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(Stroke. 2004;35:987.)
© 2004 American Heart Association, Inc.

Original Contributions

Nuclear Factor-B Contributes to Infarction After Permanent Focal Ischemia

Antti Nurmi, MSc; Perttu J. Lindsberg, MD, PhD; Milla Koistinaho, PhD; Wen Zhang, MD; Eric Juettler, MD; Marja-Liisa Karjalainen-Lindsberg, MD, PhD; Falk Weih, PhD; Norbert Frank, PhD; Markus Schwaninger, MD Jari Koistinaho, MD, PhD

From the A.I. Virtanen Institute, Department of Neurobiology, University of Kuopio, Kuopio, Finland (A.N., M.K., J.K); Department of Oncology, Kuopio University Hospital, Kuopio, Finland (J.K.); Departments of Neurology (P.J.L.) and Pathology (M.-L.K.-L.), Helsinki University Central Hospital, Helsinki, Finland; Neuroscience Program, Biomedicum, Helsinki, Finland (P.J.L.); Forschungszentrum Karlsruhe, Institute of Genetics, Karlsruhe, Germany (F.W.); and Division of Toxicology and Cancer Risk Factors, Cancer Research Center (N.F.), and Department of Neurology, University of Heidelberg, Heidelberg, Germany (W.Z., E.J., M.S.).

Reprint requests to Jari Koistinaho, MD, PhD, A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, PO Box 1627, FIN-70211, Kuopio, Finland. E-mail jari.koistinaho{at}uku.fi

	Abstract

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Background and Purpose— Activation of transcription factor nuclear factor-B (NF-B) may induce expression of either proinflammatory/apoptotic genes or antiapoptotic genes. Because a considerable number of middle cerebral artery occlusions (MCAOs) in humans are not associated with reperfusion during the first 24 hours, the role of NF-B after permanent MCAO (pMCAO) was investigated.

Methods— Mice transgenic for a NF-B–driven ß-globin reporter were exposed to pMCAO, and the expression of the reporter gene was quantified with real-time polymerase chain reaction. Mice lacking the p50 subunit of NF-B and wild-type controls were exposed to pMCAO with or without treatment with pyrrolidinedithiocarbamate (PDTC), an NF-B inhibitor. Brain sections of human stroke patients were immunostained for the activated NF-B.

Results— pMCAO increased NF-B transcriptional activity to 260% (36.9±4.5 compared with 14.4±2.6; n=10; P<0.01) in the brain; this NF-B activation was completely blocked by PDTC (17.2±2.6; n=9; P<0.05). In p50^-/- mice, pMCAO resulted in 41% (18±3.2 mm³; n=12) smaller infarcts compared with wild-type controls (32.9±3.8 mm³; n=9; P<0.05), which was comparable to the protection achieved with PDTC in wild-type mice (19.6±4.2 mm³; n=8). Pro-DTC, a PDTC analogue that does not cross the blood-brain barrier, had no effect, even though Pro-DTC and PDTC were equally protective in vitro. During the first 2 days of human stroke, NF-B was activated in neurons in the penumbral areas.

Conclusions— NF-B is induced in neurons during human stroke, and activation of NF-B in the brain may contribute to infarction in pMCAO.

Key Words: knockout micecell culture • neuroprotective agents • inflammation • stroke • transcription factor • transgene • mice • rats

	Introduction

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Inflammation plays a role in human stroke and in its animal models several hours after ischemia.^1,2 These processes are mediated by proinflammatory genes, which are induced on ischemic conditions. The importance of inflammation in stroke is supported by the neuroprotective effects of anti-inflammatory compounds in rodent stroke models.^3–5 In addition, mice deficient in proinflammatory genes, such as inducible nitric oxide synthase (iNOS),⁶ cyclooxygenase-2 (COX-2),⁷ and interleukin-1ß (IL-1ß),³ show reduced brain infarction. Finally, various mediators of inflammation are upregulated in human stroke.^8–12

Apoptosis is another major mechanism of neuronal death in stroke models, as evidenced by the use of transgenic and knockout mice and apoptosis inhibitors in ischemia studies.^13,14 Apoptosis and inflammation are also interconnected since several proinflammatory agents eventually kill neurons by apoptosis.

Transcription factor nuclear factor-B (NF-B) regulates expression of both proinflammatory genes and genes related to apoptosis.¹⁵ NF-B is activated in the brain after reperfusion.^16,17 In the periphery, NF-B is crucial for inflammation reactions.¹⁸ In the brain, the role of NF-B is unclear because the lack of the p50 subunit of NF-B is protective in the transient middle cerebral artery occlusion (MCAO) model¹⁶ but increases excitotoxic damage in the hippocampus.¹⁹ Activation of NF-B also promotes neuronal survival in various models.²⁰

Because a considerable number of MCAOs in humans are not associated with reperfusion during the first 24 hours, we studied the role of NF-B in permanent MCAO (pMCAO) models. We also determined whether NF-B is activated in the salvageable penumbral region during the first days of human stroke.

	Materials and Methods

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Immunocytochemistry
Human specimens of infarcted brains were collected at autopsy from patients who had died at 23 hours (age 89 years; postmortem time [PMT] 40 hours), 28 hours (age 67 years; PMT 40 hours), and 38 hours (age 67 years; PMT 20 hours) after the onset of ischemic stroke symptoms. The control samples were from patients aged 61, 41, and 60 years with PMT 11, 14, and 21 hours. The tissue blocks were formalin-fixed and embedded in paraffin. Then 5-µm-thin sections were deparaffinized and subjected to antigen retrieval by three 5-minute periods of microwave in citrate buffer at pH 6. After removal of endogenous peroxidase reactivity with methanol/hydrogen peroxide and blocking with normal serum, the sections were incubated with a mouse monoclonal antibody against the activated p65 subunit of the human NF-B heterodimer (Chemicon) with a dilution of 1:200. A Vectastain avidin-biotin kit was used according to the manufacturer’s instructions before application of the chromogen diaminobenzidine. To visualize cell nuclei, the sections were dipped in Meyer’s Hemalum. The study had the approval of the local research ethics committee.

Immunohistochemical staining of COX-2 was studied 24 hours after pMCAO with the use of rabbit polyclonal COX-2 antibody (1:300; Cayman), as previously deascribed.²¹

B5-Reporter Gene Assay
Transgenic mice containing a ß-globin reporter under transcriptional control of 3 NF-B binding sites were used.²² After perfusion with Ringer’s solution 24 hours after pMCAO, the ischemic and the corresponding contralateral cortices were quickly dissected and frozen. RNA was extracted with the RNA pure kit (Peqlab) according to the manufacturer’s instructions. Then 10 µg RNA was transcribed with MMLV reverse transcriptase and random hexamers. For polymerase chain reaction (PCR), the following primers were used: ß-globin transgene 5', AGC TGC ATG TGG ATC CTG AGA; ß-globin transgene 3', GAT AGG CAG CCT GCA CTG GT; cyclophilin 5', AGG TCC TGG CAT CTT GTC CAT; cyclophilin 3', GAA CCG TTT GTG TTT GGT CCA. The Taqman probe was labeled with 6-FAM and had the following sequence: CTG GTC TGT GTG CTG GCC CAT CAC T. PCR was performed according to the following protocol: 10 minutes at 95°C, 15 seconds at 95°C, and 1 minute at 60°C (40 cycles). Amplification was quantified with a Gene Amp 5700 sequence detector and the Taqman kit (Perkin Elmer). The quantification of the ß-globin transgene PCR was normalized to the quantification of the cyclophilin PCR in individual samples.

Animal Experiments
All animal studies were approved by the Animal Care and Use Committee of the University of Kuopio and followed the National Institutes of Health guidelines for animal care. pMCAO was produced as previously described²³ with the use of p50^-/- and their 129/SV X C57BL/6 wild-type controls (Jackson Laboratories), BALB/c mice (University of Kuopio), or spontaneously hypertensive rats (M&B A/S). Infarct volumes were quantified with the use of 2,3,5-triphenyltetrazolium chloride staining. NF-B inhibitors pyrrolidine dithiocarbamate ammonium salt (PDTC) (Sigma) and proline dithiocarbamate (Pro-DTC) were dissolved in saline (pH 7.4) and given either 100 or 200 mg/kg IP 2 hours after the onset of ischemia and again 12 hours later. Controls animals received saline. For physiological parameters, the right common carotid artery was cannulated to withdraw 50 to 100 µL blood for analysis with an ABL-5 blood gas analyzer (Radiometer) and a EuroFlash blood glucose analyzer (Johnson&Johnson).

Primary Cortical Cultures
Cortices were prepared from 17-day-old mouse embryos (BALB/c) as previously described.²⁴ On day 12, 10 µmol/L PDTC or pro-DTC was added in the culture medium 30 minutes before the cultures were exposed to 50 µmol/L N-methyl-D-aspartate (NMDA) for 5 minutes. Twenty-four hours later the medium was collected and prepared cell-free by centrifugation, and the release of LDH was measured immediately with a Sigma Kinetic LDH kit and a Multiskan MS ELISA reader (Labsystems). The culture experiments were repeated 3 times.

Statistical Analysis
Differences between groups were determined by Student t test (2-group comparisons) or with ANOVA combined with the Bonferroni post hoc test. In case of unequal variances, 2-group comparisons were performed with the Mann-Whitney U test. P<0.05 was considered statistically significant.

	Results

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NF-B Is Induced in Neurons During the First 2 Days of Human Stroke
Comparison of penumbras of 3 patients with stroke and 3 control patients demonstrated that 1 to 2 days after MCAO in humans, activated NF-B is induced and translocated to nuclei of neurons (Figure 1). At 23 hours an intense cytoplasmic staining of NF-B in neurons was observed (Figure 1A). At 28 hours both cytoplasmic and nuclear NF-B staining was detected (Figure 1B). Finally, at 38 hours nuclear staining dominated neuronal NF-B expression (Figure 1C). No or hardly detectable NF-B staining was seen in brain samples of the corresponding cortical brain areas from 3 control patients (Figure 1D to 1F).

View larger version (177K): [in this window] [in a new window]   Figure 1. Photomicrographs of immunohistochemical NF-B p65 stainings in the brain sections of 3 human patients with recent antemortem cerebral ischemia caused by MCAO and 3 control patients without cerebral ischemia. Ischemic brain samples were obtained from the peripheral brain areas surrounding the necrotic infarct core. A, Staining from an 89-year-old patient who experienced embolic MCAO and died 23 hours later. PMT was 12 hours. Note the pericytoplasmic vacuolization, cell pyknosis, and intense cytoplasmic staining pattern against NF-B in these cortical cerebral neurons, as illustrated in the insert located in the right bottom corner. B, One day and 4 hours after onset of ischemic symptoms caused by thrombotic MCAO in a 75-year-old patient. PMT was 40 hours. Note the cytoplasmic NF-B staining, which starts to extend to the nucleus, as illustrated in the insert. C, One day and 14 hours after onset of ischemic symptoms caused by thromboembolic MCAO in a 67-year-old patient. PMT was 20 hours. Note that nuclear staining dominates the neuronal NF-B expression pattern. D to F, Control stainings with the isotype-specific IgG of each case shown above in respective order, indicating the specificity of the stainings. G to J, Stainings performed under identical circumstances from brain samples of corresponding cortical brain areas from 3 patients who experienced sudden death of noncerebral cause and without prior cerebral ischemia. The ages of the patients were 61, 41, and 60 years, and PMTs were 11, 14, and 21 hours, respectively. Bar=20 µm.

NF-B Is Induced in pMCAO and Is Inhibited by PDTC
pMCAO increased NF-B–driven transcriptional activity of the ß-globin transgene to 260% in the brain at 24 hours (Figure 2A). When 200 mg/kg PDTC was given 2 and 12 hours after the onset of ischemia, NF-B activation was blocked completely.

View larger version (25K): [in this window] [in a new window]   Figure 2. A, B5-transgenic mice expressing human ß-globin gene under exclusive control of NF-B. Data show ischemia-induced expression of ß-globin gene 24 hours after permanent pMCAO in the ischemic (isch) hemisphere when measured with real-time PCR. PDTC treatment, started at 2 hours, completely abolishes NF-B–driven gene expression to the level of nonischemic (non-isch) hemisphere (vehicle, n=10; PDTC 200 mg/kg, n=9). *P<0.05, **P<0.01, ANOVA. B, p50-/- mice are protected against permanent ischemia (p50-/- vehicle, n=12; wild-type [wt] vehicle, n=9). PDTC treatment (200 mg/kg, starting at 2 hours) is protective in wild-type animals (n=8) but not in p50-/- mice (n=12).

Lack of p50^-/- Subunit of NF-B and PDTC Protect Against pMCAO
In p50^-/- knockout mice, pMCAO for 72 hours resulted in 41% smaller infarcts compared with wild-type controls (Figure 2B). An exactly comparable protection was achieved with PDTC in wild-type controls. PDTC treatment did not offer any additional reduction in infarct size in p50^-/- mice. This protection was not strain dependent because in BALB/c male mice the infarct volume was decreased by 33% when PDTC treatment was started 2 hours after permanent vessel occlusion (Figure 3). PDTC treatment also prevented induction of COX-2 immunoreactivity in the cortex adjacent to the infarct core 24 hours after pMCAO (Figure 4). The protective effect of PDTC was not species specific because 28% reduction in the infarct size was achieved in spontaneously hypertensive rats when the treatment was started 2 hours after the insult (not shown).

View larger version (37K): [in this window] [in a new window]   Figure 3. Quantitative analysis of BALB/c mouse brains 72 hours after permanent vessel occlusion, treated with vehicle (n=6), 100 mg/kg PDTC (n=5), 200 mg/kg PDTC (n=7), or 200 mg/kg Pro-DTC (n=6) starting 2 hours after onset of ischemia. *P<0.05, ANOVA.

View larger version (82K): [in this window] [in a new window]   Figure 4. Photomicrographs of immunohistochemical COX-2 staining in cortex adjacent to infarct core of a vehicle-treated (A) and a PDTC-treated (200 mg/kg) (B) mouse 24 hours after pMCAO. Note reduction of COX-2 neuronal staining in PDTC-treated mouse. Bar=85 µm.

Pro-DTC Protects Neurons in Culture Similar to PDTC but Does Not Reduce Infarct Volume
To investigate whether inhibition of possible NF-B activation in peripheral tissues and circulating inflammatory cells plays a role in PDTC neuroprotection, we evaluated the effect of Pro-DTC, a hydrophilic PDTC derivative that has been shown previously not to cross the blood-brain barrier but to efficiently block NF-B activation in peripheral tissues.²⁵ In primary cortical culture, the LDH release induced by 50 µmol/L NMDA was reduced by PDTC by 31.8±7.7% (P<0.05) and by Pro-DTC by 29.2±3.9% (P<0.05) when the drugs were administered at 10 µmol/L concentration. However, Pro-DTC did not reduce the volume of focal infarcts in BALB/c mice (Figure 3).

Physiological Variables
The physiological variables were normal in p50^-/- mice. In wild-type mice, PDTC slightly increased the plasma glucose level in sham animals but not 6 or 24 hours after ischemia (Table).

View this table: [in this window] [in a new window]   Physiological Variables in Mouse Permanent MCAO Studies

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
NF-B has been implicated in processes ranging from control of cell proliferation and apoptosis to various intracellular and extracellular stresses, such as oxidative stress, and inflammatory mediators.²⁶ Particularly in the brain, NF-B regulates expression of antiapoptotic, proapoptotic, and proinflammatory genes, thereby playing a dual role in neuronal survival.^14,15 Transient MCAO is a strong inducer of NF-B,^16,17 and the mice lacking the p50 subunit of NF-B develop significantly smaller infarcts 22 hours after transient focal ischemia.¹⁷ In the pMCAO model, NF-B activation has not been successfully or convincingly demonstrated.²⁷ Here we show for the first time that NF-B is induced in neurons in the penumbra of human stroke. Consistent with human stroke, in which no reperfusion normally occurs during the first 24 hours, we also demonstrate that NF-B is activated in pMCAO and that mice lacking the p50^-/- subunit of NF-B develop significantly smaller infarcts than wild-type mice. The detrimental role of NF-B in the brain after pMCAO is further supported by our results that PDTC, which is an antioxidant and established NF-B inhibitor, prevents NF-B induction and reduces infarction size. Moreover, in other studies nonspecific inhibitors of NF-B have been shown to be protective in pMCAO models.²⁸ Therefore, NF-B, especially its p50 subunit, appears to play a detrimental role in brain ischemia, even when it is not followed by reperfusion and production of reactive oxygen species, a well-known stimulus of NF-B.²⁶ When it is considered that PDTC was beneficial when administered 2 hours after the stroke, our results suggest that NF-B may indeed be a good target for stroke therapy.

PDTC has been characterized both in vitro and in vivo as a relatively selective inhibitor of NF-B activation by preventing degradation of NF-B inhibitor peptide I-Ba.^25,29 PDTC has metal-chelating and antioxidant properties, both of which can mediate NF-B inhibition, even though PDTC may also inhibit copper- or zinc-containing enzymes and prevent oxidation of the protein unrelated to NF-B activation.³⁰ Because moderate oxidative stress is triggered in pMCAO as well and antioxidants are protective in pMCAO models,³¹ the possibility that PDTC also exerts its neuroprotective effect by preventing oxidative damage separately from its NF-B–inhibiting effect cannot be ruled out. In fact, PDTC was recently shown to inhibit NF-B independent of its antioxidant properties, and it was shown that reactive oxygen species are not important for activation of NF-B.³² Our results that PDTC completely prevents NF-B activation in the brain and protects against pMCAO to the same extent as lack of the p50 subunit of NF-B support, but do not prove, the idea that the protective effect of PDTC is mediated through inhibition of NF-B activation in pMCAO.

While evidence exists that activation of NF-B induce proinflammatory genes, such as IL-1ß, COX-2, tumor necrosis factor- (TNF-), matrix metalloproteinase-9, and inducible nitric oxide synthase, all of which promote inflammatory tissue injury, NF-B also regulates potentially neuroprotective genes such as Bcl-2, manganese superoxide dismutase, and inhibitor-of-apoptosis proteins.¹⁵ It is conceivable that both potentially beneficial and detrimental genes are inhibited in the brain of p50^-/- mice and animals treated with PDTC. Hence, our results indicate that inhibition of proinflammatory genes may override the potentially detrimental effect of inhibited neuroprotective genes early during acute ischemic injury. Because inflammation may be beneficial during the later recovery phase of stroke,¹ a long-term downregulation of proinflammatory and potentially neuroprotective proteins is not necessarily beneficial.

In studies on transient MCAO in the rat and in the present study of human MCAO, NF-B activation was found to occur mainly in neurons. Our preliminary data suggest that activated NF-B localizes in neurons in mouse pMCAO as well. Even though microglia and infiltrating leukocytes are thought to be the major inflammatory cells in stroke, several NF-B–driven proinflammatory genes, such as COX-2 and TNF-, are expressed in human neurons after stroke.^10,11 Together with previous reports, our results suggest that ischemic neurons produce COX-2, TNF-, and IL-1ß through activation of NF-B. This conclusion is consistent with the evidence of functional NF-B–responsive elements in IL-1ß, TNF-, and COX-2 promoters.^18,33,34 However, we cannot exclude the possibility that NF-B–mediated synthesis of proinflammatory agents also occurs in activated microglia or other nonneuronal cells. In fact, our preliminary findings also indicate that mild NF-B activation is detected in microglia after permanent focal ischemia in mice (A. Nurmi, MSc, et al, 2004). Activation of NF-B in cells outside of the blood-brain barrier may not play a role because Pro-DTC, which does not cross the blood-brain barrier, did not have an effect on infarction even though it was equally protective with PDTC against NMDA neurotoxicity.

Our results suggest that the therapeutic benefit of NF-B inhibition is not species and strain dependent. Because both NF-B activation (this study) and induction of IL-1ß, TNF-, and COX-2 genes^8–11 occur during the first days of human stroke, these results provide therapeutic information relevant to human disease. The PDTC analogue dithiocarbamate has been shown to inhibit HIV progression in patients, suggesting clinical usefulness of PDTC.³⁵ PDTC and its structurally modified derivatives with possible greater efficacy may represent potential therapies against stroke.

	Acknowledgments

 
This study was supported by the Sigrid Juselius Foundation, National Technology Agency, Finland, and the European Union (grant QLK3-CT-2000-00463).

Received December 15, 2003; accepted December 29, 2003.

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