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Journal of Cerebral Blood Flow & Metabolism (2006), 1–12
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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 Singh1, Patrick G Sullivan1, Ying Deng, Lamin H Mbye and Edward D Hall
Spinal Cord & Brain Injury Research Center and Department of Anatomy & Neurobiology, University of
Kentucky Medical Center, Lexington, Kentucky, USA
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 3 h 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 posttraumatic 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-KNO2 + KOH) may be particularly effective for
the treatment of TBI, although the therapeutic window for this neuroprotective approach might only
be 3 h.
Journal of Cerebral Blood Flow & Metabolism advance online publication, 15 March 2006; doi:10.1038/sj.jcbfm.9600297
Keywords: mitochondria; mitochondrial permeability transition; oxidative damage; traumatic brain injury
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
Correspondence: Dr ED Hall, Spinal Cord & Brain Injury Research
Center, University of Kentucky Medical Center, B383 BBSRB, 741
S. Limestone Street, Lexington, KY 40536-0509, USA.
E-mail: [email protected]
1
These authors contributed equally to this work.
This research was supported by funding from the NIH (NS 1R01
NS46566 and NS048191) and the Kentucky Spinal Cord & Head
Injury Research Trust (KSCHIRT).
Received 8 November 2005; revised 23 December 2005; accepted
25 January 2006
and apoptotic processes in mammalian cells. Disruption of mitochondrial membrane potential (Dcm)
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,
Mitochondrial dysfunction in TBI
IN Singh et al
2
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 posttraumatic 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 3 h 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 posttraumatic mitochondrial dysfunction. Based on its
time course, a major player in this oxidative damage
scenario appears to be the ROS peroxynitrite (ONOO).
Journal of Cerebral Blood Flow & Metabolism (2006), 1–12
Materials and methods
Materials
Mannitol, sucrose, bovine serum albumin (BSA), ethyleneglycol tetraacetate (EGTA), N-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid (HEPES) potassium salt,
potassium phosphate monobasic anhydrous (KH2PO4),
magnesium chloride (MgCl2), malate, pyruvate, adenosine
50 -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 371C 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.
Mitochondrial dysfunction in TBI
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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 PotterElvejhem 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 41C. First, the
homogenate was centrifuged twice at 1300g for 3 mins in
an Eppendorf microcentrifuge at 41C 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 41C 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 41C. 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
41C. The supernatants were carefully removed, the pellet
resuspended in isolation buffer without EGTA and
centrifuged at 13,000 g for 10 mins at 41C. 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 41C
to yield a tighter pellet. The final mitochondrial pellet was
resuspended in isolation buffer without EGTA to yield a
concentration of B10 mg/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 371C as
described previously (Sullivan et al, 2000a, 2003; Jiang
et al, 2001; Sensi et al, 2003). In all, 25 to 40 mg of isolated
mitochondrial protein was placed in the chamber containing 250 mL 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 mmol/
3
L ADP were added to the mitochondria to initiate state III
respiratory rate for 2 mins, followed by the addition of
2 mmol/L oligomycin to monitor state IV respiration rate
for an additional 2 mins. For the measurement of uncoupled respiratory rate, 2 mmol/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 371C. 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 (Dcm) 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 150 nmol/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 mmol/L ADP
at 2 mins and 1 mmol/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 mL/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 mg of mitochondrial protein was transferred to a Protran (0.2 mm) nitrocellulose membrane
(Schleicher & Schuell, Dassel, Germany) by a Minifold II
Journal of Cerebral Blood Flow & Metabolism (2006), 1–12
Mitochondrial dysfunction in TBI
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vacuum slot blot apparatus (Schleicher & Schuell). Each
slot was washed with 300 mL 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 41C. 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 41C. 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
41C. 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 41C before being embedded
for electron microscopy. The fixed mitochondrial pellets
were then washed overnight at 41C 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 601C. 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 mean7s.e.m. For singlevariable comparisons, Student’s t-test was used. For
multiple-variable comparisons, data were analyzed by
one-way analysis of variance (ANOVA) followed by posthoc analysis using the Student–Neuman–Keul’s (SNK)
test.
Journal of Cerebral Blood Flow & Metabolism (2006), 1–12
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.
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 6 h, 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.
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
Mitochondrial dysfunction in TBI
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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 mg) 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 mL. 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 150 mmol/L ADP (state III). Data are presented as mean7s.e.m.
Statistical differences (one-way ANOVA and Student–Neuman—Keuls post hoc test): *P < 0.0001 versus sham; #P < 0.001 versus
sham.
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 3 h, 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.
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
Journal of Cerebral Blood Flow & Metabolism (2006), 1–12
Mitochondrial dysfunction in TBI
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Pyr+Mal ADP-1
ADP-2
200
Oligomycin FCCP
180
Oxygen (nmols O2/ml)
160
140
3 hrs Post-Injury
120
Succinate
100
80
Sham (Control)
60
40
20
0
0
0.5
1
1.5
2
2.5
3
Time (min)
3.5
4
4.5
5
5.5
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.
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).
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
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 mg) was suspended in respiration buffer, in a final volume of
250 mL, 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 mmol/L) or oligomycin (state IV), while using the
oxidative substrates, pyruvate and malate. Data are presented as mean7s.e.m. Statistical differences (one-way ANOVA and SNK
post hoc test): *P < 0.0001 versus sham; #P < 0.001 versus sham.
Journal of Cerebral Blood Flow & Metabolism (2006), 1–12
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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 371C. The extramitochondrial concentrations of Ca2 + were measured using the
fluorescent Ca2 + -sensitive indicator CaG5N. Mitochondria
(0.1 mg/mL) were incubated with 5 mmol/L pyruvate and
2.5 mmol/L malate, 150 mmol/L ADP, and 1 mmol/L oligomycin.
At 5 mins after the final addition (oligomycin), 32 mmol/L
calcium, at a rate of 0.5 mL/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 mean7s.e.m. Statistical differences (oneway ANOVA and SNK post hoc test): *P < 0.05 versus sham.
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.
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 posttraumatic 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 KNO 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 (KNO2) and hydroxyl
radical (KOH). Probably more important physiologically, ONOO will react with carbon dioxide (CO2)
to form nitrosoperoxocarbonate (ONOOCO2), which
can decompose into KNO2 and carbonate radical
(KCO3). Each of the ONOO-derived radicals (KOH,
K
NO2, and KCO3) 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
Journal of Cerebral Blood Flow & Metabolism (2006), 1–12
Mitochondrial dysfunction in TBI
IN Singh et al
8
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 (3 h) animals. Approximately 2.5 mg 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 mean7s.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.
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
Journal of Cerebral Blood Flow & Metabolism (2006), 1–12
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
K
NO appear to be important in neuronal degeneration. For instance, KNO production in mitochondria
occurs during apoptosis (Bustamante et al, 2002),
Mitochondrial dysfunction in TBI
IN Singh et al
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 KNO2 and KCO3 (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 (BeitYannai et al, 1996; Zhang et al, 1998).
9
Dissection of the Time Course of Oxidative Damage
and Mitochondrial Functional Impairment
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), 3 h (C) and 12 h (D) postinjury were
fixed in 4% glutaraldehyde at 41C before being embedded for
electron microscopy. The scale bar represents 500 nm.
and mtNOS stimulation can cause cytochrome c
release (Ghafourifar et al, 1999). Deregulation of
mitochondrial KNO 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,
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 posttraumatic 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 KOH 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 KOH 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
Journal of Cerebral Blood Flow & Metabolism (2006), 1–12
Mitochondrial dysfunction in TBI
IN Singh et al
10
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 3 h:
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 normallooking 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 inJournal of Cerebral Blood Flow & Metabolism (2006), 1–12
creased, 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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