(Stroke. 2001;32:506.)
© 2001 American Heart Association, Inc.
Original Contributions |
Increased Cytochrome c–Mediated DNA Fragmentation and Cell Death in Manganese–Superoxide Dismutase–Deficient Mice After Exposure to Subarachnoid Hemolysate
From the Department of Neurosurgery (P.G.M., M.F., A.L., Y.M-F., P.H.C.), Departments of Neurology and Neurological Sciences (M.F., A.L., Y.M-F., P.H.C.), and Program in Neurosciences (M.F., A.L., Y.M-F., P.H.C.), Stanford University School of Medicine, and Surgical Service, Palo Alto Veterans Affairs Health Care System (P.G.M.) (Calif).
Correspondence to Paul G. Matz, MD, Surgical Service (112N), Palo Alto VA Medical Center, 3801 Miranda Ave, Palo Alto, CA 94304. E-mail matzpg{at}yahoo.com
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Abstract |
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Background and Purpose—We sought to investigate the mechanisms for oxidative injury caused by subarachnoid hemolysate, a pro-oxidant.
Methods—Injection of 50 µL of subarachnoid hemolysate or saline was performed in CD1 mice (n=75), mutant mice deficient in Mn–superoxide dismutase (Sod2+/-; n=23), and their wild-type littermates (n=23). Subcellular location of cytochrome c was studied by immunocytochemistry, immunofluorescence, and immunoblotting of cellular fractions. DNA fragmentation was assessed though DNA laddering and terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end-labeling (TUNEL). Cell death was examined through basic histology.
Results—Cytochrome c immunoreactivity was present in the cytosol of neurons at 2 hours after hemolysate injection and increased by 4 hours compared with saline-injected animals (P<0.02). Cytosolic cytochrome c was more abundant in Sod2+/- mutants. DNA fragmentation was evident at 24 hours, but not 4 hours, after hemolysate injection as determined by DNA laddering and TUNEL staining (P<0.02). DNA fragmentation colocalized to cells with cytosolic cytochrome c and iron. In Sod2+/- mutants, the extent of fragmentation was increased as determined by TUNEL staining (52% increase; P<0.02) and DNA laddering (optical density=0.819 versus 0.391; P<0.01). Cell death was evident on basic histology as early as 4 hours after hemolysate injection. No cell death was evident in controls. In Sod2+/- mutants, cell death was increased by 51% compared with wild-type littermates (P<0.05).
Conclusions—These results demonstrate that subarachnoid blood products are associated with the presence of cytochrome c in the cytosol and subsequent cell death in neurons. It appears that Mn–superoxide dismutase plays a role in preventing cell death after exposure to subarachnoid blood products.
Key Words: hemoglobin • iron • stroke, experimental • subarachnoid hemorrhage • superoxide dismutase • mice
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Introduction |
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Oxidative stress has been implicated in the pathogenesis of cell injury after subarachnoid blood hemorrhage.1 2 3 Lysis of extravasated, subarachnoid red blood cells permits the release of blood constituents, including free hemoglobin.4 Hemoglobin is an iron-containing protein that is rapidly distributed throughout the subarachnoid space and has been linked to the development of lipid peroxidation, oxidative cell membrane injury, DNA damage, and cell death.5 6 7 8 9 10
Oxidative stress in brain has been associated with superoxide production, mutagenesis of nuclear genes, irreversible and reversible DNA damage, and mitochondrial injury.11 12 13 14 15 16 17 18 Cytochrome c, a mitochondrial membrane protein, functions in the respiratory chain to transport electrons from coenzyme QH2–cytochrome c reductase to the cytochrome oxidase.19 Recent attention has focused on its relationship to DNA fragmentation.20 Injury severe enough to release cytochrome c into the cytosol has been linked to subsequent fragmentation of DNA and cell death.15 Within the cytosol, cytochrome c activates caspase-3, which has been shown to trigger apoptosis.20 21 The presence of mitochondrial Mn–superoxide dismutase (Mn-SOD) appears to attenuate cytochrome c translocation after oxidative injury. This same protein is associated with protection from DNA fragmentation and cell death.15
Recently, cell death and DNA fragmentation have been described after intracranial hemorrhage.22 23 Prior studies have suggested that some cell damage may be a direct consequence of blood toxicity and independent of intracranial pressure changes and secondary ischemia.2 24 Pretreatment with antioxidants has been shown to attenuate cellular and vascular damage after exposure to subarachnoid blood.2 3 Although deposition of blood products in the subarachnoid space has been associated with oxidative injury, the specific subcellular location of their action has not been defined. It is also uncertain if their effect is deleterious enough to produce mitochondrial injury and to perturb the localization of cytochrome c. Therefore, we decided to investigate whether cytochrome c is translocated to cytosol after exposure to subarachnoid hemolysate ("heme") before cell death. We also investigated cell injury after exposure to heme in mutant mice with a heterozygous knockout gene encoding Mn-SOD (Sod2+/-). We hypothesized that reduced expression of Mn-SOD would be associated with cytosolic cytochrome c, DNA fragmentation, and cell death after exposure to subarachnoid blood products.
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Materials and Methods |
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Animal Preparation
For the first part of the study, CD1 male mice (aged 3 months; weight, 35 to 40 g) were studied. Because homozygous knockout mutants (Sod2-/-) showed neonatal lethality due to dilated cardiomyopathy,25 26 heterozygous knockout mutants (Sod2+/-) were used for the second part of this study. The Sod2+/- mice with a CD1/SV129 background were backcrossed with CD1 mice for 10 generations. These mice and their wild-type (Wt) littermates with an identical genetic background were studied. In prior studies that investigated oxidative stress from focal stroke, these same Sod2+/- mutants had Mn-SOD activity (0.1 U/mg protein) that was approximately 50% that of their Wt littermates (0.2 U/mg protein).26 In these same animals, CuZn-SOD activity was similar (5 U/mg protein) between Sod2+/- mutants and Wt littermates.26 For this study, Mn-SOD activity was measured in Sod2+/- mutants (n=3) and Wt littermates (n=3) by previously described methods.27 The Sod2+/- mutants had a level of Mn-SOD activity that was 33% less than that of Wt littermates (P<0.05).
Experimental Model of Blood Exposure
All procedures were in accordance with the National
Institutes of Health Guide for the Care and Use of Laboratory Animals
and were approved by Stanford University’s Administrative Panel on
Laboratory Animal Care. Subarachnoid injection of heme or
saline was performed in male CD1 mice (n=75),
Sod2+/- mutants (n=23), and
Wt littermates (n=23) by a method previously
described.23 Experimental
animals were anesthetized with a mixture of 68% nitrous oxide,
30% oxygen, and 2% isoflurane by mask ventilation. The rectal
temperature was controlled at 37±0.5°C with a homeothermic blanket.
After cannulation, 50 µL of autologous blood was withdrawn from the
femoral artery and placed into a sterile container. Blood was lysed by
freezing and thawing in dry ice. An equivalent volume of normal saline
was then replaced intraperitoneally.
Animals were then placed into a stereotaxic frame (Kopf Instruments). The posterior scalp was incised on the midline, and the skull was exposed at the junction of the parietal and occipital bones. A small burr hole was made 2 mm to the right of midline. The heme was then placed in a sterile 1-mL syringe with a 30-gauge needle. The needle was introduced through the burr hole and placed into the subarachnoid space over the dorsal aspect of the cortex beneath the coronal suture. Heme (n=69) or sterile saline for controls (n=52) was then slowly injected over 1 minute. The needle was removed, and the burr hole was filled with sterile bone wax. The incision was then closed.
Cytochrome
c Immunocytochemistry
At 2, 4, or 24 hours after heme or saline injection,
subjects (n=6 for each group) were anesthetized and killed by
transcardiac perfusion with heparinized 0.1 mol/L PBS (pH
7.4) followed by 3.7% formaldehyde. After endogenous
peroxidase and nonspecific binding were blocked, sections (25 µm)
were reacted with a rabbit polyclonal anti–cytochrome
c antibody (1/100, Santa Cruz
Biotechnologies) for 48 hours at 4°C. The rabbit polyclonal
anti–cytochrome c antibody has
been shown to detect cytosolic but not mitochondrial cytochrome
c. This antibody is not able to
penetrate consistently through the mitochondrial membrane;
consequently, minimal immunostaining is observed in
normal animals, and appearance of mitochondrial staining is distinctly
different from cytosolic
staining.28 29 30
Sections were reacted with biotin-conjugated goat anti-rabbit IgG antibody (1/300, Vector Laboratories) and visualized with the use of avidin-biotin horseradish peroxidase (ABC kit, Vector Laboratories) followed by 0.02% diaminobenzidine and 0.06% H2O2. The sections were counterstained with methyl green. As a negative control, some sections were incubated without primary antibody.
Cytochrome
c Immunofluorescent
Double Labeling
After reaction with the primary anti–cytochrome
c antibody, sections were
reacted with goat anti-rabbit Fab fragments (1/50, Jackson
Immunoresearch) followed by Texas red–conjugated donkey anti-goat
antibody (1/100, Jackson Immunoresearch) in PBS. Sections were then
incubated with a rabbit anti-neurofilament antibody (1/500, Serotec)
for 48 hours at 4°C followed by a biotin-conjugated goat anti-rabbit
antibody (1/200, Vector Laboratories) and
fluorescein-conjugated avidin D (1/200, Vector
Laboratories).
Western Blot Analysis of
Cytochrome c
Protein extraction of both the mitochondrial and
cytosolic fractions was performed as
described.20 28
Animals were killed at 2 or 4 hours after heme or saline injection (n=6
for each group). Approximately 50 mg of neocortex was removed from the
hemorrhagic or saline-injected hemisphere. Tissue was
homogenized in 7 volumes of cold suspension buffer with
protease inhibitors. Homogenates were
centrifuged at 750g at
4°C. Supernatants were then centrifuged at
8000g at 4°C for 20 minutes.
The 8000g pellets were
resuspended and used as the mitochondrial fraction. Supernatants were
further centrifuged at
100 000g at 4°C for 1 hour.
The final cytosolic supernatants were then removed. Protein
concentrations were determined by the Bradford method
(Bio-Rad).
Proteins (6.2 µg of cytosolic fraction or 2.6 µg of mitochondrial fraction) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 10% to 20% Tris-glycine gel (Novex) and transferred to a polyvinylidene difluoride membrane (Novex). Western blots were performed with the use of a rabbit polyclonal antibody against mouse cytochrome c (Santa Cruz Biotechnologies) at a dilution of 1:1000 or 1 µg/mL of 20E8C12 mouse monoclonal antibody to cytochrome oxidase subunit IV (COX, Molecular Probes). Immunoblots were visualized with the use of enhanced chemiluminescence Western blotting detection reagents (Amersham). A densitometric analysis was made on the mitochondrial fractions and cytosolic fractions from both hemorrhage and control hemispheres. The films were scanned by GS-700 imaging densitometer (Bio-Rad), and the results were quantified with the use of the Multi-analyst software program (Bio-Rad). Western blot analysis of ß-actin was performed with the use of a mouse monoclonal antibody with horseradish peroxidase–conjugated anti-mouse IgG reagents (Amersham).
In Situ Labeling of DNA
Fragmentation
At 4 or 24 hours after heme or saline injection,
subjects (n=6 CD1/saline, CD1/heme,
Sod2+/-/heme, Wt/heme,
Sod2+/-/saline, Wt/saline)
were anesthetized and killed by transcardiac
perfusion. Sections (25 µm) were reacted with Proteinase K (20 mg/L,
Boehringer Mannheim) in 0.01 mol/L Tris-HCl (pH 8.0) and
equilibrated with 1x terminal deoxynucleotidyl
transferase (TdT) buffer (Life Technologies) for 15 minutes. Sections
were then reacted with TdT enzyme (375 U/mL buffer, Life Technologies)
and biotinylated 16-dUTP (60 nmol/mL buffer, Boehringer
Mannheim) in 1x TdT buffer at 37°C for 60 minutes. Slides were
washed with 2x SSC (150 mmol/L sodium chloride, 15 mmol/L
sodium citrate, pH 7.4) followed by 2% bovine serum albumin.
Staining was visualized with the use of avidin-biotin horseradish
peroxidase solution (ABC kit, Vector Laboratories) followed by 0.025%
diaminobenzidine, 0.04 mol/L nickel sulfate, and 0.075%
H2O2 in 0.175 mol/L
sodium acetate. Sections were counterstained with methyl
green.
Cytochrome
c
Immunostaining With In Situ Double Labeling of DNA
Fragmentation
Adjacent sections were removed from subjects killed
at 24 hours after heme or saline injection (n=6 each group). Sections
were stained first for cytochrome
c and subsequently for DNA
fragmentation with the techniques described
above.
Iron Histology With In Situ Double
Labeling of DNA Fragmentation
To clarify the role of iron deposition, adjacent
sections were removed from subjects killed at 24 hours after heme or
saline injection (n=6 in each group). Sections were washed in deionized
water and reacted with 0.024 mol/L potassium ferrocyanide (Sigma) in
0.37% HCl for 15 minutes.31
Sections were then reacted with 0.025% diaminobenzidine, 0.075%
H2O2, and 0.1% Tween-20
for 15 minutes. Sections were then stained for DNA
fragmentation.
Gel Electrophoresis
At 4 or 24 hours after heme or saline injection,
subjects (n=6 CD1/saline, CD1/heme,
Sod2+/-/heme, Wt/heme,
Sod2+/-/saline, Wt/saline)
were anesthetized and killed by decapitation. Approximately 40
to 50 mg of neocortex was dissected from the region underlying the area
of injection. Samples were incubated in 0.6 mL of lysis buffer (0.5%
SDS, 0.01 mol/L Tris-HCl, and 0.1 mol/l EDTA) with 0.6 mg Proteinase K
at 55°C for 20 hours. The DNA was extracted with equal volumes of
phenol and phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated
in 0.2 mol/L sodium chloride in 100% ethanol at -80°C for 24
hours. DNA was resuspended in DNase-free H2O
(Sigma Chemical), and concentration was measured with the use of
To-Pro-1 dye (Molecular Probes).
Gel electrophoresis for detecting DNA laddering was performed as described.28 Before electrophoresis, 1 µg of DNA was incubated with 50 mg/L DNase-free RNase (Boehringer Mannheim) and then reacted with Klenow enzyme and dNTP in 1x Klenow buffer (Trevigen). Samples were mixed with loading buffer and subjected to electrophoresis on a 1.5% agarose gel. The gel was washed sequentially with 0.25 mol/L HCl, 0.4 mol/L NaOH/0.8 mol/L NaCl, and 0.5 mol/L Tris buffer, pH 7.5. DNA was transferred to a nylon membrane overnight in 10x SSC and reacted with streptavidin–horseradish peroxidase conjugate (Trevigen) for 30 minutes. Bands were visualized by the chemiluminescence method with the use of PeroxyGlow (Trevigen) followed by exposure to chemiluminescence film. Bands were compared by densitometry, as described above.
Histological Assessment of Cell
Death
At 4 or 24 hours after heme or saline injection (n=6
CD1/saline, CD1/heme,
Sod2+/-/heme, Wt/heme,
Sod2+/-/saline, Wt/saline),
subjects were anesthetized and killed. After
transcardiac perfusion fixation, 25-µm sections were
mounted on slides and dried overnight. Sections were then dehydrated,
delipidized, rehydrated, and stained with cresyl violet for 5 minutes.
Sections were dehydrated and coverslipped.
Statistical Analysis
For TdT-mediated dUTP-biotin nick end-labeling
(TUNEL) staining and basic histology, 12 serial sections were taken at
300-µm intervals beginning at the coronal suture and continuing
posteriorly. This encompassed a region of 3.6 mm adjacent to the
area of heme injection. Counts of TUNEL-positive cells and dead cells
were then performed by a blinded reviewer using a light microscope
equipped with a 250x250-µm grid. Counts were completed for all 12
sections in each animal. Thereafter, the codes were broken, and average
cell counts for Wt and
Sod2+/- mutants were compared
with an unpaired Student’s t
test (2-tailed). For assessment of mortality and evaluation of the
numbers of mice with cytochrome
c–immunopositive cells,
comparisons were made with a 2-tailed Fisher exact test. Finally, for
densitometric analysis, average optical densities were obtained
from Sod2+/- mutants (n=6)
and Wt littermates (n=6). Average optical densities were also obtained
from heme- (n=6) and saline-injected (n=6) subjects for cytochrome
c and COX analysis.
Results were compared with an unpaired Student’s
t test (2-tailed). Significance
was determined by a P value
<0.05.
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Results |
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After injection of heme, an 8% mortality rate was observed. Differences in survival were not statistically significant compared with saline-injected animals or between Sod2+/- mutants and Wt littermates. With heme injection, subarachnoid blood was evident in the region overlying the dorsal, right neocortex beneath the parietal bone. The appearance of the heme was similar in Sod2+/- mutants and Wt mice (Figure 1
) and was consistent with prior reports
using a similar
method.23
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Cytochrome
c Immunocytochemistry,
Immunofluorescence, and
Immunoblotting
Minimal cytochrome
c immunoreactivity was detected
in saline-injected animals at 2, 4, and 24 hours
(Figure 2A), a result consistent with the minimal
immunoreactivity of mitochondrial cytochrome
c.28 29
Cytochrome c immunoreactivity
was only evident in sparse, small fragments, consistent with
the occasional staining of mitochondria. This result compares with
prior studies and is consistent with the fact that primary
antibodies do not usually penetrate through mitochondrial
membranes.28 29
Sparse immunoreactivity was evident at 2 hours when the primary
antibody was eliminated, suggesting some immunoreactivity of the
secondary antibody
(Figure 2B). However, after heme injection, significant
cytosolic cytochrome c
immunoreactivity was observed in the ipsilateral neocortex (5/6 animals
at 2, 4, and 24 hours;
Figure 2C through 2E). These results were significant when
compared with controls for each interval
(P<0.02).
Double-immunofluorescent labeling demonstrated these cells to
be neurons
(Figure 2F and 2G). Immunostaining decreased
with distance from the injection site.
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After saline injection, COX and cytochrome
c immunoblots of
mitochondrial fractions demonstrated positive bands characteristic of
these proteins, while no positive bands were present in the cytosol
(Figure 3A). After either heme or saline injection, COX
immunoblotting was observed in the mitochondrial
fractions but not in the cytosolic fractions
(Figure 3A). The intensity
(Figure 3B) of the mitochondrial COX bands did not differ
between heme and saline. In contrast, the intensity of mitochondrial
cytochrome c
immunoblotting (optical density=0.146±0.013;
Figure 3B) was significantly diminished after heme injection
compared with the saline-injected animals (optical
density=0.182±0.028; P<0.05).
After heme injection, cytochrome
c
immunoblotting was evident in the cytosolic fractions
(Figure 3A and 3C). When compared with controls at 2 and 4
hours (optical density=0.105±0.010 and 0.102±0.017, respectively),
the degree of cytochrome c
immunoblotting
(Figure 3D) was significant at 2 (optical
density=0.241±0.052; P<0.02)
and 4 hours (optical density=0.396±0.165;
P<0.02).
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Cytosolic cytochrome
c was also observed in
Sod2+/- mutants after heme
injection
(Figure 3E
). The optical density of these
immunoblots increased
(Figure 3D and 3E) in
Sod2+/- mutants (optical
density=0.641±0.221) compared with Wt littermates at 4 hours (optical
density=0.351±0.145; P<0.05).
ß-Actin immunoblotting of cytosolic fractions showed
bands of similar intensity between saline and heme subjects
(Figure 3C and 3E).
DNA Fragmentation
DNA fragmentation was analyzed with the in situ
labeling of DNA breaks (TUNEL) at 4 and 24 hours after heme or saline
injection. DNA fragmentation was not evident after injection of saline
at 4 or 24 hours (0/6 in each group;
Figure 4A). At 4 hours after heme exposure, no
TUNEL-positive cells were observed adjacent to the area of injection
(Figure 4B). At 24 hours after heme exposure, DNA
fragmentation was observed in the ipsilateral neocortex in 5 of 6
subjects
(Figure 4C). TUNEL-positive cells were observed adjacent to
the site of heme injection and had morphologies consistent with
both apoptosis and
necrosis.32 33
The difference in the number of TUNEL-positive subjects at 4 and 24
hours after SAH (0/6 versus 5/6;
P<0.02) was statistically
significant and suggested that the onset of DNA fragmentation was a
delayed phenomenon.
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DNA fragmentation was not evident after injection of
saline in either Sod2+/-
mutants or Wt mice
(Figure 4D). DNA fragmentation was observed in the
ipsilateral neocortex of
Sod2+/- mutants and Wt mice
after injection of heme
(Figure 4E and 4F). The abundance of TUNEL-positive cells
differed significantly between
Sod2+/- mutants and Wt mice
(Figure 4G).
Sod2+/- mutants had a
significantly greater number of TUNEL-positive cells per square
millimeter (±SD) (1052±247) than Wt mutants (692±154;
P<0.02).
Colocalization of fragmented DNA and either cytochrome
c or cytosolic ferric iron was
investigated 24 hours after heme injection through double staining. At
24 hours, cytochrome
c–immunopositive cells were
also TUNEL-positive
(Figure 4H). Additionally, if cells were stained for
intracellular ferric iron with Prussian
blue,31 the accumulation of
intracellular iron occurred primarily in TUNEL-positive cells
(Figure 4I). However, some cells were TUNEL-positive without
iron, while others stained positive for iron but not TUNEL
(Figure 4I).
To investigate further the time course of DNA fragmentation
as characterized by intranucleosomal DNA fragments, we analyzed
by gel electrophoresis DNA extracted from neocortex 4 or 24 hours after
heme or saline injection. No DNA laddering was evident in the
saline-injected animals at 24 hours, nor was it present 4 hours
after heme injection
(Figure 5A). DNA laddering was present in the
neocortex 24 hours after heme injection
(Figure 5A). In Wt mice and
Sod2+/- mutants, DNA
laddering was not evident 24 hours after injection of saline (not
shown). DNA laddering was present in neocortex 24 hours after heme
injection in Wt mice and
Sod2+/- mutants
(Figure 5B). The intensity of the DNA laddering ±SD was
significantly greater
(Figure 5C) in
Sod2+/- mutants (optical
density=0.819±0.318) than in Wt littermates (optical
density=0.391±0.055;
P<0.01).
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Cell Death
The presence of cell death was evaluated by
histological assessment after cresyl violet staining.
Irreversibly damaged neocortical cells demonstrated shrunken or
condensed nuclei, often with a prominent nucleolus. After injection of
saline, no cell death was evident in neocortex in either Wt mice or
Sod2+/- mutants
(Figure 6A and 6B). Cell death was evident at 4 hours after
injection of heme in Wt mice
(Figure 6C). Cell death was abundant at 24 hours in both Wt
mice and Sod2+/- mutants.
Necrotic cells were evident closest to the site of heme injection
(Figure 6C through 6E). The abundance of dead cells per
square millimeter (±SD) was significantly greater in
Sod2+/- mutants (1603±466)
than in Wt littermates (1057±364;
P<0.05;
Figure 6F).
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Cytochrome c Immunostaining and Immunoblotting
After subarachnoid injection, blood products have been observed to disseminate throughout the brain within a few hours and to be sequestered by neurons and microglia.5 Intracranial blood products have been associated with the development of DNA fragmentation in neocortical regions of highest blood concentration.22 23 In our study cytochrome c immunoreactivity was observed at 2 hours in neocortical neurons closest to the site of heme injection. Because cytochrome c is a water-soluble protein, the diffuse intracellular immunoreactivity was presumably a consequence of protein dispersion throughout the cytosol after mitochondrial perturbation. Cytochrome c immunoreactivity was not observed in the contralateral hemisphere after heme injection, nor was it apparent in the brains of saline-injected animals. This result was consistent with recent observations that, with the use of current immunocytochemical methods, mitochondrial cytochrome c itself does not produce immunopositivity.28 29
The cytochrome c protein is synthesized in the endoplasmic reticulum but becomes associated with the mitochondria.19 Immunoblotting of subcellular fractions demonstrated the cytosolic presence of cytochrome c after heme exposure. Presumably, the cytosolic appearance of the cytochrome c protein could have arisen from impaired transport or increased synthesis after heme exposure. Although the endoplasmic reticulum and ribosomes were removed by ultracentrifugation and the increase in cytosolic cytochrome c was matched by a decrease in mitochondrial cytochrome c, a distinct possibility existed that protein transport to the mitochondria was impaired and that such impairment accounted for the presence of the cytosolic cytochrome c. Certainly, disruption of the mitochondrial membrane could have permitted cytochrome c to enter the cytosol. However, a severe disruption of the mitochondrial membrane presumably would have allowed COX to enter the cytosol, and our results with COX immunoblotting did not support this hypothesis. The last possibility is that cytochrome c was translocated to the cytosol of neurons after exposure to heme. Exploring the specific mechanism by which oxidative stress increases cytosolic cytochrome c will form the basis for further studies.
TUNEL Staining and DNA
Electrophoresis
In this study we primarily observed a pattern of TUNEL
staining consistent with the intranucleosomal DNA fragmentation
of apoptosis. TUNEL-positive cells were observed 24 hours after
heme but not at 4 hours. Prior studies have demonstrated that the
presence of TUNEL-positive cells and DNA laddering in the brain does
not necessarily equate with archetypal apoptosis, although
their presence is consistent with some form of DNA
fragmentation.34
TUNEL-positive cells may also represent cell death by necrosis,
albeit with a different
appearance.32 33
In this study some TUNEL-positive cells were indeed observed that were
consistent with necrosis. The presence of DNA laddering on gel
electrophoresis, however, suggested that most TUNEL-positive cells in
neocortex had undergone DNA fragmentation in a pattern
consistent with apoptosis. After we used both of these
methods to examine DNA fragmentation, it was evident that this
phenomenon did not occur early but was rather delayed in onset after
both heme exposure and the presence of cytochrome
c in the
cytosol.
DNA Fragmentation and Mn-SOD
Recent evidence has shown DNA fragmentation to be
linked to elevated superoxide production in
mitochondria.12 13 35
One of the principal scavengers of this anion is Mn-SOD. Reduction of
its expression has been associated with an increase in DNA
fragmentation and cell injury after focal
ischemia.15 25
In our study reduction of Mn-SOD expression was associated with the
presence of cytochrome c in the
cytosol, DNA fragmentation, and cell death.
After subarachnoid injection, blood products are rapidly distributed throughout the brain, and hemoglobin is sequestered by neurons and microglia.5 Hemoglobin has been linked to cell injury and oxidative stress.7 10 DNA fragmentation has been observed in areas of highest blood concentration.22 23 In our study DNA fragmentation was associated with the presence of both ferric iron and cytochrome c in the cytosol. It is conceivable that the sequestration of blood products by the brain could directly produce significant mitochondrial oxidative stress. Superoxide anions may form the toxic hydroxyl radical through the Haber-Weiss reaction in the presence of hydrogen peroxide and iron.36 The hydroxyl radical and oxidative stress have been shown to mediate DNA damage.8 17 18 Decreased levels of Mn-SOD may have permitted greater superoxide production and, in the presence of iron, greater hydroxyl radical formation.
Cell Death and Mn-SOD
In addition to the degree of DNA fragmentation, overall
cell death was significantly increased in
Sod2+/- mutants compared with
Wt littermates after exposure to subarachnoid heme. Cell death
may arise as a consequence of necrosis or DNA fragmentation after an
oxidative insult.32 Reactive
oxygen species have been implicated in both
pathways.12 17 18 25 37
As described above, it was possible that superoxide in the presence of
the iron carried by heme may have formed the reactive hydroxyl radical
as a mediator of cell
death.36
Another possible route to cell death and DNA damage may have been through formation of peroxynitrite from nitric oxide and superoxide.12 17 38 Peroxynitrite may damage cells through the formation of the hydroxyl radical and nitrogen dioxide, which may attack endothelial membranes.38 Diminished levels of Mn-SOD may have permitted increased formation of peroxynitrite, which would ultimately have led to DNA damage, DNA fragmentation, and cell death.12
In the endothelium, it was also possible that increased formation of peroxynitrite may have reduced the amount of nitric oxide available for endothelial relaxation. This mechanism could have exacerbated vasospasm and produced secondary ischemia, especially since heme has been a known spasmogen.39 It remains uncertain how blood products begin to increase mitochondrial reactive oxygen species. Investigation of the specific mechanisms will form the basis for future studies.
Reduced expression of Mn-SOD may also have exacerbated brain injury after heme exposure through glutamate neurotoxicity. Intracranial blood has been associated with elevated levels of glutamate in regions around the hematoma.40 Excitotoxicity has been associated with increased mitochondrial reactive oxygen species, mitochondrial injury, necrosis, and apoptosis.12 13 37 Consequently, after exposure to heme, the glutamate neurotoxicity cascade could have developed with subsequent superoxide formation, cell death, and DNA fragmentation. It is conceivable that blocking the excitotoxicity cascade may protect against cell injury after exposure to high concentrations of blood products. Exploring this mechanism of neuroprotection will form the basis for future studies.
In summary, our results have demonstrated that cytochrome c is present in the cytosol of neurons as early as 2 hours after exposure to heme and that such presence precedes cell death by DNA fragmentation. Reduction of Mn-SOD increases the abundance of cytochrome c in the cytosol, DNA fragmentation, and overall cell death after exposure to subarachnoid heme.
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Acknowledgments |
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This study was supported by a Bayer Research Fellowship (Dr Matz) and in part by grants NS14543, NS25372, NS36147, and NS38653 (Dr Chan). Dr Chan is a recipient of a Javits Neuroscience Investigator Award.
Received July 6, 2000; revision received October 26, 2000; accepted October 26, 2000.
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References |
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1. Gaetani P, Pasqualin A, Baena y Rodriguez R, Borasio E, Marzatico F. Oxidative stress in the human brain after subarachnoid hemorrhage. J Neurosurg. 1998;89:748–754.[Medline] [Order article via Infotrieve]
2. Turner CP, Panter SS, Sharp FR. Anti-oxidants prevent focal rat brain injury as assessed by induction of heat shock proteins (HSP70, HO-1/HSP32, HSP47) following subarachnoid injections of lysed blood. Mol Brain Res. 1999;65:87–102.[Medline] [Order article via Infotrieve]
3.
Kamii H, Kato I,
Kinouchi H, Chan PH, Epstein CJ, Akabane A, Okamoto H, Yoshimoto T.
Amelioration of vasospasm after subarachnoid hemorrhage
in transgenic mice overexpressing CuZn-superoxide dismutase.
Stroke. 1999;30:867–872.
4.
MacDonald RL, Weir
BK. A review of hemoglobin and the pathogenesis of cerebral vasospasm.
Stroke. 1991;22:971–982.
5. Turner CP, Bergeron M, Matz P, Zegna A, Noble LJ, Panter SS, Sharp FR. Heme oxygenase-1 is induced in glia throughout brain by subarachnoid hemoglobin. J Cereb Blood Flow Metab. 1998;18:257–273.[Medline] [Order article via Infotrieve]
6.
Braughler JM,
Duncan LA, Chase RL. The involvement of iron in lipid peroxidation.
J Biol Chem. 1986;261:10282–10289.
7. Gutteridge JM. The antioxidant activity of haptoglobin towards haemoglobin-stimulated lipid peroxidation. Biochim Biophys Acta. 1987;917:219–223.[Medline] [Order article via Infotrieve]
8.
Imlay JA, Chin SM,
Linn S. Toxic DNA damage by hydrogen peroxide through the Fenton
reaction in vivo and in vitro.
Science. 1988;240:640–642.
9.
Balla J, Jacob HS,
Balla G, Nath K, Eaton JW, Vercellotti GM.
Endothelial-cell heme uptake from heme proteins:
induction of sensitization and desensitization to oxidant damage.
Proc Natl Acad Sci
U S A. 1993;90:9285–9289.
10. Regan R, Panter SS. Neurotoxicity of hemoglobin in cortical cell culture. Neurosci Lett. 1993;153:219–222.[Medline] [Order article via Infotrieve]
11. Kontos HA, Wei EP. Superoxide production in experimental brain injury. J Neurosurg. 1986;64:803–807.[Medline] [Order article via Infotrieve]
12.
Bonfoco E,
Krainc D, Ankarcrona M, Nicotera P, Lipton SA. Apoptosis and
necrosis: two distinct events induced, respectively, by mild and
intense insults with
N-methyl-D-aspartate or
nitric oxide/superoxide in cortical cell cultures.
Proc Natl Acad Sci
U S A. 1995;92:7162–7166.
13.
Dugan LL, Sensi
SL, Canzoniero LM, Handran SD, Rothman SM, Lin TS, Goldberg MP, Choi
DW. Mitochondrial production of reactive oxygen species in
cortical neurons following exposure to
N-methyl-D-aspartate.
J Neurosci. 1995;15:6377–6388.
14. Xiong Y, Gu Q, Peterson PL, Muizelaar JP, Lee CP. Mitochondrial dysfunction and calcium perturbation induced by traumatic brain injury. J Neurotrauma. 1997;14:23–34.[Medline] [Order article via Infotrieve]
15.
Fujimura M,
Morita-Fujimura Y, Kawase M, Copin J-C, Calagui B, Epstein CJ, Chan PH.
Manganese superoxide dismutase mediates the early release of
mitochondrial cytochrome c and
subsequent DNA fragmentation after permanent focal cerebral
ischemia in mice. J
Neurosci. 1999;19:3414–3422.
16.
Liu PK, Hsu CY,
Dizdaroglu M, Floyd RA, Kow YW, Karakaya A, Rabow LE, Cui JK. Damage,
repair, and mutagenesis in nuclear genes after mouse forebrain
ischemia-reperfusion. J
Neurosci. 1996;16:6795–6806.
17.
Huang D, Shenoy
A, Cui J, Huang W, Liu PK. In
situ detection of AP sites and DNA strand breaks bearing
3'-phosphate termini in ischemic mouse brain.
FASEB J. 2000;14:407–417.
18.
Cui J, Holmes EH,
Greene TG, Liu PK. Oxidative DNA damage precedes DNA fragmentation
after experimental stroke in rat brain.
FASEB J. 2000;14:955–967.
19. Boyer PD, Chance B, Ernster L, Mitchell P, Racker E, Slater EC. Oxidative phosphorylation and photophosphorylation. Annu Rev Biochem. 1977;46:955–1026.[Medline] [Order article via Infotrieve]
20. Liu X, Kim CN, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell. 1996;86:147–157.[Medline] [Order article via Infotrieve]
21.
Slee EA, Harte
MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC,
Nicholson DW, Alnemri ES, Green DR, Martin SJ. Ordering the cytochrome
c-initiated caspase cascade:
hierarchical activation of caspases-2,-3,-6,-7,-8,-10 in a
caspase-9-dependent manner. J Cell
Biol. 1999;144:281–292.
22. Matsushita K, Meng W, Wang X, Asahi M, Asahi K, Moskowitz MA, Lo EH. Evidence for apoptosis after intracerebral hemorrhage in rat striatum. J Cereb Blood Flow Metab. 2000;20:396–404.[Medline] [Order article via Infotrieve]
23. Matz PG, Fujimura M, Chan PH. Subarachnoid hemolysate produces DNA fragmentation in a pattern similar to apoptosis in mouse brain. Brain Res. 2000;858:312–319.[Medline] [Order article via Infotrieve]
24.
Matz P,
Weinstein P, States B, Honkaniemi J, Sharp FR. Subarachnoid
injections of lysed blood induce the
hsp70 stress gene and produce
DNA fragmentation in focal areas of the rat brain.
Stroke. 1996;27:504–513.
25.
Murakami K, Kondo
T, Kawase M, Li Y, Sato S, Chen SF, Chan PH. Mitochondrial
susceptibility to oxidative stress exacerbates cerebral infarction that
follows permanent focal cerebral ischemia in mutant mice with
manganese superoxide dismutase deficiency.
J Neurosci. 1998;18:3659–3668.
26. Chan PH, Epstein CJ, Li Y, Huang TT, Carlson E, Kondo T, Murakami K, Mikawa S, Chen SF, Reola L. Studies of neuronal injury mechanism in focal stroke using mitochondrial manganese superoxide dismutase-deficient mice. In: Krieglstein J, ed. Pharmacology of Cerebral Ischemia. Stuttgart, Germany: Medpharm Scientific Publishers; 1996.
27. Crapo JD, McCord JM, Fridovich I. Preparation and assay of superoxide dismutases. Methods Enzymol. 1978;53:382–393.[Medline] [Order article via Infotrieve]
28. Fujimura M, Morita-Fujimura Y, Murakami K, Kawase M, Chan PH. Cytosolic redistribution of cytochrome c after transient focal cerebral ischemia in rats. J Cereb Blood Flow Metab. 1998;18:1239–1247.[Medline] [Order article via Infotrieve]
29.
Hortelano S,
Alvarez AM, Bosca L. Nitric oxide induces tyrosine nitration and
release of cytochrome c
preceding an increase of mitochondrial transmembrane potential in
macrophages. FASEB J. 1999;13:2311–2317.
30. Sugawara T, Fujimura M, Morita-Fujimura Y, Kawase M, Chan PH. Mitochondrial release of cytochrome c corresponds to the selective vulnerability of hippocampal CA1 neurons in rats after transient global cerebral ischemia. J Neurosci. 1999;19:RC39.
31. Koeppen AH, Dickson AC, McEvoy JA. The cellular reactions to experimental intracerebral hemorrhage. J Neurol Sci. 1995;134(suppl):102–112.
32. Charriaut-Marlangue C, Ben-Ari Y. A cautionary note on the use of the TUNEL stain to determine apoptosis. Neuroreport. 1995;7:61–64.[Medline] [Order article via Infotrieve]
33. Li Y, Chopp M, Jiang N, Yao F, Zaloga C. Temporal profile of in situ DNA fragmentation after transient middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab. 1995;15:389–397.[Medline] [Order article via Infotrieve]
34. MacManus J, Fliss H, Preston E, Rasquinha I, Tuor U. Cerebral ischemia produces laddered DNA fragments distinct from cardiac ischemia and archetypal apoptosis. J Cereb Blood Flow Metab. 1999;19:502–510.[Medline] [Order article via Infotrieve]
35.
Cai J, Jones DP.
Superoxide in apoptosis: mitochondrial generation triggered by
cytochrome c loss.
J Biol Chem. 1998;273:11401–11404.
36. Huie RE, Neta P. Chemistry of reactive oxygen species. In: Gilber DL, Colton CA, eds. Reactive Oxygen Species in Biological Systems: An Interdisciplinary Approach. New York, NY: Kluwer Academic; 1999.
37. Ankarcrona M, Dypbukt JM, Bonfoco E, Zhivotovsky B, Orrenius S, Lipton SA, Nicotera P. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron. 1995;15:961–973.[Medline] [Order article via Infotrieve]
38.
Beckman JS,
Beckman TW, Chen J, Marshall PA, Freeman BA. Apparent hydroxyl radical
production by peroxynitrite: implications for
endothelial injury from nitric oxide and superoxide.
Proc Natl Acad Sci
U S A. 1990;87:1620–1624.
39.
Delgado TJ,
Brismar J, Svendgaard NA. Subarachnoid hemorrhage in
the rat: angiography and fluorescent microscopy of the major
cerebral arteries. Stroke. 1985;16:595–602.
40. Bullock R, Zauner A, Woodward JJ, Myseros J, Choi SC, Ward JD, Marmarou A, Young HF. Factors affecting excitatory amino acid release following severe human head injury. J Neurosurg. 1998;89:507–518. [Medline] [Order article via Infotrieve]
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