Neuroglobin mRNA expression after transient global brain ischemia and prolonged hypoxia in cell culture

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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

Neuroglobin mRNA expression after transient global brain ischemia and prolonged hypoxia in cell culture Rainald Schmidt-Kastner a,b , Mark Haberkamp c , Christoph Schmitz b , Thomas Hankeln d , Thorsten Burmester e,⁎ a

Department of Neurology, University of Miami School of Medicine, Miami, FL 33101, USA Department of Psychiatry and Neuropsychology, Division of Cellular Neuroscience, Maastricht University, Maastricht, The Netherlands c Institute of Zoology, Johannes Gutenberg University of Mainz, Mainz, Germany d Institute of Molecular Genetics, Johannes Gutenberg University of Mainz, Mainz, Germany e Institute of Zoology, Biozentrum Grindel, University of Hamburg, Martin-Luther-King-Platz 3, D-20146 Hamburg, Germany b

A R T I C LE I N FO

AB S T R A C T

Article history:

Neuroglobin is a nerve-specific respiratory protein that has been proposed to play an

Accepted 16 May 2006

important role in the protection of brain neurons from ischemic and hypoxic injuries. Here,

Available online 21 June 2006

we investigated the regulation of neuroglobin expression after transient global ischemia in the rat brain using mRNA in situ hybridization and under hypoxic stress in cultured

Keywords:

neuronal cell lines (PC12, HN33) by quantitative RT-PCR. While neuroglobin mRNA

Neuroglobin

expression was significantly enhanced in cell culture after severe prolonged hypoxia (0–

Gene expression

1% O2 for 24 h), we did not find any significant increases in neuroglobin mRNA levels in the

Hypoxia

rat brain after transient global ischemia. Vegf and Glut1 mRNAs showed increases in the

Ischemia

hippocampus as expected. Therefore, it is unlikely that neuroglobin is instrumental in the acute response of neurons to hypoxic or ischemic insults, for which the mammalian brain is

Abbreviations:

not adapted.

Glut1, glucose transporter 1

© 2006 Elsevier B.V. All rights reserved.

GV, grey value ISH, in situ hybridization Ngb, neuroglobin Vegf, vascular endothelial growth factor

1.

Introduction

A respiratory protein referred to as “neuroglobin” (Ngb) was recently identified in the brain of a variety of vertebrate species (Burmester et al., 2000, 2004; Burmester and Hankeln, 2004; Fuchs et al., 2004; Kugelstadt et al., 2004). This finding was not unprecedented, since myoglobin-like oxygen storage molecules were found in the nervous system of some invertebrates, where they are believed to sustain energy

metabolism during peaks of functional activity (Kraus and Colacino, 1986; Wittenberg, 1992; Weber and Vinogradov, 2001). However, at present, the physiological role of Ngb is not well understood (Burmester and Hankeln, 2004, Hankeln et al., 2005; Pesce et al., 2002). Analogous to myoglobin in striated and cardiac muscles, Ngb may increase the availability of oxygen to brain tissue. By inference, local oxygen storage by Ngb may be an additional mechanism to meet the oxygen demands of neurons under conditions of rapid synaptic

⁎ Corresponding author. Fax: +49 40 42838 3937. E-mail address: [email protected] (T. Burmester). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.05.047

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activation in the mammalian brain. In line with this hypothesis, Ngb was found at high concentrations in the metabolically active retina, where it is located adjacent to the mitochondria (Schmidt et al., 2003; Bentmann et al., 2005). There is a fine line between discovering such an oxygen storage molecule in the brain and postulating an important role in brain ischemia and stroke (Burmester et al., 2000). If Ngb stores oxygen, then tissue levels may be increased under conditions of hypoxia and ischemia; i.e., Ngb may play a role of autoprotection. Evidence for a regulation of Ngb was presented for immature neurons in vitro (Sun et al., 2001) and for the cortex after focal ischemia (Sun et al., 2003). However, experiments with hypoxia in mice did not provide evidence for increased expression (Hundahl et al., 2005; Mammen et al., 2002). In view of the growing interest in the role of Ngb in brain function and pathology (Burmester et al., 2000; Sun et al., 2001, 2003; Burmester and Hankeln, 2004; Hankeln et al., 2005), we asked whether Ngb expression changes in vivo after transient global brain ischemia or in vitro in neuronal cell lines in response to changing oxygen levels.

2.

Results

2.1.

Ngb in situ hybridization studies on rat brains

At the section level of the hippocampus, a focally high expression of Ngb mRNA was seen in the medial hypothalamus and amygdala region. In the hippocampus and cortex of control and sham-operated animals, hybridization signals were low. These findings are in line with the description of Mammen et al. (2002) using autoradiographic ISH methods in the mouse brain. Strong hybridization signals were seen in the

Fig. 1 – Quantification of Ngb mRNA levels in different structures and at different time points after transient global brain ischemia in the rat, showing the absence of upregulation. Data are shown as percent of grey value in the medial hypothalamus where strong constitutive Ngb expression was present in the same section (means ± SD). Statistical analysis was carried out with ANOVA followed by t test (**P < 0.01). CA1, CA3 = regions of the hippocampus; CTX = cortex; DG = dentate gyrus; GV = grey values.

Fig. 2 – Cell viability under hypoxia. HN33 and PC12 cells were kept for 24 h or 48 h at 1% O2. The viability of the cell lines (compared to the normoxia control) was assayed by an XTT test; the observed changes were not significant.

hypothalamic region at all time points which were used to normalize the changes in the hippocampus and the cortex after ischemia in each section. No increase of Ngb mRNA hybridization signals was seen in the hippocampus at any time point after global brain ischemia. ANOVA of the relative GVs in the hippocampus during the first day after ischemia did not indicate significant changes over time (P > 0.05) although there was a trend for a decline (Fig. 1). Low Ngb mRNA signals were observed over the cortex of sham controls, which were even reduced at 30 min (ANOVA, P < 0.05; t test P < 0.01). When all GV data from 30 min to 24 h after ischemia were combined (n = 27) and compared to the sham-group (n = 8) in each area, the Ngb mRNA signals were significantly lower in CA3, DG and CTX after ischemia (t test P < 0.01). Ngb mRNA hybridization signals remained low in the CA1 area at 48 or 72 h after global brain ischemia when highly activated astrocytes occurred in this region (unpublished observations). For comparison, changes in the expression of Vegf and Glut1 were evaluated in the hippocampus (Pichiule et al., 1999; Jin et al., 2000; Chavez and LaManna, 2002). Vegf mRNA signals were modestly elevated in the hippocampus at 3 h and 6 h, and strong increases were seen at 24 h. Signals were variable by 48 h and declined at 3 and 7 days. Statistical analysis of relative GVs for Vegf mRNA showed that the increases at 24 h were significant in CA1 (166 ± 37% of sham; ANOVA, P = 0.01, t test P = 0.01) and CA3 (154 ± 30%; ANOVA, P = 0.02, t test P = 0.01). Signals for Glut1 mRNA were moderate in the hippocampus of sham controls and became diffusely elevated over the hippocampus between 30 min and 24 h after ischemia. Changes at 6 h (127 ± 2%) and 24 h (150 ± 12%) were highly significant (ANOVA, P < 0.001, t tests P < 0.001). Variable signals were noted in the 48- and 72 h animals due to cell damage in CA1.

2.2.

Ngb expression levels in hypoxic cell culture

We employed HN33 and PC12 cell lines for hypoxia studies. HN33 is an immortalized neuronal cell line that derives from the hippocampus of juvenile mice (Lee et al., 1990); PC12 is a standard adrenal gland pheochromocytoma cell line from rat with neuronal characteristics. The viability of the cell lines under hypoxia was estimated by standard tests which

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measure the activity of lactate dehydrogenase (LDH test) or the activity of mitochondrial dehydrogenases (XTT test). The LDH test turned out to be unsuitable for our purposes because the activity of LDH increases under hypoxia. The XTT test showed that both HN33 and PC12 cells survived for 48 h at 1% O2. While the viability of PC12 cells remained unchanged under hypoxia, we noted a slight decrease of HN33 viability, although this reduction was statistically not significant (Fig. 2). Prolonged severe hypoxia (48 h at 0.3% O2) decreased the activity of dehydrogenases, although both cell lines survived (data not shown). Quantitative real-time RT-PCR experiments showed that Ngb was expressed in both cell lines, although total Ngb mRNA levels were low and <0.6% (HN33) and <0.1% (PC12) compared to ARP mRNA which served as internal standard. ARP expression levels were not influenced by changes in oxygen concentration (data not shown; see also: Simpson et al., 2000). To follow the kinetics of Ngb expression under hypoxia, we kept HN33 cells at a constant level of 1% O2 (Fig. 3). Measurements showed that it took about 2 h until the cell medium was equilibrated to the desired oxygen level. Therefore, we determined the Ngb mRNA expression levels after 4, 8, 16 and 24 h. We first noted a slight decrease of Ngb mRNA after 4- and 8-h hypoxia, although this deviation was not significant. After 24-h hypoxia, a significant increase of Ngb expression was observed (ANOVA P < 0.01, t test P < 0.05), whereas longer incubation times (>24 h) had only minor further effects (data not shown). The amount of Ngb mRNA was then monitored in the PC12 and HN33 cell lines at different atmospheric oxygen levels (Fig. 4). Ngb expression levels were largely unaffected by 24 h of hypoxia up to 2% O2 for HN33 and 1% O2 for PC12. Only at more severe hypoxia, the amount of Ngb mRNA significantly increased in both cell lines, with a much stronger response

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Fig. 4 – Ngb expression levels in HN33 (n = 6) and PC12 cells (n = 3) at different oxygen levels. Ngb mRNA quantities were determined by real-time RT-PCR employing the TaqMan method and normalized according to the level of ARP mRNA, a non-regulated reference gene. All transcription rates were estimated with reference to Ngb expression at normoxia for each cell line (20% O2). The left hand columns (white) are the results for the HN33 cells, the right hand columns (black) represent the data obtained from the PC12 cell line. The bars are standard deviations. The significance of the data was estimated with a Student's t test: ***P < 0.001; *P < 0.05.

in HN33 cells. Higher oxygen concentrations with 30% and 40% O2 (hyperoxia) slightly increased Ngb mRNA expression in PC12 but not in HN33 cells.

3.

Discussion

The mammalian brain relies on a continuous supply with oxygen, and a reduction of the cerebral blood flow as in stroke and other cerebrovascular diseases causes severe and irreversible injury (Siesjö, 1992; Ginsberg, 2003; Lutz et al., 2003; Sharp, 2000; Sharp and Bernaudin, 2004). Since Ngb is an oxygen-binding molecule expressed in neurons, it has been suggested that an increased expression of Ngb may occur after brain hypoxia or ischemia as a regulatory response, and that Ngb might protect the brain from these insults (Sun et al., 2001, 2003).

3.1. Neither ischemia nor hypoxia increase Ngb levels in the mammalian brain

Fig. 3 – Quantification of Ngb expression levels in HN33 cells at 1% oxygen at different times of hypoxia. Ngb mRNA quantities were determined by real-time RT-PCR employing the TaqMan method and normalized according to the level of ARP mRNA, a non-regulated reference gene. All transcription rates were estimated with reference to Ngb expression at normoxia (20%). *P < 0.05 (t test).

At the in vivo level, we investigated the brain by ISH employing a specific antisense oligonucleotide-probe to rat Ngb mRNA. We focused on the hippocampus which is the most vulnerable brain region in global brain ischemia (SchmidtKastner and Freund, 1991). Vascular endothelial growth factor (Vegf) and glucose transporter 1 (Glut1) mRNAs were used as a reference for classical hypoxia-regulated genes (Sharp and Bernaudin, 2004; Semenza, 2002) that were shown to be upregulated after global brain ischemia (Pichiule et al., 1999;

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Jin et al., 2000; Chavez and LaManna, 2002). Ngb signals were found to be low in the rat hippocampus, which is in line with anatomical descriptions for the mouse brain (Mammen et al., 2002). The latter study reported a focally high Ngb mRNA pattern in the mouse brain, with expression in medial parts of the amygdala, lateral septum, midline portions of the thalamus, the preoptic area, hypothalamus, distinct brainstem nuclei and the subfornical organ (Mammen et al., 2002). At the section level of the hippocampus, we also noted distinct hybridization signals in the medial hypothalamus and amygdala in the rat. Thus, two studies using radioactive ISH methods yielded similar results with different probes to Ngb. Low levels of Ngb mRNA were also seen with Northern blots in samples of the human hippocampus as compared to other brain areas (Burmester et al., 2000). Other studies using nonradioactive methods for detection of Ngb mRNA (Reuss et al., 2002) or immunohistochemistry for Ngb (Wystub et al., 2003; Hundahl et al., 2005) showed more widespread neuronspecific expression. Studies in the post-ischemic brain did not indicate an increase in the expression levels of Ngb in the hippocampus or cortex, and the constitutive signals in the hypothalamus were unchanged. Parallel studies showed upregulation of Vegf and Glut1 mRNAs during the first day of recirculation in the hippocampus, demonstrating the expected activation of hypoxia-induced gene expression by the ischemic insult (Pichiule et al., 1999; Jin et al., 2000; Chavez and LaManna, 2002). Ngb mRNA expression was also unaffected by severe hypoxia in the forebrain and cerebellum of mice (Mammen et al., 2002; Hundahl et al., 2005). Moreover, none of the extensive microarray studies on brain ischemia have reported changes in Ngb expression (unpublished observations). Many globins show enhanced expression in animals that live at low oxygen concentrations (Weber and Vinogradov, 2001; Wittenberg, 1992), and at the first glance one might expect an increased level of Ngb expression under such conditions. However, in rodents Ngb does not exhibit any response to hypoxia (Mammen et al., 2002) or ischemia (this study) in vivo. By contrast, a recent study showed that Ngb mRNA and protein levels strongly increase in brains of zebrafish in that had been kept under hypoxia (Roesner et al., 2006). One should consider that – in contrast to many fish species – the brain of most adult mammals hardly ever faces hypoxic situations in nature. Therefore, it is unlikely that the normal mammalian brain is adapted to a shortage in oxygen supply (Lutz et al., 2003). Even if Ngb is instrumental in the oxygen metabolism of the mammalian brain, one cannot expect any response to artificial situations like hypoxia or ischemia.

3.2.

Ngb is hypoxia-inducible in neuronal cells lines

In immortalized mammalian neuronal cell lines, Ngb mRNA levels significantly increased under severe hypoxia, reaching a maximum of 2.3-fold expression in HN33 cells after 24 h at 0.3% O2 compared to the atmospheric normoxia control (Fig. 3). These figures are largely compatible with previous studies employing neuronal primary culture or HN33 cells (Sun et al., 2001; Fordel et al., 2004). Slight variations in induction times and levels can be explained by different technical setups and experimental protocols. Although these data

show that Ngb is hypoxia-responsive in vitro, it is uncertain how the hypoxia response might be conveyed. Hypoxiainducible factor 1 (HIF-1) is known as the master regulator of cellular hypoxia-response (Semenza et al., 1996), and a sequence search of the mammalian Ngb promoter region actually showed the presence of hypoxia-responsive elements (HREs) that could bind HIF-1 (Wystub et al., 2004). However, a comparative study of mammalian Ngb genes did not reveal any conservation of HREs, a prerequisite that has been considered essential for a functional HRE (Wystub et al., 2004). Therefore, it remains uncertain whether Ngb is actually controlled by HIF, or whether its hypoxia inducibility in cell culture is transferred by other means, such as the mitogen-activated protein kinase (MAPK) signal transduction pathway (Zhu et al., 2002) or hypoxia-inducible protein binding sites (HIPBS) (Wystub et al., 2004). Expression kinetics showed a slow increase of Ngb mRNA in HN33 cells (Fig. 2), which is in line with previous data (Fordel et al., 2004). We also noted a slight decrease of Ngb mRNA as an immediate hypoxia response; these results are hardly compatible with the idea of Ngb being involved in acute hypoxia or ischemia reaction (see above). Moreover, the rather slow response of Ngb mRNA levels to the hypoxia stimulus provides additional evidence that its gene is not under direct control of HIF.

3.3.

Functional implications

A direct comparison between the in vivo and the in vitro experiments is difficult, but some suggestions can be made. Oxygen levels are 12% O2 in arterial blood and 3% O2 in tissues in general, and the brain shows low and non-uniform levels of 1–5% O2 (Sharp and Bernaudin, 2004). Tissue oxygen levels decline in the hippocampus during severe, incomplete forebrain ischemia (Freund et al., 1989; Block et al., 1993), but they may not have reached 0% O2 by the end of the occlusion which typically lasts 10 to 20 min. Another explanation for the absence of an upregulation in the transient, global ischemia model may be the intrinsically slow response of the Ngb regulation which is seen in the in vitro studies. Thus, the hypoxic threshold for the induction of Ngb mRNA will not be crossed even by severe global ischemia as in cardiac arrest followed by resuscitation under clinical conditions. One can combine the anatomical Ngb expression pattern (Mammen et al., 2002; Schmidt et al., 2003; Hankeln et al., 2004) with physiological information (Burmester and Hankeln, 2004; Bentmann et al., 2005; Hankeln et al., 2005) to propose that Ngb may serve as a cellular oxygen buffer in areas of the nervous system where high physiological fluctuations of oxygen levels can be expected. In fact, Ngb mRNA expression is low in the cortex and hippocampus where local blood flow and thereby oxygen delivery are controlled by autoregulation and flow/ metabolism coupling. Other evidence for the fluctuation concept comes from the retina where high expression of Ngb was found in the photoreceptor layer (Schmidt et al., 2003). Since capillaries and photoreceptors cannot co-exist for optical reasons, the photoreceptor layer is supplied with oxygen by diffusion from the choroid vessels, which have a high basal blood flow and lack autoregulation (Yu and Cringle, 2001). Therefore, oxygen levels in the photoreceptor layer vary

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with physiological activity levels. Brain regions close to the choroid plexus in the lateral ventricle (e.g., lateral septum) and cerebrospinal fluid (CSF) spaces (e.g., medial thalamus, hypothalamus) may be exposed to variable oxygenation due to diffusion of oxygen between brain and CSF spaces. Vessels in the subfornical organ and in ventral hypothalamic regions lack blood-brain barrier properties (Broadwell and Brightman, 1976) which could be associated with unusual blood flow regulation and variable levels of oxygen in the surrounding brain tissue.

3.4.

Conclusions

We have investigated the effect of reduced oxygen availability on neuronal cells in vitro and in vivo. While low oxygen levels induce Ngb expression in neuronal cell lines (Sun et al., 2001; Fordel et al., 2004; this study), in vivo studies agree that neither hypoxia (Mammen et al., 2002) nor ischemia (this study) induces an increase of Ngb in the mammalian brain. However, these considerations do not contradict the idea that Ngb is neuroprotective in vivo by controlling distribution of oxygen at the cellular level (Burmester et al., 2000; Sun et al., 2001, 2003). Nevertheless, other functions for Ngb, such as the detoxification of reactive oxygen species, are still conceivable and may be compatible with the data.

4.

Experimental procedures

4.1.

Rat brain ischemia

Transient global brain ischemia for 12.5 min was induced by two-vessel occlusion and systemic hypotension using normoglycemic and normothermic conditions (Schmidt-Kastner et al., 2004). The procedures were approved by the University of Miami's Animal Care and Use Committee. Male Wistar rats (Rattus norvegicus) weighing between 250 and 350 g were fasted overnight, initially anesthetized with 3% halothane, and were intubated and ventilated mechanically with mixtures of 0.5– 1% halothane, 70% nitrous oxide and a balance of oxygen, after immobilization with pancuronium bromide (0.75 mg/kg, i.v.). Following catherization of femoral arteries with polyethylene tubing blood pressure measurements and sampling for arterial blood gases and plasma glucose were carried out; ventilatory adjustments kept the arterial pCO2 and pO2 in the normal range. A loop of close-fitting polyethylene tubing contained within a dual-bore Silastic tubing was placed around each common carotid artery. Skull temperature was measured with a small thermistor inserted under the skin and kept constant between 36.5 and 37.0 °C. Rectal temperature was separately maintained at 37.0 °C before and during ischemia. To induce severe incomplete forebrain ischemia, blood was gradually withdrawn into a heparinized syringe to reduce systemic blood pressure to 45–50 mm Hg, and the carotid ligatures were tightened bilaterally for 12.5 min. Mean arterial blood pressure was held at 45–50 mm Hg by controlled exsanguination. For recirculation, the carotid ligatures were removed, and blood pressure restored by reinfusing the warmed shed blood. After removal of catheters, closure of all skin incisions and application of 1% lidocaine, rats were returned to cages at room

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temperature with free access to water and food. During sham surgery, the vascular occlusion and hypotension were omitted. Following reinduction of anesthesia with 4% halothane, brains were removed under RNase free conditions, frozen in chilled methylbutane and stored at −80 °C until sectioning. The groups consisted of n = 2–4 animals with recirculation for 30 min, 1, 3, 6, 24, 48 and 72 h, plus plain control or shamcontrol animals (combined as “sham controls”). Previous work in this global ischemia model has documented that CA1 neurons die after about 2 days of recirculation (SchmidtKastner et al., 2004), and that various changes in mRNA levels occur within the first hour of recirculation.

4.2.

In situ hybridization (ISH)

Frontal sections through the hippocampus were prepared in a cryostat at 12-μm thickness and two parallel sections were placed on each slide. The ISH procedure followed established protocols for synthetic oligonucleotide probes (SchmidtKastner et al., 2000). Based on a mouse antisense probe established before (Hankeln, unpublished results), an antisense probe to rat NGB was designed (GTC CTC TAC GTT GGT CAC AGC AGC ATC AAT CAC AAG CAT; AF334379, bp: 205–243) using Lasergene (DNA Star, Wisconsin, MI) and BLAST (NCBI). As a reference for hypoxia-increased gene expression, we probed sections with antisense probes to Vegf mRNA (TCT GGA TTA AGG ACT GTT CTG TCA ACG GTG ACG ATG ATG G; AF062644, bp 400–435) and to Glut-1 mRNA: GGC TGG CGG TAG GCG GGT GAG CGG AAC AGC TCC AAG ATG GTG AC; M13979, bp 977–1020). Probes were obtained from Invitrogen (Carlsbad, CA). Films were exposed for 4 weeks for Ngb mRNA and for 2 weeks for Vegf mRNA and for Glut1 mRNA. It was important to obtain a homogenous exposure of control and experimental animals from different recirculation time points on the same film; this limited the experiments to n = 2–4 slides (4– 8 sections) per time point for Ngb mRNA. Autoradiograms were scanned on a flat bed scanner (CanoScan 8400F, Canon, New York). Images were imported into ImageJ (Image Java; http://rsb.info.nih.gov/ij/) and an analysis of grey values (GV; 0–256 scale) was performed to obtain an estimate of the hybridization levels. After inversion of the images, GV measurements for Ngb mRNA signals were taken in the hippocampus (CA1, CA3 and dentate gyrus = DG), in the dorso-lateral cortex (Ctx) and over Ngb mRNA-rich areas in the medial hypothalamus in the same section. GVs in different areas were tabulated for each hemisphere separately, and then the mean hemispheric GVs were determined for groups with 30 min, 1, 3, 6 and 24 h of survival and for sham controls (n = 4–8 sections, each). The 48-h and 72-h time points were not used to exclude a trivial influence of cell death on the analysis. The mean GVs of film background were subtracted from GV measurements in the tissue. To compensate for variations among sections, the GV signals in the hippocampus and cortex were expressed as percent of GV signal of the strongly positive hypothalamic area in the same section. In an additional analysis, GV data from 30 min to 24 h after ischemia were combined (n = 27) and compared to the sham-group (n = 8) in each area using t tests (P < 0.05). Hybridization signals for Vegf mRNA were measured in the CA1, CA3 and DG areas of the hippocampus at 6 and 24 h (n = 4, each) and analyzed as

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percent of sham control after subtraction of background values. The diffuse Glut1 mRNA signals were evaluated in one window covering the hippocampus at 6 and 24 h (n = 4 each). Background values were subtracted and mean hemispheric data were expressed as percent of sham control. ANOVA tests (P < 0.05) were carried out in each structure separately which were followed by t tests with Bonferroni corrections for multiple comparisons (P < 0.01 for the Ngb mRNA data; and P < 0.025 for the Vegf and Glut1 mRNA data). Data are presented as means ± SD.

4.3.

Cell culture methods and hypoxia experiments

The HN33 cell line was a gift from D.A. Greenberg (Buck Institute, Novato, USA), PC12 cells (ATCC Number: CRL-1721) were kindly provided by S. Saaler-Reinhardt (University of Mainz, Germany). The cell lines were kept at 37 °C in a humidified normoxic atmosphere and 5% CO2 in Dulbecco's modified Eagle medium with L-glutamine and 4.5 g/l glucose (PAA Laboratories), supplemented with 10% (HN33) or 15% (PC12) fetal calf serum, 100 IU penicillin and 100 μg/ml streptomycin. All gases were obtained from Air Liquide (Krefeld, Germany). For time-dependent hypoxia experiments, the HN33 or PC12 cells (passage number < 30) were transferred to a CB 150 incubator (WTB Binder, Germany) and kept for the desired time at 1% oxygen. For the experiments with varying oxygen levels, the cells were first incubated for 24 h at 37 °C at normoxia. Then the oxygen concentration was changed to the desired level (0.3, 1, 2, 10, 20, 30 and 40%) and the cells were kept for an additional 24 h. It should be noted that about 0.3% O2 was the technical minimum achieved in this system.

4.4.

Cell viability assay

Cell viability was assayed employing the XTT test (Cell Proliferation Kit II; Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's instructions. Cells were grown in a 12 well tissue culture plate for 24 or 48 h at the desired oxygen level. The tetrazolium ring of XTT (Na 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4methoxy-6-nitro) is cleaved by dehydrogenases of viable cells to form soluble orange formazan, which can be detected spectrophotometrically. After adding XTT, the cells were incubated for 2 h, and the increase in formazan absorbance was read at a wavelength of 485 nM in a microplate reader (SpectraFluor Plus Infinite 200, Tecan, Crailsheim, Germany).

4.5.

RNA isolation and real-time reverse transcription PCR

Total RNA from cultured cell lines was isolated using the RNeasy Mini kit for fibrous tissue from Qiagen (Hilden, Germany) according to the manufacturer's instructions. RNA concentrations were photometrically determined. Reverse transcription (RT) was carried out with 1 μg total RNA per 20 μl reaction employing the Superscript II H− reverse transcriptase (Invitrogen, Karlsruhe, Germany) and an oligo-(dT16)primer. The real-time RT-PCR experiments were performed with an ABI 7000 SDS. In each experiment, we used the

amount of cDNA equivalent to 100 ng total RNA in a 20 μl reaction. Ngb expression levels were investigated by a TaqMan assay using a pair of oligonucleotide primers (5′GAAGCATCGGGCAGTG-3′ and 5′-AGGCACTTCTCCAGCATGTAGAG-3′) and a MGB (Minor Groove Binder) probe designed by the Primer Express Software (Applied Biosystems, Darmstadt, Germany). The MGB probe was 6-FAM-labeled at the 5′-end and non-fluorescent quencher (NFQ) was attached to the 3′-end (6-FAM-5′-CTCAGCTCCTTCTCGACAGT-3′-NFQ). The final primer concentration during PCR was 0.19 μM, the concentration of the TaqMan probe was 0.2 μM. After activation of the polymerase at 95 °C for 15 min, amplification was performed with a regular three-step protocol: 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s for 40 cycles, measuring the fluorescence during the last step of each cycle. Ngb expression was normalized according to the expression levels of acidic ribosomal phosphoprotein PO (ARP), which were monitored with SYBR Green. The primers were 5′-AGGGCGACCTGGAAGTCC-3′ and 5′-GCATCTGCTTGGAGCCCA-3′. SYBR Green expression analyses were followed by dissociation curves in a temperature range from 60 °C to 92 °C to analyze the specificity of the amplification reactions. Ngb expression levels (ct-values) were normalized according to that of ARP (n = 3 to 6 for each data point) and compared with that at normoxia (20% O2). Statistical evaluation was performed by calculating the mean value of the factors of regulation and their standard deviation. The significance of the data was assessed by ANOVA (P < 0.05) and by two-tailed Student's t test (P < 0.05).

Acknowledgments Studies were supported by US Public Health Service grant NS 05820 and a UM Neuroscience grant to R.S.-K., and by grants from the Deutsche Forschungsgemeinschaft (Bu956/5 and Ha2103/3) and the European Union (QLG3-CT-2002-01548) to T.H. and T.B. We thank Isabel Saul for generating the ischemia animals, Baotong Zhang, MD, PhD, for the in situ hybridization study, and Raul Busto (Prof. em.) for supervision. We gratefully acknowledge the kind gifts of HN33 cells by L. Moens (University of Antwerp, Belgium) and D.A. Greenberg (Buck Institute, Novato, USA), and of PC12 cell by S. Saaler-Reinhardt (University of Mainz, Germany). We would also like to thank S. Mitz for her help with the cell culture experiments and F. Gerlach for his skilled introduction into the technique of quantitative RT-PCR.

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