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Abstract

To elucidate the mechanism of ischemia-induced signal transduction in vivo, we investigated the effect of the targeted disruption of the α and Δ isoforms of the cAMP-responsive element-binding protein (CREB) on c-fos and heat-shock protein (hsp) 72 gene induction, Permanent focal ischemia was induced by occlusion of the middle cerebral artery of the CREB mutant mice (CREB(−/–), n = 5) and the wild-type mice (n = 6). Three hours after onset of ischemia, the neurologic score was assessed and pictorial measurements of ATP and cerebral protein synthesis (CPS) were carried out to differentiate between the ischemic core (where ATP is depleted), the ischemic penumbra (where ATP is preserved but CPS is inhibited), and the intact tissue (where both ATP and CPS are preserved). There were no significant differences in neurologic score or in ATP, pH, and CPS between the two groups, suggesting that the sensitivity of both strains to ischemia is the same. Targeted disruption of the CREB gene significantly attenuated c-fos gene induction in the periischemic ipsilateral hemisphere but had no effect on either c-fos or hsp72 mRNA expression in the penumbra. The observations demonstrate that CREB expression, despite its differential effect on c-fos, does not modulate acute focal ischemic injury.

Keywords

Focal ischemia CREB knockout mice Protein synthesis ATP Tissue pH

According to current concepts, cerebral ischemia induces a multitude of cellular degenerative reactions, including the release of excitatory amino acids, formation of oxygen free radicals, Ca²⁺ overload, activation of multiple cellular enzyme systems such as Ca²⁺-dependent proteases, and initiation of genomic responses that can affect the tissue outside the area of reduced blood flow (for review see Hossmann, 1994; Kristian and Siesjö, 1996; Paschen, 1996; Small and Buchan, 1996). Activation of proteases can cause destruction of cellular structural proteins (Matesic and Lin, 1994), and pathologic accumulation of cytosolic Ca²⁺ induced either by influx from the extracellular space or by release from intracellular stores activates, among others, the cAMP-responsive element-binding protein (CREB) by means of a Ca²⁺/calmodulin-dependent protein kinase (Kiessling and Gass, 1994).

cAMP-responsive element-binding protein is an important transcription factor in the control of many genes that are induced by an elevation in cytoplasmic cAMP or Ca²⁺ concentration. When CREB is phosphorylated on Ser-133 by either cAMP- or Ca²⁺-dependent protein kinases (Dash et al., 1991; Sheng et al., 1991), it promotes transcription from the cAMP-responsive element (CRE) (Chrivia et al., 1993; Hunter and Karin, 1992). cAMP-responsive elements lie upstream of a number of genes, including the immediate-early genes c-fos (Sassone Corsi et al., 1988) and zif/268 (Sakamoto et al., 1991), as well as genes encoding the K+ channel (Mori et al., 1993) and the a subunit of Ca²⁺/calmodulin-dependent kinase II (Olson et al., 1995). Although binding activity has not been demonstrated, CRE also lie upstream of heat-shock protein (hsp) 70 (Williams et al., 1989). It is well known that cerebral ischemia induces the immediate-early gene, c-fos (An et al., 1993; Kiessling and Gass, 1994; Kinouchi et al., 1994; Neumann et al., 1994), and hsp70 (Kinouchi et al., 1993; Massa et al., 1996; Nowak and Jacewicz, 1994). Both genes are thought to have protective effects against cerebral ischemia, c-fos because it induces biosynthesis of neurotrophic factors by increasing AP-1 binding activity (An et al., 1993; Colangelo et al., 1996) and hsp70 because it is one of the molecular chaperons that prevent the aggregation of denatured proteins and promote the refolding of damaged polypeptides (Becker and Craig, 1994; Hartl et al., 1994; McMillan et al. 1994). For these reasons, CREB should play an important role in the concert of genomic responses induced by cerebral ischemia, although only a few reports have been published so far (Salminen et al., 1995; Yoneda et al., 1994).

The recent development of knockout/transgenic technology in mice has made it easy to manipulate the expression of genes and their particular translational products (Aguzzi et al., 1996; Chan, 1996; Ryffel, 1996). It is now possible to analyze the effect of targeted disruption or overexpression of genes in vivo. Previously, a mouse mutant with a targeted disruption of the α and Δ isoforms of CREB has been reported (Blendy et al., 1996; Hummler et al., 1994). In the present study we investigated the effect of this disruption on ischemia-induced c-fos and hsp72 mRNA induction. For evaluating gene induction, we investigated 3 hours of permanent ischemia because both genes are strongly expressed at this time (Welsh et al., 1992). Moreover, to elucidate the pathophysiologic significance of c-fos and hsp72 mRNA induction by ischemia, we correlated the genomic responses with the severity of ischemic injury as assessed by neurologic scoring and pictorial evaluations of tissue pH, ATP, and cerebral protein synthesis (CPS).

MATERIALS AND METHODS

Homozygous CREB mutant mice (CREB(−/–) group, n = 5; 3 males and 2 females) and their wild-type littermates (CREB(+/+) group, n = 6; 4 males and 2 females), weighing 24 to 32 g, were used (Blendy et al., 1996). Mice were F1 hybrids from two strains that represented backcrosses of the mutation into the C57black/6J (F5) and the FVBN (F5) background (both strains were obtained from Charles River, Germany).

Animals were housed under diurnal lighting conditions and allowed access to food and water ad libitum until the day of the experiment. Anesthesia was induced by 1.5% halothane and maintained with 1% halothane in 70% N2O and 30% O2.

Animal preparation

Focal cerebral ischemia was induced by occlusion of the middle cerebral artery (MCA) using the intraluminal filament technique (Hara et al., 1996) as modified by Hata et al (Hata et al., 1998a). After a midline neck incision, the left common and external carotid arteries were isolated and ligated. A microvascular clip (FE691; Aesclap, Tuttlingen, Germany) was temporarily placed on the internal carotid artery. An 8-0 nylon monofilament (Ethilon; Ethicon, Norderstedt, Germany) coated with silicon resin (Xantopren; Bayer Dental, Osaka, Japan) was introduced through a small incision into the common carotid artery and advanced 9 mm distal to the carotid bifurcation for occlusion of MCA.

Two hours and 15 minutes after MCA occlusion, l-[4,5-³H]-leucine (150 µCi/animal, specific activity 151 Ci/mmol, Amersham, Braunschweig, Germany) was administered intraperitoneally for evaluating CPS rates. Forty-five minutes later, i.e., 3 hours after MCA occlusion, the neurologic impact was evaluated using a five-point performance score (0 = normal motor function; 1 = flexion of contralateral torso and forelimb on lifting the animal by the tail; 2 = circling to the contralateral side but normal posture at rest; 3 = leaning to contralateral side at rest; and 4 = no spontaneous motor activity) (Huang et al., 1994).

Experiments were terminated by in situ freezing (Mies et al., 1991). Brains were removed in a cold temperature cabinet (−20°C) and cut into 20-µm-thick coronal cryostat sections at -20°C. Sections were mounted on coverslips for ATP-bioluminescence, on object holders for CPS autoradiography, and on 3-aminopropyl triethoxysilane—treated slides for in situ hybridization (see below)

Regional measurement of ATP, tissue pH, and protein synthesis

Pictorial measurements of ATP and tissue pH were carried out using ATP-specific bioluminescence (Kogure and Alonso, 1978; Paschen et al., 1992) and umbelliferone fluorescence (Csiba et al., 1985), respectively. The pH images were calibrated with graded pH standards and ATP images were calibrated by taking small tissue samples from the ipsilateral ischemic and the contralateral nonischemic parietal cortex for quantitative enzymatic analysis.

For CPS autoradiography, cryostat sections were first exposed together with ³H standards to an X-ray film (Hyperfilm ³H, Amersham) for 14 days to assess total ³H radioactivity. Brain slices were then incubated in 10% trichloroacetic acid to remove labeled free leucine and metabolites other than proteins, and subsequently reexposed for the same duration to perform autoradiography of ³H-labeled proteins (Mies et al., 1991).

Probe for c-fos and hsp72 mRNAs

The sequence and specificities of oligonucleotide probes for c-fos and hsp72 have been described elsewhere (Kamii et al., 1994; Mikawa et al., 1995). The probe sequences of c-fos (45 mer) and hsp72 (30 mer) corresponded to amino acid residues 2 through 16 of rat c-fos and residues 122 through 129 of human hsp70, respectively. Each probe was 3' -end-labeled using terminal deoxynucleotidyl transferase (Gibco BRL, Eggenstein, Germany) and a 30:1 molar ratio of [³⁵S]-dATP (1,200 Ci/mmol). Specific activity was greater than 0.5 × 10⁹ dpm/µg.

In situ hybridization

In situ hybridization was performed as described previously (Wiessner et al., 1996). Briefly, coronal brain sections (20 µm) were fixed for 15 minutes in 4% paraformaldehyde/phosphate-buffered saline, pH 7.4. Sections were then treated with 0.25% acetic anhydride/triethanolamine for 10 minutes. After dehydration, 10 µL hybridization buffer containing ³⁵S-labeled oligonucleotide probe (10 pg/µL), 2× standard—sodium citrate, 50% formamide, 10% dextran sulfate, 100 µg/mL poly(A), 120 µg/mL heparin, 1 mg/mL herring sperm DNA, 5 mmol/L dithiothreitol, and 1 mg/mL bovine serum albumin were added to the sections, and they were covered with a coverslip. After overnight hybridization at 42°C, the sections were washed twice at 42°C in 2× saline—sodium citrate/50% formamide for 30 minutes. Hybridized radioactivity was visualized by film autoradiography (Hyperfilm β-max, Amersham) with an exposure time of 1 week.

Morphometric analysis of ischemia-induced metabolic disturbance and gene expression

Bioluminescence and autoradiographic images were digitized with a charge-coupled device camera system connected to an image analyzer (NIH Image Software 1.58, National Institutes of Health, Bethesda, MD, U.S.A.). The volumes of ATP depletion, tissue acidosis, and CPS inhibition were estimated using the semiautomated method described by Swanson et al. (1990) with modification. Briefly, ATP depletion was defined as the decline to less than 1.0 µmol/g, and tissue acidosis as the fall of pH to less than 6.4. The threshold for CPS inhibition was set to the lowest CPS value of the nonischemic hemisphere excluding fiber tracts. The areas of ATP depletion and CPS inhibition were measured on each section by subtracting the area of the nonlesioned ipsilateral hemisphere from that of the contralateral hemisphere. The volumes of ATP depletion, tissue acidosis, and CPS inhibition were calculated by integration of the lesion areas at five levels of forebrain. The areas of preserved ATP and protein synthesis were outlined and superimposed to demarcate penumbral tissue in which protein synthesis was suppressed but ATP was preserved. At the level of the caudate-putamen, the signal intensities for c-fos and hsp72 mRNA were measured on film autoradiograms in cortical regions of interest, located in the core, the metabolic penumbra, and normal brain tissue, as defined by ATP and CPS imaging.

Optical densities of in situ hybridization signals of c-fos and hsp72 mRNA were measured at the level of caudate-putamen in cortical regions of interest, located in the core, the metabolic penumbra, and in normal brain tissue, as defined by ATP and CPS imaging. Optical densities were calibrated using Kodak Step Tablet No. 810ST606 standards according to the instructions of the NIH Image program. The autoradiographic procedures were standardized by using the same film (Hyperfilm β-max), exposure time, and film development for all experiments. Furthermore, sections of wild-type and CREB knockouts were exposed on the same film, and measurements from penumbra, periinfarct, and normal cortex were taken from the same tissue section.

Temperature control

Rectal temperature was maintained between 36.5° and 37.0°C by a heating lamp and a heating pad connected to a thermistor (YSI, Yellow Spring, OH, U.S.A.).

Statistics

All values are given as means ± SD. Differences in neurologic scores and areas of cortical c-fos and hsp72 mRNA expression were evaluated using the Mann-Whitney U-test. Differences in metabolic parameters and optical densities of mRNA were compared using two-way analysis of variance followed by Bonferroni's multiple comparison test. A P value of less than 0.05 was considered to indicate statistical significance.

RESULTS

Neurologic score

Focal ischemia resulted in marked neurologic disturbances 3 hours after MCA occlusion. All animals had developed at least grade 1 deficits, but there was no difference between the two groups (CREB(+/+) group, 2.33 ± 0.47 [mean ± SD]; CREB(−/–) group, 2.20 ± 0.98).

Regional brain tissue ATP, pH, and CPS

Representative images of the tissue content of ATP, tissue pH, and CPS in the wild-type mouse are shown in Fig. 1. ATP was depleted in the frontoparietal cortex, the lateral part of caudate-putamen, and the piriform cortex. Acidosis and suppression of CPS were present in the same areas but the changes extended distinctly more into the frontal, medial, and baso-occipital parts of the MCA territory. Quantitative measurements revealed significant reductions of ATP in the piriform cortex and the somatosensory area of the frontoparietal cortex at the level of striatum, in the temporal and piriform cortex at the level of the dorsal hippocampus, and in the lateral caudate-putamen. Changes of CPS and tissue pH extended further into the globus pallidus at the level of the striatum, into the parietal cortex at the level of the dorsal hippocampus, and into the temporal and entorhinal cortex at the level of the substantia nigra (Table 1). These changes were the same as in the CREB mutant mouse (Fig. 2 and Table 2).

FIG. 1.

Representative ATP images, pH images, and ³H-leucine autoradiograms of brain sections of the wild-type mouse subjected to focal ischemia by middle cerebral artery occlusion for 3 hours. Numbers under each column indicate the distance in millimeters caudal to the frontal pole.

FIG. 2.

Representative ATP images, pH images, and ³H-leucine autoradiograms of brain sections of the CREB mutant mouse subjected to focal ischemia by middle cerebral artery occlusion for 3 hours. Numbers under each column indicate the distance in millimeters caudal to the frontal pole.

TABLE 1.

Changes of ATP and cerebral protein synthesis of the wild-type and CREB mutant mouse brain after 3 hours of middle cerebral artery occlusion

	ATP% (wild)	ATP% (mutant)	CPS% (wild)	CPS% (mutant)
Level of striatum
Anterior cingulate cortex	98.5 ± 3.3	97.6 ± 2.5	97.7 ± 4.7	95.3 ± 8.0
Motor cortex	67.6 ± 17.3	75.2 ± 13.1	55.2 ± 10.2	56.0 ± 11.3
Somatosensory cortex	4.3 ± 4.0*	7.3 ± 8.9*	1.4 ± 1.5*	1.8 ± 2.4*
Piriform cortex	4.8 ± 5.1*	10.0 ± 5.9*	1.3 ± 1.0*	2.2 ± 2.6*
Lateral caudate-putamen	21.9 ± 9.0*	24.2 ± 14.1*	2.3 ± 2.1*	4.8 ± 4.9*
Medial caudate-putamen	41.5 ± 24.8†	40.6 ± 21.2†	5.5 ± 7.9*	9.4 ± 10.6*
Globus pallidus	44.2 ± 22.3	51.8 ± 33.3	23.0 ± 12.1*	28.3 ± 18.4*
Septal nucleus	96.9 ± 1.9	98.2 ± 3.9	98.2 ± 4.4	96.9 ± 5.8
Level of dorsal hippocampus
Posterior cingulate cortex	99.1 ± 2.2	99.8 ± 2.7	100.9 ± 3.4	93.9 ± 9.9
Parietal cortex	70.8 ± 18.1	88.1 ± 15.4	44.7 ± 14.8†	43.8 ± 14.9†
Temporal cortex	14.4 ± 27.5*	19.1 ± 33.8*	0.8 ± 0.5*	4.0 ± 4.2*
Piriform cortex	17.8± 39.5*	20.4± 35.8*	12.0± 23.1 *	7.5 ± 9.2*
Hippocampus	80.7 ± 26.7	96.0 ± 5.6	51.2 ± 35.2	71.7 ± 30.3
Thalamus	75.5 ± 23.3	90.1 ± 14.0	53.2 ± 31.4	65.6 ± 29.4
Hypothalamus	62.1 ± 33.3	98.2 ± 26.2	53.7 ± 44.4	75.4 ± 22.6
Level of substantia nigra
Posterior cingulate cortex	99.3 ± 2.0	101.1 ± 3.0	80.4 ± 39.3	99.6 ± 2.0
Occipital cortex	86.1 ± 26.2	98.4 ± 5.6	54.0 ± 24.7	58.7 ± 26.3
Temporal cortex	74.9 ± 36.4	88.4 ± 30.8	4.9 ± 4.4*	20.9 ± 25.2*
Entorhinal cortex	59.8 ± 39.3	78.2 ± 36.9	17.0 ± 18.5*	13.5 ± 21.6*
Substantial nigra	75.2 ± 25.0	95.5 ± 8.8	76.6 ± 38.0	82.0 ± 16.8
Medial geniculate	83.7 ± 33.6	102.6 ± 5.0	73.2 ± 36.5	90.9 ± 14.3

ATP% (wild) and CPS% (wild) are values of the ipsilateral hemisphere of the wild-type mice (n = 6) expressed as percentage of the contralateral side. ATP% (mutant) and CPS% (mutant) are values of the ipsilateral hemisphere of the mutant mice (n = 5) expressed as percentage of the contralateral side. All values are mean ± SD. Statistical analysis was performed by two-way ANOVA followed by Bonferroni's multiple comparison test. CPS, cerebral protein synthesis

*;†

Significantly lower values than in the anterior cingulate cortex (P < 0.01; P < 0.05).

TABLE 2.

Changes of tissue pH of the wild-type and CREB mutant mouse brain after 3 hours of middle cerebral artery occlusion

	pH (wild; ipsi)	pH (wild; cont)	pH (mutant; ipsi)	pH (mutant; cont)
Level of striatum
Anterior cingulate cortex	6.82 ± 0.44	7.07 ± 0.09	6.92 ± 0.58	7.07 ± 0.13
Motor cortex	6.18 ± 0.63	7.17 ± 0.11	6.22 ± 0.69	7.12 ± 0.25
Somatosensory cortex	< 5.80*,†	7.20 ± 0.05	< 5.80*,†	7.14 ± 0.22
Piriform cortex	< 5.80*,†	7.09 ± 0.13	< 5.80*,†	7.08 ± 0.20
Lateral caudate-putamen	< 5.80*,†	7.14 ± 0.11	< 5.80*,†	7.11 ± 0.15
Medial caudate-putamen	< 5.80*,†	7.09 ± 0.13	< 5.80*,†	7.09 ± 0.20
Globus pallidus	< 5.80*,†	7.08 ± 0.14	< 5.80*,†	7.09 ± 0.09
Septal nucleus	7.06 ± 0.13	7.08 ± 0.09	7.07 ± 0.20	7.09 ± 0.16
Level of dorsal hippocampus
Posterior cingulate cortex	7.00 ± 0.13	7.08 ± 0.07	6.96 ± 0.23	7.06 ± 0.08
Parietal cortex	6.33 ± 0.62	7.12 ± 0.06	6.82 ± 0.38	7.04 ± 0.05
Temporal cortex	< 5.80*,†	7.13 ± 0.08	< 5.8*,†	7.08 ±0.04
Piriform cortex	< 5.80*,†	7.05 ± 0.17	< 5.8*,†	7.01 ±0.09
Hippocampus	6.54 ± 0.95	7.18 ± 0.07	6.92 ± 0.66	7.06 ± 0.13
Thalamus	6.04 ± 0.72	7.05 ± 0.14	6.59 ± 0.25	6.93 ± 0.05
Hypothalamus	6.13 ± 0.63	6.91 ± 0.16	6.36 ± 0.66	6.80 ± 0.13
Level of substantia nigra
Posterior cingulate cortex	6.95 ± 0.28	7.09 ± 0.13	7.15 ± 0.18	7.18 ± 0.11
Occipital cortex	6.38 ± 0.83	7.08 ± 0.17	6.74 ± 0.53	7.21 ± 0.11
Temporal cortex	< 5.80*,†	7.11 ± 0.13	< 5.8*,†	7.17 ± 0.14
Entorhinal cortex	< 5.80*,†	7.00 ± 0.09	<5.8*,†	7.02 ± 0.19
Substantial nigra	6.17 ± 0.92	6.90 ± 0.15	6.57 ± 0.51	6.92 ± 0.25
Medial geniculate	6.44 ± 0.74	6.95 ±0.11	6.91 ± 0.50	7.06 ± 0.18

pH (wild; ipsi) and pH (wild; cont) represent tissue pH values in the ischemic and the nonischemic hemisphere of the wild type mouse brain (n = 6), respectively. pH (mutant; ipsi) and pH (mutant; cont) represent tissue pH values in the ischemic and the nonischemic hemisphere of the mutant type mouse brain (n = 5), respectively. All values are mean ± SD. Statistical analysis was performed by two-way ANOVA followed by Bonferroni's multiple comparison test.

Significantly lower value than in the anterior cingulate cortex (P < 0.01).

†

Significantly lower value than in the corresponding area of the contralateral side {P < 0.01).

The volume of metabolic disturbances, measured 3 hours after MCA occlusion, differed markedly depending on the chosen parameter in both CREB(−/–) and CREB(+/+) groups. In the CREB(+/+) group, ATP was depleted in 34.5% ± 11.7% of the contralateral hemisphere, tissue acidosis was detected in 43.4% ± 12.6%, and CPS inhibition in 52.7% ± 14.0%. In the CREB(−/–) mice the corresponding values were 27.3% ± 6.0% for ATP depletion, 36.3% ± 7.0% for tissue acidosis, and 48.2% ± 7.0% for inhibition of CPS. There were no significant differences in these three parameters between the two groups (Figs. 3, 4).

FIG. 3.

Areas of disturbed cerebral protein synthesis (CPS), acidosis, and ATP depletion in coronal brain sections of mice subjected to focal ischemia for 3 hours. The lesion areas (means ± SD) are plotted against the distance from the frontal pole and expressed as percentage of ipsilateral hemisphere. The area of penumbra was calculated by subtracting the percentage of ATP depletion from that of CPS inhibition. No significant differences were observed between the CREB mutant (CREB(−/–), n = 5) and the wild-type mice (CREB(+/+), n = 6).

FIG. 4.

Lesion volumes of metabolic disturbances (inhibition of cerebral protein synthesis (CPS), acidosis, and ATP depletion) in mice subjected to focal ischemia for 3 hours. The volume of penumbra was calculated by subtracting the volume of ATP depletion from the volume of CPS inhibition. Values are means ± SD of 5 CREB mutant and 6 wild-type animals. Note absence of significant differences between the two groups.

c-fos and hsp72 mRNA expression

Three hours after MCA occlusion, the topical distribution of c-fos and hsp72 mRNA expression differed greatly. Expression of c-fos mRNA was prominent not only in the metabolic penumbra but also in more distant parts of the ipsilateral hemisphere of both CREB(−/–) and CREB(+/+) groups. Superimposition of the outlines of ATP- and CPS-preserved regions on the hybridization autoradiograms revealed that c-fos mRNA expression was present both in the penumbra and the normal nonischemic cortex, particularly in the nonischemic cingulate cortex of the ipsilateral hemisphere (Figs. 5, 6). There was no significant difference in the areas of the cortical c-fos mRNA expression (CREB(+/+), 4.11 ± 0.27 mm²; CREB(−/–), 4.24 ± 0.17 mm²) between the two groups. In the metabolic penumbra, there was also no difference in the mean optical densities of the c-fos mRNA hybridization signal between the two groups. However, in the nonischemic periinfarct cortex, the mean optical densities of the c-fos mRNA of the CREB(−/–) group was significantly (P < 0.05) reduced by 33% as compared with the CREB(+/+) group (Fig. 7).

FIG. 5.

Representative images of cerebral protein synthesis (CPS), ATP, hsp72 mRNA, and c-fos mRNA of the wild-type mouse at the level of the caudate-putamen. The outlines of intact CPS (black) and preserved ATP (gray) were superimposed on the hsp72 and c-fos mRNA images for differentiation between core and penumbra (see text for definitions). Note selective expressions of hsp72 in the penumbra as compared with the more widespread expression of c-fos.

FIG. 6.

Representative images of cerebral protein synthesis (CPS), ATP, hsp72 mRNA, and c-fos mRNA of the CREB mutant mouse at the level of the caudate-putamen. The outlines of intact CPS (black) and normal ATP (gray) were superimposed on the hsp72 and c-fos mRNA images for differentiation between core and penumbra (see text for definitions). Note similar genomic expression pattern as in the wild-type mice, except for the less intense c-fos expression in the periischemic cortex (arrowheads).

FIG. 7.

Semiquantitative measurement of hsp72 mRNA (A) and c-fos mRNA (B) in mice brain after 3 hours of middle cerebral artery occlusion. Mean optical densities (mOD) of in situ hybridization autoradiograms were measured in the ischemic core (temporoparietal cortex with suppression of both protein synthesis and ATP), penumbral cortex (parietal cortex with suppressed protein synthesis and preserved ATP), and intact cortex (ipsilateral cingulate cortex with preserved protein synthesis and ATP). Values are means ± SD of 5 CREB mutant and 6 wild-type animals. **, & indicate significant difference (P < 0.01) between core and penumbra; $ indicates significant difference (P < 0.05) between the CREB mutant and the wild-type mouse. Note differential expression of hsp72 and c-fos in penumbra and periinfarct cortex, and significant reduction of c-fos expression in the periischemic cortex of the CREB mutant mouse.

In contrast to c-fos mRNA, hsp72 mRNA was only expressed in the periinfarct rim of both CREB(−/–) and CREB(+/+) groups. Superimposition of the outlines of ATP- and CPS-preserved regions revealed that hsp72 mRNA expression was clearly restricted to the metabolic penumbra (Figs. 5, 6). Furthermore, there was no significant difference in the areas of the cortical hsp72 mRNA expression (CREB(+/+), 2.06 ±0.18 mm²; CREB(−/–), 2.14 ± 0.72 mm²) or the mean optical densities of the penumbral hsp72 mRNA hybridization signal between the two groups (Fig. 7).

DISCUSSION

In the present investigation permanent MCA occlusion in the CREB mutant and the wild-type mice led to similar tissue acidosis and disturbance of cerebral energy metabolism and protein synthesis, indicating that the sensitivity of both strains to ischemia is the same. However, in the periinfarct cortex of the CREB mutant mice, expression of the immediate-early gene c-fos was markedly attenuated as compared with the wild-type mice. This leads us to conclude that despite the different c-fos mRNA induction in the periinfarct area between the two strains, the expression of CREB does not modulate the susceptibility of brain tissue to acute ischemia.

Mechanism of c-fos induction

The major DNA elements responsible for c-fos induction are the sis-inducible element, the serum-response element (SRE), and the CRE (reviewed in Edwards, 1994; Karin et al., 1997). The sis-inducible element is recognized by the transcription factor, STAT (signal transducer and activator of transcription) (Darnell et al., 1994). These transcription factors are activated by Janus kinases through phosphorylation of tyrosine residues. They are translocated to the nucleus, bind to the sis-inducible element, and stimulate transcription of c-fos. These Janus kinase—STAT pathways can be activated by certain growth factors, cytokines, and interferons (Darnell, 1996). The serum-response element is recognized by a dimer of the serum-response factor that recruits the monomeric ternary complex factors (Treisman, 1992). A member of the E26-transformation specific family of nuclear proto-oncoproteins, Elk-1 (Hipskind et al., 1991), is phosphorylated by ERK (extracellular signal-regulated kinases), and the Elk-1–serum-response factor-SRE ternary complex is necessary for full SRE function (Edwards, 1994). As ERK are rapidly activated by most mitogens, this pathway may account for mitogen-induced c-fos transcription (Karin et al., 1997).

The CRE is recognized by CREB, which is phosphorylated on Ser-133 by protein kinase A or Ca²⁺/ calmodulin-dependent protein kinases II and IV in response to intracellular increases of cAMP or Ca²⁺. The CREB is also phosphorylated by the Ras—Raf—ERK—pp90 pathway. The phosphorylated form of CREB promotes transcription from CRE, which in turn contributes to c-fos induction (Finkbeiner et al., 1997; Ginty, 1997; Hagiwara et al., 1996).

It is reasonable to assume that induction of c-fos after MCA occlusion is mediated at least in part by CREB because ischemia and the associated periinfarct depolarization induces a sharp rise of cytosolic calcium. This interpretation is supported by the present finding that in the CREB mutant mice, c-fos induction was markedly attenuated. However, c-fos was not completely suppressed, indicating that other factors must also be involved. These could be either other transcriptional factors of the CREB family, such as CREM and ATF-1, or other pathways such as the SRE-dependent c-fos induction. In fact, both CRE and SRE are able to confer Ca²⁺-dependent induction of c-fos in vitro (Bading et al., 1993; Misra et al., 1994). Another explanation could be that the CREB splice variant, CREB-β, is upregulated in the CREB mutant mice, although this isoform is only present at low level in wild-type mice (Blendy et al., 1996). However, activator (τ) and inhibitor (α and β) forms of CREM are also upregulated, and these upregulations are thought to result in an 80% to 90% decrease in CREB activity in these mutant mice (Blendy et al., 1996; Maldonado et al., 1996). It is, therefore, likely that the observed attenuation of c-fos induction is caused by a decrease in CREB activity.

The fact that knockout of CREB alone decreases c-fos mRNA is in line with earlier conclusions that in vivo stimulation of any given element on the c-fos promoter is not sufficient to produce the entire c-fos induction (Robertson et al., 1995; Bontempi and Sharp, 1997).

Mechanism of hsp70 induction

Although the human hsp70 gene promoter possesses CRE/ATF and SRE (Williams et al., 1989), the major regulatory factor for stress-induced activation of hsp70 transcription is the heat-shock response element and the cytoplasmic heat-shock factor-1 (Morimoto et al., 1996). Various kinds of stress such as heat shock result in the formation of heat-shock factor-1 trimer, which after phosphorylation migrates to the nucleus and binds to heat-shock response element, causing the transcription of hsp70 mRNA (Baler et al., 1993). Some studies suggest that calcium or cAMP are required for activation of hsp70 transcription and that the cAMP sensitivity is conferred by both CRE/ATF and heat-shock response element (Choi et al., 1991; Price and Calderwood, 1991). On the other hand, agents that increase cAMP failed to stimulate the expression of hsp70 gene in intact cells (Murakami et al., 1991; Pizurki and Polla, 1994). In our study the mRNA of hsp72, a member of the 70-kDa heat-shock protein family, was readily induced after MCA occlusion in both the CREB mutant and the wild-type mice, and their expression patterns were similar. This suggests that ischemia-induced expression of hsp72 mRNA in mice is not under the control of CREB.

Imaging ischemic penumbra

Focal cerebral ischemia induces a complex pattern of genomic responses that may interfere in different ways with cell survival, depending on the severity and duration of ischemic injury. The interpretation of such data depends crucially on the exact differentiation between core, penumbra (Astrup et al., 1981), and normal brain tissue because the same signal transduction pathway may result in different responses, depending on the metabolic status of the tissue. Such a differentiation can be obtained by the use of multiparametric imaging techniques (Mies, 1993). According to the threshold concept of ischemic brain injury, inhibition of CPS and tissue acidosis occur at higher flow values than the breakdown of the energy metabolism state (Hossmann, 1994). Imaging of these variables on adjacent cryostat sections allows the precise demarcation between the ischemic core in which CPS is suppressed but ATP is preserved, and the intact tissue in which brain metabolism is normal. This has been previously documented in the C57black/6J mice after MCA occlusion for the same duration as in the present study (Hata et al., 1998b). The comparison of these data with the present study revealed that there are no differences in the volumes of tissue affected by metabolic disturbances among the C57black/6J mice, the CREB mutant mice, and their wild-type littermates. This suggests that the genetic background of the three strains does not modulate the sensitivity of brain tissue to acute ischemia despite the recently reported differential susceptibility to excitotoxicity (Choi, 1997; Schauwecker and Steward, 1997).

Penumbra and c-fos induction

In accordance with our previous study in the C57black/6J mice, c-fos mRNA was expressed both in the penumbra and in the more distant periinfarct cortex of the ipsilateral hemisphere. It has been documented that mRNA synthesis of the c-fos gene does not require protein synthesis (Herrera and Robertson, 1996). This explains why c-fos mRNA was expressed in the penumbra although global protein synthesis was severely suppressed. The additional expression in the more distant parts of the periinfarct surrounding has been attributed to the transient increase in cytosolic calcium evoked by the passage of periinfarct spreading depressions (for review see Gass and Herdegen, 1995; Herrera and Robertson, 1996; Hossmann, 1996).

Interestingly, penumbral c-fos mRNA expression was similar in the wild-type and the CREB mutant mice, but in the more distant areas, in particular the ipsilateral cingulate cortex, c-fos mRNA induction was significantly attenuated in the mutant mice. This suggests that in focal ischemia c-fos mRNA is induced by at least two different mechanisms, depending on the region involved.

This interpretation is supported by pharmacologic studies. Treatment with the noncompetitive N-methyl-d-aspartate (NMDA) antagonist dizolcipine, which has been shown to suppress periinfarct spreading depression (Iijima et al., 1992) and in consequence calcium flooding through NMDA receptor-operated ion channels, abolished c-fos expression in the distant but not in the adjacent periinfarct surrounding (Gass et al., 1992).

The attenuated induction of c-fos mRNA in the distant area of the CREB mutant mice suggests that in this area CREB may contribute to c-fos induction in vivo, although the SRE (not the CRE) is an important regulatory element in the c-fos induction through calcium influx via NMDA receptor-operated ion channels in vitro (Bading et al., 1993). In the periinfarct penumbra, in contrast, c-fos is expressed in the absence of CREB, which suggests that in this part of the evolving infarct SRE may play the dominant role for c-fos induction after calcium influx through NMDA receptor-operated channels, voltage-sensitive calcium channels, or both (Santella and Carafoli, 1997).

An important deduction of this observation is the independence of infarct progression from CREB expression. In the CREB mutant and the wild-type mice, the derangements of energy metabolism and protein synthesis as well as tissue acidosis were quite similar. Our study, therefore, suggests that the expression of CREB is not an important factor in the concerted molecular interactions involved in the evolution of brain infarcts during acute ischemia.

Penumbra and hsp70 induction

In both the CREB mutant and the wild-type mice, hsp72 mRNA expression strictly matched a narrow rim in the periphery of the ischemic territory in which postischemic protein synthesis was inhibited but ATP was preserved. hsp72, an inducible member of the 70-kDa heat-shock protein family, acts as a molecular chaperon that prevents the aggregation of denatured proteins and promotes the refolding of damaged polypeptides (Becker and Craig, 1994; Hartl et al., 1994). Although the precise mechanism of hsp72 gene induction after ischemia remains unknown, an obvious activator of hsp72 transcription after ischemia is the presence of denatured proteins. There are also indications from in vivo studies that inhibition of protein synthesis promotes transcription (Nowak and Jacewicz, 1994; Planas et al., 1997). Induction of hsp72 gene does not respond to periinfarct spreading depression, and a treatment of infarcts with NMDA antagonists does not suppress its induction in the penumbra (Yamashita et al., 1996).

It has been repeatedly reported that heat-stress proteins are neuroprotective. In fact, hsp72 mRNA and its protein product have been demonstrated in neurons that survive transient global (Kanemitsu et al., 1994) or permanent focal ischemia (Kinouchi et al., 1993). However, the protective effect of the stress protein is abolished whenever other metabolic disturbances supervene. As an example, the metabolic workload induced by periinfarct spreading depression results in irreversible energy failure in the periinfarct border zone despite expression of stress proteins (Back et al., 1994). Imaging of stress proteins may, therefore, be useful to evaluate progression or regression of the ischemic injury. If, for instance, hsp72 mRNA expression is detected in a region in which ATP is depleted, this constellation would indicate that ischemic injury has progressed into the penumbra region. Conversely, the expression of hsp72 mRNA in a region with preserved CPS would indicate reversal of injury. In the present study, hsp72 expression was clearly restricted to the penumbra in both the CREB mutant and wild-type mice subjected to focal cerebral ischemia for 3 hours. This is in line with the observation that the severity of ischemia was similar in both groups and suggests that CREB does not modulate the progression of injury.

CONCLUSION

The comparison of c-fos and hsp72 mRNA expression after 3 hours of MCA occlusion in the CREB mutant and the wild-type mice revealed a significant reduction in c-fos mRNA in the distant periinfarct surrounding of the CREB mutant strain. However, hsp72 mRNA expression, and the associated disturbances of energy metabolism, tissue pH, and protein synthesis did not differ between the two strains. We therefore conclude that the expression of CREB and the periinfarct induction of c-fos mRNA are not major pathogenic factors for the evolution of brain infarcts during acute ischemia.

Footnotes

Acknowledgments

The authors thank Mrs. U. Beckmann, Mrs. M. Nelles, and S. Krause for excellent technical assistance, and Mrs. D. Schewetzky for the careful preparation of the manuscript.

Abbreviations used

References

Aguzzi

Brandner

Marino

Steinbach

(1996) Transgenic and knockout mice in the study of neurodegenerative diseases. J Mol Med 74: 111–126

Lin

Liu

Xue

Hsu

(1993) Expression of c-fos and c-jun family genes after focal cerebral ischemia. Ann Neurol 33:457–464

Astrup

Siesjö

Symon

(1981) Thresholds in cerebral ischemia—the ischemic penumbra. Stroke 12:723–725

Back

Kohno

Hossmann

(1994) Cortical negative DC deflections following middle cerebral artery occlusion and KCl-induced spreading depression: effect on blood flow, tissue oxygenation, and electroencephalogram. J Cereb Blood Flow Metab 14:12–19

Bading

Ginty

Greenberg

(1993) Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways. Science 260:181–186

Baler

Dahl

Voellmy

(1993) Activation of human heat shock genes is accompanied by oligomerization, modification, and rapid translocation of heat shock transcription factor HSF1. Mol Cell Biol 13:2486–2496

Becker

Craig

(1994) Heat-shock proteins as molecular chaperons. Eur J Biochem 219: 11–23

Blendy

Kaestner

Schmid

Gass

Schutz

(1996) Targeting of the CREB gene leads to up-regulation of a novel CREB mRNA isoform. EMBO J 15:1098–1106

Bontempi

Sharp

(1997) Systemic morphine-induced Fos protein in the rat striatum and nucleus accumbens is regulated by mu opioid receptors in the substantia nigra and ventral tegmental area. J Neurosci 17:8596–8612

10.

Chan

(1996) Role of oxidants in ischemic brain damage. Stroke 27:1124–1129

11.

Choi

(1997) Background genes—out of sight, but not out of brain. Trends Neurosci 20:499–500

12.

Choi

Lin

Huang

Liu

(1991) cAMP and cAMP-dependent protein kinase regulate the human heat shock protein 70 gene promoter activity. J Biol Chem 266: 11858–11865

13.

Chrivia

Kwok

RPS

Lamb

Hagiwara

Montminy

Goodman

(1993) Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365:855–859

14.

Colangelo

Pani

Mocchetti

(1996) Correlation between increased AP-1NGF binding activity and induction of nerve growth factor transcription by multiple signal transduction pathways in C6-2B glioma cells. Mol Brain Res 35:1–10

15.

Csiba

Paschen

Mies

(1985) Regional changes in tissue pH and glucose content during cortical spreading depression in rat brain. Brain Res 336:167–170

16.

Darnell

Jr (1996) The JAK-STAT pathway: summary of initial studies and recent advances. Recent Prog Horm Res 51:391–403

17.

Darnell

Jr Kerr

Stark

(1994) Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415–1421

18.

Dash

Karl

Colicos

Prywes

Kandel

(1991) Cyclic AMP response element-binding protein is activated by calcium calmodulin-dependent as well as cyclic AMP-dependent protein kinase. Proc Natl Acad Sci USA 88:5061–5065

19.

Edwards

(1994) Cell signalling and the control of gene transcription. Trends Pharmacol Sci 15:239–244

20.

Finkbeiner

Tavazoie

Maloratsky

Jacobs

Harris Greenberg

(1997) CREB: a major mediator of neuronal neurotrophin responses. Neuron 19:1031–1047

21.

Gass

Herdegen

(1995) Neuronal expression of AP-1 proteins in excitotoxic-neurodegenerative disorders and following nerve fiber lesions. Prog Neurobiol 47:257–290

22.

Gass

Spranger

Herdegen

Bravo

Kock

Hacke

Kiessling

(1992) Induction of FOS and JUN proteins after focal ischemia in the rat: differential effect of the N-methyl-d-aspartate receptor antagonist MK-801. Acta Neuropathol 84:545–553

23.

Ginty

(1997) Calcium regulation of gene expression: isn't that spatial? Neuron 18: 183–186

24.

Hagiwara

Shimomura

Yoshida

Imaki

(1996) Gene expression and CREB phosphorylation induced by cAMP and Ca²⁺ in neuronal cells. Adv Pharmacol 36:277–285

25.

Hara

Huang

Panahian

Fishman

Moskowitz

(1996) Reduced brain edema and infarction volume in mice lacking the neuronal isoform of nitric oxide synthase after transient MCA occlusion. J Cereb Blood Flow Metab 16:605–611

26.

Hartl

Hlodan

Langer

(1994) Molecular chaperones in protein folding: the art of avoiding sticky situations. Trends Biochem Sci 19:20–25

27.

Hata

Mies

Wiessner

Fritze

Hasselbarth

Brinker

Hossmann

K-A

(1998a) A reproducible model of middle cerebral artery occlusion in mice: hemodynamical, biochemical and magnetic resonance imaging. J Cereb Blood Flow Metab 18:367–375

28.

Hata

Mies

Wiessner

Hossmann

K-A

(1998b) Differential expression of c-fos and hsp72 mRNA in focal cerebral ischemia of mice. Neuroreport 9:27–32

29.

Herrera

Robertson

(1996) Activation of c-fos in the brain. Prog Neurobiol 50:83–107

30.

Hipskind

Rao

Mueller

CGF

Reddy

ESP

Nordheim

(1991) Ets-related protein Elk-1 is homologous to the c-Fos regulatory factor P62t-C-F. Nature 354:531–534

31.

Hossmann

(1994) Viability thresholds and the penumbra of focal ischemia. Ann Neurol 36:557–565

32.

Hossmann

(1996) Periinfarct depolarizations. Cerebrovasc Brain Metab Rev 8: 195–208

33.

Huang

Panahian

Dalkara

Fishman

Moskowitz

(1994) Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science 265: 1883–1885

34.

Hummler

Cole

Blendy

Ganss

Aguzzi

Schmid

Beermann

Schuetz

(1994) Targeted mutation of the CREB gene: compensation within the CREB-ATF family of transcription factors. Proc Natl Acad Sci USA 91:5647–5651

35.

Hunter

Karin

(1992) The regulation of transcription by phosphorylation. Cell 70:375–387

36.

Iijima

Mies

Hossmann

(1992) Repeated negative DC deflections in rat cortex following middle cerebral artery occlusion are abolished by MK-801: effect on volume of ischemic injury. J Cereb Blood Flow Metab 12:727–733

37.

Kamii

Kinouchi

Sharp

Epstein

Sagar

Chan

(1994) Expression of c-fos mRNA after a mild focal cerebral ischemia in SOD-1 transgenic mice. Brain Res 662:240–244

38.

Kanemitsu

Kirino

Nakagomi

Tsujita

Iwamoto

Tomich

Tamura

(1994) Key of induced tolerance to ischaemia in gerbil hippocampal CA 1 is not at transcriptional level of hsp70 gene: in situ hybridization of hsp70 mRNA. Neurol Res 16:209–212

39.

Karin

Liu

Zandi

(1997) AP-1 function and regulation. Curr Opin Cell Biol 9:240–246

40.

Kiessling

Gass

(1994) Stimulus-transcription coupling in focal cerebral ischemia. Brain Pathol 4:77–83

41.

Kinouchi

Sharp

Koistinaho

Hicks

Kamii

Chan

(1993) Induction of heat shock hsp70 mRNA and HSP70 kDa protein in neurons in the ‘penumbra’ following focal cerebral ischemia in the rat. Brain Res 619:334–338

42.

Kinouchi

Sharp

Chan

Koistinaho

Sagar

Yoshimoto

(1994) Induction of c-fos, junB, c-jun, and hsp70 mRNA in cortex, thalamus, basal ganglia, and hippocampus following middle cerebral artery occlusion. J Cereb Blood Flow Metab 14: 808–817

43.

Kogure

Alonso

(1978) A pictorial representation of endogenous brain ATP by a bioluminescent method. Brain Res 154:273–284

44.

Kristian

Siesjö

(1996) Calcium-related damage in ischemia. Life Sci 59:357–367

45.

Maldonado

Blendy

Tzavara

Gass

Roques

Hanoune

Schütz

(1996) Reduction of morphine abstinence in mice with a mutation in the gene encoding CREB. Science 273:657–659

46.

Massa

Swanson

Sharp

(1996) The stress gene response in brain. Cerebrovasc Brain Metab Rev 8:95–158

47.

Matesic

Lin

(1994) Microtubule-associated protein 2 as an early indicator of ischemia-induced neurodegeneration in the gerbil forebrain. J Neurochem 63:1012–1020

48.

McMillan

Gething

Sambrook

(1994) The cellular response to unfolded proteins: intercompartmental signaling. Curr Opin Biotechnol 5:540–545

49.

Mies

(1993) Autoradiographic and biochemical imaging in cerebral ischemia. Adv Exp Med Biol 333:273–285

50.

Mies

Ishimaru

Xie

Seo

Hossmann

(1991) Ischemic thresholds of cerebral protein synthesis and energy state following middle cerebral artery occlusion in rat. J Cereb Blood Flow Metab 11:753–761

51.

Mikawa

Sharp

Kamii

Kinouchi

Epstein

Chan

(1995) Expression of c-fos and hsp70 mRNA after traumatic brain injury in transgenic mice overexpressing CuZn-superoxide dismutase. Mol Brain Res 33:288–294

52.

Misra

Bonni

Miranti

Rivera

Sheng

Greenberg

(1994) L-type voltage-sensitive calcium channel activation stimulates gene expression by a serum response factor-dependent pathway. J Biol Chem 269:25483–25493

53.

Mori

Matsubara

Folco

Siegel

Koren

(1993) The transcription of a mammalian voltage-gated potassium channel is regulated by cAMP in a cell-specific manner. J Biol Chem 268:26482–26493

54.

Morimoto

Kroeger

Cotto

(1996) The transcriptional regulation of heat shock genes: a plethora of heat shock factors and regulatory conditions. EXS 77:139–163

55.

Murakami

Ohmori

Gotoh

Tsuda

Ohya

Akiya

Higashi

(1991) Down modulation of N-myc, heat-shock protein 70, and nucleolin during the differentiation of human neuroblastoma cells. J Biochem 110:146–150

56.

Neumann

Wiessner

Vogel

Back

Hossmann

(1994) Differential expression of the immediate early genes c-fos, c-jun, junB, and NGFI-B in the rat brain following transient forebrain ischemia. J Cereb Blood Flow Metab 14:206–216

57.

Nowak

Jacewicz

(1994) The heat shock/stress response in focal cerebral ischemia. Brain Pathol 4:67–76

58.

Olson

Masse

Suzuki

Chen

Alam

Kelly

(1995) Functional identification of the promoter for the gene encoding the alpha subunit of calcium-calmodulin-dependent protein kinase II. Proc Natl Acad Sci USA 92:1659–1663

59.

Paschen

(1996) Disturbances of calcium homeostasis within the endoplasmic reticulum may contribute to the development of ischemic-cell damage. Med Hypotheses 47:283–288

60.

Paschen

Mies

Hossmann

(1992) Threshold relationship between cerebral blood flow, glucose utilization, and energy metabolites during development of stroke in gerbils. Exp Neurol 117: 325–333

61.

Pizurki

Polla

(1994) cAMP modulates stress protein synthesis in human monocytes-macrophages. J Cell Physiol 161:169–177

62.

Planas

Soriano

Estrada

Sanz

Martin

Ferrer

(1997) The heat shock stress response after brain lesions: induction of 72 kDa heat shock protein (cell types involved, axonal transport, transcriptional regulation) and protein synthesis inhibition. Prog Neurobiol 51:607–636

63.

Price

Calderwood

(1991) Ca²⁺ is essential for multistep activation of the heat shock factor in permeabilized cells. Mol Cell Biol 11:3365–3368

64.

Robertson

Kerppola

Vendrell

Luk

Smeyne

Bocchiaro

Morgan

Curran

(1995) Regulation of c-fos expression in transgenic mice requires multiple interdependent transcription control elements. Neuron 14:241–252

65.

Ryffel

(1996) Gene knockout mice as investigative tools in pathophysiology. Int J Exp Pathol 77:125–141

66.

Sakamoto

Bardeleben

Yates

Raines

Golde

Gasson

(1991) 5′ Upstream sequence and genomic structure of the human primary response gene Egr-1-Tis8. Oncogene 6:867–872

67.

Salminen

Liu

Hsu

(1995) Alteration of transcription factor binding activities in the ischemic rat brain. Biochem Biophys Res Commun 212:939–944

68.

Santella

Carafoli

(1997) Calcium signaling in the cell nucleus, FASEB J 11:1091–1109

69.

Sassone Corsi

Visvader

Ferland

Mellon

Verma

(1988) Induction of proto-oncogene fos transcription through the adenylate cyclase pathway characterization of a cyclic AMP-responsive element Genes Dev 2: 1529–1538

70.

Schauwecker

Steward

(1997) Genetic determinants of susceptibility to excitotoxic cell death—implications for gene targeting approaches, Proc Natl Acad Sci USA 94:4103–4108

71.

Sheng

Thompson

Greenberg

(1991) CREB a calcium regulated transcription factor phosphorylated by calmodulin-dependent kinases, Science 252: 1427–1430

72.

Small

Buchan

(1996) Mechanisms of cerebral ischemia: intracellular cascades and therapeutic interventions. J Cardiothorac Vasc Anesth 10:139–146

73.

Swanson

Morton

Tsao

Savalos

Davidson

Sharp

(1990) A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab 10:290–293

74.

Treisman

(1992) The serum response element Trends Biochem Sci 17:423–426

75.

Welsh

Moyer

Harris

(1992) Regional expression of heat shock protein-70 mRNA and c-fos mRNA following focal ischemia in rat brain. J Cereb Blood Flow Metab 12:204–212

76.

Wiessner

Brink

Lorenz

Neumann-Haefelin

Vogel

Yamashita

(1996) Cyclin Dl messenger RNA is induced in microglia rather than neurons following transient forebrain ischaemia. Neuroscience 72:947–958

77.

Williams

McClanahan

Morimoto

(1989) E1a transactivation of the human HSP70 promoter is mediated through the basal transcriptional complex. Mol Cell Biol 9:2574–2587

78.

Yamashita

Vogel

Fritze

Back

Hossmann

Wiessner

(1996) Monitoring the temporal and spatial activation pattern of astrocytes in focal cerebral ischemia using in situ hybridization to GFAP mRNA: comparison with sgp-2 and hsp70 mRNA and the effect of glutamate receptor antagonists. Brain Res 735:285–297

79.

Yoneda

Ogita

Inoue

Mitani

Zhang

Masuda

Higashihara

Kataoka

(1994) Rapid potentiation of DNA binding activities of particular transcription factors with leucine-zipper motifs in discrete brain structures of the gerbil with transient forebrain ischemia. Brain Res 667:54–66

Attenuated c-fos mRNA Induction after Middle Cerebral Artery Occlusion in CREB Knockout Mice Does Not Modulate Focal Ischemic Injury

Abstract

Keywords

MATERIALS AND METHODS

Animals were housed under diurnal lighting conditions and allowed access to food and water ad libitum until the day of the experiment. Anesthesia was induced by 1.5% halothane and maintained with 1% halothane in 70% N2O and 30% O2.

Animal preparation

Regional measurement of ATP, tissue pH, and protein synthesis

Probe for c-fos and hsp72 mRNAs

In situ hybridization

Morphometric analysis of ischemia-induced metabolic disturbance and gene expression

Temperature control

Statistics

RESULTS

Neurologic score

Regional brain tissue ATP, pH, and CPS

c-fos and hsp72 mRNA expression

DISCUSSION

Mechanism of c-fos induction

Mechanism of hsp70 induction

Imaging ischemic penumbra

Penumbra and c-fos induction

Penumbra and hsp70 induction

CONCLUSION

Footnotes

Acknowledgments

Abbreviations used

References