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(Stroke. 1997;28:1073-1081.)
© 1997 American Heart Association, Inc.

Articles

Global Ischemia Activates Nuclear Factor-B in Forebrain Neurons of Rats

James A. Clemens, PhD; Diane T. Stephenson, BS; E. Barry Smalstig, BS; Eric P. Dixon, MS; Sheila P. Little, PhD

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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Background and Purpose After global ischemia, brain levels of hydrogen peroxide, oxygen radicals, and the cytokines tumor necrosis factor- (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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Hippocampal CA1 neurons are highly susceptible to short periods of transient global ischemia. It takes approximately 72 hours to obtain clear histological evidence of CA1 neuronal degeneration. This pattern of delayed neuronal cell death is thought to occur in victims of cardiac arrest who have been resuscitated more than 8 to 10 minutes after arrest.¹ CA1 neuronal death after global ischemia is not well understood, but evidence exists to support the hypothesis that it is apoptotic.^{2 3 4 5 6 7} A variety of agents including free radicals,^{8 9} calcium overload,¹⁰ excitotoxicity,¹¹ eicosanoids,¹² proteases,¹³ and cytokines^{14 15} have been proposed as contributory factors. Many of these agents have been shown to induce apoptotic cell death under certain conditions in vitro,^{16 17 18 19 20} and inhibitors of certain members of the above classes of compounds have been reported to reduce ischemic damage.^{21 22 23 24} It is possible that many of these agents eventually exert their cytotoxic effects through a common mechanism.

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 ischemia^{29 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, IB. On activation, NF-B dissociates from IB 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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Forebrain Global Ischemia and Brain Tissues
Transient forebrain ischemia was induced by 4-VO as described.⁴⁰ Briefly, Wistar rats (Hilltop Laboratories, Scottsdale, Pa) were prepared for forebrain ischemia under 2% halothane inhalation anesthesia by electrocauterization of the bilateral vertebral arteries and placement of atraumatic clasps around the common carotid arteries without interruption of the arterial blood flow. On the following day, forebrain ischemia was induced by tightening of the clasps for 30 minutes. Body temperature was maintained at 37°C by means of heat lamps. All animal procedures were performed in compliance with the institute's animal care and use committee. After ischemia, animals were perfused with PBS followed by ice-cold 4% buffered paraformaldehyde, or brains were excised and immediately frozen at -70°C. Before freezing, the region of the dorsal hippocampus containing the CA1 layer was carefully dissected from the remainder of the hippocampus. Twenty-two animals were evaluated with immunocytochemistry: 72-hour postischemia (n=13) and sham-operated controls (n=9). In addition, 6 animals were processed for TUNEL (72-hour postischemia, n=5; sham, n=1). For biochemical studies, a total of 37 animals were evaluated as follows: Western blot analysis (72-hour postischemia, n=7; sham-operated controls, n=13), gel-shift assay (72-hour postischemia, n=4; sham, n=4), RNA (72-hour postischemia, n=3; sham, n=3), and gel-shift Western assay (72-hour postischemia, n=5).

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 anti–NF-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 non–NF-B–related 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 ice–cooled 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 anti–NF-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 goat–anti-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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fig 1A is a photomicrograph of a hematoxylin-eosin–stained section through the hippocampal CA1 layer of a rat subjected to 4-VO. Many neurons appear shrunken and eosinophilic, while others demonstrate chromatin compaction and segregation, which is characteristic of apoptosis. Sections stained for TUNEL using the Apoptag kit showed distinctive nuclear staining characteristic of DNA fragmentation (Fig 1B). Hippocampal regions that did not degenerate were devoid of TUNEL staining (Fig 2). At 72 hours after 4-VO, degenerating neurons of the hippocampal CA1 layer demonstrated nuclear immunostaining for NF-B p50 and p65. Fig 3A is a photomicrograph of NF-B p50 immunostaining through the CA1 paramedian zone showing robust immunostaining of the nuclei of hippocampal pyramidal neurons, and Fig 3B is a photomicrograph showing the localization of NF-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). Thus, at 72 hours after 4-VO, nuclei of degenerating hippocampal CA1 neurons demonstrated nuclear immunostaining of the activated p50/p65 NF-B and TUNEL staining. Nuclear NF-B localization was never observed by immunocytochemistry in sham-operated controls (Fig 5D). In hippocampal areas not susceptible to damage induced by global ischemia, NF-B immunostaining was not observed (Fig 5C). Furthermore, nonhippocampal regions, including cerebral cortex, thalamus, hypothalamus, and amygdala, were devoid of nuclear NF-B immunoreactivity.

View larger version (127K): [in this window] [in a new window]   Figure 1. CA1 pyramidal neurons of the hippocampus show histological evidence of PCD. Sections were taken from animals killed at 72 hours after 4-VO. A, Hematoxylin-eosin–stained section through the CA1 layer shows evidence of pyknosis and karyorrhexis of neurons. Some cells show chromosome condensation and fragmentation (arrows). B, TUNEL labeling illustrates DNA fragmentation in CA1 hippocampal pyramidal neurons. Staining is localized to the nucleus. Bar=10 µm.

View larger version (91K): [in this window] [in a new window]   Figure 2. Apoptag labeling of hippocampal pyramidal neurons from a rat subjected to 4-VO. A, CA1 neurons show strong nuclear localization of peroxidase reaction product at 72 hours after ischemia. B, CA3 region from the same animal reveals no detectable staining. Sections were lightly counterstained with methyl green. The level of signal shown in B is identical to that observed in the CA1 region of sham-operated controls. Bar=60 µm.

View larger version (108K): [in this window] [in a new window]   Figure 3. Immunoreactivity for NF-B p50 and p65 is detected within hippocampal pyramidal CA1 neurons of the paramedian zone. A, Immunoperoxidase staining with antisera directed to p50 reveals distinctive nuclear staining. B, Nuclear staining is likewise observed, albeit less intensely, in CA1 neurons with antisera directed to p65 (arrows). Bar=10 µm.

View larger version (66K): [in this window] [in a new window]   Figure 4. NF-B p50 and TUNEL are colocalized in the nucleus of CA1 pyramidal neurons. Sections taken from a rat several days after ischemia were immunostained with NF-B p50 antiserum using a Texas Red–based detection system (red), followed by in situ labeling of DNA fragmentation using a fluoresceinated tag (green) and coverslipping using mounting medium containing the nuclear stain DAPI (blue). A, NF-B; B, TUNEL; C, DAPI. Arrows highlight individual neurons where NF-B and TUNEL are colocalized. Bar=10 µm.

View larger version (152K): [in this window] [in a new window]   Figure 5. NF-B p50 immunoreactivity is not detected in intact neurons. Nissl staining (A) and the adjacent section stained with anti–NF-B p50 antiserum (C) in the CA3 region from an animal at 72 hours after 4-VO. Nissl staining (B) and NF-B p50 staining (D) in the CA1 region are from a sham-operated control animal. Immunostained sections were not counterstained. Bar=20 µm.

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).

View larger version (42K): [in this window] [in a new window]   Figure 6. NF-B is induced in the dorsal hippocampus after ischemia. Western immunoblots of microdissected homogenates from dorsal hippocampus of animals at 72 hours after ischemia were evaluated in parallel with sham-operated controls. A, Western gel probed with polyclonal anti–NF-B p50 (Santa Cruz Biotechnology). Lane 1, 4-VO; lane 2, sham; and lane 3, HeLa cell nuclear extracts (H). Bands migrating below 50 kD are nonspecific. B, Western gel immunoblotted with anti–NF-B p65 (Rockland). Lanes 1 and 2, 4-VO; lanes 3 and 4, sham; and lane 5, HeLa cell nuclear extracts (H).

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-B–specific oligo probe extinguished the specific retarded band.

View larger version (59K): [in this window] [in a new window]   Figure 7. NF-B DNA binding activity by EMSA in hippocampal nuclear extracts. Pooled nuclear extracts from either 4-VO (lanes 1 and 3) or sham-operated (lanes 2 and 4) rats were prepared from microdissected dorsal hippocampus. Lanes 1, 2, and 5 were evaluated in the absence of competitor, and lanes 3 and 4 were performed in the presence of competitor. Lanes 1 and 3, 4-VO; lanes 2 and 4, sham; lane 5, HeLa nuclear extracts, which served as a positive control. Free probe can be detected at the bottom of the gel. + indicates presence of cold competitor; -, absence of cold competitor.

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.

View larger version (46K): [in this window] [in a new window]   Figure 8. Levels of NF-B p50 messenger RNA are increased at 72 hours after ischemia. Northern blot analysis of total RNA prepared from sham (A) and ischemic (B) microdissected dorsal hippocampus. Blots were probed with radiolabeled antisense cDNA to NF-B p50.

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.

View larger version (24K): [in this window] [in a new window]   Figure 9. Western-EMSA analysis demonstrates the presence of both NF-B p50 and NF-B p65 in rat dorsal hippocampus. Pooled nuclear extracts were subjected to EMSA, transferred to nitrocellulose, and probed with either p50 (A) or p65 (B) antibodies. Lane 1, 4-VO; lane 2, sham; and lane 3, HeLa nuclear extracts. Identification of p50 and p65 bands was based on molecular weight markers (not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
For clarity of discussion, we wish to briefly review the activation process for NF-B. The NF-B transcript is translated in the cytoplasm of mammalian cells. NF-B consists of a p50 and p65/RelA complex. IB- inhibitory protein (IB) traps NF-B in the cytoplasm and prevents its translocation to the nucleus. Activation consists of phosphorylation or degradation of IB 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, anti–NF-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, IB 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 IB 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-B–binding 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

 

EMSA = electrophoretic mobility shift analysis NF-B = nuclear factor-B PAGE = polyacrylamide gel electrophroesis PCD = programmed cell death PCR = polymerase chain reaction PVDF = polyvinylidene difluoride ROS = reactive oxygen species SOD = superoxide dismutase TBST = Tris-buffered saline with Tween TUNEL = terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling 4-VO = four-vessel occlusion

	Acknowledgments

 
We wish to thank Dr Warner C. Greene for a generous supply of NF-B antibodies.

Received November 7, 1996; revision received January 15, 1997; accepted February 28, 1997.

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