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Journal of Cerebral Blood Flow & Metabolism

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Research article

First published November 2006

Time Course of Post-Traumatic Mitochondrial Oxidative Damage and Dysfunction in a Mouse Model of Focal Traumatic Brain Injury: Implications for Neuroprotective Therapy

Indrapal N Singh, Patrick G Sullivan, Ying Deng, Lamin H Mbye, and Edward D Hall [email protected]View all authors and affiliations

Volume 26, Issue 11

https://doi.org/10.1038/sj.jcbfm.9600297

Abstract

In the present study, we investigate the hypothesis that mitochondrial oxidative damage and dysfunction precede the onset of neuronal loss after controlled cortical impact traumatic brain injury (TBI) in mice. Accordingly, we evaluated the time course of post-traumatic mitochondrial dysfunction in the injured cortex and hippocampus at 30 mins, 1, 3, 6, 12, 24, 48, and 72 h after severe TBI. A significant decrease in the coupling of the electron transport system with oxidative phosphorylation was observed as early as 30 mins after injury, followed by a recovery to baseline at 1 h after injury. A statistically significant (P < 0.0001) decline in the respiratory control ratio was noted at 3 h, which persisted at all subsequent time-points up to 72 h after injury in both cortical and hippocampal mitochondria. Structural damage seen in purified cortical mitochondria included severely swollen mitochondria, a disruption of the cristae and rupture of outer membranes, indicative of mitochondrial permeability transition. Consistent with this finding, cortical mitochondrial calcium-buffering capacity was severely compromised by 3h after injury, and accompanied by significant increases in mitochondrial protein oxidation and lipid peroxidation. A possible causative role for reactive nitrogen species was suggested by the rapid increase in cortical mitochondrial 3-nitrotyrosine levels shown as early as 30 mins after injury. These findings indicate that post-traumatic oxidative lipid and protein damage, mediated in part by peroxynitrite, occurs in mitochondria with concomitant ultrastructural damage and impairment of mitochondrial bioenergetics. The data also indicate that compounds which specifically scavenge peroxynitrite (ONOO) or ONOO⁻derived radicals (e.g. ONOO⁻ + H⁺ → ONOOH → ^†NO₂ + ^†OH) may be particularly effective for the treatment of TBI, although the therapeutic window for this neuroprotective approach might only be 3 h.

Introduction

Mitochondria are important in cellular bioenergetics and are sensitive to changes in the physiological state of cells. Accumulating evidence indicates that mitochondria play a pivotal role in both the necrotic and apoptotic processes in mammalian cells. Disruption of mitochondrial membrane potential (Δϕ_m) is considered to be an indicator of mitochondria damage and generally is defined as an early stage of apoptosis, preceding the efflux of macromolecules from the mitochondria (including cytochrome c, apoptosis-inducing factor, cellular inhibitor of apoptosis proteins (cIAPs), etc.) and caspase-9/caspase-3 cascade activation (Green and Reed, 1998; Spierings et al, 2005).

Mitochondria also appear to play a critical role in the secondary injury that occurs after traumatic brain injury (TBI) (Finkel, 2001; Hunot and Flavell, 2001), and mitochondrial dysfunction has been shown to be involved in excitatory amino acid (EAA)-induced neurotoxicity (White and Reynolds, 1996; Nicholls and Budd, 1998; Stout et al, 1998; Kroemer and Reed, 2000; Jiang et al, 2001; Brustovetsky et al, 2002). Mitochondrial dysfunction after TBI has been linked to impairment of brain mitochondrial electron transfer and energy transduction due to overloading of mitochondrion-associated calcium (Xiong et al, 1997), increased mitochondrial reactive oxygen species (ROS) production, oxidative damage, disruption of synaptic homeostasis (Azbill et al, 1997; Matsushita and Xiong, 1997; Sullivan et al, 1999a, b, c), and cell death (Robertson, 2004).

Most convincing with regard to the importance of mitochondrial failure in secondary brain injury, pharmacological agents that target mitochondria have been shown to be neuroprotective. Administration of cyclosporine A (CsA) after experimental TBI significantly reduces mitochondrial dysfunction (Sullivan et al, 1999c), cortical damage (Scheff and Sullivan, 1999; Sullivan et al, 2000b, c), and cytoskeleton and axonal dysfunction (Okonkwo et al, 1999; Okonkwo and Povlishock, 1999). Subsequently, Sullivan et al (2000a) have shown that dietary supplementation with creatine is also effective in ameliorating neuronal cell death by reducing mitochondrial ROS production and maintaining adenosine triphosphate levels after TBI. Although these prior studies clearly show the importance of mitochondrial dysfunction and failure in secondary brain injury, and the potential therapeutic value of pharmacological mitochondrial protection, the precise time course, characteristics and causes of post-traumatic mitochondrial dysfunction have not been fully defined. This is important to achieve to better understand the optimal mechanistic approaches and the therapeutic time window for being able to salvage mitochondrial function and achieve successful neuroprotection in the injured brain.

Thus, the current investigation was performed in the context of the mouse lateral controlled cortical impact (CCI)-TBI model (Smith et al, 1995; Raghupathi et al, 1998; Hannay et al, 1999; Sullivan et al, 1999a, c), and follows our recent definition of the time course of calpain-mediated cytoskeleton degradation and neurodegeneration in this paradigm (Hall et al, 2005). That study showed that cytoskeleton and neuronal degradation did not become apparent until 6 h after injury and do not peak until 24 to 48 h. In the present study, we report marked and sustained decreases in mitochondrial respiratory rates, respiratory control ratios (RCRs) and calcium-buffering capacity, indices of mitochondrial dysfunction, which become statistically significant by 3h and peaks by 12 h after TBI. The onset of metabolic dysfunction is coincident with mitochondrial oxidative damage, suggesting that oxygen radical-induced modification of membrane lipids and proteins might be pivotal factors in post-traumatic mitochondrial dysfunction. Based on its time course, a major player in this oxidative damage scenario appears to be the ROS peroxynitrite (ONOO⁻).

Materials and methods

Materials

Mannitol, sucrose, bovine serum albumin (BSA), ethyleneglycol tetraacetate (EGTA), N−2-hydroxyethylpiperazine-N'−2-ethanesulfonic acid (HEPES) potassium salt, potassium phosphate monobasic anhydrous (KH₂PO₄), magnesium chloride (MgCl₂), malate, pyruvate, adenosine 5'-diphosphate (ADP), oligomycin A, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and succinate were purchased from Sigma-Aldrich (St Louis, MO, USA). BCA protein assay kit was purchased from Pierce (Rockford, IL, USA).

Animals

The studies were performed using 350 young adult male CF-1 mice (Charles River, Portage, MI, USA) weighing 29 to 31 g. Ipsilateral cortical and ipsilateral hippocampal tissues from five mice were pooled as N1 and we used 3 N's (15 animals) for TBI and 1 N (5 animals) for sham at every time-point studied, as described in the present paper. All the protocols for performing surgery and TBI have been approved by the University of Kentucky Institutional Animal Care and Use Committee.

Mouse Model of Focal (Lateral Controlled Cortical Impact) Traumatic Brain Injury

Mice were initially anesthetized in a plexiglass chamber using 4.0% isoflurane and placed in a stereotaxic frame (David Kopf, Tujunga, CA, USA). During the injury procedure, anesthesia was maintained with 2.5% isoflurane delivered via a nose cone. The head was positioned in the horizontal plane with the nose bar set at zero. After a midline incision exposing the skull, a 4-mm craniotomy was made lateral to the sagittal suture and centered between the bregma and lambda. The skull cap at the craniotomy was carefully removed without damaging the underlying dura, and the exposed cortex was injured using a pneumatically controlled impactor device as described previously (Sullivan et al, 1999a, c). The impactor tip diameter was 3 mm and the impact velocity was set at 3.5 m/sec with a cortical compression of 1.0 mm. After injury, the craniotomy was closed by placement of a small disk of saline moistened Surgicil over the dura, followed by super gluing a 6 mm disk made of dental cement over the site. The animals were then placed in a Hova-Bator Incubator (model 1583; Randall Burkey Co, Boerne, TX, USA), which was set at 37°C until consciousness (i.e. return of righting reflex and mobility) was regained to prevent hypothermia. Injured mice were allowed to survive from 30 mins to 3 days depending on experimental group assignment. A sham group was included at all time-points which underwent anesthesia and a craniotomy, but no cortical impact or injury.

Isolation of Percoll-Purified Mitochondria

Brain mitochondria were extracted as described previously with some modifications (Sullivan et al, 2000a, 2003). Briefly, the mice were decapitated and the ipsilateral brain regions of interest (cortex and hippocampus) were quickly removed and pooled from five mice. The cortices and hippocampi were placed in a Potter-Elvejhem homogenizer containing five times the volume of ice-cold isolation buffer (five-fold buffer to tissue) with 1 mmol/L EGTA (215 mmol/L mannitol, 75 mmol/L sucrose, 0.1% BSA, 20 mmol/L HEPES, 1 mmol/L EGTA; pH adjusted to 7.2 with KOH), and the homogenates were subjected to differential centrifugation at 4°C. First, the homogenate was centrifuged twice at 1300g for 3 mins in an Eppendorf microcentrifuge at 4°C to remove cellular debris and nuclei. The pellet was discarded and the supernatant further centrifuged at 13,000g for 10 mins. The crude mitochondrial pellet obtained after differential centrifugation was then subjected to nitrogen decompression to release synaptic mitochondria, using a nitrogen cell disruption bomb, cooled to 4°C under a pressure of 1200 psi for 10 mins (Sullivan et al, 2003; Brown et al, 2004; Kristian et al, 2005). After nitrogen disruption the mitochondria were suspended in 3.5 mL of 15% percoll and further laid on two preformed layers consisting of 3.5 mL of 24% percoll and 3.5 mL of 40% percoll in 13 mL ultraclear tubes. The gradient was centrifuged in fixed angle rotor at 30,400g for 10 mins at 4°C. The fraction accumulated at the interphase of 40% and 24% percoll was carefully removed and diluted with isolation buffer without EGTA, then centrifuged at 12,300g for 10 mins at 4°C. The supernatants were carefully removed, the pellet resuspended in isolation buffer without EGTA and centrifuged at 13,000 × g for 10 mins at 4°C. The mitochondrial pellets at the bottom were then transferred to microcentrifuge tubes and topped off with isolation buffer without EGTA and centrifuged at 10,000g for 5 mins at 4°C to yield a tighter pellet. The final mitochondrial pellet was resuspended in isolation buffer without EGTA to yield a concentration of ~10mg/mL. The protein concentration was determined using the BCA protein assay kit measuring absorbance at 562 nm with a BioTek Synergy HT plate reader (Winooski, VT, USA).

Mitochondrial Respiration Studies

Mitochondrial respiratory rates were measured using a Clark-type electrode in a continuously stirred, sealed thermostatically controlled chamber (Oxytherm System, Hansatech Instruments Ltd) maintained at 37°Cas described previously (Sullivan et al, 2000a, 2003; Jiang et al, 2001; Sensi et al, 2003). In all, 25 to 40 μg of isolated mitochondrial protein was placed in the chamber containing 250 μL of KCl-based respiration buffer (125 mmol/L KCl, 2 mmol/L MgCl₂, 2.5 mmol/L KH₂PO₄, 0.1% BSA, 20 mmol/L HEPES at pH 7.2) and allowed to equilibrate for 1 min. This was followed by the addition of complex-I substrates, 5 mmol/L pyruvate and 2.5 mmol/L malate, to monitor state II respiratory rate. Two boluses of 150 μmol/L ADP were added to the mitochondria to initiate state III respiratory rate for 2 mins, followed by the addition of 2 μmol/L oligomycin to monitor state IV respiration rate for an additional 2 mins. For the measurement of uncoupled respiratory rate, 2 μmol/L FCCP was added to the mitochondria in the chamber and oxygen consumption was monitored for another 2 mins. This was followed by the addition of 10 mmol/L succinate to monitor complex II-driven respiration. The RCR was calculated by dividing state III oxygen consumption (defined as the rate of respiration in the presence of ADP, second bolus addition) by the state IV oxygen consumption (rate obtained in the presence of oligomycin). Fresh mitochondria were prepared for each experiment and used within 4 h.

Mitochondrial Calcium-Buffering Studies

Mitochondrial calcium-buffering measurements were performed in a Shimadzu RF-5301 spectrofluorimeter maintained at 37°C. The extra-mitochondrial concentrations of Ca²⁺ were performed using the fluorescent Ca²⁺ -sensitive indicator Calcium Green 5 N (CaG5N) (Molecular Probes, Eugene, OR, USA) at a final concentration of 100 nmol/L with excitation and emission wavelengths 506 nm (slit 5 nm) and 532 nm (slit 10 nm), respectively. The mitochondrial transmembrane potential (Δϕ_m) was estimated using the fluorescence quenching of the cationic indicator, 150 nmol/L tetramethyl rhodamine ethyl ester (TMRE), which is accumulated and quenched inside energized mitochondria. The excitation wavelength was 550 nm (slit 5 nm) and the emission wavelength 575 nm (slit 10 nm) for TMRE. The experiment was initiated by the sequential additions of mitochondria (0.1 mg/2.0 mL protein) to a 2.0 mL cuvette containing KCl-based respiration buffer (125 mmol/L KCl, 2 mmol/L MgCl₂, 2.5 mmol/L KH₂PO₄, 0.1% BSA, 20 mmol/L HEPES at pH 7.2), followed by 100 nmol/L CaG5N and 150nmol/L TMRE. The reaction was initiated by the addition of the oxidizable substrates, such as 5 mmol/L pyruvate and 2.5 mmol/L malate at 1 min, followed by the addition of 150 μmol/L ADP at 2 mins and 1 μmol/L oligomycin (final concentrations) at 3 mins, and the reaction was continued upto 5 mins. At 5 mins after the final addition, 32 mmol/L calcium, at a rate of 0.5 μL/min (equivalent to 80 nmol of calcium per mg of mitochondrial protein per min) was continuously infused into the cuvette using a syringe pump.

Measurement of Oxidative Damage Markers by Slot-Blot Analysis

A portion of isolated ipsilateral cortical and ipsilateral hippocampal mitochondria from sham (3 h), 30 mins, 3, and 12 h after injury was used for the quantitative measurements of 4-hydroxynonenal (4-HNE) as an index of lipid peroxidation, protein carbonyls, an index of protein oxidation, and 3-nitrotyrosine (3-NT) as an index of protein nitration, using the slot-blot technique. Briefly, approximately 2.5 μg of mitochondrial protein was transferred to a Protran (0.2 μm) nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) by a Minifold II vacuum slot blot apparatus (Schleicher & Schuell). Each slot was washed with 300 μL PBS and the membranes were dried. For the detection of protein tyrosine nitration, a rabbit polyclonal antityrosine antibody (Upstate Biotechnology, Milford, MA, USA) was used at a dilution of 1:2000 with overnight incubation at 4°C. For detection of HNE, a rabbit polyclonal anti-HNE antibody (Alpha Diagnostics International) was used at a dilution of 1:5000 with overnight incubation at 4°C. The levels of protein carbonyl were determined by using the OxyBlot Protein Oxidation Detection Kit (OxyBlot, Chemicon, Temecula, CA, USA) with rabbit polyclonal anti-dinitrophenyl hydrazine (DNP), with overnight incubation at 4°C. Positive bands detected with a peroxidase-coupled secondary IR-Dye 800CW goat anti-rabbit was used at a dilution of 1:500 (Vector Laboratories, CA, USA). All incubations and washing steps were performed according to the instructions given by the manufacturers. The intensity of the bands was visualized and quantitated using Li-Cor Odyssey Infrared Imager. On all blots, a blank without mitochondrial protein was employed to correct for nonspecific binding. The value of the blank was background subtracted from the values for all the other samples. In the case of 3-NT, we showed that preincubation with nitrated BSA blocked the reactivity of the antibody with the sample down to negligible levels.

Electron Microscopy

The purified mitochondrial pellets were fixed in 4% glutaraldehyde overnight at 4°C before being embedded for electron microscopy. The fixed mitochondrial pellets were then washed overnight at 4°C in 0.1 mol/L sodium cacodylate buffer, followed by 1 h secondary fixation at room temperature in 1% osmium tetroxide (in sodium cacodylate buffer). Then, the mitochondrial pellets were rinsed with distilled water and dehydrated in a gradient ethanol series up to 100% and twice in propylene oxide. The pellets were then placed in a 1:1 mixture of propylene oxide and Epon/Araldite resin and infiltrated overnight on a rotator. Next, 100% Epon/Araldite resin was added and rotated for 1 h at room temperature. Finally, fresh resin was prepared and degassed using a vacuum chamber. The mitochondrial pellets were added to the flat molds, filled with fresh resin, and baked overnight at 60°C. The 90 nm ultra-thin sections were cut using an RMC MT-7000 ultramicrotome mounted on 150 mesh copper grids and stained with uranyl acetate and lead citrate. A Zeiss 902 electron microscope was used to examine and photograph the samples.

Statistical Analysis

Results were expressed as the mean ± s.e.m. For single-variable comparisons, Student's t-test was used. For multiple-variable comparisons, data were analyzed by one-way analysis of variance (ANOVA) followed by post-hoc analysis using the Student-Neuman-Keul's (SNK) test.

Results

Total (synaptic and nonsynaptic population) ipsilateral cortical and hippocampal mitochondria were isolated using the ‘nitrogen bomb’ decompression method from sham (craniotomy, but no injury) and injured animals at 30 mins, 1, 3, 6, 12, 24, 48, and 72 h after injury. At all time-points, mitochondria isolated from either the cortices or hippocampi of sham animals were metabolically intact and wellcoupled, as shown by the RCR being > 6 (Figures 1 and 3). This indicates that in mitochondria isolated from sham animals at all time-points the electron transport system (ETS) was well coupled to oxidative phosphorylation and that any differences observed in the mitochondria from the injured animals were not due to preparation artifact.

Figure 1 Time course of mitochondrial respiratory dysfunction (A) and States III & IV respiratory rates (B) in mouse ipsilateral cortex after severe (1.0 mm) TBI. Mitochondrial oxygen consumption was measured using a Clark-type electrode in a continuously stirred, sealed chamber (Oxygraph System; Hansatech Instruments Ltd). Purified mitochondrial protein (25 to 35 μg) was suspended in respiration buffer (125 mmol/L KCl, 2 mmol/L MgCl₂, 2.5 mmol/L KH₂PO₄, 0.1% BSA, 20 mmol/L HEPES, pH 7.2) in a final volume of 250 μL. Respiratory control ratio was calculated as the ratio of oxygen consumption in the presence of 5 mmol/L pyruvate and 2.5 mmol/L malate before (state IV) and after the second addition of l50 μmol/L ADP (state III). Data are presented as mean ± s.e.m. Statistical differences (one-way ANOVA and Student-Neuman-Keuls *post hoc* test): ^*P < 0.0001 versus sham; ^*P < 0.001 versus sham.

Time Course of Cortical and Hippocampal Mitochondrial Respiratory Dysfunction After Traumatic Brain Injury

Mitochondrial bioenergetics were assessed in animals after a severe TBI at 30 mins, 1, 3, 6, 12, 24, 48, and 72 h after injury. This revealed significant differences in the RCR that were due to changes in state III and IV respiratory rates. A significant decrease in RCR was observed in ipsilateral cortical mitochondria as early as 30 mins after injury, followed by a recovery to baseline at 1 h (Figure 1A). This significant decrease in RCR at 30 mins after injury was due to a significant increase in state IV respiration, indicative of increased proton conductance across the mitochondrial inner membrane (Figure 1B), which could in part be due to the onset of mitochondrial permeability transition (mPT). However, at 1 h, states III, IV and the RCR returned back to baseline. At 3 h, a secondary significant decrease (-70% compared with sham) in RCR was noted, which was followed by a partial recovery (Figure 1A). State IV respiration was increased by 233%. At 6h, state III respiration was seen to decrease significantly (-30%). This decrease in maximal respiratory capacity progressed down to −63% at 12 h. Although the RCR partially recovered toward the sham baseline, it remained significantly suppressed in comparison to that seen in mitochondria from the sham animals up to 72 h after injury. Similarly, state IV respiration remained significantly elevated. However, state III respiration appeared to recover completely. Figure 2 shows the contrast between the typical oxymetric respiratory traces from cortical mitochondria isolated from a sham versus 3 h postinjury brain.

Figure 2 A typical oxymetric trace showing alterations in cortical mitochondrial bioenergetics after a severe TBI. Mitochondria isolated 3 h after injury from the injured cortex of mice show a reduction in RCR (rate of respiration in the presence of ADP versus rate of respiration in the absence of ADP) and a loss of ETS capacity (rate of respiration in the presence of the uncoupler FCCP), compared with sham-operated animals.

Figure 3 Time course of mitochondrial respiratory dysfunction (A) and States III & IV respiratory rates (B) in mouse ipsilateral hippocampus after severe (1.0 mm) TBI. Mitochondrial protein (25 to 35 μg) was suspended in respiration buffer, in a final volume of 250 μL, and oxygen consumption assessed under various experimental conditions. Respiratory control ratio was calculated as the ratio of oxygen consumption in the presence of ADP (second addition of 150 μmol/L) or oligomycin (state IV), while using the oxidative substrates, pyruvate and malate. Data are presented as mean ± s.e.m. Statistical differences (one-way ANOVA and SNK *post hoc* test): ^*P < 0.0001 versus sham; ^*P < 0.001 versus sham.

Results similar to those seen in cortical mitochondria were obtained with mitochondria isolated from ipsilateral hippocampus with regard to changes in the RCR (Figure 3A) and state III and IV respiration rates (Figure 3B) as a function of time after injury. The one difference observed was a significant reduction in state III respiration at 72 h after injury in the hippocampal mitochondria. This indicates that there may be a tertiary phase of mitochondrial dysfunction between 24 and 72 h after injury. Although a similar decrease in state III respiration at this time-point was measured in the cortical mitochondria, it did not reach statistical significance.

Time Course of Cortical Mitochondrial Calcium Buffering Impairment After Traumatic Brain Injury

The ability of the cortical mitochondria to buffer Ca²⁺ before undergoing mPT was compared between sham and injured animals at 30 mins, 3 and 12 h (Figure 4) after injury. Calcium uptake was not significantly impaired at 30 mins after injury. However, by 3h, a > 50% decrease (P < 0.05) in calcium buffering was observed, which persisted at 12 h after injury. The inset in Figure 4 shows differences in mitochondrial calcium uptake (i.e. calcium-buffering capacity) using the Ca²⁺ -sensitive indicator, CaG5N, during a sustained calcium exposure, in injured versus noninjured mitochondria, indicating a reduced threshold for the onset of the mPT.

Figure 4 Post-traumatic time course of changes in cortical mitochondrial calcium buffering. Mitochondrial calcium buffering was assessed in a stirred cuvette mounted in a Shimadzu RF-5301 spectrofluorimeter maintained at 37°C. The extra-mitochondrial concentrations of Ca²⁺ were measured using the fluorescent Ca²⁺ -sensitive indicator CaG5N. Mitochondria (0.1mg/mL) were incubated with 5 mmol/L pyruvate and 2.5 mmol/L malate, 150 μmol/L ADP and 1 μmol/L oligomycin. At 5 mins after the final addition (oligomycin), 32 mmol/L calcium, at a rate of 0.5 μL/min, was infused continuously into the cuvette using a syringe pump. The inset is a typical trace that shows the differences in mitochondrial calcium uptake (i.e. calcium-buffering capacity), in injured (3 h after injury) versus noninjured (sham) mitochondria, indicating a reduced threshold for mpT. Bars are the mean ± s.e.m. Statistical differences (oneway ANOVA and SNK *post hoc* test): ^*P < 0.05 versus sham.

Time Course of Mitochondrial Oxidative Injury After Traumatic Brain Injury

Based on the time course of mitochondrial dysfunction, markers of oxidative damage were analyzed at the 30-min, 3-, and 12-h postinjury time-points for ipsilateral cortical (Figure 5A) and hippocampal (Figure 5B) mitochondria. In the cortex, an increase in lipid peroxidation products (HNE; P < 0.05) and protein nitration (3-NT; P < 0.05) was seen as early as 30 mins after injury. Protein carbonyl levels were significantly (P < 0.001) elevated at 3 h after injury compared with sham animals. At 12 h, only the HNE levels remained significantly increased, whereas the protein carbonyl and 3-NT had returned to within normal limits (i.e. in comparison to sham animals).

Figure 5 Time course of lipid (HNE) and protein (protein carbonyl) oxidative damage in percoll-purified mitochondria from the ipsilateral cortex (A) and ipsilateral hippocampus (B) after a severe (1.0 mm) TBI. Distribution of HNE, protein carbonyls, and 3-NT in cortical mitochondria (A), and hippocampal mitochondria (B), of mice at 30 mins, 3 and 12 h after injury and were compared with sham (3h) animals. Approximately 2.5 μg of percoll-purified mitochondrial protein isolated from cortical and hippocampal region was applied onto Protran nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany), as described in the experimental section, and oxidative markers were quantitated using a Minifold II vacuum slot blot apparatus. Data are presented as mean ± s.e.m. Statistical differences (one-way ANOVA and SNK *post hoc* test): ^*P < 0.05 versus sham; ^**P < 0.001 versus sham; ^***P < 0.0001 versus sham; ^@P < 0.005 versus sham.

Ipsilateral hippocampal mitochondria displayed a more delayed increase in oxidative damage, with HNE and protein carbonyl not being significantly increased until 3 h after injury. At 3 h, the HNE and 3-NT remained elevated along with the appearance of a significant (P < 0.0001) increase in protein oxidation-derived carbonyl levels. By 12 h, these had returned to normal sham levels, but a delayed increase in 3-NT (P < 0.005) was shown.

Time Course of Changes in Cortical Mitochondria After Traumatic Brain Injury

Several lines of evidence in the current study implicated the onset of the catastrophic mPT after TBI. To directly assess this question, the ultrastructural integrity of cortical mitochondria isolated from sham animals was qualitatively compared with those harvested from injured brains at 30 mins, 3 and 12 h after injury using transmission electron microscopy. The photomicrographs indicate that the cortical mitochondria isolated from sham animals displayed typical tight cristae and intact outer membranes (Figure 6A). In contrast, electron microscopy of the ipsilateral cortical mitochondria from TBI animals revealed obvious structural damage as early as 30 mins (Figure 6B), which appeared to get progressively worse at 3 h (Figure 6C) and 12 h (Figure 6D) after injury. The time-dependent damage consisted of mitochondrial swelling, disruption or loss of cristae, and breakage of the outer membranes indicative of osmotic swelling due to mPT. By 12 h, few, if any, normal-looking mitochondria were visible in any given sample of ipsilateral cortical mitochondria.

Figure 6 Electron microscopic pictures of percoll-purified mitochondria from ipsilateral cortex after severe postinjury reveal mitochondrial matrix condensation, cristae disruption and swelling. Percoll-purified mitochondrial pellets from sham (3 h), (A), 30 mins, (B), 3h (C) and 12 h (D) postinjury were fixed in 4% glutaraldehyde at 4°C before being embedded for electron microscopy. The scale bar represents 500 nm.

Discussion

This study describes the time course of structural and functional alterations in isolated cortical and hippocampal mitochondria during the first 72 h after a severe level of CCI-TBI in mice. Mitochondrial dysfunction was observed as early as 30 mins after injury, as evidenced by a decrease in RCR due to a significant increase in state IV respiration in both brain regions ipsilateral to the injury. The post-traumatic mitochondrial dysfunction occurred simultaneously with an increase in markers of mitochondrial oxidative damage, including lipid peroxidation (increased HNE), protein oxidation (increased protein carbonyl), and protein nitration (3-NT). This is very intriguing considering that oxidative stress is a known inducer of mPT susceptibility (Sullivan et al, 2004a, b, 2005). Oxidative stress is also known to significantly impair mitochondrial respiration specifically by altering complex-I, nicotinamide adenine dinucleotide (reduced form)-driven respiration (Sullivan et al, 2004a, c, 2005).

Role of Peroxynitrite in Traumatic Brain Injury Pathophysiology

Fifteen years ago, Beckman and coworkers introduced the theory that the principal ROS involved in producing tissue injury in a variety of neurological disorders is ONOO⁻, which is formed by the combination of nitric oxide synthase (NOS) ONOO⁻-generated ^†NO radical and superoxide radical (Beckman, 1991). Since that time, the biochemistry of ONOO⁻, which is often referred to as a reactive nitrogen species, has been clarified. ONOO⁻-mediated oxidative damage is actually caused by ONOO⁻ decomposition products that possess potent free radical characteristics. The first involves the protonation of ONOO⁻ to form peroxynitrous acid (ONOOH), which can undergo homolytic decomposition to form the highly reactive nitrogen dioxide radical (^†NO₂) and hydroxyl radical (^†OH). Probably more important physiologically, ONOO⁻ will react with carbon dioxide (CO₂) to form nitrosoperoxocarbonate (ONOOCO₂), which can decompose into ^†NO₂ and carbonate radical (^†CO₃). Each of the ONOO⁻-derived radicals (^†OH, ^†NO₂, and ^†CO₃) can initiate LP cellular damage by abstraction of an electron from a hydrogen atom bound to an allylic carbon in polyunsaturated fatty acids or cause protein carbonylation by reaction with susceptible amino acids (e.g. lysine, cysteine, arginine). Both of these oxidative damage markers were increased in cortical and hippocampal mitochondria during the first 3 h after injury. However, the coincidental increase in protein nitration (nitrotyrosine) in the isolated mitochondria as early as 30 mins after injury signals that a major portion of the ROS-induced damage is probably caused by ONOO⁻.

The implication of ONOO⁻ in post-traumatic mitochondrial dysfunction and neurodegeneration is derived from five lines of evidence. First of all, all three NOS isoforms (endothelial, neuronal and inducible) are known to be upregulated during the first 24 h after TBI in rodents. Secondly, it has been shown that the acute treatment of injured mice or rats with NOS inhibitors can exert a neuroprotective effect and/or improve neurological recovery (Mesenge et al, 1996b, 1998a, b; Wallis et al, 1996; Wada et al, 1998a, b, 1999). Thirdly, biochemical footprints of ONOO⁻-mediated damage have been documented in rodent TBI paradigms, including an increase in 3-NT levels (Mesenge et al, 1998a, b). The notion that these markers of ONOO⁻ -mediated damage are pathophysiologically important is supported by the finding that the NOS inhibitor L-arginine analog N-nitro-L-arginine methyl ester can lessen the accumulation of 3-NT in injured brains (Mesenge et al, 1998a, b) at the same doses that improve neurological recovery (Mesenge et al, 1996a).

Fourth, recent evidence indicates that the principal oxidative damage-producing ROS that is being formed by mitochondria is most likely ONOO. For instance, nitric oxide has been shown to be present in mitochondria (Lopez-Figueroa et al, 2000; Zanella et al, 2002), and a mitochondrial NOS isoform (mtNOS) has been isolated, characterized and shown to be similar to neuronal NOS (nNOS), except for post-translational acylation with myristic acid and C-terminal phosphorylation (Kanai et al, 2001; Elfering et al, 2002). Although physiological roles for mtNOS have been suggested, mtNOS and ^†NO appear to be important in neuronal degeneration. For instance, ^†NO production in mitochondria occurs during apoptosis (Bustamante et al, 2002), and mtNOS stimulation can cause cytochrome c release (Ghafourifar et al, 1999). Deregulation of mitochondrial ^†NO generation and the aberrant production of its toxic metabolite ONOO⁻ appear to be involved in many, if not all, of the major acute and chronic neurodegenerative conditions (Heales et al, 1999). Exposure of mitochondria to Ca²⁺, which is known to cause them to become dysfunctional, leads to impairment of mitochondrial oxidative phosphorylation (Xiong et al, 1997) and ONOO⁻ generation, which in turn triggers mitochondrial Ca²⁺ release (i.e. limits their Ca²⁺ uptake or buffering capacity) (Bringold et al, 2000).

The fifth and possibly most compelling evidence supporting a role of ONOO⁻ in acute TBI comes from recent studies showing the beneficial effects of multiple compounds that possess the ability to directly scavenge ONOO⁻ or ONOO⁻-derived radicals in TBI models. For instance, penicillamine, which can stoichiometrically react with ONOO⁻ (Althaus et al, 2000), is able to improve neurological recovery of mice subjected to moderately severe diffuse TBI (Hall et al, 1999). Similarly, the brainpenetrable pyrrolopyrimidine antioxidant U-101033E, which can react with ONOO⁻ (Rohn et al, 1998), also improves the neurological recovery of injured mice (Hall et al, 1995). Furthermore, the indolamine melatonin, which is also reactive with ONOO⁻, has been reported to be neuroprotective in mice after diffuse TBI (Mesenge et al, 1998a, b) and rats after cortical contusion injury (Cirak et al, 1999; Sarrafzadeh et al, 2000). However, most promising as an anti-ONOO⁻ agent is the antioxidant tempol, which has been shown to catalytically scavenge ONOO⁻ derived ^†NO₂ and ^†CO₃ (Carroll et al, 2000; Bonini et al, 2002). Tempol has been reported to reduce post-traumatic brain edema and improve neurological recovery in a rat contusion injury model (Beit-Yannai et al, 1996; Zhang et al, 1998).

Dissection of the Time Course of Oxidative Damage and Mitochondrial Functional Impairment

The decrease in mitochondrial RCR, which first occurred at 30 mins, indicates that some mitochondrial uncoupling has taken place. The increase in state IV represents an increase in proton leak across the inner mitochondrial membrane. Although state III respiration also increased at 30 mins, its magnitude was less than the increase in state IV. Consequently, the RCR (state III/state IV) was decreased.

At 1 h after injury, mitochondrial function appeared to recover with the RCR, state III and IV respiration all returning to preinjury levels. The explanation for this early biphasic functional change could be twofold. The first possibility is that a fraction of the mitochondria in the injured cortical and hippocampal samples are undergoing mPT and thus irreversible function loss as early as 30 mins. Once they undergo mPT, they are beyond the point of salvage. The photomicrographs (Figure 6) showing that a portion of the mitochondria examined at 30 mins is severely swollen is consistent with the notion that those particular mitochondria are probably past the point of functional return. Consequently, the apparent recovery at 1 h may simply be that the initially damaged mitochondria seen at 30 mins are no longer part of the 1 h percoll isolation.

A second possibility is that an initial post-traumatic increase in intracellular and mitochondrial calcium leads to an early transient burst in ROS, which produces a reversible degree of oxidative damage and functional impairment. Indeed, a rapid increase in ^†OH production in the injured mouse (Hall et al, 1993) and rat (Smith et al, 1994) brain has been shown after severe TBI. However, in both cases, the increase in ^†OH ablated by 1 h. Some of the current findings support this explanation for the initial transient decrease in the RCR. Although an increase in mitochondrial HNE and 3-NT is seen in the 30-min cortical sample indicative of ONOO⁻ induced damage, the ability of the 30-min mitochondrial sample to buffer calcium is no different from that seen in sham, noninjured, brains. This suggests that most of the mitochondria in the 30-min postinjury sample may still be viable, although partially uncoupled and thus functionally impaired. The subsequent recovery in RCR, state III and state IV respiration may then signal that many mitochondria in the population have not actually undergone mPT, and are therefore able to recover from the initial wave of ROS production and oxidative damage.

At 3 h after injury, a secondary, more severe, wave of functional depression was seen, which was characterized by a 70% decrease in RCR (compared with sham) in both the cortical and the hippocampal mitochondria. As explained above concerning the initial 30-min decline in RCR, this secondary suppression in RCR is in part due to a significant increase in state IV respiration, which could imply that mPT was occurring in some of the isolated mitochondria, decreasing the overall RCR for the sample. Indeed, state IV was increased by 233% in the cortical mitochondria and 185% in the hippocampal sample. Maximal calcium-buffering capacity, examined in cortical mitochondria, was reduced by 53% at 3 h. This mitochondrial functional failure was paralleled by severe ultrastructural damage to the cortical mitochondria at 3 h, including swelling, dilated cristae and broken outer membranes, which are all indicative of the onset of mPT and subsequent osmotic swelling. Many fewer normal-looking mitochondria were observed in comparison to either the sham or the 30-min postinjury samples. Oxidative damage was observed in the mitochondria from both regions at 3h: increased protein carbonyl and 3-NT in the cortical sample and increased HNE and protein carbonyl in the hippocampal sample.

The increase in state IV respiration, indicative of an increased inner membrane proton conductance, was also coupled with a progressive decrease in state III respiration (oxidative phosphorylation) in the injured cortex and hippocampus at 6 and 12 h after injury. This decrease in state III respiration signals an impairment of electron transport chain and/or enzymes of the citric acid cycle. In addition, mitochondrial calcium uptake (i.e. calcium-buffering capacity) continued to be severely compromised at 12 h after injury in the injured cortex. Some evidence of mitochondrial oxidative damage persisted at the 12 h time-point (increase in HNE in cortical mitochondria, increase in 3-NT in hippocampal mitochondria). However, the overall time course of oxidative damage suggests that the peak was probably at 3 h. Ultrastructural examination of a cortical sample at 12 h revealed very few normal-looking mitochondria.

At 24 h after injury, mitochondrial oxidative phosphorylation (state III respiration) appeared to recover to sham levels in both brain regions. However, at the same time, proton conductance (state IV respiration) remained significantly increased, which resulted in a sustained reduction in RCR. The most logical explanation for this apparent recovery, in part based on the mitochondrial ultrastructural analysis, would be that after 12 h the majority of damaged mitochondria would not be present at 24 h after injury. The reason for this is that the Percoll isolation itself selects for relatively uninjured mitochondria based on density. Additionally, since the preparation contains mitochondria that are synaptic as well as nonsynaptic in origin, mitochondria from inflammatory cells that entered the CNS after TBI could also account for part of this apparent recovery (Lifshitz et al, 2003, 2004; Sullivan and Brown, 2005; Sullivan, 2005). Nevertheless, even at these later time-points a significant tertiary loss of mitochondrial bioenergetics was apparent and represented by a sustained decrease in RCR and increase in proton conductance during state IV respiration, indicating an ongoing disruption of mitochondrial functional integrity.

Ongoing studies in our laboratories are evaluating the relative efficacy of ONOO⁻-directed pharmacological agents in preserving mitochondrial function against ONOO⁻ in both in vitro and in vivo TBI models. However, the current time-course analysis suggests that if ONOO⁻ is truly a major underlying cause of mitochondrial dysfunction, the therapeutic efficacy window may only be 3 h in length. Later treatment initiation might be able to protect against the late-phase damage and dysfunction that becomes manifest between 24 and 72 h, but would miss the initial major component that appears to occur during the first several hours. Ideally, an attempt to pharmacologically protect mitochondria from ONOO⁻ -induced oxidative damage would be initiated within 3 h and then maintained for at least 48 to 72 h to optimally reduce mitochondrial dysfunction throughout its full time course and to exert the best neuroprotective effect.

Acknowledgements

The authors thank Tonya Gibson Hurst, Kristen Day and Brian Thompson for expert technical assistance.

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Journal of Cerebral Blood Flow & Metabolism

Volume 26, Issue 11

Pages: 1407 - 1418

Article first published: November 2006

Issue published: November 2006

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PubMed: 16538231