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Research Communications |
,#1,#,**
* Departments of Anesthesiology and Critical Care Medicine,
Pediatrics,
Cell Biology and Physiology,
§ Neurological Surgery,
|| Neurology, and
¶ Pathology, and the
# Safar Center for Resuscitation Research and
** Brain Trauma Research Center, University of Pittsburgh School of Medicine; and
Neurology Service (127), Department of Veterans Affairs Medical Center, Pittsburgh, Pennsylvania 15260, USA
1Correspondence: Robert S. B. Clark, M.D, Safar Center for Resuscitation Research, 3434 Fifth Avenue Pittsburgh, PA 15260. E-mail: clark{at}smtp.anes.upmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: apoptosis • Bax • BclxL • Cpp32 • interleukin-1ß converting enzyme
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Programmed cell death (PCD) is the genetic mechanism by which cells are
eliminated during development and is the physiological mechanism by
which superfluous cells are removed in the adult animal. Cells that die
via PCD often undergo distinct morphological changes known as apoptosis
and cleave their DNA into small fragments. Recently, the genes that
control PCD in the mammalian neuron have been identified. These genes
include the caspase family of cysteine proteases [including
interleukin-1ß converting enzyme (ICE) and cpp32] that promote PCD
and a family of genes that are homologous to the oncogene Bcl-2 that
either promote or suppress cell death. It has been proposed that the
interaction between Bcl-2 family members that promote (such as Bax) and
suppress (such as Bcl-2 and Bcl-xL) apoptosis
determines whether cells undergo PCD (1, 
2)
.
PCD may occur in pathological as well as physiological cell death.
Dysregulation of genes that control PCD may exacerbate cell death in
disease and result in morphological changes with both necrotic and
apoptotic characteristics (3,
4)
. One precipitant for apoptosis in
neurons is axonal injury (5,
6)
, a common event after TBI (7)
. Several
reports in experimental models suggest that PCD occurs after TBI
(8
9
10
11
12
13)
. Furthermore, dysregulation of expression of the Bcl-2 and
caspase family genes has been observed in rat TBI models. Bcl-2 is
induced in neurons in vulnerable regions of brain after TBI in rats and
many neurons lacking Bcl-2 exhibit apoptotic morphologies (11)
.
Yakovlev et al. showed that caspase-1 and caspase-3 are activated and
their expression increased after TBI in rats (9)
. In preliminary
reports, evidence for DNA fragmentation was seen in patients after
fatal head injury (14,
15)
. Evidence for PCD has also been reported in
neurodegenerative diseases including Alzheimer and Parkinson disease
(16,
18)
.
Although the results of the experimental studies in rodent models
of TBI are compelling, whether dysregulation of death regulatory genes
occurs in human head injury is not known. Verifying that mechanisms
operating in animal models are relevant to human disease is
particularly important in studies of TBI, where encouraging results in
rodent models have failed to translate into clinically useful therapies
(19)
. In this study, we show that expression of the Bcl-2 and caspase
families of cell death-regulatory proteins are altered in humans after
TBI, and provide evidence that PCD participates in neuronal cell death
seen after TBI in humans.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Patients in the study were admitted to the University of
Pittsburgh Medical Center between August 1995 and November 1996.
Clinical variables for patients after TBI are shown in Table 1
. The average age of the patients was 35.9 ± 4.4 years. The median
admission Glasgow Coma Scale score (20)
was 5.5 [3–15]. The median
Glasgow Outcome Scale score (21)
at 3 months after injury was 3
[1–5]. The study was undertaken during the prospective, randomized
evaluation of the effect of moderate hypothermia (32°C for 24 h)
vs. normothermia (37°C for 24 h) on neurological outcome after
closed head injury (22)
. Five of eight patients were enrolled in the
hypothermia study: two were in the hypothermia (patients T1 and T8) and
three were in the normothermia (patients T3, T5, and T7) treatment
groups.
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Control samples were from frozen post-mortem brain specimens
obtained from patients who died of causes not related to central
nervous system trauma. Samples were from temporal lobe cortex and were
originally collected between April and October 1996. Clinical variables
for control patients are shown in Table 2
. The average age of the patients was 52.2 ± 9.0 years. The median
post-mortem time was 8.8 ± 2.9 h.
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Antibodies
The monoclonal antibody against Bcl-2 was obtained from DAKO
(Carpinteria, CA). The polyclonal antibodies against
Bcl-xL and caspase-3 were obtained from
Transduction Laboratories (Lexington, KY). The polyclonal antibody
against Bax was obtained from Pharmingen (San Diego, CA). The
polyclonal antibodies against caspase-3 and the p10 fragment of
caspase-1 were obtained from Santa Cruz Biotechnologies (Santa Cruz,
CA). All antibodies were generated against human peptides and have been
tested for specificity in human tissues by the respective suppliers.
Western blot analysis
A sample of contused cerebral cortex from each TBI patient and
temporal lobe cortex from each control patient was analyzed by
immunoblotting. Each sample was homogenized in lysis buffer containing
0.1 M NaCl, 0.01 M Tris, 0.1 mM EDTA, and the protease inhibitors
chymostatin 2 µg/ml, leupeptin 2 µg/ml, pepstatin 2 µg/ml, and
phenylmethylsulfonyl fluoride 100 µg/ml. Lysates were centrifuged at
14,000 rpm for 30 min at 4°C and boiled in loading buffer for 5 min.
Fifty-microgram protein samples, as determined by
A280, were loaded on sodium dodecyl
sulfate-polyacrylamide gels, separated electrophoretically, and
transferred to a Hybond-ECL nitrocellulose membrane (Amersham,
Arlington Heights, IL) overnight. The transferred membranes were
incubated in either the primary antibody against Bcl-2 (1:200
dilution), Bcl-xL (1:1000 dilution), Bax (1:1000
dilution), the p10 fragment of caspase-1 (1:500 dilution), or caspase-3
(monoclonal 1:1000 dilution; polyclonal 1:500 dilution) at room
temperature for 1 h. After washing three times in
phosphate-buffered saline (PBS) containing 0.1% Tween-20, the
appropriate secondary antibody was applied at a 1:3000 dilution for
1 h. The membrane was washed in PBS containing 0.1% Tween-20
three times over 25 min, then incubated in commercial enhanced
chemiluminescence reagents (New England Nuclear Life Science Products,
Boston, MA) and exposed to Fuji RX film (Fuji, Tokyo, Japan). For each
antibody, two gels were run simultaneously with three control and four
TBI samples each, and the two corresponding membranes were exposed on
the same X-ray film. This was done so that for each protein examined
each sample was treated with identical electrophoresis and transfer
times, antibody concentrations, incubation times, exposure to
chemiluminescent reagents, and film exposure times. Autoradiogram
signals were quantified by a gel densitometric scanning program (MCID,
St. Catherines, Ontario, Canada). The relative protein levels were
determined from the relative optical densities of the corresponding
protein bands and were normalized to background values obtained on the
same lane.
A preabsorption control experiment was performed using the caspase-1 p10 antibody and the commercially available peptide (Santa Cruz Biotechnologies). A 1:10 solution of antibody to peptide was incubated at 4°C overnight. Pairs of control and TBI samples (50 µg) were run on a single gel and transferred to a nitrocellulose membrane as described above. The membrane was cut, one pair of samples was incubated in primary antibody, the other was incubated in primary antibody with peptide, and Western blot analysis was completed as described above.
Immunocytochemistry
Immunocytochemistry for Bcl-2, Bax, and caspase-3 was also
performed on sections from contused brain from four TBI patients and
temporal lobe cortex from four control patients. Previously frozen
specimens were post-fixed for 1 h in ice-cold 2%
paraformaldehyde, then snap frozen in 2-methylbutane in liquid
nitrogen. Specimens were cut in 10-µM sections using a cryostat and
mounted on glass slides. Slides were washed three times with PBS, and
then washed three times in PBS containing 0.5% bovine serum albumin
and 0.15% glycine (buffer A). Nonspecific activity was blocked with
5% normal goat serum in buffer A. Brain sections were then incubated
overnight at room temperature in a 1:100 dilution of antibody against
Bcl-2, a 1:300 dilution of antibody against Bax, or a 1:100 dilution of
antibody against caspase-3. Sections were washed three times in buffer
A and then incubated for 1 h in a 1:3000 dilution of goat
anti-mouse Cy3.18 immunoconjugate (Jackson Immunochemicals, West Grove,
PA). Sections were then washed three times in buffer A and three times
in PBS (5 min/wash), mounted in gelvatol, and coverslipped for light
microscopy. To label cell nuclei, bis-benzamide (Sigma, St.
Louis, MO) diluted in sterile water was applied to some sections for
30 s before coverslipping. Sections were examined with an Olympus
light microscope equipped for epifluorescent illumination. Images were
collected using an integrating three-chip Sony color video camera (700
x 600 pixels) equipped with a color frame grabber board. All images
were collected while integrating at 4 frames/s using
excitation/emission wavelengths of 550/565 nm (red) and 346/460 nm
(blue). To discriminate between immunolabeling and nonspecific
autofluorescence, sections were also examined using an
excitation/emission wavelength of 494/520 nm (green) with a dual-pass
cube. In sections from each specimen, the primary antibody was omitted
to assess for nonspecific binding of the secondary antibody.
Terminal deoxynucleotidyl transferase-mediated biotinylated dUTP
nick-end labeling (TUNEL)
In situ labeling of DNA fragmentation using TUNEL was
performed on separate brain sections from six TBI patients and six
control patients with the use of a diaminobenzidine colorimetric method
for light-microscopic analysis as described previously (10)
. Briefly,
frozen 20-µM coronal sections were cut using a cryostat and mounted
on glass slides. Sections were fixed in 10% neutral buffered formalin
for 10 min at room temperature followed by ethanol:acetic acid (2:1)
for 5 min at -20°C. Sections were then incubated in 3% Triton X-100
(Sigma) at room temperature for 1 h, then placed in 3%
H2O2 and 30% methanol in
PBS for 20 min. Sections were then incubated in 300 U/ml terminal
deoxynucleotidyl transferase and 20 nmol/ml biotin-16-dUTP (Boehringer
Mannheim, Indianapolis, IN) in 1 ml 1x buffer at 37°C for 90 min,
washed with PBS three times, incubated in avidin-biotin complex (ABC
standard kit, Vector Labs, Burlingame, CA), and TUNEL visualized with
diaminobenzidine (Vector Labs). After observation of TUNEL-positive
cells, coverslips were removed and sections were counterstained with
hematoxylin and re-coverslipped.
Statistical analysis
All data are presented as mean ± standard error of the
mean or median (range) where appropriate. Comparisons of relative
protein levels between TBI patients and control patients were made
using a Mann-Whitney rank sum test. Statistics were performed using
SigmaStat software (Jandel Scientific, San Rafael, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and compared them to post-mortem brain
specimens obtained from patients who died of causes not related to
central nervous system trauma (Table 2)
. In vivo
experimental models have shown that Bcl-2,
Bcl-xL, and Bax protein levels are altered after
ischemic, excitotoxic, and TBI (11,
23
24
25)
. Bcl-2 is minimally
present, whereas both Bcl-xL and Bax are
constitutively expressed in normal adult rat brain (11,
24
25
26)
.
Western blots and relative protein levels demonstrated that Bcl-2 was
minimally present in control patients, and was increased 17-fold in
patients after TBI compared with controls (Fig. 1
, P=0.020). In contrast to Bcl-2,
Bcl-xL and Bax were detected in most of the
control samples and there was no difference in relative levels of
Bcl-xL or Bax in patients after TBI compared with
controls (Fig. 1, P
=0.662 and 0.775, respectively). The
changes in expression of Bcl-2, Bcl-xL, and Bax
after TBI in humans are consistent with experimental models of brain
injury.
|
To determine cell types expressing Bcl-2 family proteins, and whether
the relative Bcl-2 family protein levels were related to protein
expression in parenchymal cells vs. hemorrhage or infiltrating
leukocytes, immunocytochemistry using antibodies against Bcl-2 and Bax
was performed on injured brain sections from patients after TBI
(patients T2, T3, T5, and T6) and in controls (patients C1, C4, C5, and
C6). Bcl-2 protein was detected in three of four TBI patients but in
none of the controls. Bcl-2 protein was detected in cells with the
morphological characteristics of neurons and endothelium, and other
cells in which morphological type could not be distinguished after TBI
(Fig. 2
A–C). Bax protein was detected in all samples
analyzed by immunocytochemistry. Bax protein was detected in cells with
morphological characteristics of neurons, in other cells in which
morphological type could not be distinguished after TBI, and in
controls (Fig. 2, D
–F). These data demonstrate
expression of Bcl-2 and Bax protein in parenchymal brain cells
including neurons.
|
Cleavage of caspase-1 and up-regulation and cleavage of caspase-3
after TBI
To provide further evidence for the initiation of the PCD cascade
after TBI, we examined expression of the caspases. Pro-caspase-1 is a
45-kDa protein that is activated when cleaved into 20- and 10-kDa
subunits. In these experiments we used an antibody generated against
the p10 fragment (amino acids 385–404) of human caspase-1 to detect
both the intact 45-kDa and the cleaved 10-kDa proteins. Caspase-3 is a
32-kDa protein that is activated when cleaved into 17- and 12-kDa
subunits. To detect caspase-3 we used a monoclonal antibody generated
against the majority of the intact protein (amino acids 1–219) to
detect the 32-kDa protein, or a polyclonal antibody generated against
the intact protein to detect the 32-kDa and the cleaved 17- and 12-kDa
proteins. Western blots showed that pro-caspase-1 was reduced in
patients after TBI compared with controls (Fig. 3
, twofold reduction, P=0.02). In contrast, the p10 fragment
of caspase-1 was increased in patients after TBI compared with controls
(Fig. 3
, 78-fold increase, P<0.001). In addition to the p45
and p10 fragments, a distinct protein band of ~28–30 kDa was also
detected in samples from both TBI and control patients (Fig. 3A
). Preabsorption studies with the peptide used to generate
the anti-caspase-1 p10 antibody showed that immunoblotting for all
three bands was specific (Fig. 3B
). Thus, the 28- to 30-kDa
protein could represent intermediate processing of caspase-1 or ICE
,
which is an alternatively spliced isoform of caspase-1 that contains
the p10 sequence and inhibits apoptosis under some experimental
conditions (27)
. These data show that pro-caspase-1 is constitutively
present and cleaved after TBI in humans. Intact caspase-3 was increased
in patients after TBI compared with controls (Fig. 3
, 14-fold increase,
P=0.02). In addition, the cleaved 12-kDa fragment of
caspase-3 was increased in patients after TBI compared with controls
(Fig. 3
, 19-fold increase, P=0.008). The 17-kDa fragment was
also detected in four patients after TBI, but not in controls. These
data show that caspase-3 is up-regulated and cleaved after TBI in
humans.
|
Immunocytochemical studies also showed that caspase-3 was
increased after TBI in humans compared with controls (Fig. 2, G
--I). Cells with the morphological appearance of
neurons and glia showed increased immunoreactivity in contused brain
after TBI. Several cells with increased caspase-3 immunoreactivity
displayed condensed and shrunken nuclei as assessed by Hoechst staining
(Fig. 2G
).
DNA fragmentation after TBI
TUNEL was performed in additional brain tissue sections to
assess for evidence of DNA fragmentation after TBI (patients T1, T2,
T3, T5, T6, and T7). Brain sections used for immunocytochemistry and
TUNEL included contused but intact tissue. There were microscopic
regions of tissue disruption and scattered microscopic hemorrhages, but
in general the tissue examined consisted of intact parenchyma with
selective cellular changes. Scattered populations of TUNEL-positive
cells were seen in most of the TBI patients examined. Both larger
TUNEL-positive cells with the morphological characteristics of neurons
and smaller TUNEL-positive cells, which could represent glia,
infiltrated leukocytes, or oligodendrocytes, were seen (Fig. 2)
. Some
TUNEL-positive cells had the morphological features of apoptosis
(shrunken, condensed, fragmented nucleus), whereas others had the
morphological features of necrosis (swollen nucleus; Fig. 2
). Areas of
hemorrhage were seen in several of the sections; however,
TUNEL-positive cells were primarily within the brain parenchyma.
TUNEL-positive cells were also seen in cortical samples from two out of
six control patients (patients C4 and C5); these patients had
conditions for which this finding was not unexpected (Table 2)
.
TUNEL-positive cells in control patients primarily had morphological
features of necrosis (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The caspase family consists of 13 currently identified cysteine
proteases that also participate in the regulation and execution of cell
death (28
29
30)
. Activated caspase-3 (Yama/Apopain/Cpp32) cleaves
proteins important in the regulation of apoptosis, DNA repair, and
cytoskeletal integrity, such as the inhibitor of caspase-activated
deoxyribonuclease, DNA-dependent protein kinase, nuclear lamin,
spectrin, and actin (31
32
33)
. caspase-1 (ICE) cleaves pro-interleukin-1
to active interleukin-1ß, a process that is important in the
inflammatory response (30)
and possibly reparative processes (34)
.
Caspases modulate cell death and brain injury in animal models of
stroke (35
36
37
38)
and TBI. Yakovlev et al. (9)
showed that caspase-1 and
caspase-3 are activated and increased after experimental TBI in rats.
The current finding that expression of caspase-3 is increased and that
caspase-1 and caspase-3 are cleaved after TBI provide strong evidence
that PCD is activated after TBI in humans and may contribute to
secondary cell injury and cell death. Caspase activation may also
produce beneficial effects after TBI, such as attenuating cellular
energy depletion via cleavage of poly(ADP)-ribose polymerase, an
important mechanism in models of cerebral ischemia (39,
40)
.
In vivo experimental models have shown that Bcl-2 family
protein levels are altered after ischemic and traumatic insults (11,
23
24
25)
. After TBI in rats, total Bcl-2 protein is increased in injured
brain. Many neurons lacking Bcl-2 exhibit apoptotic morphologies,
whereas those expressing Bcl-2 do not demonstrate biochemical or
morphological features of apoptosis (11)
. Thus, Bcl-2 protein
expression is induced in neurons that are injured yet appear to
survive. When Bcl-2 protein is overexpressed in vivo,
neurons are resistant to ischemic injury (41
42
43)
. These studies
support the hypothesis that Bcl-2 protects neurons from injury. The
present study in humans also demonstrates increased neuronal expression
of Bcl-2. Thus, Bcl-2 expression could be an important factor that
promotes survival of neurons injured after trauma.
Although Bcl-2 expression was increased, expression of Bax and
Bcl-xL were not significantly increased after
TBI. These results do not exclude the possibility that Bax and
Bcl-xL also regulate neuronal death after TBI
because translocation of Bax and Bcl-xL from
cytosolic to mitochondrial locations, rather than alterations in
protein expression levels itself is the key mechanism by which these
Bcl-2 family genes control PCD (44)
. Furthermore, the interaction of
Bcl-2 protein death-antagonists to death-agonists has been proposed to
determine whether a cell survives or dies after injury (1,
2,
45,
46)
. It is important to point out; however, that Bax may act
independently to promote cell death (47)
, and whether intracellular
heterodimeriztion occurs between Bax and Bcl-2 or
Bcl-xL, is controversial (48,
49)
.
This study, showing caspase activation and cleavage of DNA,
demonstrates that trauma can initiate the PCD cascade in human brain.
In the setting of trauma, PCD could be a maladaptive response that
exacerbates injury. In animal models of ischemia and trauma, PCD that
occurs within hours to days after injury appears to be deleterious to
outcome. On the other hand, PCD could also be a physiological response
to injury, participating in the remodeling of neuronal circuits or the
culling of injured or dysfunctional cells after injury. There is
progressive loss of cortical volume after TBI for weeks to months after
injury (50)
, despite clinical improvement in patients during this
interval, supporting the latter possibility.
Whether PCD that occurs after brain injury is maladaptive or beneficial
has been addressed in several studies in animal models of stroke and
trauma. Overexpression of Bcl-2 reduces neurological tissue damage in
rodents after cerebral ischemia (42,
43)
. Raghupathi et al. have shown
that mice overexpressing Bcl-2 have a reduction in lesion volume and a
modest improvement in motor function after TBI (51)
. Yakovlev et al.
reported that the caspase inhibitor
N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone
reduced post-traumatic apoptosis and improved neurological recovery
after TBI (9)
. Caspase inhibitors have also been shown to be effective
in animal models of cerebral ischemia and excitotoxicity (35,
37)
.
These studies and the current findings suggest that PCD may exacerbate
acute neurological damage after TBI in humans, beyond the damage
produced from direct mechanical injury.
The use of experimental paradigms utilizing human tissues can demonstrate mechanistic evidence that corroborates in vitro and animal studies; however, inherent limitations in the use of clinical materials exist. Control brain samples, where normal brain tissue was removed under similar conditions, are not available. Furthermore, tissue samples from patients were removed at different times after injury. The samples may not be representative of changes in gene expression in the brains as a whole. For example, the samples may vary in their proximity to contusions, and the small sample size forbids histological analysis of adjacent tissue or comprehensive multi-label studies that are more feasible in experimental models. There are also differences in handling of tissues from patient samples compared to the autopsy control samples. Trauma patient samples were frozen within minutes after removal, whereas autopsy brains were much more slowly preserved. However, it is the trauma specimens that show evidence of caspase cleavage and DNA fragmentation, thus one cannot attribute the changes found in trauma brain tissue to post-mortem factors alone. Furthermore, the expression patterns of the proteins examined in this study are consistent with those seen in experimental models of brain injury where ideal controls were available. Nonetheless, differences in time between injury and surgery, region, severity, type of injury, and other factors cannot be controlled in this clinical study, possibly affecting detection of death regulatory genes. The results of this study need to be interpreted with the above limitations in mind.
In conclusion, several key steps in the PCD cascade have been identified in human brain after TBI. These findings demonstrate that genes that orchestrate PCD during central nervous system development can be reactivated in mature brain after trauma, and provide a rationale for the further development of pharmacological and molecular therapies targeting PCD after TBI, stroke, and other types of acute brain injury.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication December 8, 1998.
Revision received January 12, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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