(Stroke. 1997;28:1073-1081.)
© 1997 American Heart Association, Inc.
Articles |
B in Forebrain Neurons of Rats
From Eli Lilly and Company, CNS Division, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Ind.
Correspondence to Dr James A. Clemens, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285.
| Abstract |
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(TNF-
) and
interleukin-1ß (IL-1ß) are increased. Oxygen radicals, TNF-
, and
IL-1ß are known to activate nuclear factor-
B (NF-
B) in
vitro. The present study was performed to determine whether NF-
B
was activated in vivo by global ischemia in hippocampal
CA1 neurons.
Methods Adult male rats were subjected to 30 minutes of
four-vessel occlusion and killed 72 hours later. Levels of NF-
B p50
and p65 subunits in hippocampus were determined by immunocytochemistry,
Western blot, and gel-shift analysis. Specific labeling of DNA
strand breaks was demonstrated by means of an Apoptag apoptosis
detection kit.
Results Labeling of DNA strand breaks was present at 72
hours. Chromatin compaction and segregation, a characteristic of
apoptosis, was observed in sections stained with hematoxylin
and eosin. NF-
B p50 and p65 immunoreactivity localized only to
nuclei of CA1 neurons at 72 hours after reperfusion. Induction of the
activated p50 and p65 subunits was confirmed by Western blot
and electromobility shift analysis. The results demonstrate
that NF-
B is activated selectively in hippocampal CA1
neurons at 72 hours after four-vessel occlusion, which is at the
approximate time of CA1 neuronal cell death.
Conclusions Transient forebrain ischemia resulted
in a marked activation of nuclear NF-
B in the highly vulnerable CA1
sector. Intense nuclear localization of NF-
B was associated only
with dying neurons; regions of the hippocampus that were not vulnerable
to four-vessel occlusion did not exhibit nuclear NF-
B localization.
The elevation of NF-
B in degenerating CA1 neurons may be associated
mechanistically with apoptotic or necrotic cell death.
Key Words: apoptosis cerebral ischemia, global hippocampus
| Introduction |
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One common mechanism might be the generation of toxic ROS. In rats subjected to transient global ischemia by the 4-VO technique, large amounts of hydrogen peroxide were released during the early reperfusion period.25 With use of the salicylate trapping technique, hydroxyl radicals were observed during reperfusion after global ischemia.26 27 28 The nitric oxide radical is also known to increase after ischemia29 30 and can lead to the production of the toxic hydroxyl radical.31 32 33 Furthermore, factors that are known to increase after global ischemia such as cytokines,34 excitatory amino acids,35 or high intracellular calcium can also stimulate production of ROS, and antioxidants22 can reduce ischemic damage. The possibility then exists that high amounts of ROS generated during reperfusion after global ischemia may initiate PCD of CA1 neurons.
Levels of ROS are tightly regulated by an abundance of
physiological antioxidants.36 ROS also
regulate the activity of the transcription factors,
NF-
B,37 and activator
protein-1.38 NF-
B is an oxidative stress-responsive
transcription factor39 that can be activated by
hydrogen peroxide, ROS, tumor necrosis factor-
, interleukin-1ß,
ultraviolet light, or HIV. When activated, this multisubunit
transcription factor induces the expression of genes encoding
acute-phase proteins, cell adhesion molecules, cell-surface receptors,
and cytokines. NF-
B is composed of a dimer of 50- and 65-kD
subunits that is retained in the cytoplasm of most cells by an
inhibitory protein, I
B. On activation, NF-
B
dissociates from I
B and translocates as a p50/p65 dimer to the
nucleus, where it can initiate gene transcription. Most of the known
stimuli that activate NF-
B can be blocked by
antioxidants.39
Because of the association of ROS with reperfusion injury, and the link
between ROS and other agents that are produced as a result of
ischemia/reperfusion and activation of NF-
B, we decided to
determine whether activated NF-
B could be detected in the
hippocampus of rats subjected to transient global forebrain
ischemia. We also attempted to visualize evidence for DNA
fragmentation and apoptosis.
| Materials and Methods |
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Antibodies
Polyclonal antisera generated to specific regions of NF-
B
were used in the various assays. Antisera used for immunocytochemistry
Ab392 and Ab567 were kindly supplied by Dr Warner Greene. Ab392 was
made against the N-terminal peptide of
BF-1, the p50 homodimer;
Ab567 was raised against the N-terminal peptide of p65. These antisera
have been characterized previously.41 42 For Western blot
analysis, antiserum 392 (described above) and a commercially
available peptide antiserum were used. This latter antiserum was raised
to an epitope corresponding to a peptide of 15 amino acids in length
mapping at the nuclear localization sequence region of NF-
B p50
(sc-114, Santa Cruz Biotechnology). The p65 antisera used for Western
blots was obtained from Rockland.
Immunocytochemistry
Forebrains were postfixed by immersion for 24 hours in fixative
and cryoprotected in 30% sucrose. Tissues were rapidly frozen in
isopentane chilled with dry ice and serially sectioned in the coronal
plane throughout the rostrocaudal extent of the hippocampus. Sections
(20 µm) were thaw-mounted onto gelatin-coated slides and stored
at -70°C. For anatomic localization of the lesioned areas, tissue
sections were stained with cresyl violet for Nissl substance.
Immunocytochemisty was performed using the avidin-biotin-peroxidase
system (ABC kit, Vector Labs). Briefly, tissue sections were incubated
with serum to block nonspecific staining followed by treatment
overnight with primary antiNF-
B p50 antiserum. Sections were
stained with the ABC immunoperoxidase system according to the
recommendations of the manufacturer. The reaction product was
visualized by development with 3,3'-diaminobenzidine and
H2O2. Negative controls included incubating
adjacent sections with antisera directed against other
nonNF-
Brelated proteins (eg, glial fibrillary acidic protein).
Stained sections were dehydrated, mounted in Permount, and examined
using a Nikon Microphot equipped with Nomarsky optics.
Sections from a parallel series of animals were processed and stained
for in situ DNA fragments based on the TUNEL technique. Animals at 72
hours after ischemia were decapitated and frozen in dry
icecooled isopentane. Tissue sections were cut and stained using the
Apoptag kit (Oncor) according to the manufacturer's recommendations.
For colocalization of NF-
B and TUNEL, fresh frozen sections through
the dorsal hippocampus were postfixed for 30 minutes with 4% buffered
paraformaldehyde, washed, and incubated in
antiNF-
B p50 antiserum (1:250), followed by detection with biotin
anti-rabbit, followed by avidin Texas Red (Vector Labs). After a
washing with PBS, sections were stained using the in situ cell death
kit according to the manufacturer's recommendations (Boehringer
Mannheim Biochemicals). Slides were coverslipped using Vectashield
mounting medium containing DAPI (Vector Labs) as nuclear
counterstain.
Cell Extracts and Western Blot Analysis
Cells were harvested by homogenization with
a Polytron in ice-cold buffer A (10 mmol/L HEPES, pH 7.9, 1.5
mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L DTT,
1 mmol/L PMSF, 1 µg/mL leupeptin, 1 µg/mL pepstatin). After 15
minutes on ice, cells were passed through a 25-gauge needle. The nuclei
were collected by centrifugation at 600g for
10 minutes, then resuspended in buffer C (20 mmol/L HEPES, pH 7.9,
25% glycerol, 0.42 mmol/L NaCl, 1.5 mmol/L
MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L PMSF, 0.5
mmol/L DTT, 1% NP-40). The nuclear extracts were microfuged at 4°C
for 10 minutes, collected, and microdialyzed against buffer D (20
mmol/L HEPES, pH 7.9, 20% glycerol, 0.1 mol/L KCl, 0.2 mmol/L
EDTA, 0.5 mmol/L PMSF, 0.5 mmol/L DTT) to the same
concentration.43
Equal amounts of extracts based on protein assay were heated to 100°C
for 5 minutes in Laemmli sample buffer, run on 12.5% SDS-PAGE gels,
and electroblotted onto PVDF membrane. Western blots were performed
using the Amersham ECL kit following the manufacturer's instructions.
The primary NF-
B p50 antibody was used at 1 µg/mL in TBST/1% dry
milk buffer (10 mmol/L Tris, pH 8.0, 150 mmol/L NaCl, 0.05%
Tween-20). The secondary antibody was goatanti-rabbit Ig F(ab')
conjugated to horseradish peroxidase (Jackson Immunoresearch) used at
1:5000 dilution in TBST buffer.
Northern Blot Analysis
Total RNA was isolated from three 4-VO or three sham rats using
the acid guanidium thiocyanate-phenol-chloroform extraction procedure
(Chomczynski and Sacchi, 1987). Polyadenylated RNA was selected
by affinity chromatography using oligo-d(T) oligotex
matrix (Qiagen). To ensure equal loading of the RNA samples for
Northern blotting, semiquantitative reverse-transcription PCR for the
housekeeping gene GAPDH was performed on an aliquot of each sample.
Poly A RNA was electrophoresed in a 1.2% agarose gel containing 2.2
mol/L formaldehyde and 20 mmol/L HEPES, pH 7.9 buffer, and then
transferred overnight onto a positive nylon filter (Micron Separations
Inc). The filter was hybridized in 1 mmol/L EDTA, 0.5 mol/L
NaH2PO4, pH 7.2, 7% SDS. Complementary DNA
template for labeling NF-
B p50 was generated by PCR using total HeLa
cDNA primers (5'-atg gca gaa gat gat cca tat ttg-3'-sense; 5'-tcc acc
ttc tgc ttg caa ata ggc-3'-antisense). The p50 PCR fragment was
verified by sequencing. Random primed 33P-labeled NF-
B
p50 cDNA fragment was added (8.5x107 total cpm) and
hybridized at 65°C for 18 hours. The Northern blot was washed twice
at 65°C for 30 minutes with 1 mmol/L EDTA, 40 mmol/L
NaHPO4, pH 7.2, 1% SDS. Autoradiography
was performed at -70°C for 72 hours.
EMSA
NF-
B DNA binding probes were generated by 5' end-labeling
with
-[32P]ATP and T4 polynucleotide
kinase to double-stranded cDNA encoding
5'-CAACGGCAGGGGAATTCCCCTCTCCTT-3'. Binding reactions were prepared
in a final volume of 20 µL containing 10 mmol/L HEPES, pH 7.5,
50 mmol/L KCl, 1 mmol/L EDTA, 50 µg/mL poly(dI-dC), 0.1%
deoxycholate, 5% glycerol, and 0.1 mmol/L DTT. Nuclear extracts
(20 µg), buffer, and 1x105 cpm of radiolabeled NF-
B
probe were incubated at room temperature for 15 minutes. Competition
reactions were prepared as described, except 0.1 µg/mL of cold
double-strand DNA oligonucleotides was added 15 minutes
before the radiolabeled probe. Bound complexes were separated from free
probe by electrophoresis in 5% Tris-buffered EDTA nondenaturing
polyacrylamide gels, then visualized by
autoradiography.
Shift-Western blots were analyzed by electrotransfer onto stacked membranes at a fixed current (0.5 mA) in 0.5x Tris-buffered EDTA. The first filter below the gel was nitrocellulose, followed by PVDF membrane. Radiolabeled components from the PVDF membrane were detected by autoradiography, whereas proteins bound to the nitrocellulose were immunodetected as described for Western blotting.44
| Results |
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B p50 and p65. Fig 3A
B p50
immunostaining through the CA1 paramedian zone showing
robust immunostaining of the nuclei of hippocampal
pyramidal neurons, and Fig 3B
B p65 immunostaining of the
nuclei of degenerating CA1 neurons. Double labeling revealed that
NF-
B immunoreactivity and TUNEL staining could be observed in the
same neuron (Fig 4
B
and TUNEL staining. Nuclear NF-
B localization was never observed by
immunocytochemistry in sham-operated controls (Fig 5D
B immunostaining was not
observed (Fig 5C
B immunoreactivity.
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A number of approaches were used to characterize the state of the
NF-
B complex in affected tissue: Western blot, EMSA, Northern blot,
and gel-shift Western analysis. Western blot analysis
of homogenates prepared from ischemic and
sham-operated animals was performed using anti NF-
B p50 and p65
antibodies. A substantial increase in translation or translocation of
NF-
B p50 and p65 was observed in extracts from the dorsal
hippocampus of ischemic rats but not of sham-operated controls
(Fig 6
).
|
Fig 7
shows an EMSA of pooled nuclear brain extracts
from the hippocampus from ischemic and sham-operated rats using
the NF-
B DNA binding consensus sequence. As a result of
ischemia, there was an induction of NF-
B DNA-binding
activity in pooled nuclear hippocampal tissue. Competition assays using
an excess of nonradiolabeled NF-
Bspecific oligo probe extinguished
the specific retarded band.
|
Levels of NF-
B mRNA were also measured in ischemic and
sham-operated controls. Fig 8
shows the Northern blot
analysis of total RNA of ischemic and sham hippocampal
extracts using an NF-
B antisense cDNA probe. It appears that NF-
B
messenger RNA levels increase during ischemia.
|
A gel-shift Western assay was used to determine whether p50 and p65
were components of the NF-
B DNA-binding activity. The results of the
gel-shift Western assay are shown in Fig 9
. As shown in
this figure, the transcription factor complex contains factors having
epitopes to both p50 and p65, suggesting the presence of NF-
B.
|
| Discussion |
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B. The NF-
B transcript is translated in
the cytoplasm of mammalian cells. NF-
B consists of a p50 and
p65/RelA complex. I
B-
inhibitory protein (I
B)
traps NF-
B in the cytoplasm and prevents its translocation to the
nucleus. Activation consists of phosphorylation or
degradation of I
B from the complex in the cytoplasm. On activation,
NF-
B is translocated to the nucleus of the cell.45
The results of this study demonstrate induction of activated
NF-
B after global forebrain ischemia. The presence of active
NF-
B was confirmed by nuclear localization in the degenerating
pyramidal neurons using antibodies to NF-
B p50 and p65,
Western blot of microdissected tissue extracts, and gel-shift
analysis of nuclear extracts. The classic NF-
B complex
consisting of the heterodimer p50/p65 was induced at 72 hours.
Specificity for induced NF-
B bands in Western blot analysis
was determined by using a well-characterized, antiNF-
B p50
antibody from Santa Cruz.46 The specific NF-
B p50
comigrated with a 50-kD band found in HeLa nuclear extracts. Because
p50 homodimers can also exist, we monitored p65 as well. Western blot
analysis and immunocytochemistry revealed a parallel increase
in p65 after ischemia. The nuclear NF-
B immunoreactivity, as
visualized by immunocytochemistry, at 72 hours was restricted to the
degenerating CA1 hippocampal pyramidal neurons and was not
seen in areas that did not degenerate. Other hippocampal subfields,
cerebral cortex, thalamus, hypothalamus, and other brain stem areas
were free of immunoreactivity. These results indicate that a unique
association exists between hippocampal pyramidal neuron
degeneration and NF-
B activation. We concluded that NF-
B was
induced in a pattern closely related to neuronal death during global
forebrain ischemia.
We observed DNA fragmentation and histological evidence of apoptosis at 72 hours after 30 minutes of 4-VO by in situ labeling of DNA fragmentation. Although in situ labeling of DNA fragmentation using the TUNEL assay is observed with different kinds of cell death, and its detection in situ cannot be considered as a specific marker of apoptosis,47 other observations provide additional evidence for apoptotic death of CA1 neurons after global ischemia. Earlier observations in models of global ischemia demonstrated DNA fragmentation by in situ as well as biochemical evidence of DNA laddering, which is typical of nuclear DNA fragmentation into oligonucleosomal fragments after transient forebrain ischemia.2 3 4 5 6 7 Additional evidence is provided by our histological evidence of apoptosis in hippocampal CA1 neurons. At 72 hours after 4-VO, some hippocampal CA1 pyramidal cells demonstrated chromatin compaction and segregation, which is characteristic of PCD, whereas morphological changes such as eosinophilic shrunken cytoplasm and pyknotic nuclei were observed in other neurons. The eosinophilic shrunken neurons may have eventually demonstrated chromatin compaction and segregation as evidence of apoptosis.
Other proteins (c-fos, c-jun, heat shock
proteins, etc) were previously investigated for their ability to serve
as markers associated with neuronal cell death, but none seemed to be
able to clearly differentiate neurons that were destined to die from
those that would survive.48 49 50 On the other hand, NF-
B
activation at 72 hours after ischemia was associated only with
degenerating neurons. A portion of these neurons appeared to be
undergoing PCD. Many of the agents known to be produced in the brain
after transient ischemia (such as reactive oxygen
intermediates, cytokines, tumor necrosis factor-
, and
interleukin-1ß) have already been shown to activate NF-
B
in vitro.39 51 52 In addition, studies in vitro found that
the stimuli capable of activating NF-
B could be blocked by
antioxidants.39 It is possible that cytokines,
glutamate, or other toxic factors that are capable of leading to the
production of reactive oxygen intermediates are responsible for
the activation of NF-
B observed in this study.
NF-
B activation as evidenced by nuclear localization by
immunocytochemistry was never observed in the sham-operated controls.
However, low levels of constitutive NF-
B activity were observed in
sham-operated controls by EMSA. While the nuclear localization of
NF-
B to dying neurons at 72 hours after 4-VO is suggestive of a role
for NF-
B in neuronal cell death, it may have other functions. The
presence of activated NF-
B in degenerating neurons at 72
hours after ischemia is likely to be a marker of neuronal cell
death, but at the present time we cannot conclude that it is a
causative factor.
NF-
B is able to stimulate transcription of genes that could be
considered to be either protective in nature or deleterious. There are
NF-
B consensus sequences on the genes for Mn SOD and Cu/Zn SOD of
numerous species, including humans and rats.53 The
increase in NF-
B may be a futile attempt to induce transcription of
protective factors such as Mn SOD or Cu/Zn SOD; however, because of the
long-lasting inhibition of synthesis of many proteins in this area, the
protective factors could not be produced. In addition, I
B may be
unable to be synthesized to inactivate the already
activated NF-
B. Thus, NF-
B remained activated.
Future studies will address this possibility by evaluating SOD and
I
B after global forebrain ischemia.
The possibility of a deleterious role for NF-
B must be considered.
NF-
B can cause transcription of the inducible nitric oxide synthase
gene,54 and NF-
B consensus sequences are present in
the promoter region of the neuronal nitric oxide synthase
gene.55 Furthermore, nitric oxide is known to be a factor
in neurotoxicity.29 The 5'-flanking region of the
cytosolic phospholipase A2 gene contains consensus NF-
B
sequences.56 We have recently reported induction of
cytosolic phospholipase A2 after global
ischemia.57 Transcription of this gene could
result in the eventual production of eicosanoid products
that are neurotoxic. Evidence that transcription factors of the
NF-
B/Rel family are involved in PCD is continuously increasing.
Potential target genes for NF-
B are among the genes induced on
apoptosis. They include p53,58
c-myc,59 Fas/Apo-1 ligand,60 61
and interleukin-1ß converting enzyme.62 Stimuli that
activate NF-
B can transcriptionally activate these
death genes, and where examined, their upstream promoter regions
contain potential NF-
Bbinding motifs. In contrast, the activity of
NF-
B is downregulated by the antiapoptotic protein
bcl-2.63 Recently, it was shown that bcl-2 and bcl-x
long-form mRNA were expressed after global ischemia in both
surviving and dying neurons, but their proteins were expressed
primarily in neurons destined to survive.64 Thus, the
proapoptotic influence of NF-
B could continue unopposed.
In conclusion, our studies establish that activated NF-
B
appears in the nucleus of CA1 pyramidal cells during
degeneration and may serve as a marker of dying neurons in this brain
area or may be involved mechanistically in events leading to neuronal
death. Differential expression of both p50 and p65 was seen in the CA1
region of ischemic brain. The NF-
B immunoreactivity was
restricted to regions susceptible to damage and was not present in
other hippocampal subfields. We also show histological
evidence that CA1 pyramidal neurons demonstrate chromatin
compaction and segregation, which is characteristic of an early stage
of apoptosis. Moreover, our studies agree with others
demonstrating that CA1 neurons show in situ labeling of DNA fragments
based on the TUNEL technique after transient global forebrain
ischemia. However, further studies elucidating the specific
role of NF-
B in neuronal cell death will be conducted.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
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B antibodies. Received November 7, 1996; revision received January 15, 1997; accepted February 28, 1997.
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