Sage Journals: Discover world-class research

Abstract

We have characterized the induction of the mitogen-inducible form of cyclo-oxygenase, COX-2, during focal cerebral ischemia following permanent middle cerebral artery occlusion (MCAO) in the rat. Marked unilateral induction of COX-2 mRNA was detected in ischemic regions ipsilateral to the occlusion. A significant increase in COX-2 mRNA was detected in “core” and “penumbra” regions of the cerebral cortex between 4 and 24 h after occlusion; this was most marked at 4 h in the penumbra region, in which a 19-fold increase above untreated control levels was detected. A smaller but significant induction was also detected at 4 h in the caudate. A correlation was demonstrated between the extent of COX-2 mRNA induction in cortical regions at 4 h and the severity of tissue damage subsequently detected at 24 h post MCAO. MK-801 significantly attenuated the induction of COX-2 mRNA in the penumbra region at 4 h. The demonstration of COX-2 induction following experimental ischemia highlights the importance of this reaction and its products and by-products, for example, free radicals, in the tissue response to this insult.

Keywords

Cyclo-oxygenase mRNA Focal cerebral ischemia Cerebral cortex Gene expression Glutamate

Although the mechanisms that give rise to ischemic brain damage have not been fully determined, there is mounting evidence implicating the involvement of glutamate, calcium, and free radicals. It has emerged that excitatory responses mediated through the rapid action of glutamate also have profound effects on gene activation, producing long-term cellular responses. These responses are associated with the induction of immediate early genes (IEGs), which show a distinct time course of activation (Dragunow and Robertson, 1987a,b; Saffen et al.; Bartel et al., 1989; Cole et al., 1989; Janssen-Timmen et al, 1989; Sonnenberg et al., 1989; Simonato et al., 1991; Colląo-Moraes et al., 1994). Many of these IEGs (e.g., c-fos, c-jun, zif-268) in turn regulate the action of a number of other genes, thus initiating a cascade of gene activation (Sheng and Greenberg, 1990; Nathans et al, 1991). An ischemia/reperfusion study has demonstrated, using nuclear run-on assays, that the elevated levels of c-fos, jun-B, and c-jun mRNA are likely to be due to an increased rate of transcription (An et al., 1993). The involvement of these IEGs in the regulation of gene expression in ischemia is also supported by an increase in DNA binding activity of the transcriptional factor AP-1 (formed from fos and jun dimers), determined directly by mobility shift assays.

Identifying the targets of these IEGs is necessary to characterise the cellular response to synaptic activation. Recently, using molecular cloning strategies to identify genes induced by excitatory synaptic activity in brain neurones, Yamagata et al. (1993) identified a mitogen-inducible cyclo-oxygenase (COX) that was markedly activated by synaptic activity. Cyclo-oxygenase, or prostaglandin H-synthetase, is the rate-limiting enzyme in the biosynthetic pathway leading to the production of prostaglandins (PG) and thromboxanes from arachidonic acid. This pathway leads to the formation of PGG₂ from arachidonic acid and two molecules of oxygen, which is then reduced to PGH₂, and in turn acts as the precursor of various eicosanoids including PGE₂, PGF_2cα, PGD₂, prostacyclin, and thromboxane A₂. The importance of this enzyme in ischemic damage is indicated by the observation that pretreatment with a COX inhibitor markedly decreases the ischemic damage affecting the CA1 region of the hippocampus in gerbils (Sasaki et al., 1988).

Two isoforms of COX have been identified: COX-1, a constitutively expressed gene (De Witt and Smith, 1988; Funck et al., 1991), and, more recently, a mitogen-inducible form, COX-2, whose expression in mouse fibroblasts is induced by growth factors, phorbol esters, and serum (Herschman, 1991; Kujubu et al., 1991; Xie et al., 1991; O'Banion et al., 1992). COX-1 and COX-2 exhibit approximately 75% amino acid sequence homology (Funk et al., 1991; Kujubu et al., 1991), with the important catalytic amino acid residues conserved (Shimokawa et al., 1990). Neuronal expression of COX-2 is rapidly and transiently induced by seizures or N-methyl-d-aspartate(NMDA)-dependent synaptic activity in discrete neuronal populations in the rat cerebral cortex, but no expression is detected in glia or vascular endothelial cells (Yamagata et al., 1993). Recently, unilateral kainate injection into the nucleus basalis, a model known to produce a selective induction of IEGs (Wood and de Belleroche, 1991; Colląo-Moraes and de Belleroche, 1995), has also been shown to cause a marked transient induction of COX-2 mRNA in ipsilateral cortex. This effect is also sensitive to inhibition with the NMDA antagonist, MK-801 (Adams et al., 1996).

In this study, we characterized the time course of COX-2 mRNA induction during focal cerebral ischemia using the intravascular suture model of middle cerebral artery occlusion (MCAO) in the rat. The pharmacological sensitivity of the response was investigated by using the NMDA receptor antagonist MK-801.

METHODS

Animal model of MCAO and reperfusion

Male Sprague-Dawley rats weighing 300 ± 25 g were anaesthetized with 4% halothane in a 1:1 mixture of nitrous oxide and oxygen after an overnight fast. With the halothane then maintained at 1.5% in an open anaesthetic system without tracheotomy, the origin of the left middle cerebral artery (MCA) was occluded with an intravascular suture following the method of Koizumi et al. (1986), described in an English version by Nagasawa and Kogure (1989). Briefly, this involved introducing a 4/0 nylon suture (Ethylon, Ethicon Ltd., U.K.) with 7 mm of its shaft from the tip thickened with silicone rubber (Silasatic sealant 732 RTV, Merck Ltd., U.K.) to an outside diameter of 0.28 mm, into the left external carotid artery in a retrograde fashion toward the carotid bifurcation, and then directed distally up the left internal carotid artery to a distance of 17.5 ± 0.5 mm from the carotid bifurcation to the tip of the suture. Postmortem examination of the brains for the exact location of the intravascular suture in pilot studies (Calląo-Moraes et al., unpublished data) showed that the suture fully occluded the left anterior portion of the circle of Willis, from the anastomosis of the left internal carotid artery to the origin of the left anterior cerebral artery (ACA). In the process, the origins of the left MCA and penetrating branches supplying the caudate nucleus/putamen were blocked off. Perfusion of the left ACA territory is maintained by an azygous continuation of the union of both left and right ACA in the rat brain (Green, 1968). Access to the left posterior cerebral artery (PCA) from the basilar artery via the posterior communicating artery was uninterrupted. The suture was secured at two points: first, to the stump of the ligated left external carotid artery; second, within the ligated internal carotid artery, proximal to the pterygopalatine branch. Rectal temperature was maintained throughout at 37.0 ± 0.5°C with a heat lamp, and fluid loss during surgery was compensated by a postoperative i.p. injection of saline (10 ml/kg). The cutaneous wound was sutured and cleaned, and the rats were left to recover with free access to food and water.

To investigate the time course of COX-2 mRNA induction during permanent cerebral ischemia, the rats were killed by cervical dislocation at various times between 2 and 24 h postocclusion (3–6 rats at each time point). A separate group of four animals was pretreated with MK-801 (3 mg/kg) 20 min before MCAO, killed 4 h after occlusion, and similarly compared with animals receiving a vehicle injection before MCAO and killed at the same time.

Immediately after cervical dislocation, the brain was rapidly removed, anatomically dissected, and frozen in liquid nitrogen. Up to five regions, both ipsilateral and contralateral to MCAO, were selected. These were cortical tissue from the centre of the MCA territory and the caudate nucleus/putamen (both regions shown to be part of the ischemic “core” from previous ink perfusion studies) (Colląo-Moraes et al., 1994; unpublished data); and three cortical regions surrounding the “core”, including frontal cortex and dorsomedial cortex, both containing tissue supplied by the ACA and MCA, and occipital cortex containing tissue supplied by both the MCA and PCA. The last three regions contain tissue at the border zone between MCA and adjacent arterial territories, where the depth of ischemia may be only moderate, representing an ischemia penumbra where neurones are alive but nonfunctional but whose survival is limited to only a few hours (Hossmann, 1994). In most of the RNA analyses, the dorsomedial cortex sample was used as a source of such moderately ischemic tissue, and for this purpose it has been referred to as the penumbra throughout this study. Similarly, the sample of cortex from the centre of the MCA territory has been referred to as the core. These samples were stored at −70°C until required. Nonoperated rats were similarly investigated and served as baseline controls.

Histochemical measurement of cerebral infarction

To compare the induction of COX-2 mRNA with brain tissue damage in this animal model, the extent of cerebral infarction was measured using the histochemical technique of triphenyltetrazolium chloride (TTC) perfusion staining as previously described (Park et al., 1988). Following 24 h of permanent ischemia in a separate group of 12 rats, TTC (2% in saline) was perfused by a transcardiac route to the brain with the rats under terminal anaesthesia (i.p. 0.8 g/kg of urethane), followed by perfusion of formal saline. The brains were removed and sectioned into 1-mm-thick coronal slices. Unstained areas of cerebral gray matter in photographs of the slices were then measured by computerized planimetry and integrated volumetrically.

RNA extraction and detection

Total RNA was isolated from tissue samples by acid guanidinium thiocyanate-phenol-chloroform extraction (Chomczynski and Sacchi, 1987). The resulting RNA was analysed by Northern blotting and slot blotting as previously described (de Belleroche et al., 1990). Tissue levels of COX-2 mRNA were quantitated by reference to ß-tubulin mRNA using cDNA probes for detection. We used a 449-bp EcoRI fragment of pCRII to detect COX-1 mRNA, a 608-bp EcoRI fragment of pCRII to detect COX-2 mRNA (Feng et al., 1993; donated by Simon Tate, Glaxo Group Research), and an 800-bp fragment of ß-tubulin (Hall et al., 1983) to detect ß-tubulin mRNA.

Northern and slot blot filters were preincubated in hybridization buffer [50% deionized formamide, 5 × SSPE (0.9 M NaCl, 0.05 M sodium pyrophosfate, pH 7.4, 5 mM EDTA), 5 × Denhardt's solution (0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone), 0.5% (wt/vol) sodium dodecyl sulfate, 100 μg/ml salmon sperm DNA] for 2 h at 42°C. Filters were then hybridized overnight at 42°C in hybridization buffer containing [³²P]-labeled cDNA (3–5 × 10⁶ dpm/ml) to a specific activity of 2.5–3.5 × 10⁹ dpm/μg with [α-³²P]-dCTP using the oligolabeling method of Feinberg and Vogelstein (1983, 1984). Blots were washed with 3 × SSC, 0.1% SDS for 30 min at 65°C and 1 × SSC, 0.1% SDS for a further 30 min at 65°C and exposed to Hyperfilm-MP (Amersham International PLC) with intensifying screens at −70°C.

The COX-2 cDNA probe hybridized to a single mRNA species of 4.2 kb as previously reported (Yamagata et al., 1993). The COX-1 cDNA probe hybridized predominantly to a single mRNA species of 2.8 kb and a minor species of 5.1 kb (O'Banion et al., 1992). The intensity of hybridization on slot blots was measured with a scanning densitometer (Joyce Loebl Chromoscan) at 530 nm. The results are expressed as a ratio of the COX-2 mRNA signal: ß-tubulin mRNA signal. Statistical analysis was carried out by analysis of variance (ANOVA) and multiple t tests (Student's t test).

RESULTS

Time course of the induction of COX-2 mRNA during permanent cerebral ischemia and its sensitivity to MK-801

Marked unilateral induction of COX-2 mRNA was detected in the core ischemic region ipsilateral to the occlusion as shown by Northern analysis (Fig. 1). A single species of mRNA of 4.2 kb was detected at 2, 8, and 24 h. Levels of COX-2 mRNA in nonoperated control animals (t₀ time = 0) were not clearly detectable despite greater RNA loading for these samples (Fig. 1). Similarly, there was no induction of COX-2 mRNA detected in contralateral cerebral cortex by this method. Quantitation of COX-2 mRNA was carried out by slot-blot analysis with reference to levels of ß-tubulin mRNA in the same samples. A significant increase in COX-2 mRNA was detected in the core and penumbra regions between 4 and 24 h after occlusion, most marked at 4 h in the penumbra region, where a 19-fold increase above control t₀ levels was detected (Fig. 2). A smaller but significant induction was also detected at 4 h in the caudate (which also undergoes ischemic changes in this model). Although MCAO caused a small induction in COX-2 mRNA levels in the contralateral cortex, they were not significantly elevated compared with t₀ control levels (Fig. 2). Northern analysis of COX-1 mRNA was carried out in parallel, but no significant increase was detected in the brain following MCAO (data not shown), indicating the specificity of the induction of COX-2 mRNA.

Fig. 1.

Effect of permanent left middle cerebral artery occlusion MCAO on levels of cyclo-oxygenase (COX)-2 mRNA related to levels of ß-tubulin mRNA. MCAO territory (core) cerebral cortex ipsilateral (L) and contralateral (R) to the occlusion was dissected at various times (2, 8, and 24 h) after occlusion and frozen in liquid nitrogen. Then mRNA was prepared and separated by electrophoresis, blotted, and hybridized with cDNA probes for COX-2 and ß-tubulin. Loading in nonoperated control rat samples t₀ controls, shown by 0, was greatest to allow detection of low basal levels of COX-2 mRNA. A marked induction of COX-2 mRNA was detected in ipsilateral cortex at 2, 8, and 24 h after ischemia. The contralateral and ipsilateral cortex at each time point was derived from the same rat. Quantitation was carried out at each time point (n = 3–6) by slot-blot analysis (see Fig. 2).

Fig. 2.

Time course of induction of cyclo-oxygenase (COX)-2 mRNA following permanent left middle cerebral artery occlusion (MCAO). At various time points (4, 8, and 24 h) after occlusion, MCA territory [core (top)], and border zone (penumbra) cortex (middle) and caudate (bottom), ipsilateral (□) and contralateral () to the occlusion, were dissected and used for preparation of mRNA. Quantitation of COX-2 mRNA was carried out by slot-blot analysis by reference to levels of ß-tubulin mRNA. Values are means with the SDs shown by error bars for three to six rats at each time point. Analysis of variance showed a significant effect of time in ipsilateral cortex in core, penumbra, and caudate regions (p = 0.003, p = 0.003, and p = 0.039, respectively) and compared with contralateral cortex in the core and penumbra regions (p = 0.0001 and p = 0.001, respectively), †, ††, and ††† indicate that MCAO significantly increased levels of COX-2 mRNA compared with t₀ controls (p < 0.003, p < 0.001 and p < 0.0001). * and ** indicate that the values in ipsilateral cortex were significantly greater than contralateral cortex of the same rats (p < 0.025 and p < 0.005).

MK-801 significantly attenuated the induction of COX-2 mRNA in the penumbra region at 4 h (p < 0.025, Table 1). The induction of COX-2 mRNA in the core region was smaller than that in the penumbra and showed only a slight attenuation by MK-801. No effect was detected in caudate.

Table 1.

Effect of treatment with MK-801 (3 mg/kg) on the induction of COX-2 mRNA after MCAO^a

	COX-2 mRNA/ß-tubulin mRNAM
	Core		Penumbra		Caudate
	I	C	I	C	I	C
Control	0.20 ± 0.13	0.24 ± 0.20	0.08 ± 0.04	0.08 ± 0.03	0.38 ± 0.16	0.44 ± 0.24
MCAO + vehicle	0.68 ± 0.34^b,c	0.27 ± 0.19	1.86 ± 0.81^b,c	0.34 ± 0.30	1.34 ±0.62^d	0.57 ± 0.41
MCAO + MK-801	0.48 ± 0.15	0.23 ± 0.19	0.73 ± 0.37^b,e	0.20 ± 0.04	0.57 ± 0.80	0.54 ± 0.13

COX-2, cyclo-oxygenase 2; MCAO, middle cerebral artery occlusion; I, ipsilateral; C, core.

MK-801 was administered at the time of MCAO and ipsilateral and contralateral core and penumbral regions of cerebral cortex and caudate were removed at 4 h. Controls received the appropriate vehicle described in the Methods. Values (COX-2 mRNA: ß-tubulin mRNA) are means from three to six rats ± SD.

Indicates that MCAO significantly increased levels of COX-2 mRNA compared to controls (p < 0.001).

c,d

Indicate that the value in ipsilateral cortex is significantly greater than contralateral cortex of the same animals (p < 0.005, and p < 0.002).

Indicates that drug treatment caused a significant reduction in the induction of COX-2 mRNA (p < 0.025).

Correlation between changes in gene expression and infarct volume

All five tissue regions contained infarcted tissue at 24 h. The volume of infarction in the frontal cortex region, measured in the anterior four 1-mm coronal slices, represented 17.7% of the total volume of cortex for that region. The percentage volume of cortical infarction in the next five 1-mm coronal slices, containing the central MCA territory and dorsomedial cortex region (core and penumbra combined) was 52.4%. In the caudate nucleus/putamen, 79.6% of the tissue was infarcted, and the posterior five 1-mm coronal slices, the occipital cortex region, contained 25.8% infarcted cortex (Table 2). A correlation was seen between the extent of COX-2 mRNA induction in cortical regions at 4 h with the severity of tissue damage subsequently detected [coefficient of correlation (r) = 0.901], both being highest in the core/penumbra regions compared with the frontal and occipital regions; however, this relationship was not borne out in the caudate, where the greatest proportion of tissue volume was affected, but only intermediate levels of COX-2 mRNA were induced.

Table 2.

Relationship between infarct volume and COX-2 mRNA induction^a

	Core and penumbral cortex	Occipital cortex	Frontal cortex	Caudate
Volume (mm³)	156.9 ± 9.8	98.0 ± 5.6	88.8 ± 5.0	54.4 ± 3.4
Infarct volume (mm³)	82.1 ± 20.4	25.3 ± 19.2	15.7 ± 10.5	43.3 ± 11.0
% Infarct (24 h)	52.4 ± 13.6	25.8 ± 17.3	17.7 ± 11.7	79.6 ± 18.0
COX-2 mRNA (4 h)	0.9 1 ± 0.44	0.74 ± 0.18	0.45 ± 0.45	0.58 ± 0. 10
β-tubulin mRNA

COX-2, cyclo-oxygenase 2.

The mean volumes (mm³) of sampled brain regions and the extent of infarction were measured by triphenyltetrazoluim chloride staining following 24 h of permanent middle cerebral artery occlusion (MCAO) in 12 rats. Values are means ± SD. The maximal induction of COX-2 mRNA relative to ß-tubulin mRNA at 4 h in the defined regions ipsilateral to MCAO was determined as the difference between the ipsilateral and contralateral region of the same animal. Values are means ± SD.

DISCUSSION

Experimental cerebral ischemia causes a profound effect on the expression of a number of immediate early genes/early response genes and heat-shock proteins. The candidates recruited and the temporal pattern of these responses are similar to those seen during the intense synaptic activation that occurs in seizures, kindling, and excitotoxin injection and appears to be initiated by glutamate, as NMDA receptor antagonists block these IEG responses to excitation and ischemia (Dragunow et al., 1990; Wood and de Belleroche, 1991; Colląo-Moraes et al, 1994; Colląo-Moraes and de Belleroche, 1995; Adams et al., 1996). Further, inhibition of NMDA receptor synthesis, by treatment with antisense oligodeoxynucleotides to NR-1 mRNA (the universal NMDA receptor subunit), protects cortical neurones form neurotoxicity elicited by NMDA in vitro and reduces infarct volume produced by focal ischemia in spontaneously hypertensive rats (Wahlestedt et al., 1993). Understanding the functional consequence of these changes requires the identification of their gene targets in the case of transcription factors because a number of neuronal genes with the appropriate responsive elements in their promoter sequences could potentially be affected. The identification of COX-2 mRNA in this response to experimental ischemia highlights the potential importance of this reaction and its products in the tissue response to this insult subject to the demonstration of a parallel increase in COX-2 protein levels. The enzyme catalyses the key regulatory step in the biosynthesis of prostaglandins and thromboxanes, which are abundant in the CNS and are believed to play an important role in neuronal signaling (Narumiya et al, 1982; Shimizu and Wolfe, 1990). A further consequence of increased COX-2 activity is the generation of highly reactive oxygen free-radical species, with their potent damaging effects on lipids, proteins, and DNA (Siesjö, 1989). Thus, through this mechanism, COX-2 induction could be responsible for a major component of tissue damage resulting from ischemia and would represent an important target for treatment of the pathological consequences of this condition. The induction of COX-2 mRNA could also be part of a protective response to generate local inflammatory reactions, such as vasodilatation and neutrophil recruitment.

Although levels of COX-2 mRNA were raised in most ischemic regions, that is, the cortical “core” and the caudate, higher levels were found in the penumbra and frontal cortex at 4 h. These two regions would have contained zones of moderate ischemia with varying degrees of collateral perfusion from the anterior cerebral artery, and such raised levels of COX-2 enzyme would use the residual supply of oxygen during the acute period of ischemia and, in turn, generate cytotoxic free radicals to augment tissue damage. By 24 h, moderately ischemic neurones are dead (Hossmann, 1994). Reperfusion of ischemic tissue would provide conditions for enhanced oxygen-derived free-radical damage. Reducing glutamate action by the NMDA antagonist, MK-801, produced a powerful inhibitory effect on COX-2 induction during ischemia, particularly in the penumbra region, where the most marked induction of COX-2 was seen.

Both the rat and murine COX-2 promoter share a number of elements that confer phorbol ester and serum inducibility (Sirois and Richards, 1993; Fletcher et al., 1992). An important mediator of a number of neuronal responses is the cAMP-responsive element binding protein (CREB), which is phosphorylated by both a cAMP-dependent protein kinase (Sheng and Greenberg, 1990) and a Ca²⁺-dependent protein kinase, probably Ca²⁺/calmodulin-dependent protein kinase II (Sheng et al., 1990). The consensus sequence for CRE to which the phosphorylated protein binds (Xie et al., 1994) is present in the mouse and rat COX-2 promoter (Fletcher et al., 1992; Sirois et al, 1993). The sensitivity of COX-2 induction to a glutamate receptor antagonist could reflect the role of CREB, whose activity would be stimulated by increased intracellular cAMP and Ca²⁺, especially in the penumbra region. Levels of Ca²⁺ would be elevated by NMDA receptor gating Ca²⁺ channels, depolarisation, and neurotransmitter activation of G-protein receptors coupled to phospholipase C and IP₃ production, whereas levels of cAMP would be elevated by neurotransmitter activation of adenylyl cyclase-linked G-protein receptors. A cis-acting region of murine COX-2 has also been identified (−300 to −371 from the transcription start site) that is responsive to PAF and retinoic acid (RE), acting both independently and synergistically (Bazan et al., 1994); inhibition of intracellular PAF binding inhibits the induction of zif-268 mRNA and COX-2 mRNA in the rat brain (Marcheselli and Bazan, 1994; Bazan et al., 1994), indicating that PAF generated during ischemia may also stimulate COX-2 mRNA production.

In conclusion, the identification of COX-2 mRNA induction during experimental cerebral ischemia in this study highlights the importance of COX-2 and its products and by-products (e.g., free radicals) in the tissue response to this insult.

Footnotes

Abbreviations used

Acknowledgment:

We thank the BBSRC (SERC) for the award of a studentship to Y.C.M. and Bayer AG for financial support.

References

Adams

Colląo-Moraes

de Belleroche

(1996) Cyclooxygenase-2 induction in cerebral cortex: an intracellular response to synaptic excitation. J Neurochem 66:6–13.

Lin

T-N

Liu

J.S.X.J.J.

Y-Y

Hsu

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

Battel

Shen

Lau

Greenberg

(1989) Growth factors membrane depolarisation activate distinct programs of early response gene expression: dissociation of fos jun induction. Genes Dev 3:304–313.

Bazan

Fletcher

Herschman

Mukherjee

(1994) Platelet-activating factor retinoic acid synergistically activate the inducible prostaglandin synthase gene. Proc Natl Acad Sci USA 91: 5252–5256.

Chomczynski

Sacchi

(1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol chloroform extraction. Anal Biochem 162:156–159.

Cole

Saffen

Baraban

Morley

(1989) Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation. Nature, 340:474–476.

Colląo-Moraes

Aspey

de Belleroche

Harrison

MJG

(1994) Focal ischaemia causes an extensive induction of immediate early genes that are sensitive to MK-801. Stroke 25:1855–1861.

Colląo-Moraes

de Belleroche

(1995) Differential temporal patterns of expression of immediate early genes in cerebral cortex induced by intracerebral excitotoxin injection: sensitivity to dexamethasone and MK-801. Neuropharmacology 34:521–531.

de Belleroche

Bandopadhyay

King

Malcolm

ADB

O'Brien

Premi

Rashid

(1990) Regional distribution of cholecystokinin messenger RNA in rat brain during development: quantitation correlation with cholecystokinin immunoreactivity. Neuropeptides 15:201–212.

10.

De Witt

Smith

(1988) Primary structure of prostaglandin G/H synthase from sheep vesicular gl determined from the complementary DNA sequence. Proc Natl Acad Sci USA 85:1412–1416.

11.

Dragunow

Faull

RLM

Jansen

KLR

(1990) MK-801, an antagonist of NMDA receptors, inhibits injury-induced c-fos protein accumulation in rat brain. Neurosci Lett 109:128–133.

12.

Dragunow

Robertson

(1987a) Kindling stimulation induces c-fos protein(s) in granule cells of the rat dentate gyrus. Nature 329:411–142.

13.

Dragunow

Robertson

(1987b) Generalized seizures induce c-fos protein(s) in mammalian neurones. Neurosci Lett 82:157–161.

14.

Feinberg

Vogelstein

(1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13.

15.

Feinberg

Vogelstein

(1984) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity: addendum. Anal Biochem 137:266–267.

16.

Feng

Sun

Xia

Tang

Chanmugam

Soyoola

Wilson

Hwang

(1993) Cloning two isoforms of rat cyclooxygenase: differential regulation of their expression. Arch Biochem Biophys 307:361–368.

17.

Fletcher

Kujubu

Perrin

Herschman

(1992) Structure of the mitogen-inducible TIS10 gene demonstration that the TIS10-encoded protein is a functional prostaglandin G/H synthase. J Biol Chem 267:4338–4344.

18.

Funk

Kennedy

Pong

Fitzgerald

(1991) Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression and gene chromosomal assignment. FASEB J 5:2304.

19.

Green

(1968) Anatomy of the rat. Trans Am Phil Soc XXVII, Hafner Publishing Co., London.

20.

Hall

Dudley

Dobner

Lewis

Cowan

(1983) Identification of two beta tubulin isotopes. Mol Cell Biol 3:854–862.

21.

Herschman

(1991) Primary response genes induced by growth factors tumor promoters. Annu Rev Biochem 60:281–389.

22.

Hossmann

K-A

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

23.

Janssen-Timmen

Lemaire

Mattei

Relevant

Charnay

(1989) Structure chromosome mapping regulation of the mouse zinc finger gene krox-24: evidence for a common regulatory pathway for immediate early serum response genes. Gene 80:325–336.

24.

Koizumi

Yoshida

Nakazawa

Ooreda

(1986) Experimental studies of ischaemic brain edema: a new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischaemic area. Jpn J Stroke 8:1–8.

25.

Kujubu

Fletcher

Varnum

Lim

Herschman

(1991) TIS 10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells encodes a novel prostaglandin synthase/cyclooxygenase homologue. J Biol Chem 266:12866–12872.

26.

Marcheselli

Bazan

(1994) Platelet activating factor is a messenger in the electroconvulsive shock-induced transcriptional activation of c-fos zif-268 in the hippocampus. J Neurochem Res 37:54–61.

27.

Nagasawa

Kogure

(1989) Correlation between cerebral blood flow and histologic changes in a new rat model of middle cerebral artery occlusion. Stroke 20:1037–1043.

28.

Narumiya

Ogforochi

Nakao

Hayashi

(1982) Prostaglandin D2 in rat brain, spinal cord pituitary: basal level regional distribution. Life Sci 31:2093–2103.

29.

Nathans

Christy

Dubois

Lanahan

Saners

Nakabeppu

(1991) Transcription factors induced by growth-signalling agents. In: Origins of Human Cancer: A Comprehensive Review, Cold Spring Harbor, NY, Cold Spring Harbor Laboratories, pp 353–364.

30.

O'Banion

Winn

Young

(1992) cDNA cloning functional activity of a glucocorticoid regulated inflammatory cyclooxygenase. Proc Natl Acad Sci USA 89:4888–4892.

31.

Park

Mendelow

Graham

McCulloch

Teasdale

(1988) Correlation of triphenyltetrazolium chloride perfusion staining with conventional neurohistology in the detection of early brain ischaemia. Neuropathol Appl Neurobiol 14:289–298.

32.

Saffen

Cole

Worley

Christy

Ryder

Baraban

(1988) Convulsant-induced increase in transcription factor messenger RNAs in rat brain. Proc Natl Acad Sci USA 85:7795–7799.

33.

Sasaki

Nakagomi

Kirino

Tamura

Noguchi

Saito

Takakura

(1988) Indomethacin ameliorates ischemic neuronal damage in the gerbil hippocampal CA1 sector. Stroke 19:1399–1403.

34.

Sheng

Greenberg

(1990) The regulation function of c-fos other IEGs in the nervous system. Neuron 4:477–485.

35.

Sheng

McFadden

Greenberg

(1990) Membrane depolarisation calcium induce c-fos transcription via phosphorylation of transcription factor CREB. Neuron 4:571–582.

36.

Shimizu

Wolfe

(1990) Arachidonic acid cascade signal transduction. J Neurochem 55:1–15.

37.

Shimokawa

Kulmacz

De Witt

Smith

(1990) Tryosine 385 of prostaglandin endoperoxide synthase is required for cyclooxygenase catalysis. J Biol Chem 265:20073–20076.

38.

Siesjö

(1989) Free radicals brain damage. Cerebrovasc Brain Metab Rev 1:165–211.

39.

Simonato

Hosford

Labiner

Shin

Mansbach

McNamara

(1991) Differential expression of immediate early genes in the hippocampus in the kindling model of epilepsy. Mol Brain Res 11:115–124.

40.

Sirois

Levy L Simmons

Richards

(1993) Characterization of hormonal regulation of the promoter of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells, J Biol Chem 268:12199–12206.

41.

Sirois

Richards

(1993) Transcriptional regulation of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. J Biol Chem 268:21931–21938.

42.

Sonnenberg

Mitchelmore

Macgregor-Leon

Hempstead

Morgan

Curran

(1989) Glutamate receptor agonists increase the expression of Fos, Fra AP-1 DNA binding activity in the mammalian brain. J Neurosci Res 24:72–80.

43.

Wahlestedt

Golanov

Yamamoto

Yee

Ericson

Yoo

Inturrisi

Reis

(1993) Antisense oligodeoxynucleotides to NMDA-R1 receptor channel protect cortical neurones form excitotoxicity reduce focal ischaemic infarctions. Nature 363:260–263.

44.

Wood

de Belleroche

(1991) Induction of c-fos mRNA in cerebral cortex by excitotoxin stimulation of cortical inputs: involvement of N-methyl-D-aspartate receptors. Brain Res 545:183–190.

45.

Xie

Chipman

Robertson

Erikson

Simmons

(1991) Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc Natl Acad Sci USA 88:2692–2696.

46.

Xie

Fletcher

Andersen

Herschman

(1994) v-src Induction of the TIS10/PGS2 prostaglandin synthase gene is mediated by an ATF/CRE transcription response element. Mol Cell Biol 14:6531–6539.

47.

Yamagata

Andreasson

Kaufmann

Barnes

Worley

(1993) Expression of a mitogen-inducible cyclooxygenase in brain neurones: regulation by synaptic activity and glucocorticoids. Neuron 11:371–386.

Cyclo-oxygenase-2 Messenger RNA Induction in Focal Cerebral Ischemia

Abstract

Keywords

METHODS

Animal model of MCAO and reperfusion

Histochemical measurement of cerebral infarction

RNA extraction and detection

RESULTS

Time course of the induction of COX-2 mRNA during permanent cerebral ischemia and its sensitivity to MK-801

Correlation between changes in gene expression and infarct volume

DISCUSSION

Footnotes

Abbreviations used

Acknowledgment:

References