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1
* Institute for Cognitive and Computational Sciences,
Interdisciplinary Program in Neuroscience, and
Department of Neurology, Georgetown University Medical Center, Washington, DC 20007, USA
1Correspondence: EP-04 Research Building, 3970 Reservoir Road, N.W., Washington, D.C. 20007, USA. E-mail: fadena{at}giccs.georgetown.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: neuronal injury • ischemia • caspases • 3-nitropropionic acid
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Such cell death may occur through either necrosis or
apoptosis (2
, 3)
. Necrosis is characterized by loss of
membrane integrity, disruption of intracellular organelles and
lysosomes, cellular swelling, and cell lysis; in contrast, apoptotic
cell death is typified by preservation of membrane integrity and
organelles, reduction of intracellular volume, and nuclear condensation
and fragmentation (4)
.
Considerable experimental evidence suggests that both necrotic and
apoptotic cell death occur after in vivo ischemia
(5
6
7)
or in vivo TBI (2
, 3)
.
Recent studies indicate that activation of cysteine proteases
(caspases), in particular the caspase-3-like family, is associated with
apoptotic cell death after TBI (3)
or cerebral ischemia
(8
9
10
11)
.
Neuronal apoptosis has been reported using an in vitro
oxygen-glucose deprivation model after blockade of inotropic glutamate
receptors in the presence of a severe injury stimulus (12
, 13)
. In addition, both necrosis and apoptosis have been observed
after severe `chemical ischemia' in vitro (14
, 15)
. Although recently reported (16)
, apoptosis of
cortical neurons has not been a feature of more traditional in
vitro `ischemia' models or a significant feature of traumatic
neuronal injury in vitro.
Ischemia and/or metabolic impairment has been reported to accompany
traumatic central nervous system injury in animals (17
, 18)
and in humans (19
20
21)
. We therefore we
hypothesized that the relative lack of apoptotic cell death noted in
earlier in vitro studies may be attributable to the lack of
an underlying metabolic impairment that usually occurs in the setting
of traumatic injury in vivo.
In the present study we used mechanical trauma in the presence of metabolic impairment produced by hypoglycemia and administration of the succinate dehydrogenase/complex II inhibitor 3-nitropropionic acid (3NP) to injure rat neuronal-glial cortical cultures. We examined whether this combined insult would induce both necrotic and apoptotic cell death, as occurs after TBI in animals, and whether caspases known to be involved in ischemic and/or traumatic injury in vivo play a role in such cell death in vitro. To address these questions, we tested for the presence of apoptosis as defined by nuclear fragmentation and condensation, DNA fragmentation into oligonucleosomes, ability of a protein synthesis inhibitor to protect against this injury, and activation of caspases. In addition, we examined the effect of caspase inhibitors on combined injury in the presence and absence of inotropic glutamate receptor antagonists.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Briefly, dissociated cortices from 1- to
3-day-old rats were seeded in 96-well Primaria microplates (Falcon,
Lincoln Park, N.J.) and allowed to grow to confluency. Isolated
embryonic cortices were dissociated and individual cells (2–2.5 x 106 cells/ml) were seeded on 10 day in
vitro (DIV) confluent glial cultures. Cortices were dissociated in
Hank's balanced salt solution without calcium or magnesium (Mediatech,
Herndon, Va.) supplemented with 10 mM HEPES (pH 7.0; Biofluids,
Rockville, Md.) and 1 mM sodium pyruvate (Biofluids). Cultures were fed
twice per week by replacement of one-third of media with minimal
essential medium with Earle's salts (Mediatech) supplemented with 10%
equine serum (HyClone Laboratories, Logan, Utah), 27.5 mM HEPES (pH
7.2), 2 mM glutamine (Biofluids), 20 mM glucose (Biofluids), and 1%
antibiotic-antimycotic solution (Biofluids). Ara-C (10 µM; Sigma, St.
Louis, Mo.) was added during the first feeding to stop further glial
proliferation. After 10 DIV, glutamine concentration was reduced to 1
mM and equine serum was omitted. Cultures were incubated at 37°C in
humid atmosphere with 4% CO2. Neuronal-glial
cultures were used at 19–21 DIV. Glial cultures were treated
identically to neuronal-glial cultures with the exception of the
addition of embryonic cortical neurons. Neuronal cultures were prepared from 18-day Sprague-Dawley rat embryonic cortices as outlined above. Instead of addition of isolated cells to confluent glial cultures, cell suspension was seeded at 1 x 106 cells/ml onto Corning microplates precoated with 10 µg/ml poly-D-lysine (Sigma)by dilution with neurobasal medium (NBM; Life Technologies, Grand Island, N.Y.) supplemented with 25 µM glutamate (Sigma), 0.5 mM glutamine, 1% antibiotic-antimycotic solution, and 2% B27 supplement (Life Technologies). Neuronal cultures were fed on day 4 in vitro with NBM supplemented with 0.5 mM glutamine, 1% antibiotic-antimycotic solution, and 2% B27 supplement. Cultures were incubated at 37°C in humid atmosphere with 4% CO2. Neurons were used at 11–14 DIV.
Qualitative reverse-transcription polymerase chain reaction
(RT-PCR)
The presence of caspase-1 and -3 mRNA was analyzed by
qualitative RT-PCR, using a previously described method (22
, 23)
. In brief, total RNA was isolated from cultures by acidic
phenol extraction and RNA concentrations were estimated
spectrometrically. RNA was then treated with RNase-free DNase I
(Promega, Madison, Wis.) for 1 h at 37°C. Total RNA (20 µg)
was reverse transcribed using M-MLV RT (Life Technologies) and an
oligo-dT primer and a random primer (Life Technologies). PCR was
performed on one-tenth of the resulting cDNA using 30 pmol of the
following oligonucleotides: 5'-CACATTGAAGTGCCCAAGCT-3' (caspase-1 sense
primer), 5'-TCCAAGTCACAAGACCAGGC-3' (caspase-1 antisense primer),
5'-GGTATTGAGACAGACAGTGG-3' (caspase-3 sense primer), and
5'-CATGGGATCTGTTTCTTTGC-3' (caspase-3 antisense primer).
PCR was performed using 30 cycles and the following program: initial denaturing at 95°C for 2 min, subsequent denaturing at 94°C for 2 s, annealing at 55°C for 15 s, primer extension at 72°C for 45 s, and final primer extension at 72°C for 2 min. One-third of the reaction volume was loaded onto a 1.5% agarose gel in 1 x TBE buffer containing 0.5 µg/ml ethidium bromide and electrophoresis was performed at 5 mV/cm.
Induction of in vitro mechanical trauma
The induction of injury and the cellular response to this trauma
model have been described in detail (22)
. All treatments
were added directly to media 30 min prior to injury. Media from
neuronal-glial cultures (19–21 DIV) was replaced with a balanced salt
solution (BSS) containing 116 mM NaCl, 5.4 mM KCl, 0.8 mM
MgSO4, 1.8 mM CaCl2, 1.0 mM
NaH2PO4, 26.2 mM
NaHCO3, 0.01 mM glycine, and 10 mg/l phenol red.
Control (uninjured) cultures or those subjected to trauma without
metabolic impairment were supplemented with 5.5 mM glucose. Ten
millimolar 3NP was added to cultures used for trauma plus 3NP with
glucose deprivation (trauma + 3NP/GD) immediately prior to injury.
Injury was induced by a specially designed punch device that produces
28 parallel cuts 1.2 mm in length at 0.5 mm intervals. Immediately
after injury, cultures were returned to 37°C and 4%
CO2 and incubated for 60 min. Cultures were then
washed with BSS, supplemented with 5.5 mM glucose and 1%
antibiotic-antimycotic solution, and incubated at 37°C for 24 h.
Control uninjured sister cultures were treated identically with the
exception of trauma and were used to estimate basal lactate
dehydrogenase (LDH) release.
Cell death assessment
Total cell death was estimated using LDH release, which has been
widely used as a biochemical measure of cell injury
(24
25
26)
and accurately reflects cell death measured by
trypan blue counts or by increases in ethidium homodimer-1 fluorescence
in this trauma model (22
, 27)
. Briefly, 24 h after
trauma, 75 µl media was transferred to a 96-well microplate and
diluted with 150 µl LDH assay reagent containing 5 mM ß-NAD, 25 mM
lactic acid, 0.03% bovine serum albumin (BSA), 100 mM Trizma, and
0.9% NaCl (pH 8.45), all from Sigma. Spectrophotometric analysis was
performed at room temperature using a Ceres 900 microplate reader
(Biotek Instruments, Inc., Winooski, Vt.) measuring optical density at
340 nm over 250 s at 5 s intervals (50 readings per sample).
Linear regression analysis provided an estimate of LDH activity. Basal
LDH activity levels were subtracted from treatments prior to analysis.
DNA fragmentation analysis
DNA was extracted and analyzed as described previously
(28)
. In brief, cells were lysed in 7 M guanidine
hydrochloride (Life Technologies), added directly to Wizard Minipreps
DNA Purification Resin (Promega), and centrifuged at 2000 x
g. Pellet was resuspended in washing solution containing 90
mM NaCl, 9 mM Tris-HCl (pH 7.4), 2.25 mM EDTA, and 55% ethanol, all
from Sigma. DNA solution was passed through a Wizard Minicolumn
(Promega) mounted onto a vacuum manifold and washed with 3 ml washing
solution. Columns were dried by centrifugation at 5000 x
g for 2 min. DNA was eluted from the Minicolumn with 50 µl
deionized water and centrifugation at 5000 x g for 2
min. RNA was removed by incubation with 2 µg RNase A (5 Prime
3
Prime, Boulder, Colo.) for 15 min at 37°C. DNA was loaded onto a
1.5% agarose gel (U.S. Biochemicals, Cleveland, Ohio) in TBE buffer
(Digene Diagnostics, Beltsville, Md.) containing 0.5 µg/ml ethidium
bromide. Electrophoresis was performed at 5 V/cm, after which DNA was
visualized by 300 nm transillumination on a Speedlight Gel
Documentation System (Hoefer, San Francisco, Calif.).
Hoechst 33258 staining
Hoechst 33258 (Sigma) was diluted in deionized water to 10 mg/ml
and stored at -20°C. Hoechst 33258 was added to culture media to a
final concentration of 2 µg/ml and incubated at 37°C for 10 min.
Cultures were washed with BSS and examined using a Nikon TE300
microscope (Nikon, Melville, N.Y.), with excitation at 360 nm and
emission at 460 nm. Images were captured using an Optronics DEI-750
digital camera (Optronics, Goleta, Calif.) and Scion Image 1.62a
software. Images used for quantitative counts were obtained after
Hoechst 33258 staining by randomly selecting a field at 400x
magnification in each culture well and then positioning the image so
that cuts induced by the trauma device were placed at the upper and
lower limits of the field. Cells that exhibited condensed or fragmented
nuclei were counted in a defined area that remained constant between
experiments.
Caspase activity assay
Culture media was replaced 24 h after injury with lysis
buffer containing 10 mM HEPES-KOH (pH 7.4, 100 mM NaCl, 5 mM DTT, and
0.1% CHAPS, all from Sigma. Microplates were sealed using Storage Mat
II (Corning) to prevent volume loss and placed at -80°C. After
thawing on ice, cell lysates were collected and combined (21 wells per
sample). Samples were triturated and placed at -80°C. Samples were
thawed on ice and centrifuged at 13,000 x g for 30 min
at 4°C. Supernatants were either used immediately or stored at
-80°C. Protein concentration was estimated by Bradford's method
using a BSA standard. To assay for caspase-1-like or caspase-3-like
activity, 20 µg protein was incubated in a microtiter plate with the
florigenic substrate Ac-YVAD-AMC or Ac-DEVD-AMC (20 µM; Bachem),
respectively. Free aminomethylcoumarin accumulation was measured over
time using a CytoFluor II Fluorescence Multi-well Plate Reader
(PerSeptive Biosystems, Inc., Framington, Mass.) with excitation at 360
nm and emission at 460 nm. Specific activity was determined by linear
regression analysis.
Drugs
3NP was purchased from Sigma. The noncompetitive NMDA receptor
antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]
cyclohepten-5,10-imine (MK-801) and the AMPA/kainate receptor
antagonist 6-nitro-7-sulfamoylbenzo[F]quinoxaline-2,3-dione (NBQX)
were obtained from Tocris Cookson (St. Louis, Mo.). z-DEVD-fmk and
z-YVAD-fmk were obtained from Enzyme Systems Products (Dublin, Calif.).
BAF was purchased from Bachem; cycloheximide was obtained from Sigma.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Using this injury model,
ischemia-like conditions were induced by the addition of 3NP, a
selective inhibitor of succinate dehydrogenase/complex II, with
simultaneous GD (glucose deprivation). Neuronal-glial cultures
incubated in the presence of 10 mM 3NP with GD for 60 min did not
demonstrate an increase in LDH release 24 h after exposure when
compared to control sister cultures (P=0.906, Student's
t test, n=32). In contrast, neuronal-glial
cultures subjected to mechanical trauma in the presence of 10 mM 3NP
with GD (trauma + 3NP/GD) for 60 min showed a highly significant
increase in LDH release 24 h postinjury compared with sister
cultures that received traumatic injury in the absence of metabolic
impairment (Fig. 1
A).
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Role of inotropic glutamate receptors in injury induced by trauma +
3NP/GD
To examine the involvement of inotropic glutamate
receptor-mediated cell death in injury induced by trauma + 3NP/GD,
neuronal-glial cultures were treated for 30 min prior to injury and for
24 h postinjury with selective antagonists. Inhibition of NMDA
receptors by MK801 (10 µM) produced marked protection against trauma
+ 3NP/GD-induced LDH release (Fig. 1B
). This injury was
insensitive to selective inhibition of AMPA/KA receptors by NBQX (10
µM; Fig. 1B
).
Induction of apoptosis by trauma + 3NP/GD
To investigate the presence of apoptotic cell death after trauma +
3NP/GD, cultures were stained 24 h after injury with the nuclear
dye Hoechst 33258. As shown in Fig. 2
, numerous cells display condensed or fragmented nuclear morphology,
consistent with apoptotic cell death after staining with Hoechst 33258,
in contrast to the normal diffuse nuclear staining seen in healthy
cells. Furthermore, DNA fragmentation was visible after gel
electrophoresis of DNA samples isolated from neuronal-glial cultures
subjected to trauma + 3NP/GD (Fig. 3
). Less prominent DNA fragmentation was visible in cultures injured by
trauma in the absence of metabolic impairment, whereas no fragmentation
was detectable in samples isolated from sister uninjured cultures (Fig. 3)
.
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Quantitative data were obtained by counting the number of cells
in a defined area that displayed apoptotic-like nuclear morphology as
visualized by Hoechst 33258 staining. Using this method, trauma induced
an approximately twofold increase and trauma + 3NP/GD induced a
fourfold increase over basal levels, which was significantly greater
than either basal or trauma-induced levels (Fig. 4
A).
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Addition of the protein synthesis inhibitor cycloheximide (10
µg/ml) significantly attenuated trauma + 3NP/GD-induced cell death
(Fig. 4B
), further suggesting the presence of significant
apoptotic cell death.
Role of caspases in trauma + 3NP/GD injury
Qualitative RT-PCR was used to determine the expression of
caspase-1 and caspase-3 mRNA in neuronal, glial, and neuronal-glial
cultures. Neuronal-glial and glial cultures express both caspase-1 and
caspase-3 (Fig. 5
A). In contrast, neuronal cultures were positive only for
caspase-3 mRNA (Fig. 6
A). Consistent with the relative increase in the number of
cells with apoptotic nuclear morphology, protein extracts from trauma +
3NP/GD induced significant increases in caspase-3-like activity over
basal levels (Fig. 5B
). In contrast, caspase-1-like activity
was not significantly altered from basal levels after trauma or trauma
+ 3NP/GD (Fig. 6C
).
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To determine the functional relevance of injury-induced caspase-3-like
activity, neuronal-glial cultures were treated with relatively
selective caspase inhibitors during trauma or trauma + 3NP/GD. Both the
pan-caspase inhibitor BAF (100 µM) and the caspase-3 selective
inhibitor z-DEVD-fmk (160 µM) provided significant protection against
trauma + 3NP/GD-induced LDH release (Fig. 6A
). Consistent
with selective induction of caspase-3-like activity, trauma +
3NP/GD-induced cell death was insensitive to inhibition of
caspase-1-like activity by z-YVAD-fmk (160 µM; Fig. 6A
).
Administration of z-DEVD-fmk during trauma + 3NP/GD also significantly
decreased the number of apoptotic cells compared with trauma + 3NP/GD
alone (Fig. 6B
). In contrast to trauma + 3NP/GD injury,
trauma without metabolic impairment was insensitive to caspase
inhibition by BAF (100 µM), z-DEVD-fmk (160 µM), or z-YVAD-fmk (160
µM; data not shown).
Additive protection by concurrent inhibition of NMDA receptors and
caspase-3-like activity
To determine whether NMDA receptor-mediated cell death represented
a distinct pathway from caspase-3-sensitive cell death, the effects of
concurrent inhibition of both NMDA receptors and caspase-3-like
activity were examined during trauma + 3NP/GD. Significantly greater
neuroprotection was observed with the simultaneous application of MK801
(10 µM) and BAF (100 µM) than with application by either agent
alone (Fig. 7
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. We also present evidence that although apoptosis has
not been reported to be a feature of in vitro traumatic
injury, modest apoptotic cell death is also induced by traumatic injury
alone; however, blockade of apoptotic cell death by caspase inhibitors
does not provide significant neuroprotection in this model.
Ischemic conditions have most commonly been produced in
vitro through the use of an anaerobic chamber or through
`chemical ischemia' induced by inhibitors of the tricarboxylic acid
cycle or oxidative phosphorylation. Various metabolic `poisons' have
been used to generate chemical ischemia, including cyanide (29
, 30)
, azide (31)
, and 3NP (15)
.
Unfortunately cyanide and azide have other cellular effects in addition
to inhibition of oxidative phosphorylation and ATP synthesis
(32
33
34)
. In contrast, 3NP is a selective inhibitor of
succinate dehydrogenase/complex II, with no other reported cellular
effects (35
, 36)
. Therefore, 3NP was used to induce
chemical ischemia during trauma. Consistent with other in
vitro `ischemia' models, cultures were also deprived of glucose
during injury.
Incubation of control neuronal-glial cultures with 3NP (10 mM) with
concurrent glucose deprivation (GD) for 60 min was without effect on
LDH release over the next 24 h. The addition of traumatic injury
to this subthreshold metabolic impairment induced a marked increase in
cell death. Consistent with in vivo TBI (37
, 38)
and with mechanical trauma in vitro
(22)
, NMDA receptor inhibition by MK801 (10 µM) was
highly protective against trauma + 3NP/GD, whereas AMPA/kainate
receptor inhibition was without significant effect. Thus, trauma +
3NP/GD induces significant necrotic cell death, as evidenced by the
protection afforded by NMDA receptor inhibition.
Trauma + 3NP/GD induces significant apoptotic as well as necrotic cell
death. Hoechst 33258 staining was used to visualize nuclear morphology.
We have found that the number of apoptotic cells assayed by counting
cells that exhibit apoptotic nuclear morphology after Hoechst 33258
staining correlates well with other methods of estimating apoptotic
cell death, such as TUNEL staining, in another model of neuronal injury
that induces significant levels of apoptosis (unpublished
observations). A small number of cells in control cultures were
undergoing apoptotic cell death as assayed by Hoechst 33258 staining.
This low basal level of apoptosis in control cultures is consistent
with the use of postmitotic neuronal cultures and has been reported by
others (15)
. Approximately a fourfold increase in the
number of cells with apoptotic-like nuclear morphology under control
conditions was detected 24 h after trauma + 3NP/GD; mechanical
trauma in the absence of chemical ischemia produced a less substantial
rise over basal levels. Furthermore, DNA extracted from cultures
subjected to trauma + 3NP/GD was characterized by the presence of
oligonucleosomal fragmentation as assayed by gel electrophoresis. In
addition, trauma + 3NP/GD-induced cell death was significantly
decreased by the protein synthesis inhibitor cycloheximide. This effect
was modest, which was expected as significant levels of necrotic cell
death occur in this injury model.
Whereas chronic exposure to 3NP for 24 h induces both apoptosis
and necrosis (14
, 15)
, acute treatment of neuronal-glial
cultures with 3NP does not induce detectable cell death over control
levels. Taken together, the data presented here suggest that mild
metabolic impairment sensitizes cells to trauma-mediated apoptosis.
Application of glutamate to cultured cells may induce necrosis and/or
apoptosis, with the type of resultant cell death depending on
mitochondrial function (39)
. Specifically, cell death that
occurs within hours of glutamate administration is accompanied by rapid
loss of mitochondrial membrane potential and necrotic cell death,
whereas a portion of those cells surviving the early necrotic phase
recover mitochondrial function and subsequently undergo apoptosis
(39)
. By analogy, cells that do not recover mitochondrial
function after `reperfusion,' i.e., replacement of media containing
3NP with media supplemented with glucose, after trauma + 3NP/GD may
undergo necrotic cell death, whereas a portion of those cells that
recover mitochondrial function may subsequently die by apoptosis.
In accordance with in vivo TBI (3)
, trauma +
3NP/GD induced selective increases in caspase-3-like but not
caspase-1-like activity. Caspase-3 activation has been shown to
colocalize with neurons exhibiting DNA fragmentation after in
vivo traumatic brain injury, suggesting that activation of
caspase-3 may serve as a marker for apoptotic cell death
(40)
. Inhibition of caspase-3-like activity by z-DEVD-fmk
significantly attenuated cell death after injury, but the selective
caspase-1-like inhibitor z-YVAD-fmk was without effect. In
vitro dose-response curves have previously been generated for both
z-DEVD-fmk and z-YVAD-fmk, and the doses used in the present study
produce maximal inhibition (23)
. Although mechanical
trauma induced a twofold increase in cells with apoptotic-like nuclear
morphology, caspase inhibitors had no effect on such cell death. Thus,
apoptosis may significantly contribute to total cell death only in
injury induced by the combined insult of trauma and chemical ischemia.
Both caspase-1 and caspase-3 mRNA were detected by qualitative RT-PCR
in rat cortical neuronal-glial cultures. Caspase-1 expression was not
detected in cortical neurons seeded without a glial layer, suggesting
that cortical neurons may not express caspase-1. Similarly, rat
cerebellar granule neurons also express caspase-3 but not caspase-1
mRNA (23)
. Thus, selective activation of caspase-3, but
not caspase-1 activity, and protection by z-DEVD-fmk, but not
z-YVAD-fmk, after trauma + 3NP/GD may reflect the relatively selective
neuronal injury in this model (27)
.
As necrosis and apoptosis appear to represent two distinct forms of cell death, we examined the effect of concurrent inhibition of NMDA receptors and caspase-3-like activity to determine whether the protective effects are additive or synergistic. This combination provided significantly greater protection against trauma + 3NP/GD than either treatment alone, suggesting that two independent cell death pathways are induced by trauma + 3NP/GD: NMDA receptor-mediated and caspase-3-dependent cell death. As both cell death pathways are induced by TBI in vivo, a combination of NMDA receptor blockade and caspase-3 inhibition may be an effective therapeutic strategy for the treatment of acute central nervous system injuries.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication January 12, 1999. Revised for publication April 22, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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