Cell death and proliferation in NF-κB p50 knockout mouse after cerebral ischemia

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Cell death and proliferation in NF-κB p50 knockout mouse after cerebral ischemia Jimei Li a,b , Zhongyang Lu b , Wen-Lei Li b , Shan Ping Yu b,c , Ling Wei b,⁎ a

Department of Neurology, Beijing Friendship Hospital, Capital Medical University, Beijing, China Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, USA c Department of Pharmaceutical and Biomedical Sciences, Medical University of South Carolina, Charleston, SC, USA b

A R T I C LE I N FO

AB S T R A C T

Article history:

The transcription factor NF-κB is a key regulator of inflammation and cell survival. NF-κB

Accepted 27 June 2008

activation increases following cerebral ischemia. We previously showed accelerated aging

Available online 14 July 2008

process in NF-κB p50 subunit knockout (p50−/−) mice under physiological condition. The present investigation concerned the role of NF-κB p50 gene in ischemia-induced neuronal

Keywords:

cell death. In an animal model of permanent middle cerebral artery occlusion (MCAO),

NF-κB

infarct formation, apoptotic cell death and cell proliferation were examined in adult wild

p50 subunit

type (WT) and p50−/− mice. The ischemic infarct volume was significantly larger in p50−/−

Cell death

mice than that in WT mice. Consistently, the numbers of cells in the penumbra region

Apoptosis

positive to terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end-

Neuron

labeling (TUNEL) and caspase-3 staining were significantly more in p50−/− mice than that in

Ischemic stroke

WT mice. To identify proliferation after cerebral ischemia, bromodeoxyurindine (BrdU) was intraperitoneal injected daily after MCAO. Ischemia increased BrdU positive cells in the penumbra, subventricular zone, corpus callosum, and cerebral cortex, while cell proliferation was hampered in p50−/− mice. These results suggest that NF-κB signaling is a neuroprotective mechanism and may play a role in cell proliferation in the stroke model of permanent MCAO. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

Nuclear factor-κB (NF-κB) is a downstream signal of the TNFα pathway and a ubiquitous transcription factor in virtually all cell types (Baeuerle and Baltimore, 1996; Baeuerle and Henkel, 1994; Ghosh et al., 2000). Functional NF-κB complexes are present in the nervous system, including neurons, astrocytes, microglia and oligodendrocytes (Kaltschmidt et al., 1994; Kingston et al., 1999; (Meberg et al., 1996). Activation

of NF-κB is regulated by its inhibitor (IκB) in the cytoplasm. Upon activating signals, IκB is phosphorylated and proteolytically degraded, resulting in NF-κB translocation to the nucleus (Baldwin, 1996). Among the five NF-κB subunits in mammalian cells, p50, p52, p65 (RelA), RelB and cRel, the p50 subunit is believed to be the major functional subunit (Yu et al., 1999). p50 and p65 homodimers are found in adult brain and contributes to NF-κB transcriptional activity in the developing brain (Denis-Donini et al., 2005). Previous work

⁎ Corresponding author. Current address: Department of Anesthesiology, Emory University, Atlanta, GA 30322. E-mail address: [email protected] (L. Wei). Abbreviations: MCAO, Middle cerebral artery occlusion; BrdU, bromodeoxyurindine; TUNEL, terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end-labeling; NF-κB, Nuclear factor-κB; CCAs, common carotid arteries; TTC, 2, 3, 5-triphenyltetrazolium chloride; GFAP, Glial fibrillary acidic protein; FAM, APO LOGIXTM carboxyfluorescein 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.06.130

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indicated that NF-κB is activated in responding to situations of infection, stress and injury (Kopp and Ghosh, 1995; Uberti et al., 1998). Stroke is one of the most common causes of human death and the chief cause of disability. The outcome and infarction size after focal cerebral ischemia is determined in part by scattered and/or delayed neuronal cell death in the ischemic penumbra region, mainly mediated by programmed cell death or apoptosis (Dugan et al., 1996; Li et al., 1995; Namura et al., 1998). NF-κB can regulate expression of inflammatory genes and genes related to apoptosis, and may plays an important role in protecting cells from apoptosis (Bruce-Keller et al., 1998; Doi et al., 1997; Mattson and Camandola, 2001). Contribution of NF-κB p50 subutnit to infarction formation was also reported in permanent and transient ischemic stroke of mouse models (Nurmi et al., 2004; Schneider et al., 1999). On the other hand, mice lacking the p50 subunit of NF-κB showed enhanced vulnerability to kainate-induced damage to hippocampal pyramidal neurons (Yu et al., 1999). Thus, NF-κB signaling can be either pro- or anti-ischemic injury (Baichwal and Baeuerle, 1997). Our recent investigation showed that although p50−/− mice appeared normal at birth, degenerative alterations and accelerated aging process were obvious in p50−/− mice of 6 and 10 months old. These include less body weight, increased spontaneous cell death in the brain, fewer myelinated axons of the optic nerve, abnormal capillaries, and abnormal ultrastructural changes in neuronal and non-neuronal cells (Lu et al., 2006). It was thus suggested that NF-κB plays an important role in neurovascular development, cell survival, and the aging process in the CNS. In the present investigation, we focused on the role of NF-κB in ischemia-induced brain damage; especially, we were interested to know whether the lack of p50 gene would affect the outcome of ischemic stroke in adult animals. In human strokes, reperfusion normally do not occur during the first 24 h (Nurmi et al., 2004), therefore the permanent MCA occlusion model may have more clinical significance. In this investigation, we specifically wanted to verify the role of NF-κB in a permanent ischemia. Specific attention was given to the influence on the penumbra region after stroke.

2.

Results

2.1. Enlarged ischemic infarct volume in NF-κB p50 subunit deficient mice We first examined ischemic damage in 2-month old young adult mice deficient in the p50 subunit of NF-κB. The p50−/− mice at this developmental stage appeared normal (Lu et al., 2006). Focal ischemia was induced by permanent right side MCAO and 10-min ligation of both central cerebral arteries (CCAs). Three days after ischemia, brain slices were staining with TTC to reveal cell death and infarction in the cerebral cortex (Fig. 1A). Compared to wild-type (WT) mice, the size of the ischemic infarct in p50−/− mice was significantly larger at each stereotaxic level (Fig. 1B). Consistently, p50−/− mice showed greater infarct volume than that in WT mice (Fig. 1C).

Fig. 1 – Increased ischemic infarct in p50−/− mice: (A) Cerebral infarction in brain sections 3 days after focal ischemia stained with TTC. (B) Focal ischemia induced infarction areas at each stereotaxic level 3 days after ischemia. (C) Infarct volume 3 days after focal ischemia in 2-month old adult mice.*Significant difference from WT mice (n = 15 mice in each group; p < 0.05).

2.2. Enhanced cell death and apoptosis in the ischemic brain of p50−/− mice TUNEL staining was used to detect DNA fragmentation and cell death in the 2-month old mouse brain 3 and 7 days after ischemia (Fig. 2A). Consistent to the infarct volume measurement, more TUNEL-positive cells were detected in the penumbra region of p50−/− mice than that in WT mice (Fig. 2B). The total cells in the penumbra showed no changes at 1 and 3 days, but more cells existed in WT mice than that in p50−/− mice at 7 days after ischemia (Fig 2C). As a result, in p50−/− mice, the number of NeuN-positive cells was significantly lower than that in WT mice (Fig. 2D). Moreover, fewer GFAP-positive cells were seen in p50−/− mice at 14 days after cerebral ischemia (Fig. 3). Caspase-3 activation serves as a sensitive marker of apoptosis. We detected caspase-3-positive neurons in the penumbra region of p50−/− and WT mice 1 day after focal ischemia (Fig. 4A). The numbers of caspase-3-positive cells in p50−/− mice were greater than that in WT mice at 1 and 3 days after focal ischemia (Fig. 4B).

2.3.

Cell proliferation in the ischemic brain

In immunofluorescence experiments, BrdU-positive cells were identified in the subventricular zone (SVZ), corpus

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Fig. 2 – Increased ischemic cell death in p50−/− mice: TUNEL staining was used to detect cell death in the post-ischemic brain sections from WT and p50−/− mice. (A) TUNEL-positive cells (green) in penumbra regions 3, and 7 days after cerebral ischemia in adult mice. There were more TUNEL positive cells in p50−/− mice than that in WT mice. Bar = 20 µm. (B) Average number of TUNEL positive cells per survey field different days after ischemia. (C) Total cells per survey field in the ipsilateral cortex different days after ischemia. (D) Reduced number of NeuN positive cells was detected in p50−/− mice compared to WT mice. n ≥ 5; *significant difference from WT controls (p < 0.05).

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Fig. 3 – Reduced astrocyte reaction in p50−/−cortex after ischemia: GFAP staining in penumbra regions 14 days after cerebral ischemia. GFAP (red) and NeuN (blue) double immunofluorescence staining showed more GFAP positive cells in WT mice than that in p50−/− mice. Bar = 20 µm; Magnification × 20.

callosum (CC), cortex (CX) and penumbra regions at 1–14 days after ischemia (Fig. 5A). BrdU-positive cells in the SVZ were increased at 3 days in both groups, and did not appear much different between WT and p50−/− mice (Fig. 5B). More BrdU-positive cells were seen in the ipsilateral side

than that in contralateral side in CC and CX (Figs. 5C and E). In corpus callosum, ischemia induced increases in BrdUpositive cells were seen in ipsilateral side 3 to 14 days postischemia, and significant more BrdU-positive cells were detected at 7 days post-ischemia in WT mice than that in

Fig. 4 – Caspase-3 staining in penumbra regions after ischemia: Immunostaining of activated caspase-3 was applied for apoptotic cells. (A) Caspase-3 (green) and NeuN (red) double immunofluorescence staining 3 days after MCAO. Bar = 20 µm. (B) Summery of caspase-3-positive cells in penumbra 1 and 3 days after ischemia. More caspase-3 positive cells were detected in p50−/− mice than in WT mice. n = 5; *significant difference from WT mice (p < 0.05).

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p50−/− mice (Fig. 5C). In the cortex, ischemia induced increases in BrdU-positive cells were seen both in contralateral and ipsilateral sides, with more increases in ipsilateral

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cortex 3 to 14 days post-ischemia. The BrdU/NeuN double staining detected that more newly generated neuronal cells emerged in the penumbra region. BrdU/NeuN-positive cells

Fig. 5 – Cell proliferation in the post-ischemic brain of p50−/− and WT mice. Cell proliferation was labeled with BrdU that was daily injected (50 mg/kg, i.p.) from 1 day after ischemia and until the day of sacrifice. BrdU immunoreactivity was detected in subventricular zone (SVZ), corpus callosum (CC), penumbra and cortex (CX) regions 3 days after cerebral ischemia. (A) Illustration of BrdU (red) and NeuN (blue) double staining in the penumbra region 3 days after cerebral ischemia in WT mice. Bar = 40 µm. (B to E) BrdU-positive cells in different brain regions after ischemia. No significant difference in proliferation activity was seen in the SVZ area (B). However, less BrdU-positive cells were seen in various brain regions of p50−/− mice. No BrdU/NeuN positive cell was detected from penumbra (D) and CX (E) at 1 day after cerebral ischemia. The increased numbers of BrdU/NeuN-positive cells were then detected 3 to 14 days after ischemia in the penumbra and the cortex. Knocking out of p50 suppressed BrdU-positive cells, the inhibitory effect in the ipsilateral cortex was delayed, appeared at day 14 post-ischemia. n ≥ 5; *significant difference from WT mice (p < 0.05).

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Fig. 6 – Proliferation of astrocytes and microglias after ischemia in the penumbra. Astrocytes and microglias were stained with Iba1 and GFAP, respectively. Seven days after ischemia, there was a marked increased in Iba1-positive microglia cells in WT (A) and p50 knockout (B) mice compared with that in WT sham controls (C). GAFP/BrdU-positive cells were also increased, however, less increase was seen in p50−/− mice (D and E). The bar graph in F summarizes the cell counts for microglia proliferation, showing less increase in p50−/− mice. N = 3; *significant difference vs. WT (p < 0.05). Bar = 40 µm.

in the penumbra increased more evidently at 14 days after ischemia. At each time point, less neuronal proliferation activity was seen in p50−/− mice (Fig. 5D). Less increases in BrdU/NeuN positive cells were seen in ipsilateral and contralateral cortex of p50−/− mice (Fig. 5E). We next examined the effect of ischemia on proliferation of non-neuronal cells. Double staining of BrdU with Iba1 and GFAP was performed to identify proliferation of microglia cells and astrocytes in control and post-ischemic brains. Compared with non-ischemic controls, proliferation of microglia cells and astrocytes was significantly increased 7 days after the ischemic insult; however, the increase was much less prominent in p50−/− mice (Fig. 6).

3.

Discussion

In present study, we show that knockout of the p50 subunit of NF-κB results in significant increases in cell death and infarct volume after permanent MCAO in mice. This is consistent with our previous report that the p50 gene was neuroprotective and anti-apoptotic in these animals (Lu et al., 2006). NF-κB 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 (Kingston et al., 1999). In the brain, NF-κB regulates expression of anti-apoptotic, pro-apoptotic, and pro-inflammatory genes, playing a dual role in neuronal survival. Regarding to the dual role of NF-κB in neurodegenerative diseases, it is suggested that activation of NF-κB in

neurons promotes their survival, whereas activation in glial and immune cells mediates pathological inflammatory processes (Camandola and Mattson, 2007). Transient MCAO is a strong inducer of NF-κB (Schneider et al., 1999; Stephenson et al., 2000). The lack of the p50 subunit of NF-κB is protective in the transient MCAO model, but increases excitotoxic damage in the hippocampus (Nurmi et al., 2004; Schneider et al., 1999). In the permanent MCAO model, decreased NF-κB activity may exacerbate ischemia induced neuronal cell death (Irving et al., 2000). Consistent with this and our investigations, Duckworth et al. recently showed that p50−/− mice manifest more damage after permanent MCAO (Duckworth et al., 2006). Some studies in cultured cells report that NF-κB activation has a protective effect (Barger et al., 1995; Bruce-Keller et al., 1998; Guo et al., 1997; Lezoualc'h et al., 1998; Liu et al., 1996; Taglialatela et al., 1997; Tamatani et al., 1999; Van Antwerp et al., 1996). On the other hand, inhibition of NF-κB binding may decrease damage from myocardial infarction (Morishita et al., 1997) and reduce infarct size after MCAO (Phillips et al., 1998). In addition to p50, p65 is another NF-κB member that may play a complementary role in the absence of p50. It may be possible that the different results reported with p50 modification are partly due to changes in p65. NF-κB plays important roles in the CNS, mainly represented by p50/RelA heterodimers in astrocytes, Schwann cells, oligodendrosytes and microglia (Carter et al., 1996; Nakajima et al., 1998; Sparacio et al., 1992; Vollgraf et al., 1999). The consequence of this event may be twofold. The reduced number of glial cells decreases some inflammatory response and scare formation after ischemia. On the other hand,

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astrocytes exhibit several functions that are essential for normal neuronal activity, including glutamate uptake, ion buffering, water transport, and metabolic and trophic supports. Thus, many of these astrocyte functions can promote neuronal survival during ischemia and promote neurite outgrowth and regeneration in the post-injury period (Swanson et al., 2004). In our study, we found that many GFAP-positive cells surrounded stroke area in WT mice 14 days after cerebral ischemia whereas fewer astrocytes were seen in p50 knockout mice. This is consistent with the observation that proliferation of GFAP-positive astrocytes was deficient in p50−/− mice. The reduced number of astrocytes may explain, at least partially, the increased neuronal injury and may affect neural repair in the post-ischemic brain. Knocking out p50 suppressed the inflammatory response; there was significantly less proliferation of microglia cells in the penumbra after ischemia. The significance of the suppressed immune response remains to be further elucidated; it is likely that it contributes to the aggravated brain damage. BrdU is a uridine analog that can be incorporated into replicating DNA and is commonly used to visualize cell proliferation. Newly proliferated cells appear to migrate from the SVZ to the lesioned area through corpus callosum after stroke (Arvidsson et al., 2002). NF-κB genes may control a variety of events correlated with the generation of new neurons and glia and/or with oriented migration processes in the SVZ/RMS (Denis-Donini et al., 2005). In present investigation, we did not detect significant changes of BrdU positive cells in the SVZ. However, we saw less BrdU-positive cells at different times in corpus callosum and penumbra regions of p50−/− mice after stroke. These results suggest that although the p50 subunit of NF-κB did not directly affect cell proliferation in the SVZ, it may hamper cell proliferation in other brain regions. Alternatively, the lack of p50 might impair cell migration, differentiation and/or increased these cells' susceptibility to cell death/apoptosis.

4.

Experimental produres

4.1.

Experimental animals

Adult p50-knockout (p50−/−, B6, 129P-Nfkb1) and wild type (p50+/+, B6, 129PF2) mice (2 month-old, body weight = 25–30 g, n = 50 from Jackson Laboratories (Bar Harbor, ME, USA) were used in the experiments. Animals were maintained at room temperature (∼21 °C) with a 12-h light/dark cycle in the pathogen-free Laboratory Animal Center for Research at Medicine University of South Carolina.

4.2. Permanent focal cerebral ischemia and infarct assessment Mice were anesthetized by ip. injection of chloral hydrate at 400 mg/kg. Anesthesia and surgical procedures were performed in accordance with institutional guidelines. Focal ischemia, confined to the cerebral cortex in the right middle cerebral artery (MCA) territory of male mice were induced by ligation of the right MCA and both common carotid arteries (CCAs). After 10 min, both CCA clips were released. Rectal temperature was

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closely monitored and maintained at 37 ± 0.3 °C during MCAO and for up to 1 h after ischemia via an Digi-Sense® temperature controller (Cole-Parmer Instrument Co, Chicago, IL) linked to a heating pad and a heating lamp. Free access to food and water was allowed after recovery from anesthesia. All mice were kept in air-ventilated cages with room temperature maintained at 24 ± 0.5 °C. Animals received intraperitoneal injections of 50 mg/ kg bromodeoxyuridine (BrdU, Sigma, St. Louis, MO) daily. Different (1, 3, 7 and 14) days after MCAO, animals were killed with an overdose of pentobarbital (100 mg/kg) followed by intracardiac perfusion of 200 ml 0.9% NaCl. The brains were then sliced into 1-mm coronal sections. Cortical infarct volume was morphometrically measured after staining with 2% 2, 3, 5triphenyltetrazolium chloride (TTC, Sigma, St Louis, USA) in phosphate-buffered saline (pH 7.4) at 37 °C for 20 min and then stored in 10% neutral-buffered formalin. The cross-sectional area of the TTC-unstained region was determined with the use of an image analyzer (DUMAS, Drexel University). The indirect method (subtraction of residual right hemisphere cortical volume from cortical volume of the intact left hemisphere) was used for infarct volumes calculation. Surgery and infarct measurements were performed under double-blind conditions.

4.3. Immunofluorescence of NeuN, BrdU, Iba1 and GFAP-positive cells Coronal brain sections (8-µm) were cut on a freezing microtome and fixed with 10% formalin, blocked with 1% fish gel and incubated with mouse anti-NeuN antibody (Chemicon international Inc., Temecula, USA), mouse anti-Iba1, or mouse anti-Glial fibrillary acidic protein (GFAP) antibody (Biomeda, Foster City, USA) overnight. For BrdU immunostaining, brain sections were incubated in 2 N HCl at 37 °C for 1 h and rinsed with boric acid (pH 8.5). After incubation, all sections were incubated with a primary anti-BrdU antibody (Abcam Inc., Cambridge, MA). Alexa Fluor 488 anti-rat and anti-mouse Cy3 or Cy5 were used as secondary antibody. Results were visualized by a fluorescence microscopy (Olympus America Inc., New York, USA) and verified under a confocal microscope (BX61; Olympus, Tokyo, Japan).

4.4. Terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) staining A TUNEL staining kit (DeadEnd™ Fluorometric TUNEL system, Promega, Madison, USA) was used to assess cell death by catalytically incorporating fluorenscein-12-dUTP at 3′-OH DNA ends using the terminal deoxynucleotidyl transferase and recombinant enzyme (rTdT). Brain sections were placed in equilibration buffer and incubated with nucleotide mix and rTdT enzyme at 37 °C for 1 h, and stopped reaction with 2× SSC. Hoechst 33342 (Molecular Probes, Eugene, Oregon, USA) was used to stain neuronal nuclei. Results were visualized by the Olympus fluorescence microscopy.

4.5.

Staining of activated caspase-3

Detection of active caspase-3 in brain tissues was performed using an APO LOGIXTM carboxyfluorescein (FAM) caspase detection kit (Cell Technology, Minneapolis, MN, USA). Fresh

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frozen brain sections were fixed with 10% formalin for 1 min. The brief 1-min fixation helped to prevent fall off the sections during staining and did not affect the caspase activity assay. Tissues were incubated with 30× working dilution FAM-Peptide-FMK for 1 h at room temperature; then washed twice with 1× working dilution wash buffer. Hoechst 33342 staining was used to identify neuronal nuclei. Fluorescent signals were observed under the Olympus fluorescent microscope.

4.6.

Quantification of immunocytostaining positive cells

The TUNEL positive cells and Caspase-3 positive cells were counted in ischemic penumbra region. For each staining, counting was performed on 3 randomly selected non-overlapping 20× fields per 10-µm thick section. Cell counts for each animal were performed in the same volume of wild type and p50−/− brains. Design-based stereology and systematic random sampling were employed to ensure accurate and non-redundant cell counting. Every section under analysis was a minimum distance of 150 µm from the next, and a total of 6 sections that spanned the entire infarct region of interest were randomly selected for cell counting. For each section under analysis, the region of interest in the ipsilateral hemisphere and the corresponding location was selected in the contralateral hemisphere. Similarly, agematched sham control brain sections were selected at random from similar areas of the cortex. Cell counts were performed using ImageJ (National Institutes of Health, Bethesda, MD), compiled, and analyzed (Abramoff et al., 2004). Cell courts were performed without knowledge of the genotype of mice.

4.7.

Statistical analysis

Data were analyzed by unpaired Student's t-test and Fisher's test with least significant difference. Multiple comparisons were analyzed using one-way ANOVA followed by a post-hoc Tukey test, performed by the computer software Microsoft Excel (Seattle, WA). Differences were considered significant at p < 0.05. All values represent mean ± SEM.

Acknowledgments This work was supported by NIH grants NS 37372, NS 045155, and NS 045810, and American Heart Association and Bugher Foundation (AHA-Bugher) Awards 0170064N and 0170063N. It was also supported by the National Natural Science Foundation of China (30772302) and Beijing Natural Science foundation (7082029). REFERENCES

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