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(Stroke. 1999;30:2472-2478.)
© 1999 American Heart Association, Inc.

Original Contributions

Nuclear Factor-B and Cell Death After Experimental Intracerebral Hemorrhage in Rats

Susan L. Hickenbottom, MD; James C. Grotta, MD; Roger Strong, MS; Larry A. Denner, PhD Jaroslaw Aronowski, PhD

From the Stroke Program, Department of Neurology, University of Texas–Houston Medical School, and the Apoptosis Program, Department of Cell Biology, Texas Biotechnology Corporation (L.A.D.), Houston.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Background and Purpose—Nuclear factor-B (NF-B) is a ubiquitous transcription factor that, when activated, translocates to the nucleus, binds to DNA, and promotes transcription of many target genes. Its activation has been demonstrated in chronic inflammatory conditions, cerebral ischemia, and apoptotic cell death. The present study evaluated the presence and activation of NF-B in relation to cell death surrounding intracerebral hemorrhage (ICH).

Methods—Striatal ICH was induced in rats by the double blood injection method. Animals were killed 2, 8, and 24 hours and 4 days after ICH. To examine changes in NF-B protein, Western blot was performed on brain extract. We determined NF-B activity using electrophoretic mobility shift assay (EMSA) and immunohistochemistry, using an antibody that only recognizes active NF-B. DNA fragmentation was detected with terminal deoxynucleotidyl transferase–mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL) staining.

Results—Western blot analysis of the NF-B p65 subunit showed that there was no difference in p65 protein levels in the control, 2-hour, 8-hour, or 24-hour groups. However, ipsilateral perilesional samples from the 4-day group revealed a 1.8- to 2.5-fold increase compared with the contralateral hemisphere. Western blotting showed no differences in the inhibitor of NF-B, IB, in any group. EMSA showed 1.3-, 2.1-, and 3.6-fold increased NF-B activation in the ipsilateral striatum from the 8-hour, 24-hour, and 4-day groups, respectively, compared with the contralateral hemisphere. Immunohistochemistry, in which an activation-dependent anti–NF-B antibody was used, demonstrated perivascular NF-B activation as early as 2 hours after ICH with more generalized activation at 8 hours, in agreement with the EMSA results. NF-B activation colocalized to cells containing fragmented DNA measured by TUNEL.

Conclusions—The present study suggests a relationship between NF-B and the pathobiology of perilesional cell death after ICH.

Key Words: cell death • DNA fragmentation • intracerebral hemorrhage • NF-kappa B • rats

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Evidence for an inflammatory response after intracerebral hemorrhage (ICH) has been reported both in humans and in experimental models of hemorrhage. In 1961, Wisniewski¹ described the infiltration of polymorphonuclear leukocytes into hemorrhagic brain parenchyma 2 to 4 days after ICH. This reaction was preceded by polymorphonuclear leukocyte accumulation in vessels as early as 6 hours after ICH. These early human pathological observations have been confirmed by more recent animal models of ICH, in which many investigators have documented robust polymorphonuclear leukocyte and monocyte infiltration into perihemorrhagic areas at 1 to 7 days after ICH.^{1 2 3 4 5 6} Despite substantial evidence for such an inflammatory reaction, there are a limited number of studies devoted to examining the pathological role of inflammatory mediators after experimental ICH.

Nuclear factor-B (NF-B) is a ubiquitous transcription factor and a member of a family of proteins that are critical regulators of a variety of responses, including inflammation.⁷ NF-B exists as a dimer predominantly composed of the p50 and p65 (RelA) subunits, as well as other members of the NF-B/Rel family, such as RelB, c-Rel, and p52. In unstimulated cells, inactive NF-B is sequestered in the cytoplasm by the inhibitory proteins IBs, which prevent its translocation to the nucleus. In response to various external pathogenic stimuli, including cytokines, reactive oxygen species, and viruses,^{8 9} specific kinases phosphorylate IB, leading to its proteolysis and dissociation from NF-B. The free, newly activated NF-B migrates into the cell nucleus, where it binds to specific NF-B response elements in the promoters of target genes. This results in the transcriptional induction of genes for many proinflammatory substances, such as cytokines, chemokines, adhesion molecules, and inflammatory enzymes.⁷

NF-B has also been implicated in the inflammatory response associated with many other pathologies. For example, NF-B activation has been reported in chronic immune diseases⁷ and in global and focal cerebral ischemia.^{10 11 12} Inhibition of NF-B has been correlated with amelioration of excitotoxic^{13 14} and ischemia-induced¹¹ neuronal death. Because inflammatory responses may be involved in cell damage and death after ICH and because NF-B has been implicated to play a role in other pathological inflammatory processes, we evaluated the activation of NF-B and cell death in a rat model of ICH.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Double Blood Injection Model and Brain Tissue Specimen Preparation
ICH was induced by the double blood injection model, as described recently.¹⁵ Briefly, male Sprague-Dawley rats (weight, 250 to 350 g) under chloral hydrate anesthesia (0.5 g/kg IP) were immobilized in a stereotaxic frame, a 1-mm-diameter burr hole was produced in the skull (0.7 mm anterior and 3 mm lateral to bregma), and a 22-gauge stainless steel cannula was inserted for blood injection in the caudate putamen (5 mm deep to bregma). Hemorrhage was induced first by an injection of 15 µL of autologous blood (over 3 minutes), followed 7 minutes later by 30 µL injected over 5 minutes. Both blood samples were drawn from the femoral artery within 60 seconds before injection under arterial pressure through a shunt between the femoral artery and the injection cannula. As determined in a separate group of animals, blood removal did not affect blood pressure. The injection cannula was slowly withdrawn 10 minutes after the second injection. During the first 3 hours of recovery after surgery, core body temperature was sustained at 36.5°C with the use of a feed-forward temperature controller (YSI, model 72) that utilized a heating lamp and warming blanket. Sham animals were subjected to the same manipulations as ICH rats, but no blood was injected. For biochemical studies, animals were fatally anesthetized with chloral hydrate (1.0 g/kg IP) and cooled to 30°C by cold water immersion. At specified times, brains were excised, immediately placed in ice-cold PBS, and subdissected.

For immunohistochemistry, animals were cooled by cold water immersion to 30°C under chloral hydrate anesthesia and then perfused with 250 mL ice-cold saline under constant pressure of 100 mm Hg. Brains were quickly removed, snap frozen in -75°C 2-methylbutane, and stored in a -80°C freezer before cryosectioning.

All procedures were in compliance with the National Institutes of Health and institutional guidelines for the humane care of animals.

Cellular Extraction and Western Blot Analysis
Cells were harvested by homogenization with a Potter homogenizer in ice-cold hypotonic lysis buffer (75 mg tissue per 1 mL) (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 1.5 mmol/L MgCl₂, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L DTT, 0.5 mmol/L PMSF, 2 µg/mL leupeptin, 2 µg/mL aprotinin, 0.5 mg/mL benzamidine). Equal amounts of extracts based on protein assay (micro BCA; Pierce) were run on 10% SDS-PAGE gels and electroblotted onto nitrocellulose membrane. Membranes were blocked with 5% milk plus 1% bovine serum albumin to reduce nonspecific binding. The primary polyclonal anti–NF-B p65 antibody (Santa Cruz Biotechnology, C20, 1:1000) or primary anti-IB antibody (Santa Cruz Biotechnology, C21, 1:1000) in TBS-NP-40 (150 mmol/L NaCl, 10 mmol/L Tris, pH 8.0, 0.05% NP-40) with 0.5% BSA was used. The secondary antibody was goat-anti-rabbit IgG conjugated to horseradish peroxidase (Promega) used at 1:5000 in TBS-NP40 buffer. Detection of immunopositive bands was performed with the Amersham ECL kit according to the manufacturer's instructions. Semiquantification of immunostaining intensity visualized on x-ray film was performed by analyses of optical density with the use of the computer-assisted Bio-Rad GS-670 Imaging Densitometer and Molecular Analyst program.

Nuclear Extraction and EMSA
Brain tissue was harvested and homogenized in the aforementioned manner. After 15 minutes on ice, 10% NP-40 was added to the homogenate to a final concentration of 3.125%, and the mixture was vortexed and microfuged (10 000 rpm) for 1 minute at 4°C. The nuclear pellet was resuspended in ice-cold hypertonic nuclear extraction buffer (20 mmol/L HEPES, pH 7.9, 420 mmol/L NaCl, 1.5 mmol/L MgCl₂, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 0.5 mmol/L PMSF, 2 µg/mL leupeptin, 2 µg/mL aprotinin, 0.5 mg/mL benzamidine), incubated on ice for 30 minutes with intermittent vortexing, and microfuged (10 000 rpm) for 5 minutes at 4°C. The supernatant containing the nuclear extract was collected, and 4 µg of nuclear extract was incubated for 15 minutes at 37°C with 16 fmol of ³²P–end-labeled 45-mer double-stranded NF-B oligonucleotide from the HIV-LTR, 5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGAGGCGTGGG-3' containing 2 (underlined) NF-B binding sites. The specificity of NF-B binding to the wild-type probe was determined with mutated, 5'-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3', oligonucleotide. Binding reactions were prepared in a final volume of 20 µL containing 2 µg poly(dI-dC), 25 mmol/L HEPES, pH 7.9, 0.5 mmol/L EDTA, 0.5 mmol/L DTT, 1% NP-40, 5% glycerol, and 50 mmol/L NaCl. Bound complexes were separated on 7.5% acrylamide gel with Tris-glycine running buffer, then visualized on x-ray film with autoradiography. Optical density was assessed with the use of the computer-assisted Bio-Rad GS-670 Imaging Densitometer and Molecular Analyst program. No NF-B binding to the mutated probe was detected in our experiments (data not included), confirming specificity of interaction between NF-B and wild-type oligonucleotide in our experiments.

Immunohistochemistry
Coronal cryosections (10 µm thick) were cut at -19°C with the use of a Leica model CM1800 cryostat. Sections were collected on glass microscope slides, dried overnight at 37°C, and treated with 100% methanol at -10°C for 10 minutes before immunostaining. After overnight blocking at 4°C in PBS containing 2% normal goat serum and 0.5% NP-40, the cryosections were probed with an activation-specific monoclonal antibody for NF-B p65 subunit (Boehringer Mannheim, 1 µg/mL), the epitope of which is available for binding only after IB dissociation. Secondary anti-mouse antibody conjugated to CY-3 (Sigma, 1:500) was applied according to the manufacturer's instructions. Both primary and secondary antibody incubations were performed for 60 minutes at room temperature in PBS containing 1% bovine serum albumin. DNA fragmentation was analyzed with a cell death detection kit (Boehringer Mannheim) with the use of FITC to visualize positively labeled cells. Fluorescent preparations were mounted in 50% glycerol with 0.1% phenylenediamine to reduce fading. For double labeling immunofluorescence, the green excitation was trimmed with a 530-nm-long pass filter, and FITC emission was trimmed with a 520-nm-long pass interference filter to prevent crossover between the fluorochromes. To analyze fluorescence (immunohistochemistry and TUNEL), we used an Olympus Vanox photomicroscope using blue or green epifluorescence. Images were captured with a Hamamatsu color 3CCD. Digitized images were processed in Photoshop by Adobe. We examined 3 rats at 2 hours and 3 rats at 8 hours after ICH. Multiple sections from each rat were examined independently by 2 investigators (J.A. and L.A.D.).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Time-Dependent Changes in p65 and IB After ICH
Western blot analysis (semiquantitative densitometry in duplicate on each sample) for p65 and for IB was performed with the use of homogenates of subcortical and cortical tissue, respectively, representing the perilesional region and more distal periphery of the hemorrhage, as well as equivalent portions of the contralateral hemisphere (Figure 1), at 2 hours (n=3), 8 hours (n=3), 24 hours (n=3), or 4 days (n=3) after ICH. No difference in p65 immunoreactivity was revealed in either ipsilateral or contralateral perilesional or periphery samples at 2 hours, 8 hours (Figure 2A), or 24 hours after ICH. However, ipsilateral perilesional samples taken 4 days after ICH demonstrated a significant (P<0.05) 1.8- to 2.5-fold increase in p65 immunoreactivity, indicating an increase in the amount of NF-B compared with the contralateral side of the same animal (Figure 2B). No difference in immunoreactivity was found for IB at any time after ICH (Figure 2C).

View larger version (89K): [in this window] [in a new window]   Figure 1. Coronal section through the rat brain subjected to experimental ICH produced by the double blood injection method. Demarcated is the portion of the brain representing ipsilateral (IPSI) ICH perilesional core. Remaining ipsilateral brain tissue was considered as ICH periphery. Homologous tissue of contralateral (CONTRA) hemisphere was analyzed as a control.

View larger version (58K): [in this window] [in a new window]   Figure 2. Representative immunoblot of p65 NF-B subunit (A and B) and IB (C) in the perilesional core (C) and periphery (P) of ipsilateral (IPSI) and contralateral (CONTRA) brain tissue at 8 hours (A) and 4 days (B and C) after ICH. Forty micrograms of each homogenate protein was separated on 10% SDS-PAGE, followed by electroblotting onto nitrocellulose and immunostaining. Note increased immunoreactivity to p65 at 4 days in the ICH core.

Time-Dependent Changes in NF-B Activity After ICH
Activation of NF-B was determined by EMSA at 2 hours (n=3), 8 hours (n=3), 24 hours (n=3), and 4 days (n=4) after ICH and then confirmed with immunohistochemistry at selected times. Sham-operated animals were used as controls (n=3).

At 8 hours, 24 hours, and 4 days after ICH, EMSA demonstrated a 1.29±0.12-, 2.10±0.47-, and 3.57±0.67-fold relative increase, respectively (P<0.05), in DNA binding activity in ipsilateral perilesional samples and a 1.40±0.16-, 1.28±0.09-, and 1.80±0.66-fold increase (P<0.05) in ipsilateral peripheral samples compared with equivalent areas from the contralateral side of the same animal (Figure 3). No difference in DNA binding between the ipsilateral and contralateral side was detected 2 hours after ICH. Although increased DNA binding activity of this transcription factor was seen as early as 8 hours after ICH, activation of NF-B persisted for several days and was much more pronounced at 4 days after ICH. It is interesting to note that NF-B activation in the hemisphere ipsilateral to the hemorrhage 4 days after ICH was expressed mostly by amplification of the signal in the lower band in the perilesional zone but in the upper band in the periphery of the hemorrhage (Figure 3).

View larger version (62K): [in this window] [in a new window]   Figure 3. Detection of NF-B DNA binding activity in the rat brain 8 hours and 4 days after ICH. Equal amounts of nuclear protein extract prepared from the perilesional core (C) and periphery (P) of ICH from both ipsilateral (IPSI) and homologous contralateral (CONTRA) hemisphere regions were incubated with the 32P-labeled oligonucleotide probe containing the NF-B binding site before electrophoretic separation (EMSA). Upper (U), middle (M), and lower (L) bands represent NF-B with different dimer composition and distinct electrophoretic mobility. Darker band reflects more NF-B DNA binding activity.

To obtain information on the distribution of NF-B activation, we used an antibody that recognizes epitope of p65 subunit of NF-B that is not accessible in the inactive enzyme. Immunocytochemical detection of activated NF-B demonstrated focal activation as early as 2 hours after ICH. This very early activation was localized solely to the blood vessels in close proximity to the hemorrhage and did not include other brain cells such as astrocytes, neurons, or microglia; it was detected in 2 of 3 animals analyzed (Figure 4A). At 8 hours after ICH, more widespread activation of NF-B was present in 3 of 3 rats analyzed, extending to the adjacent ipsilateral peripheral cortex (Figure 4B). At the same time (8 hours after ICH), TUNEL-positive cells were detected throughout neuropil surrounding the hemorrhage (n=3) (Figure 4C), suggesting a link between NF-B activation and DNA fragmentation. To further investigate the possibility of such a link, we analyzed sections for colocalization of NF-B activation and TUNEL and found that, while some cells were only positive for NF-B activation, nearly all TUNEL-positive cells were also positive for activated NF-B (Figure 4D).

View larger version (51K): [in this window] [in a new window]   Figure 4. A, Representative immunofluorescence (CY-3) illustrating perivascular activation of NF-B/p65 in ipsilateral striatum 2 hours after ICH. B, More widespread immunofluorescence (CY-3), indicating that NF-B activation was observed 8 hours after ICH in the ipsilateral (Ipsi) cerebral cortex. Note selective NF-B activation in the ipsilateral and not contralateral (Contra) cortex. C, TUNEL staining (FITC; green/yellow) around hemorrhage in the caudate nucleus 8 hours after ICH. Blood is localized to the top of the figure, with the brain tissue occupying the lower half of the image. D. TUNEL (A), NF-B/p65 (B), and double TUNEL plus NF-B/p65 labeling (C) in the caudate nucleus proximal to ICH 4 hours after blood injection, indicating colocalization of activated NF-B/p65 and DNA fragmentation.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
ICH remains a devastating clinical entity. The incidence of ICH is estimated to be 15 to 35 per 100 000,^{16 17} and the case-fatality rate approaches 50%.¹⁸ Neither surgical nor medical therapy has been shown to reduce morbidity or mortality after ICH.^{17 18 19 20 21 22 23} Primary damage after ICH results from the mass effect of the hematoma, which causes local compression of microvasculature and which also creates midline shift, leading to compression of distant structures with consequent shearing, microhemorrhage, and eventual herniation.^{24 25} Until recently it was thought that maximal hemorrhage volume was reached within a few minutes of the onset of ICH²⁶ ; however, it is now appreciated that in some instances mass effect substantially increases over the first few hours after ICH.^{27 28} Secondary injury is thought to arise from tissue reaction to invasion of blood products in the area around the hematoma, resulting in inflammation, ischemia, and edema.^{29 30 31 32} The present investigation suggests that inflammatory responses may be initiated during the first hours after ICH, thus presenting potential early therapeutic targets to modulate the pathobiology of ICH.

The results of this study demonstrate activation of NF-B after ICH. The activation of NF-B was confirmed by EMSA on nuclear extracts and by immunohistochemistry with the use of an activation-specific antibody. Focal perivascular activation of the NF-B complex was seen as early as 2 hours after ICH, while more widespread perilesional activation was observed at 8 hours. In addition, EMSA demonstrated that activation of NF-B surrounding the hemorrhage and in the more peripheral cortex ipsilateral to the hemorrhage persisted, and in fact intensified, for several days after ICH. At 4 days after ICH, NF-B complexes were predominantly composed of the higher mobility lower band in the perilesional zone and lower mobility upper band in the peripheral cortex. The presence of NF-B complexes composed of different subunits suggests transcriptional regulation of different target genes near the hematoma compared with the peripheral cortex during the course of ICH. We plan to identify the proteins in these complexes using supershift assay with antibodies to various subunits of NF-B. Finally, increased protein levels of the p65 subunit of NF-B were detected around the hemorrhage at 4 days after ICH, suggesting increased synthesis or decreased breakdown of p65 at the hemorrhagic site.

Our results also demonstrate a correlation between NF-B activation and DNA fragmentation after ICH. Irrespective of whether TUNEL represents apoptosis and/or necrosis, DNA fragmentation is an important feature of cell death. It is important to note that NF-B was active in TUNEL-negative and TUNEL-positive cells. These results suggest that the activation signal is not a consequence of DNA fragmentation. Furthermore, since all TUNEL-positive cells contained active NF-B, it appears likely that once NF-B is active, the cells are destined to die. Finally, the lack of TUNEL-positive cells within the hemorrhage itself suggests that the dying cells were indigenous to the brain and did not infiltrate from the circulation.

The roles of NF-B activation in cell death are pleiotrophic. In several studies, NF-B activation has been shown to be either proapoptotic^{13 33 34 35} or antiapoptotic.^{36 37 38 39 40 41} Furthermore, a recent study of global cerebral ischemia found that transient activation of NF-B may be neuroprotective, while more persistent activation could be responsible for the induction of proteins that lead to neuronal cell death.¹¹ While other investigators have colocalized TUNEL and active NF-B after global cerebral ischemia,¹⁰ similar to data presented here for ICH, these studies could necessarily only demonstrate a correlation and not a causal relationship between these events.

Delineating the relationship between NF-B activation, DNA fragmentation, and cell death in both ischemic and hemorrhagic cerebrovascular disease may result in the identification of target molecules for the development of therapeutic interventions. For example, it was recently demonstrated that aspirin and its metabolite sodium salicylate, but not indomethacin, protected neurons from death after an excitotoxic insult in tissue culture and hippocampal slices, an effect positively correlating with inhibition of NF-B activity.¹³ These anti-inflammatory drugs specifically inhibit phosphorylation of IB by IB-kinase-ß (IKK-ß), preventing IB dissociation from NF-B, which is necessary for NF-B nuclear translocation⁴² and transcriptional regulation of target genes. Free radicals are also proposed to lead to NF-B activation.⁴³ Thus, administration of the antioxidant LY231617 had a protective effect on survival of CA1 hippocampal neurons in an animal subjected to global ischemia.¹¹ Similar to salicylates, this neuroprotection positively correlated with inhibition of nuclear translocation of ischemia-activated NF-B. Finally, a selective NF-B inhibitor, SN50, which prevents NF-B nuclear translocation, reduced intranucleosomal DNA fragmentation and striatal cell death after excitotoxin-induced insult.¹⁴ If we assume that NF-B plays a detrimental role in ICH pathology similar to that suggested with ischemia and excitotoxicity,^{10 11 12 13 14} salicylates or other antioxidant-based neuroprotective strategies could be attempted in the treatment of ICH. The results of the present study suggest that such interventions would preferentially need to be undertaken early in the course of ICH, before generalized activation of NF-B and expression of proinflammatory genes. Other therapeutic options might include early surgical evacuation of the hematoma, within the first hours after hemorrhage, to remove toxic blood products that would initiate the inflammatory cascade. Of course, these hypotheses can be addressed experimentally, and such studies are currently in progress in our laboratory.

	Acknowledgments

 
This study was supported in part by a National Institutes of Health fellowship training grant to the University of Texas–Houston Medical School Stroke Program (Dr Hickenbottom).

	Footnotes

 
Reprint requests to Dr Jaroslaw Aronowski, University of Texas, Medical School, Department of Neurology, 6431 Fannin, Room 7.044, Houston, TX 77030.

Received May 18, 1999; revision received July 21, 1999; accepted August 12, 1999.

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Editorial Comment

R. Loch Macdonald, MD, PhD, Guest Editor

Section of Neurosurgery, University of Chicago Medical Center, Chicago, Illinois

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Hickenbottom et al created deep ICHs in rats and studied the brain tissue around the hematomas 2, 8, and 24 hours and 4 days later. There was activation of NF-B in the brain around the hemorrhage 8 hours to 4 days after the hemorrhage and an increase in NF-B protein by 4 days after hemorrhage. Cells with activated NF-B contained fragmented DNA, suggesting that they were dying, perhaps by apoptosis. NF-B is a transcription factor that, when activated, increases transcription of a number of different genes, including some involved in inflammation. The findings are of great interest in terms of the pathophysiology of brain damage from ICH. There must be some brain damage incurred immediately at the time of intracerebral bleeding, probably due to mechanical disruption of cell bodies and axons and perhaps ischemia from increased local pressure and from toxic effects of blood products. It is usually postulated that there is then continuing brain damage over time that is mediated by different processes, probably varying depending on the time after hemorrhage, location of hemorrhage, and other factors. These secondary processes are classically hoped to be remediable by clot removal and optimization of the patient's clinical condition. Hickenbottom and colleagues provide evidence that changes in a transcription factor, NF-B, occur in the brain after ICH. There are a number of questions that need to be addressed, but one could theorize that cells are dying or detrimental processes are occurring in a delayed fashion after the hemorrhage and that these processes are in part mediated by altered gene expression. Does this lead to detrimental or beneficial effects, or both? What genes does it activate in this disease? Blood products activate immediate early genes in smooth muscle cells and may do the same in the various cells in the brain.¹ The expression of many genes probably is altered. The availability of complementary DNA arrays had made it possible to screen tissues to determine changes in gene expression, but unfortunately even simple manipulations such as readdition of serum to serum-starved fibroblasts leads to activation of hundreds of genes.² This study provides the impetus to begin investigating the responses of the neurons and glia around an ICH and to determine how to modulate them to keep these cells alive or maybe even to allow regeneration.

Several points need to be kept in mind, however. The cellular localization of the activation of NF-B was not determined and will be an important area for further investigation. Are these cells neurons, glia, infiltrating inflammatory cells, or something else? The cause and effect relation between NF-B activation and DNA fragmentation is not established. Also, there are important limitations to the use of rats in the study of human disease, especially when changes in gene expression are investigated. For example, the regulation of immediate early and stress-response genes may differ greatly between humans and rats. Hemoglobin, one of the more abundant proteins in blood, is metabolized in part by heme oxygenases. Heme oxygenase-1, an inducible form, is regulated by different stimuli in rats compared with humans. It is a heat-shock protein in rats but not in humans.³ This protein is mentioned because it may be induced around ICHs in response to heme and hemoglobin released from the blood clot.⁴ Inducible nitric oxide synthase, which is believed to be an important mediator of inflammatory processes, such as occur after ICH, is inducible in rat macrophages by endotoxin and various cytokines, whereas it is not in primate tissues.⁵ There may be many more differences.

The clinical applications of the present findings are remote at present but are mentioned by the authors at the end of the discussion. Aspirin is discussed as a potential neuroprotectant. In addition to the inhibition of cyclooxygenase, aspirin was reported to increase ferritin synthesis in endothelial cells.⁶ Ferritin is an iron-binding protein that sequesters iron and may reduce oxidative stress in cells that are exposed to excess iron. Iron is abundant in the hemoglobin of blood clots, and hemorrhages in the subarachnoid or intracerebral space induce heme oxygenase-1 and ferritin in the brain and cerebral vessels.^{4 7} One could speculate that these reactions might be important after ICH. Aspirin might not be the ideal way to induce ferritin protein after human ICH, although it might be a useful tool to investigate the role of changes in various proteins after ICH. In any case, it is hoped that studies along the lines of those of Hickenbottom et al will lead to a better understanding of the mechanisms of brain damage associated with ICH and to new therapies.

Received May 18, 1999; revision received July 21, 1999; accepted August 12, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
1. Wang X, Marton LS, Weir BKA, Macdonald RL. Immediate early gene expression in vascular smooth-muscle cells synergistically induced by hemolysate components. J Neurosurg. 1999;90:1083–1090.[Medline] [Order article via Infotrieve]

2. Iyer VR, Eisen MB, Ross DT, Schuler G, Moore T, Lee JCF, Trent JM, Staudt LM, Hudson J Jr, Boguski MS, Lashkari D, Shalon D, Botstein D, Brown PO. The transcriptional program in the response of human fibroblasts to serum. Science. 1999;283:83–87.[Abstract/Free Full Text]

3. Maines MD. The heme oxygenase system: a regulator of second messenger gases. Ann Rev Pharmacol Toxicol. 1997;37:514–554.

4. Matz PG, Weinstein PR, Sharp FR. Heme oxygenase-1 and heat shock protein 70 induction in glia and neurons throughout rat brain after experimental intracerebral hemorrhage. Neurosurgery. 1997;40:152–160.[Medline] [Order article via Infotrieve]

5. Jesch NK, Dörger M, Enders G, Rieder G, Vogelmeier C, Messmer K, Krombach F. Expression of inducible nitric oxide synthase and formation of nitric oxide by alveolar macrophages: an interspecies comparison. Environ Health Perspect. 1997;105(suppl 5):1297–1300.

6. Oberle S, Polte T, Abate A, Podhaisky HP, Schroder H. Aspirin increases ferritin synthesis in endothelial cells: a novel antioxidant pathway. Circ Res. 1998;82:1016–1020.[Abstract/Free Full Text]

7. Macdonald RL, Weir BK, Marton LS, Windmeyer E, Johns L, Kowalczuk A, Lin G. Heme oxygenase-1 and ferritin proteins are increased in cerebral arteries after subarachnoid hemorrhage in monkeys. Surg Forum. 1998;49:501–503.

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