin (10 μM) as a corresponding inhibitor of NOX, xanthine oxidase, mitochondrial respiration, and cyclooxygenase function for O₂^.− production.

NOX activity was evaluated in 96-well plates. In a dark area of work, plates were placed in the luminometer (SpectraMax®)) to calculate basal luminescence, then after addition of NADPH (0.2 mM), kinetic readings were taken for 15 min. Blank readings were subtracted from the PMS-added well’s results of each sample. The changes in chemiluminescence/min/mg protein in each sample were calculated for percent changes versus sham operated rats (% control). Samples were also analyzed in the presence of NOX inhibitor diphenyliodonium and quinacrine (100 μM and 1.0 mM, respectively) and readings were subtracted from the values in the absence of inhibitors. Subsequently, measured chemiluminescence was due to O₂^.− produced with NOX.

Estimation of NO in hippocampus

Assessment of total NO was completed as the analysis of total nitrite/nitrate present in samples according to the method described earlier [71]. In brief, 100 μl of nitrate standards (in serial dilution) and samples were added to 400 μl of carbonate buffer in test tubes, followed by a small amount of (~0.15 g) cadmium beads. The tubes were then incubated at room temperature for 1h with thorough shaking. The reaction was stopped by the addition of 100 μl of NaOH (0.35 M), and vortexed with the addition of 400 μl of ZnSO₄ (120 mM). The tubes were allowed to stand for 10 min and centrifuged at 4000g for 10 min. Clear supernatant (150 μl aliquots) was transferred into the 96 well plates in triplicate. Griess reagent (equal volumes of 1% naphthalene diamine dihydrochloride in distilled water and a mixture of 10% sulfanilamide and 50% concentrated H₃PO₄) (150 μl ) was added with gentle mixing. After 10 min, the absorbance was taken at 545nm against blank containing reaction mixture but no biological sample or nitrate standard. The absorbance values of each sample calculated first for the nmol-NO/mg protein and then results converted into percent change versus sham operates.

Western-blot analysis for eNOS and NOX proteins

NOX associated key proteins (p67^Phox, p47^Phox, and p-p40^Phox) and p-eNOS was analyzed as previously described using the Western-blot method [50,56]. In the present study, β-actin, GAPDH, and Na⁺/K⁺-ATPase were used as a corresponding standard protein markers of cytoskeleton, cytosol, and cell membrane. Each P2 fraction (mentioned above) was mixed with corresponding PMS and diluted 1:1 (v/v) in lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1% Triton X-100, 10% glycerol, and protease inhibitor cocktail). All the samples were centrifuged at 13,000g for 10 min at 4°C and then collected supernatants further centrifuged for 1h at 100,000g at 4°C. The obtained pellets and supernatants were designated as the membrane and cytosolic fraction [48]. Equal amounts of protein (50μg) were loaded with the appropriate marker (Bio-Rad) on a gradient gel (4-20% Tris-HCl), followed by transfer to a nitrocellulose membrane using a semi-dry transfer system (Bio-Rad) in transfer buffer (25 mM Tris, 150 mM glycine, 20% MeOH) for 2 h at 15 volt. NOX protein analyzing membranes were blocked with 5% milk, and p-eNOS assessing blots were blocked with 3% bovine serum albumin (BSA), in Tris/saline buffer-Tween (TBST). Primary antibodies of p-67^Phox, p-47^Phox, p-40^Phox, p-eNOS β-actin, GAPDH, and Na⁺/K⁺-ATPase were added at a concentration of 1:1000 and incubated overnight at 4°C. The blots were washed three times in TBST and incubated for 1 h with alkaline phosphatase conjugated secondary antibodies in a 1:8000 dilution. All the membranes were washed three times in TBST for 10 min and developed in Sigma Fast tablets (BCIP/NBT substrate). Blots were then dried, scanned with Adobe Photoshop, and quantified with Scion Image (PC version of Macintosh-compatible NIH Image).

Statistical analysis

Time-dependent changes in enzymatic and non-enzymatic oxidative/nitrosative markers, NOX proteins, and p-eNOS are reported as mean ± SD. The values of each variable normalized first with the results in contralateral sample (own control) and then compared with sham operated animals. Possible differences between group means were evaluated with a one-way ANOVA (Graph Pad Prizm) followed by Dunnett’s test. Association between both the enzyme’s function, O₂^.− and NO production, and translocation of the NOX subunits were examined using Spearman correlation. For significant differences, alpha was set at 0.05.

Results

Time course of NOX activity and O₂^.− levels

NOX produces O₂^.− radicals during oxidative metabolism of NADPH as a cell function. The time course analysis of NOX function revealed a significant time-dependent up-regulation in the hippocampus following TBI [F (8, 45) = 5.903, p < 0.0001] (Fig. 1A). The earliest significant NOX up-regulation was observed at 6 h (160%) after injury. This increased activation continued until at least 72h post TBI. At 96 h post injury, although NOX activity remained elevated it was no longer statistically different from sham operated animals.

Figure 1.

Time course for the activity of NADPH Oxidase (NOX) and levels of superoxide (O₂^.−) in the ipsilateral hippocampus following a moderate cortical contusion. (A) A significant elevation in the NOX activity occurred at 6 h after injury, with the maximum NOX activity observed at 24 h. Levels of NOX derived O₂^.− also demonstrated a time-dependent increase with significant changes within 6 h post trauma and maximum increases at 24 h post injury. (B) Each bar represents the group mean ± SD of six animals/group. ^*p < 0.05 versus sham operates.

The level of O₂^.− also demonstrated a significant time-dependent injury effect in the hippocampus [F (8, 45) = 5.698, p < 0.0001] (Fig. 1B). The amount of O₂^.− was significantly increased by 6 h post trauma (149%) and remained significantly elevated at 48h, with peak levels observed at 24 h (197%) post TBI. The contralateral hippocampus failed to demonstrate any significant (p > 0.1) change in either NOX function or levels of O₂^.−.

Time course of NOX subunits

The cytosolic NOX subunits p-67^Phox, p-47^Phox, and p-40^Phox were investigated in both the ipsilateral and contralateral hippocampus after a moderate TBI. Western-blot analysis of both the cytosolic and membrane fractions demonstrated a time-dependent translocation (Fig. 2A&B) of NOX subunits in the ipsilateral hippocampus. The contralateral hippocampus failed to demonstrate any significant change in any of these NOX proteins.

Figure 2.

Key cytosolic NOX subunit proteins were quantitatively assessed in the hippocampus with immunobloting followed by Western-blot. (A) The levels of cytosolic p67^Phox, p47^Phox, and p40^Phox were increased in membrane fractions and (B) declined in cytosolic fraction, demonstrating a substantial translocation of these subunits. Na⁺/K⁺-ATPase and GAPDH show equal protein used for membrane and cytosolic Western-blots.

Time dependent changes in different NOX proteins in ipsilateral hippocampus membrane fraction following a moderate cortical contusion. Cytosolic NOX protein, p67^Phox (C), was significantly increased early (6 h) in the membrane fraction of the hippocampus following TBI. The maximum increase of p67^Phox in the membrane fraction was at 24 h and continued at 96 h post TBI. In a similar fashion, other key cytosolic NOX protein, p47^Phox (E), and p40^Phox (G) were also increased in membrane fraction of ipsilateral hippocampus as compared sham animals. Each bar represents the group mean ± SD of six animals/group. ^*p < 0.05 versus sham operates.

Levels of NOX proteins, p67^Phox (D), p47^Phox (F), and p40^Phox (H) significantly decreased in cytosolic fraction of the hippocampus following TBI. Result indicates there was a time dependently sifting/translocation of cytosolic component to the membrane for the up-regulation of NOX and increase production of O₂⁻ after TBI. Each bar represents the group mean ± SD of six animals/group. ^*p < 0.05 versus sham operated rats.

Quantitative analysis of NOX protein p-67^Phox revealed a time-dependent injury effect in the hippocampus, suggesting translocation of this subunit. Levels of p-67^Phox demonstrated a significant increase in membrane [F (8, 45) = 3.807, p < 0.005] as early as 6 h post injury (Fig. 2C) and time dependent decline in the cytosolic fraction [F (8, 45) = 5.419, p < 0.0001] (Fig. 2D). The decline in the cytosolic p-67^Phox was apparent even at 96 h after head trauma. Similar changes were observed for the cytosolic NOX protein p-47^Phox, which showed a time-dependent increase [F (8, 45) = 2.458, p < 0.05] in the membrane (Fig. 2E) and decrease [F (8, 45) = 4.900, p < 0.0005] in the cytosolic fraction (Fig. 2F). In the membrane fraction, both the p-67^Phox and p-47^Phox subunits were maximal increased at 24h post TBI.

The cytosolic phosphorprotein p-40^Phox is required to assemble NOX subunits and participates in its function. In the membrane fraction, p-40^Phox was also increased by 50% (overall variability prevented significance of the main effect) compared to sham controls (Fig. 2G) and demonstrated a significant decline in the cytosol fraction [F (8, 45) = 5.205, p < 0.0001] (Fig. 2H). Like p67^Phox and p-47^Phox, levels of p40^Phox demonstrated an early post trauma translocation of this important subunit.

Time course of p-eNOS and the total NO

The p-eNOS activation is the catalyzed production of NO that participates in pathophysiological mechanisms after TBI. Including participation in SIC, p-eNOS derived NO might also work to maintain blood flow in the adjacent area, penumbra region, of the impact [68]. Quantitative analysis revealed that p-eNOS increases in a time-dependent fashion following TBI [F (8, 45) = 8.705, p > 0.0001] (Fig. 3A & B). This elevation occurs as early as 1h post trauma and is maintained for at least 96h (p < 0.01). Maximum levels in the ipsilateral hippocampus were observed at 12-48 h (~225%).

Figure 3.

Time-dependent changes in hippocampal phosphorylated eNOS (p-eNOS) and level total nitric oxide (NO) after a moderate cortical contusion. (A) Western-blot analysis shows p-eNOS was significant increase in the ipsilateral hippocampus, as early as 1 h and maintained elevated levels at 96 h post trauma (B). Total NO levels in ipsilateral hippocampus were significantly increased at 24 h after TBI (C). Each bar represents the group mean ± SD of six animals/group. ^*p < 0.05 versus sham operated rats.

NO was evaluated in the same samples and displayed a delayed time-dependent increase [F (8, 45) = 3.313, p < 0.005] (Fig. 3C). Peak elevation of NO was observed at 24 h after TBI with levels approaching pre injury values at 72 h post trauma.

NOX up-regulation, p-eNOS, upsurge of O₂⁻ and NO

Association between the time dependent changes in individual NOX proteins (increase in membrane), NOX function and produced O₂^.− was assessed in the hippocampus following TBI. Time dependent increase of p-67^Phox and p-47^Phox proteins in the membrane fraction was strongly associated (p < 0.05) with both the increased NOX activity and surge of O₂^.− (Fig. 4A, B, C & D). However, increased p-40^Phox in the membrane fraction was significantly associated with the increased NOX activity (p < 0.05), but it failed to show a strong association with increased O₂^.− levels (Fig. 4E & F).

Figure 4.

Scatterplot (A, C, E) showing the relationship between key NOX subunits (p-67^Phox, p-47^Phox, & p-40^Phox) in the membrane fraction and changes in NOX activity Increases in the subunits significantly correlated with the elevation in total NOX activity. Scatterplot (B, D, F) shows the relationship between key NOX subunits (p-67^Phox, p-47^Phox, & p-40^Phox) in the membrane fraction and changes in O₂^.− activity. Lines are shown to represent the direction of the correlation.

Across all the injured rats, it is observed that increased p-eNOS was almost in parallel with the NOX up-regulation after TBI. NOX up-regulation was strongly associated with the increased levels of O₂^.− (p < 0.001) and appeared to be a major source of O₂^.− increase in the ipsilateral hippocampus (Fig. 5A). Notably, there p-eNOS was failed to show strong association with total NO (p = 0.130) and appeared to be a non-significant contributor in NO increase after TBI (Fig. 5B). Though, these results are not to show cause and effect, but the correlation analysis among the changes in NOX and level of total NO shows there was a substantial associations between them (Fig. 5C). NOX up-regulation after injury mirrored total NO production. Increased levels of total NO was significantly associated with the upsurge of O₂⁻ (p < 0.01), as the levels of O₂⁻ increased so did the levels of NO (Fig. 5D).

Figure 5.

Scatterplot showing the significant relationship between levels of NOX activity and O₂^.− in the ipsilateral hippocampus following a moderate TBI (A). As the NOX levels increased they were paralleled by an increase in O₂^.−. NOX levels also significantly correlated with changes in the levels of NO (C). Levels of NO in the hippocampus following the moderate TBI significantly associated with increased O₂^.−. The analysis failed to demonstrate a significant correlation between p-eNOS levels and NO following the injury (B). Lines are shown to represent the direction of the correlation.

Discussion

This study evaluated very early changes in NOX in the hippocampus following TBI. As part of this evaluation, changes in the levels of several NOX subunits were quantified in both the cytosol and cell membrane fractions extracted from the hippocampus. Recent studies have shown significant NOX activation occurs between 48-72 h after a diffuse brain injury [49] and remains active even at 28 days after a focal brain injury [41]. In the present study, we focused on the first 96 hours after a defined cortical contusion. Previous studies from the laboratory have characterized the overall oxidative stress response in both the cortex and hippocampus [10-12,15]. Multiple different markers demonstrated the fact that the response following brain injury peaks between 12 and 24 h, but it is unclear what the source is of that oxidative stress. Here we demonstrate that NOX may be a major contributor to overall oxidative stress and is significantly elevated at 6 h post trauma similar to other markers [11]. NOX up-regulation remains for an extended period of time post injury, thus contributing to SIC and the spreading of neuronal injury. NOX is dedicated to the specific and deliberate production of O₂^.− [46,47,54,55,57]. Increased O₂^.− acts as a key component in oxidative stress and neuroinflammation [14,26,28,32] mediated SIC, that contributes to neuronal loss [11], neuronal circuitry failure [72,73] and eventually behavioral problems after TBI [23,49,74-76].

In our study, both NOX activity and its derived O₂^.− increased in a time dependent fashion, with the maximum values at 24 h. NOX up-regulation and O₂^.− production occurred early after injury and remains elevated for an extended period of time after head trauma. A recent study reported NOX activation and O₂^.− production maximal (peak value) at 1 h after injury. With the minimum values at 6h, both NOX activity and O₂^.− were significantly elevated at 96 h as compared to sham control [25]. Reasons for these differences may reflect the fact that a different animal (mice) model was used. It is also possible that the detection procedure may be lacking inhibitors for other sources of O₂^.− and performed by estimation of florescence of hydroethidine (redox-reaction) [64] administered intravenously before injury [25]. Such an early peak (at 1 h) of O₂⁻ is most probably associated with mitochondrial dysfunction that is known to occur within 30 min after TBI [77].

The present study showed a strong correlation in cytosolic NOX subunit translocation and O₂⁻ production. Activation of NOX results from the translocation and assembly of the cytosolic proteins (p67^Phox, p47^Phox, and p-40^Phox) into membrane subunits. Increases in the membrane fraction did not exactly mirror the declines in the cytosolic fraction. For example, at 48 h, while cytosolic levels of subunits remained significantly low, levels in the membrane fraction began to decline. Such a difference may illuminate possible degradation of cytosolic proteins. It is well known that the activation of NOX catalyzes O₂⁻ production [51-53], and consumes the secondary energy molecules NADPH. Increased NOX derived O₂⁻ in endothelial and smooth muscle cells contribute to a compromised blood brain barrier resulting in impaired cerebral blood flow [37,78-80]. Mechanistically, NOX activation is associated with increased intracellular Ca²⁺ [46,47], protein kinase C (PKC) functions [81-83] and participates in the cell signaling processes. NOX derived O₂^.− contributes to the expression of matrix metalloproteinase-9 (MMP-9) and the activation of extracellular signal-regulated kinase (ERK) [81,82], c-Jun-N-terminal kinase (JNK) and nuclear factor kappa-B (NF-kB) [82-84]. Consequently, NOX up-regulation has been implicated in microglia activation/proliferation [21,63,85], inflammation [39,40,86,87] and mediated apoptotic neuronal death [44,45,88]. Activated microglia produces both O₂^.− and NO, as a mechanistic tool in neurodegenerative processes [26,32,34,39]. Up-regulation of NOX showed a positive significant correlation with increased levels of NO. Our study is the first to show a significant association between the increase in NO and O₂^.− in the hippocampus following TBI.

In the present set of experiments we found a time-dependent activation, phosphorylation of eNOS (p-eNOS). This activation occurred very early (1 h) and remained significantly higher than uninjured animals up to 96 h while significant elevation in the total NO was observed at 24 h after TBI. It was surprising that p-eNOS did not strongly associated with the total NO levels supporting the idea that p-eNOS is not driving the increase in NO. These result suggest that perhaps either 1) early after injury eNOS derived NO have been consumed in the various biological processes [67-69] or 2) a significant elevation in total NO occurred after the expression/activation of iNOS and nNOS [66]. Oxygen radicals can also become a controlling factor for NO, such as, O₂^.− can favor NO production [89] and also control their bioavailability [90]. Several studies have shown that NO and its metabolites contribute to pathology [34-36,91,92] and can be considered as a biomarker in severe-TBI [91]. Endothelial NOS derived NO participates in various physiological functions [67-69] including regulation of blood flow [67,90], arterial pressure [93], and signal transduction [94]. Similar to NOX, eNOS is also activated with the increased intracellular Ca²⁺ and NO function involves the activation of ERK [64,68], NF-kB [40,84] and protein kinase B (Akt) [68,70,84]. In the reaction of O₂^.−, NO generates various other ROS/RNS that may cause protein damages [2,11], i.e., protein’s nitration [2,3], hence, play in SIC after TBI.

Conclusions

This study revealed an early, time dependent, up-regulation of NOX and p-eNOS after TBI. These enzymes increase O₂^.− and NO production, and play an important role in SIC. The weak association between p-eNOS and total NO suggests that targeting eNOS activation after TBI may not be an effective therapy. A strong association between the up-regulation of NOX and level of O₂⁻ suggests that the NOX is a major source of free radicals in the ipsilateral hippocampus. Targeting NOX function is recognized for therapeutic intervention following TBI. Our result suggests that treatment should begin early (within the first 12 hours) that may target translocation of cytosolic subunit into membrane for the modulation of NOX.

Figure 6.

Figure 7.

Abbreviations

Akt

protein kinase B

Akt

protein kinase B

BCIP/NBT

5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium

CCI

controlled cortical impact

EDTA

ethylenediaminetetraacetic acid

ERK

extracellular signal-regulated kinase

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

GTPase

guanosine triphosphatase

JNK

c-Jun-N-terminal kinase

MMP-9

matrix metalloproteinase

Na⁺/K⁺-APTase

sodium-potassium adenosine triphosphatase

NF-kB

nuclear factor kappa-B

PKC

protein kinase-C

PMS

post mitochondrial supernatant

SIC

secondary injury cascades