(Stroke. 1999;30:321-327.)
© 1999 American Heart Association, Inc.
Original Contributions |
Intrathecal Expression of Proteins Regulating Apoptosis in Acute Stroke
From the Departments of Rheumatology and Clinical Immunology (E.T., A.T.), Clinical Neurosciences (Section of Neurology) (E.T., L.R., C.B.), and Radiology (C.J., S.E.), University of Göteborg, Göteborg, Sweden.
Correspondence to Dr Elisabeth Tarkowski, Departments of Rheumatology and Clinical Immunology, Guldhedsgatan 10, S-413 46 Göteborg, Sweden. E-mail andrej.tarkowski{at}microbio.gu.se
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Abstract |
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Background and Purpose—The neuronal death that accompanies an ischemic stroke has previously been attributed to a necrotic process. However, numerous studies in experimental models of ischemia have recently indicated that programmed cell death, also called apoptosis, may contribute to neuronal death. The aim of the present study was to investigate the intrathecal levels of proteins regulating apoptosis in acute stroke and to relate these levels to brain damage and to production of proinflammatory and anti-inflammatory cytokines.
Methods—Thirty stroke patients were studied prospectively on days
0 to 4, 7 to 9, 21 to 26, and after day 90 with clinical
evaluation, radiological assessment, and analysis of
cerebrospinal fluid (CSF) levels of soluble (s) Fas/APO-1 and sbcl-2, 2
proteins that regulate apoptosis. In addition, analysis
of the intrathecal levels of cytokines interleukin
(IL)-1ß, IL-6, IL-8, IL-10, granulocyte-macrophage
colony-stimulating factor (GM-CSF), and tumor necrosis
factor-
was performed. Nineteen CSF samples from healthy subjects
were used for control purposes. The patients were examined with MRI 1
to 3 months after stroke onset for measurement of infarct volume
Results—Significantly decreased CSF levels of sFas/APO-1 were
observed during the entire observation period, with a maximal decrease
on day 21 after the onset of stroke. The intrathecal levels
of sFas/APO-1 were significantly negatively correlated with the volume
of brain infarct and with the neurological deficit 3 weeks and 3 months
after the onset of the stroke. In addition, the intrathecal
levels of sFas/APO-1 were significantly correlated with the levels of
IL-1ß, IL-6, IL-10, and GM-CSF 3 weeks after the onset of the
disease. The intrathecal levels of sbcl-2 were
significantly decreased during the first 3 days after stroke onset and
at the same time were positively correlated with the levels of IL-6 and
tumor necrosis factor-.
Conclusions—Our study demonstrates decreased intrathecal levels of proteins with antiapoptotic properties, suggesting that patients with acute stroke display a propensity toward apoptosis. Control of factors regulating apoptosis may lead to decreased delayed brain damage in stroke.
Key Words: apoptosis • brain • cerebrospinal fluid • cytokines • magnetic resonance imaging • proteins • stroke
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Introduction |
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Stroke is a common disease, leading to a high mortality and disability rate and consequently referred to as "the big crippler." The clinical outcome of the disease is not only dependent on the extent of the brain lesion but also on its localization. Thus, one of the aims of the research of the last decade has been to delineate the pathophysiological mechanisms that lead to neuronal cell death after compromised blood circulation and energy supply. Neuronal cell death can occur acutely in the core of the infarcted area if the blood flow is <10 mL/100 g tissue per minute and may extend to the penumbral area even after reestablishment of the blood circulation.1 Several factors, such as acidosis,2 changes in calcium homeostasis,2 increased outflow of excitatory amino acids,3 and free oxygen radical–mediated damage,4 have been implicated in both acute and delayed cell death. In addition, a growing body of evidence stresses the role of inflammatory mechanisms in the pathophysiology of ischemic brain damage.5 6
The neuronal death that accompanies an ischemic stroke has previously been attributed to necrotic processes in the brain tissue. However, numerous studies in experimental models of ischemia have now reported that apoptosis contributes to neuronal death (reviewed by Chalmers-Redman et al7 ). Apoptosis requires the activation of a "cell death" gene program, and many of the extracellular signals that regulate apoptosis have been identified. For example, interaction between the Fas/APO-1 molecule, a cell surface protein, with its ligand (Fas-L) leads to programmed cell death.8 Soluble (s) Fas/APO-1, a molecule lacking the transmembrane domain of Fas/APO-1, blocks apoptosis by inhibiting interaction between Fas/APO-1 and Fas-L on the cell surface.9 Fas expression has been detected on B and T cells and on neutrophils. It has been suggested that the Fas/Fas-L pathway is one of the major mechanisms for T-cell–mediated cytotoxicity.8 10 11 Importantly, it has been recently demonstrated by in situ hybridization that the expression of Fas/APO-1 was induced in murine brain after transient global cerebral ischemia.12 In this respect, inhibition of apoptosis after brain ischemia in a rat model of focal ischemia reduces the size of brain infarct.13 Another gene product, bcl-2, has been shown to suppress apoptosis14 15 and to protect primary neuronal cell cultures from apoptosis induced by nerve growth factor depletion.16 17 Overexpression of bcl-2 in transgenic mice protects neurons from naturally occurring death in experimental cerebral ischemia.17 18 Macrophages and T lymphocytes kill target cells by inducing apoptosis, one of the potential mechanisms whereby the inflammatory cells invading the infarcted brain area participate in neuronal cell death.8 10 19 Interestingly, Chopp et al20 have demonstrated in a recent study that antibodies against adhesion molecules not only reduce inflammatory response and volume of infarction in an experimental stroke model but also reduce apoptosis.
We recently demonstrated that stroke patients displayed an intrathecal production of proinflammatory cytokines, such as interleukin (IL)-1ß, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and of the anti-inflammatory cytokine IL-10 within the first 24 hours after the onset of symptoms, supporting the notion of localized immune response to the acute brain lesion in humans.21 22 Some of these cytokines (eg, IL-1ß and IL-8) stimulate influx of leukocytes to the infarcted brain, a prerequisite for Fas/APO-1– and bcl-2–mediated apoptosis. The aim of the present study was to analyze the intrathecal release of sFas/APO-1 and sbcl-2, 2 proteins that suppress apoptosis, and to relate their levels to the size of brain lesions and the intrathecal release of proinflammatory and anti-inflammatory cytokines during stroke.
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Subjects and Methods |
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Patients
Thirty patients (23 men and 7 women; age range, 40 to 81 years; mean±SD age, 64±11 years), all patients at Sahlgrenska University Hospital, Department of Neurology, were consecutively incorporated into the study. All patients had experienced stroke
3 days before
inclusion. No patients had a history of previous stroke. Patients with
malignant and autoimmune diseases or severe infections or taking
immunosuppressive drugs were excluded. All patients were evaluated by a
standardized examination of motor and sensory deficit,
peripheral reflexes, muscular tone, and cranial nerve
function. Occurrence of dysphasia and tactile neglect symptoms was also
evaluated. Furthermore, the patients were examined according to the
Scandinavian Stroke Scale23 at the inclusion in the study
and then 1 week, 3 weeks, and 3 months after onset. The Scandinavian
Stroke Scale has a score ranging from 58 (normal neurological status)
to 0 (maximal neurological deficit of the scale). The degree of
disability was evaluated by the Barthel Index24 3 months
after onset (score ranging from 100=no disability to 0=complete
dependence for activities of daily life). All patients were examined
with CT of the brain during the first days after onset of stroke.
Twenty patients were reexamined with MRI and the remaining 10 patients
with CT 
1 month after stroke onset. Stroke patients were stratified into minor and major stroke groups, according to the classification of Hachinski.25 The definition of minor stroke requires that the patient is discharged and sent home, walks without assistance, and copes unaided with such self-care activity as eating, dressing, and toileting within 1 month after the disease onset.26 In contrast, patients with major stroke had stable and usually severe neurological deficit.
Cerebrospinal fluid (CSF) samples were obtained for analysis of cytokine levels on days 0 to 4, 7 to 9, 21 to 26, and 3 months after disease onset21 22 and for analysis of Fas/APO-1 and bcl-2 (present study). In 10 patients, serum and CSF samples could be obtained twice within the first 4 days after the stroke onset. CSF samples from 19 control individuals without any neurological disease or deficit aged 49 to 78 years (mean±SEM age, 65±2 years) were obtained to establish the reference values of Fas/APO-1 and bcl-2.
The study was approved by the Ethics Committee of the University of Göteborg.
Reagents and Procedures
Analysis of Soluble Fas/APO-1 and bcl-2
Levels of Fas/APO-1 and of bcl-2 in CSF and serum samples were
estimated by a sandwich ELISA (Calbiochem). The detection levels
for Fas/APO-1 and bcl-2 were 0.07 U/mL and 1 U/mL, respectively.
All values below the detection levels were considered negative.
Cytokine Analysis
Levels of GM-CSF, IL-8, and IL-10 in CSF samples were estimated
by an ELISA with the use of monoclonal antibodies for coating and
developing steps, as previously described.22 Levels of
tumor necrosis factor- (TNF-) in CSF samples were estimated by an
ELISA (Predicta, Genzyme).
The detection levels for GM-CSF, IL-8, IL-10, and TNF- were 40, 3,
25, and 2 pg/mL, respectively. The CSF was diluted 10 times for IL-8
and TNF- determinations and 5 times for GM-CSF and IL-10
measurements. All values below the detection levels were regarded as
negative.
Levels of IL-1ß in CSF and serum samples were estimated by an ELISA (Quantikine R&D Systems). The normal levels of serum IL-1ß are <3.5 pg/mL according to the manufacturer's data.
IL-6 Assay
Cell line B13.29, which is dependent on IL-6 for growth, has
been previously described.27 For IL-6 determinations, the
more sensitive subclone B9 was used.28 29 B9 cells were
harvested from tissue culture flasks, seeded into microtiter plates
(Nunc) at a concentration of 5000 cells per well, and cultured in
Iscove's medium supplemented with 5x10-5 mol/L
2-mercaptoethanol, 5% fetal calf serum (Seralab), penicillin (100
U/mL), and streptomycin (100 µg/mL), and CSF or serum samples were
added. [3H]thymidine was added after 68 hours
of culturing, and the cells were harvested 4 hours later. The samples
were tested in 2-fold dilutions and compared with a recombinant human
IL-6 standard (Genzyme). B9 cells were previously shown not to react
with several recombinant cytokines, including IL-1, IL-1ß,
IL-2, IL-3, IL-5, GM-CSF, TNF-, and interferon-
. There was only
weak reactivity with IL-4.29
To assess the specificity of the bioassay, we used a highly purified
monoclonal antibody specific for human IL-6 (Genzyme) in a
neutralization assay. Preincubation of 10 µg/mL of this antibody with
either recombinant IL-6 or CSF from stroke patients containing IL-6 (1
hour, 37°C) reduces proliferative responses of B9 indicator cells by
on average 
95%.
MRI and CT Scan Analyses
To evaluate the final extent and localization of brain lesions,
neuroimaging was performed 4 weeks after the onset of stroke. This
delay in imaging was chosen to obtain a better delineation of the
permanent damage. The neuroimaging techniques used were CT as well as
multiplanar MRI. The CT scans were routinely performed parallel to the
canthomeatal plane, ie, a gantry tilt +10° from Reid's baseline
using 5-mm (posterior fossa) and 10-mm
(supratentorial) slice thickness. The MRI
examinations (Philips Gyroscan T5-II) were performed with conventional
fast spin-echo technique to obtain axial proton-density and T2-weighted
images of the brain. If a lesion was identified, a 3-dimensional volume
sampling was also performed with the use of a TI-weighted coronal fast
gradient-echo technique ( repetition time, 30 ms; echo time, 13 ms;
flip angle, 30°) with 3-mm slice thickness (scan
matrix=205x256 and field of view=220). All scans were evaluated to
correlate each lesion with its anatomic location. The evaluation of the
scans was done by 2 experienced neuroradiologists without knowledge of
clinical data. Sixty-six important anatomic brain structures were
defined in the scans according to Kretschmann and
Weinrich,30 and all the lesions were related to these
structures. Volume measurements of the lesions were done by means of a
3-dimensional reconstruction program in the workstation environment
(Philips Gyroview). This technique entails the use of a proper
segmentation and subsequent seeding within the lesion. The imaging
sequence used for 3-dimensional sampling resulted in a sharp contrast
between these ischemic lesions and the surrounding normal
tissue, making lesion segmentation easy. The volume of the lesion thus
created is automatically given when reconstruction is finished.
Statistical Analysis
Statistical analysis was performed by the Mann-Whitney
U test. The 
2 test was used to
analyze categorical data. Spearman's rank order correlation
test was used to calculate the correlations between different
parameters. A P value <0.05 was considered
statistically significant.
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Results |
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Clinical Findings
Twenty-three patients included in the study displayed varying degrees of hemiparesis when examined at the onset of stroke. Eighteen of the hemiparetic patients were able to perform some voluntary movements, whereas 5 had complete paralysis. Seven displayed no motor deficit but showed other stroke-related symptoms, such as isolated hemisensory deficit or aphasia.
Twelve patients displayed impaired sensory function; 4 of these patients displayed neglect symptoms. Eight had dysphasia. Thirteen patients had neurological signs on the right side of the body, 16 on the left side, and 1 on both sides. Twenty-four patients were classified as having a minor and 6 as having a major stroke.
Use of the Scandinavian Stroke Scale showed that the average neurological deficit was limited during the first days after disease onset (mean±SD, 47.9±2.3) and diminished in course of time (day 7 to 9, 50.9±2.4; day 21 to 26, 53.8±1.9; after 3 months, 54.2±1.9). The degree of disability, measured 3 months after the onset of stroke by the Barthel Index, was also low (92.6±3.4), indicating that the majority of the patients could perform daily life activities without any help.
Radiological Findings
Thirteen patients exhibited a single infarct, 13 exhibited
multiple infarcts, and 4 had no pathological MRI changes in support of
infarct. Seventeen patients displayed white matter lesions in the
unaffected brain hemisphere, whereas in the remaining 13 patients such
changes were not found. Thirteen patients had a large infarct (ie, the
sum of the largest transverse and sagittal diameter divided by 2 was
>1.5 cm), and 13 had a small infarct (ie, the sum of the largest
transverse and sagittal diameter divided by 2 was <1.5 cm). The 4
patients with no radiologically visible infarct changes were assumed to
have a small infarct. Fourteen patients had an infarct mainly (>50%)
located in the gray matter, and 10 had an infarct mainly located in the
white matter. Nine patients had a cortical lesion, and 17 patients had
a subcortical lesion.
MRI analysis of brain infarcts in 20 stroke patients revealed that the mean±SEM lesion volume was 2.2±0.8 mL.
CSF Levels of sFas/APO-1
Only 9 of the 30 stroke patients but 16 of the 19 controls
exhibited detectable levels of sFas/APO-1 in the CSF within the first
days after disease onset (P<0.001). The
intrathecal levels of sFas/APO-1 were significantly
decreased in stroke patients compared with controls during the entire
observation period, with a maximal decrease at day 21 after onset
(Figure 1
).
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CSF Levels of sFas/APO-1 in Relation to Radiological
Findings
The CSF levels of sFas/APO-1 3 months after stroke onset were
significantly negatively correlated (r=-0.47;
P<0.05) with the volume of the brain lesion measured by
MRI. No statistically significant correlation was found between the
sFas/APO-1 levels in CSF and the brain lesion volume during the first 3
weeks after the onset of the disease (day 0 to 4: r=-0.38,
P=NS; day 7: r=-0.13, P=NS; day 21:
r=-0.42, P=NS).
Patients with a brain lesion affecting mainly (>50%) the gray matter
did not show any statistically significant differences in CSF
sFas/APO-1 levels compared with patients with mainly white matter
lesions (Table 1). No
statistically significant difference in CSF levels of sFas/APO-1 was
found between patients with a single brain lesion and patients with
multiple infarcts or between patients with white matter damage in the
hemisphere contralateral to the ischemic brain lesion and
patients without white matter lesions (data not shown). Patients with
cortical lesions and patients with subcortical lesions did not display
any statistically significant differences in sFas/APO-1 levels in the
CSF (Table 1). Patients with left-sided brain lesions and
patients with right-sided brain lesions exhibited similar levels of
sFas/APO-1 levels in the CSF (data not shown).
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sFas/APO-1 Levels in the CSF in Relation to Clinical
Findings
Interestingly, there was a significant correlation between the
levels of sFas/APO-1 in CSF and the Scandinavian Stroke Scale score 3
weeks after stroke onset (r=0.42, P=0.05),
indicating that stroke patients with low score, ie, pronounced
neurological deficit, had low CSF levels of sFas/APO-1. When
analyzed separately, patients with a minor and major stroke did
not show any statistically significant differences in the sFas/APO-1
levels in CSF (data not shown). No statistically significant
correlation between Barthel Index and CSF levels of sFas/APO-1 was
found (r=0.18, P=NS).
CSF Levels of sFas/APO-1 in Relation to Intrathecal
Cytokine Production
Three weeks after stroke onset, the level of sFas/APO-1 in CSF was
significantly and positively correlated with the levels of the
proinflammatory cytokines IL-1ß (r=0.69,
P=0.0001), IL-6 (r=0.44, P=0.042), and
GM-CSF (r=0.52, P=0.012) and with the level of
the anti-inflammatory cytokine IL-10 (r=0.46,
P=0.029). In contrast, the sFas/APO-1 levels in CSF were
negatively correlated (r=-048, P=0.022) with CSF
levels of the inflammatory cytokine TNF- 3 weeks after
stroke onset (Table 2). No statistically
significant correlation was found between the CSF levels of sFas/APO-1
and the cytokines during the first week after the stroke onset
(Table 2).
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CSF Levels of sFas/APO-1 in Relation to CSF:Serum Albumin
Ratios
CSF:serum albumin ratios and IgG index were investigated
for every patient and at each time point. During the first week after
stroke onset, 16 of the 30 patients showed no CSF:serum albumin
ratio increases (day 1 to 4, 0.0061±0.0003; day 7, 0.0058±0.0003
[mean±SEM]), suggesting an intact blood-brain barrier, whereas the
14 remaining patients exhibited a small increase of CSF:serum
albumin ratio (day 1 to 4, 0.0099±0.0005; day 7,
0.0104±0.0006), suggesting modest blood-brain barrier damage. However,
when analyzed separately, these 2 groups of patients displayed
no statistically significant differences in CSF levels of sFas/APO-1
during the entire observation period (data not shown). IgG index
remained unaltered throughout the course of stroke (data not
shown).
CSF Levels of sbcl-2
Only 13 of the 27 stroke patients but all of the 19 controls
exhibited detectable levels of sbcl-2 in the CSF within the first 4
days after disease onset (P<0.001). sbcl-2 levels in the
CSF of stroke patients were not significantly lower than in the
controls on the day of stroke onset (stroke patients at day 0,
5.76±1.54 U/mL; controls, 5.85±0.85 U/mL; P=NS) but
decreased rapidly to the lowest levels on day 2 (stroke patients at day
2, 1.58±0.84 U/mL; controls, 5.85±0.85 U/mL; P=0.001) and
become partially normalized thereafter. Overall, the
intrathecal levels of sbcl-2 were not statistically
significantly decreased in stroke patients compared with the controls
during the first 4 days of the disease (Figure 2).
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CSF Levels of bcl-2 in Relation to Radiological Findings
The bcl-2 levels in CSF were highly significantly correlated with
the volume of brain lesion during the first 4 days after the onset of
the disease (day 0 to 4, r=0.68, P=0.003). When
analyzed separately, patients with small brain lesions
displayed significantly lower levels of bcl-2 in CSF during the first 4
days after stroke onset than the patients with large brain lesions
(P=0.006) (Table 1
) and controls
(P=0.0001).
Interestingly, patients with subcortical lesions showed initially
significantly lower levels of bcl-2 than the patients with cortical
lesions (P=0.0.15) (Table 1) and controls
(P=0.009). In addition, patients with a brain lesion
affecting mainly the white matter displayed significantly lower levels
of bcl-2 in CSF compared with patients with mainly gray matter lesions
(P=0.02) (Table 1) and compared with controls
(P=0.006) 3 months after stroke onset. In contrast, no
statistically significant difference in CSF levels of bcl-2 was found
between patients with a single brain lesion and patients with multiple
infarcts or between patients with white matter damage in the hemisphere
contralateral to the ischemic brain lesion and patients without
white matter lesions (data not shown). Patients with right-sided or
left-sided brain lesion displayed almost equal levels of CSF bcl-2
(data not shown).
sbcl-2 Levels in CSF in Relation to Clinical Findings
Patients with a minor versus major stroke did not show any
statistically significant differences in the sbcl-2 levels in CSF (data
not shown). No statistically significant correlation between the
Scandinavian Stroke Scale or the Barthel Index and CSF levels of sbcl-2
was found (data not shown).
CSF Levels of bcl-2 in Relation to Intrathecally
Produced Cytokines
A significant correlation was found between the CSF levels of
bcl-2 and the CSF levels of the proinflammatory cytokines IL-6
(r=0.43, P=0.026) and TNF- (r=0.41,
P=0.036) measured during the first days of the stroke. In
contrast, GM-CSF levels were negatively correlated with the levels of
bcl-2 at 3 weeks (r=-0.48, P=0.031) and 3 months
(r=-0.37, P=0.002) after stroke onset (Table 3).
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CSF Levels of sbcl-2 in Relation to CSF:Serum Albumin
Ratios
Patients with intact blood brain-barrier and patients with damaged
blood-brain barrier displayed no statistically significant differences
in the intrathecal levels of sbcl-2 during the first 3
weeks after the onset of the disease (data not shown). However, 3
months after stroke onset, the difference in CSF levels of sbcl-2
became significant between these 2 groups (patients without blood-brain
barrier damage, 6.7±1.4 U/mL; patients with blood-brain barrier
damage, 2.2±1.1 U/mL; P=0.034). IgG index remained
unaltered throughout the course of stroke (data not shown).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Our study has shown a significant decrease of sFas/APO-1 and sbcl-2, 2 proteins that suppress apoptosis, in the CSF of patients with acute stroke. In addition, the CSF levels of sFas/APO-1 were negatively correlated with the degree of neurological deficit and with the volume of brain infarct 3 weeks and 3 months, respectively, after the onset of the disease.
A growing body of evidence points out the role of apoptosis as contributing to the cell death occurring in the brain as a consequence of ischemia.7 31 For example, the expression of Fas/APO-1 mRNA is induced in some neuronal and glial cells after brain ischemia, suggesting that apoptotic cell death is mediated by the Fas antigen in the postischemic brain.12 The detection of the Fas-L in the CSF in patients after severe head trauma suggests strongly that the Fas/Fas-L pathway may have a pivotal role in causing local tissue destruction in brain.32 Several mechanisms may downregulate Fas-mediated cell death. For example, Cheng et al9 detected a soluble form of Fas/APO-1 protein lacking a transmembrane domain in the CSF of patients with systemic lupus erythematosus. sFas/APO-1 is generated by alternative splicing of Fas mRNA and blocks apoptosis by inhibiting binding between Fas/APO-1 and Fas-L or sFas-L.9 The Fas-triggered cell death may be partially inhibited by overexpression of bcl-233 and completely inhibited by coexpression of bcl-2 and its binding protein BAG-1.34 Consequently, a decrease of proteins downregulating Fas-mediated apoptosis, seen in our study, together with the increased expression of Fas/APO-1 in the brain tissue after an ischemic lesion, as observed by Matsuyama et al,12 may result in increased apoptosis, leading to aggravated tissue damage during stroke. Thus, the decreased levels of molecules inhibiting apoptosis observed in our study could be associated with a poorer prognosis. Indeed, the present study demonstrated an inverse correlation between the CSF levels of sFas/APO-1 and the volume of brain infarct on one hand and the degree of neurological deficit on the other, supporting this hypothesis.
An important issue is whether the decreased levels of proteins downregulating apoptosis observed in the present study are due to decreased production, increased consumption, or both. Interestingly, on the day of stroke onset, bcl-2 levels in the CSF were similar to the levels of bcl-2 in controls and then decreased rapidly but transiently. For a number of reasons we believe that increased consumption may be the reason for low intrathecal bcl-2 levels. First, the decrease was observed very rapidly, with kinetics that were more typical for consumption rather than for decreased production. In addition, several experimental studies have shown early-onset apoptosis as a consequence of stroke.7 In this process, sbcl-2 is consumed because it binds to neurite extension in neurons, thereby counteracting the apoptotic process.15 Similarly, the levels of Fas/APO-1 are also significantly decreased early (but also late) during the stroke, suggesting receptor-mediated consumption rather than decreased production of this molecule.
Another intriguing issue is the positive correlation between CSF levels
of sFas/APO-1 and bcl-2 and locally produced cytokines. A
possible explanation for this correlation is the presence of
apoptotic cells known to modulate the production of
cytokines by immunocompetent cells. In this aspect, Voll et
al35 demonstrated that the presence of apoptotic
cells increases the secretion of IL-10 and decreased secretion of
TNF- by monocytes. Cytokines are known to modulate
apoptosis. In this respect, Holmin36 demonstrated
that injection of IL-ß in rat brain elicited massive DNA
fragmentation (ie, apoptosis) within 24 hours, whereas
intracerebral injection of TNF- did not. The
IL-1ß–mediated apoptosis was primarily confined to neural
cells but also to the invading inflammatory cells. Our study points to
a highly significant correlation between the intrathecal
levels of sFas/APO-1 and IL-1ß. In contrast, TNF-, a powerful
cytokine inducing apoptosis in the extraneural
compartment of the body, has been demonstrated to protect rat
hippocampal, septal, and cortical cells against
metabolic-excitotoxic insults37 and to
facilitate regeneration of injured axons.38 More
importantly, TNF- and -ß protect neurons against amyloid
ß-protein–triggered toxicity.39 In the
present study we observed a significant correlation between the
intrathecal bcl-2 levels and the intrathecal
TNF- levels during the first week of the stroke and later,
suggesting that this cytokine might have neuroprotective
properties by downregulating the process of apoptosis. Indeed,
we recently demonstrated that TNF- triggers in vitro human neuronal
cell to produce bcl-2.40
In conclusion, our study demonstrates decreased intrathecal levels of proteins with antiapoptotic properties, suggesting a propensity toward apoptosis in patients with acute stroke. In addition, the CSF levels of sFas/APO-1 were negatively correlated with the volume of brain infarct and with the degree of neurological deficit 3 months after the onset of the disease, suggesting that the intracerebral decrease of sFas/APO-1 levels is associated with a poorer prognosis. We conclude that control of factors regulating apoptosis may facilitate future attempts to decrease delayed brain damage in stroke.
Received April 17, 1998; revision received October 22, 1998; accepted November 12, 1998.
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References |
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1. Kogure K, Kato H. Neurochemistry of stroke. In: Barnett HJM, Mohr JP, Stein BM, Yatsu FM, eds. Stroke: Pathophysiology, Diagnosis, and Management. 2nd ed. New York, NY: Churchill Livingstone; 1992:3–28.
2. Siesjö BK. A new perspective on ischemic brain damage? Prog Brain Res. 1993;96:1–12.[Medline] [Order article via Infotrieve]
3. Diemer NH, Valente E, Bruhn T, Berg M, Jörgensen MB, Johansen FF. Glutamate receptor transmission and ischemic nerve cell damage: evidence for involvement of excitotoxic mechanisms. Prog Brain Res.. 1993;96:105–124.[Medline] [Order article via Infotrieve]
4. Watson BD. Evaluation of the concomitance of lipid peroxidation in experimental models of cerebral ischemia and stroke. Prog Brain Res.. 1993;96:69–96.[Medline] [Order article via Infotrieve]
5.
Kochanek PM, Hallenbeck JM. Polymorphonuclear
leukocytes and monocytes/macrophages in the pathogenesis of
cerebral ischemia and stroke. Stroke. 1992;23:1367–1379.
6.
Lindsberg PJ, Carpen O, Paetau A,
Karjalainen-Lindsberg M-J, Kaste M. Endothelial ICAM-1
expression associated with inflammatory cell response in human
ischemic stroke. Circulation. 1996;94:939–945.
7. Chalmers-Redman RME, Fraser AD, Ju WYH, Wadia J, Tatton NA, Tatton WG. Mechanisms of nerve cell death: apoptosis or necrosis after cerebral ischemia. In: Green AR, Cross AJ, eds. Neuroprotective Agents and Cerebral Ischemia. San Diego, Calif: Academic Press; 1997:1–25.
8.
Nagata S, Golstein P. The Fas death factor.
Science. 1995;267:1449–1456.
9.
Cheng J, Zhou T, Liu C, Shapiro JP, Brauer MJ, Kiefer
MC, Barr PJ, Mountz JD. Protection from Fas-mediated apoptosis
by a soluble form of the Fas molecule. Science. 1994;263:1759–1762.
10.
Kagi D, Vignaux F, Lederman B, Burki K, Depraetere V,
Nagata S, Hengartner H, Golstein P. Fas and perforin pathways as major
mechanisms of T cell-mediated cytotoxicity. Science. 1994;265:528–530.
11.
Liles WC, Kiener PA, Ledbetter JA, Aruffo A, Klebanoff
SJ. Differential expression of Fas (CD95) and Fas ligand on normal
human phagocytes: implications for the regulation of apoptosis
in neutrophils. J Exp Med. 1996;184:429–440.
12. Matsuyama T, Hata R, Yamamoto Y, Tagaya M, Akita H, Uno H, Wanaka A, Furuyama J-I, Sugita M. Localization of Fas antigen mRNA induced in postischemic murine forebrain by in situ hybridization. Mol Brain Res. 1995;34:166–172.[Medline] [Order article via Infotrieve]
13.
Linnik MD, Zobrist RH, Hatfield MD. Evidence supporting
a role for programmed cell death in focal cerebral ischemia in
rats. Stroke. 1993;24:2002–2009.
14. Hockenberry D, Nunez G, Milliman C, Schreiber R, Korsmeyer S. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature. 1990;348:334–336.[Medline] [Order article via Infotrieve]
15.
Borner C, Martinou I, Mattmann, C, Irmler M, Schaerer
E, Martinou J-C, Tschopp J. The protein bcl-2a does not require
membrane attachment, but two conserved domains to suppress
apoptosis. J Cell Biol. 1994;126:1059–1068.
16. Allsopp TE, Wyatt A, Paterson HF, Davies AM. The protooncogene bcl-2 can selectively rescue neurotrophic-factor dependent neurons from apoptosis. Cell. 1993;73:295–307.[Medline] [Order article via Infotrieve]
17. Martinou J, Dubois-Dauphin M, Staple J, Frankowski H, Missotten M, Albertini P, Talabot D, Catsicas S, Pietra C. Overexpression of bcl-2 in transgenic mice protects neurons from naturally occurring death and experimental ischemia. Neuron. 1994;13:1017–1030.[Medline] [Order article via Infotrieve]
18.
Linnik MD, Zahos P, Geschwind MD, Federoff HJ.
Expression of bcl-2 from a defective herpes simplex virus-1 limits
neuronal death in focal cerebral ischemia. Stroke. 1995;26:1670–1675.
19. Aliprantis AO, Diez-Roux G, Mulder LCF, Zychlinsky A, Lang RA. Do macrophages kill through apoptosis? Immunol Today.. 1996;17:573–576.[Medline] [Order article via Infotrieve]
20. Chopp M, Li Y, Jiang N, Zhang RL, Prostak J. Antibodies against adhesion molecules reduce apoptosis after middle cerebral artery occlusion in rat brain. J Cereb Blood Flow Metab. 1996;16:578–584.[Medline] [Order article via Infotrieve]
21.
Tarkowski E, Rosengren L, Blomstrand C, Wikkelsö
C, Jensen C, Ekholm S, Tarkowski A. Early intrathecal
production of interleukin-6 predicts the size of brain lesion
in stroke. Stroke. 1995;26:1393–1398.
22. Tarkowski E, Rosengren L, Blomstrand C, Wikkelsö C, Jensen C, Ekholm S, Tarkowski A. Intrathecal release of pro- and antiinflammatory cytokines in stroke. Clin Exp Immunol. 1997;110:492–499.[Medline] [Order article via Infotrieve]
23.
Scandinavian Stroke Study Group. Multicenter trial of
hemodilution in ischemic stroke: background and study protocol.
Stroke. 1985;16:885–890.
24. Mahoney FI, Barthel DW. Functional evaluation: the Barthel Index. Md State Med J. 1965;24:61–69.
25. Hachinski V. Classification of stroke for clinical trials. Stroke. 1990;21:27–29.
26. Blomstrand C, Olsson JE, Nilsson B, von Arbin M, Britton M, Elwin CE, Elmers C, Norrving B, Rosen A, Samuelsson K, Strandberg K, Wahlgren NG. Swedish Aspirin Low-dose Trial (SALT) of 75 mg aspirin as secondary prophylaxis after cerebrovascular ischaemic events. Lancet. 1991;338:1345–1349.[Medline] [Order article via Infotrieve]
27. Landorp PM, Aarden LA, Calafat J, Zeijlemaker WP. A growth factor dependent B-cell hybridoma. Curr Top Microbiol Immunol. 1986;132:105–113.[Medline] [Order article via Infotrieve]
28. Aarden LA, de Groot ER, Shaap OL, Lansdorp PM. Production of hybridoma growth factor by human monocytes. Eur J Immunol. 1987;17:1411–1416.[Medline] [Order article via Infotrieve]
29. Helle M, Boeije L, Aarden LA. Functional discrimination between interleukin-6 and interleukin-1. Eur J Immunol. 1988;18:1535–1540.[Medline] [Order article via Infotrieve]
30. Kretschmann H-J, Weinrich V. Cranial Neuroimaging and Clinical Anatomy. 2nd ed. Stuttgart, Germany: Thieme; 1992:1–100.
31.
Bariniga M. Stroke-damaged neurons may commit cellular
suicide. Science. 1998;281:1302–1303.
32. Ertel W, Keel M, Stocker R, Imhof H-G, Leist M, Steckholzer U, Tanaka M, Trentz O, Nagata S. Detectable concentrations of Fas ligand in cerebrospinal fluid after severe head injury. J Neuroimmunol. 1997;80:93–96.[Medline] [Order article via Infotrieve]
33. Itoh N, Tsujimoto Y, Nagata S. Effect of bcl-2 on Fas antigen mediated cell death. J Immunol. 1993;151:621–627.[Abstract]
34. Takayama S, Sato T, Krajewski S, Kochel K, Irie S, Millan JA, Reed JC. Cloning and functional analysis of BAG-1: a novel bcl-2-binding protein with anti-cell death activity. Cell. 1995;80:279–284.[Medline] [Order article via Infotrieve]
35. Voll ER, Hermann M, Roth EA, Stach C, Kalden JR. Immunosuppressive effects of apoptotic cells. Nature. 1997;390:350–351.[Medline] [Order article via Infotrieve]
36. Holmin S. Inflammation in Brain Contusion [thesis]. Stockholm, Sweden: Stockholm University; 1997.
37. Cheng B, Christakos S, Mattson MP. Tumor necrosis factors protect neurons against metabolic-excitotoxic insults and promote maintenance of calcium homeostasis. Neuron. 1994;12:139–153.[Medline] [Order article via Infotrieve]
38. Schwartz M, Solomon A, Lavie V, Ben BS, Belkin M, Cohen A. Tumor necrosis factor facilitates regeneration of injured central nervous system axons. Brain Res. 1991;545:334–338.[Medline] [Order article via Infotrieve]
39.
Barger SW, Hörster D, Furukawa K, Goodman Y,
Krieglstein J, Mattson MP. Tumor necrosis factors and ß
protect neurons against amyloid ß-peptide toxicity: evidence for
involvement of a 
B-binding factor and attenuation of peroxide and
Ca2+ accumulation. Proc Natl Acad Sci
U S A. 1995;92:9328–9332.
40.
Tarkowski E, Blennow K, Wallin A, Tarkowski A.
Intracerebral production of tumor necrosis
factor- in Alzheimer disease and vascular dementia.
J Neuroimmunol. 1998;90:31.
Abstract.
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