METHODS

Animals

Studies were approved by the Animal Care and Use Committee of the University of Minnesota. Experiments were conducted in 14 C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME), 11 129/SVeV (SV129) mice (Jackson Laboratories) and 27 iNOS -/- mice. All mice were males. As described in detail elsewhere (Iadecola et al., 1997), iNOS -/- mice were obtained from a colony established from breeding pairs provided by Dr. C. Nathan and J. Mudgett (MacMicking et al., 1995). Mice were studied at age 8 to 9 weeks.

Middle cerebral artery occlusion

Focal cerebral ischemia was produced by occlusion of the MCA as previously described (Iadecola et al., 1997; Zhang et al., 1997). Briefly, mice were anesthetized with halothane (induction 5%, maintenance 1%) in an oxygen-nitrogen mixture. Body temperature was maintained at 37 0.5 °C by a thermostatically controlled infrared lamp. A 2-mm hole was drilled at a site superior and lateral to the left foramen ovale to expose the left MCA. The MCA was elevated and cauterized distal to the origin of the lenticulostriate branches. Wounds were sutured, and mice were returned to their cages and closely monitored until they recovered from anesthesia. Rectal temperature was measured and controlled until mice regained full consciousness. Thereafter, rectal temperature was measured at 24-hour intervals (before and 2 hours after each NS398 administration) until the time of they were killed. All mice survived until they were killed for determination of infarct volume.

Determination of infarct volume

Mice were killed for measurement of infarct volume 96 hours after MCA occlusion, shortly after the last neurologic evaluation. As described in detail elsewhere (Iadecola et al., 1997; Zhang et al., 1997), brains were removed and frozen in cooled isopentane (-30°C). Coronal forebrain sections (thickness, 30 m) were serially cut in a cryostat, collected at 150 m intervals, and stained with thionin. Infarct volume was determined by an image analyzer (MCID, Imaging Research Inc.). To factor out the contribution of ischemic swelling to the volume of the lesion, infarct volume in cerebral neocortex was corrected for swelling using the procedure developed by Lin et al. (Iadecola et al., 1997; Lin et al., 1993; Zhang et al., 1997).

Neurologic evaluation

Neurologic deficits were assessed by a neurologic scoring system using a modification of the postural reflex test, as previously described (Bederson et al., 1986; Iadecola et al., 1997). The examiner was not aware of the identity of the mice. Neurologic scores were: 0, normal motor function; 1, flexion of torso and contralateral forelimb when mouse was lifted by the tail; 2, circling to the contralateral side when mouse was held by the tail on a flat surface, but normal posture at rest; 3, leaning to the contralateral side at rest; 4, no spontaneous motor activity. Mice were evaluated before MCA occlusion and at 24-hour intervals up to 96 hours after MCA occlusion.

Monitoring of CBF

Techniques used for monitoring CBF in anesthetized mice have been described in detail previously (Iadecola et al., 1997). Mice were anesthetized with halothane (maintenance 1%), and the femoral artery and trachea were cannulated. Mice were artificially ventilated with an oxygen-nitrogen mixture by a mechanical ventilator (SAR-830, CWI Inc., Ardmore, PA, U.S.A.). The oxygen concentration in the mixture was adjusted to maintain arterial PO₂ between 120 and 150 mm Hg. End-tidal CO₂ was continuously monitored using a CO₂ analyzer (Capstar-100, CWI Inc.) and maintained at 2.6% to 2.7% which corresponds to a PCO₂ of 33 to 35 mm Hg (Iadecola et al., 1997). The physiologic parameters of the mice studied were: mean arterial pressure: 95 8 mm Hg; PCO₂: 33.4 1.5 mm Hg; PO₂: 139 15 mm Hg; pH: 7.42 0.03. CBF was monitored by a laser-Doppler flow probe (Vasamedic, St. Paul, MN, U.S.A.) placed through a burr hole drilled over the parietal cortex. The zero value for CBF was obtained after the mice were killed and was used to calculate percent changes in CBF (Iadecola et al., 1997).

Experimental protocol

Effect of NS-398 on infarct volume. C57BL/6, 129/SVeV, and iNOS null mice were studied. The COX-2 inhibitor NS398 (20 mg/kg; intraperitoneally) or vehicle was administered twice daily starting 6 hours after MCA occlusion. NS398 was diluted in distilled water at pH 10. Vehicle consisted of distilled water only (pH = 10). The dose of NS398 used was found in previous experiments to inhibit postischemic prostaglandin accumulation (Nogawa et al., 1998; Nogawa et al., 1997). NS398 does not affect NOS activity (Zingarelli et al., 1997). Rectal temperature was monitored daily. Mice were killed 96 hours after MCA occlusion, and brains were processed for measurement of infarct volume.

Effect of 7-NI on infarct volume in iNOS -/- mice. The relatively selective neuronal NOS (nNOS) inhibitor 7-nitroindazole (7-NI; 50 mg/kg; n = 6) or vehicle (oil; n = 5) was administered intraperitoneally 5 minutes after MCA occlusion as previously reported (Kamii et al., 1996). This dose of 7-NI inhibits forebrain NOS activity by 74% in rodents (Iadecola and Zhang, 1996). Mice were killed 96 hours after MCA occlusion for determination of infarct volume.

Effect of NS398 on CBF in iNOS -/- mice. In these experiments, the effect of NS398 on resting CBF and on the vasodilation produced by hypercapnia were studied in iNOS -/- (n = 6). Hypercapnia was induced by introducing CO₂ in the circuit of the ventilator (Iadecola et al., 1997). First, resting CBF and the increase in CBF produced by hypercapnia (pCO₂: 50 to 60 mm Hg) were established. Then, NS398 was administered (20 mg/kg; intraperitoneally) and effects on resting CBF and hypercapnic vasodilation studied 30 to 45 minutes later.

Data analysis

Data in text, table, and figures are expressed as mean SD. Comparisons among multiple groups were statistically evaluated by one-way analysis of variance followed by post hoc Tukey-Kramer honestly significant difference test (Systat, Evanston, IL, U.S.A.). Two-group comparisons were analyzed by the paired or unpaired two-tailed t test as appropriate. Neurologic scores were evaluated by nonparametric statistical procedures. Two-group comparisons were analyzed by the Mann-Whitney U analysis (Kirk, 1982), whereas multiple comparisons were analyzed by the Kruskal-Wallis test followed by post hoc Tukey-Kramer honestly significant different test (Kirk, 1982). For all statistical procedures, differences were considered significant at P < .05.

RESULTS

Effect of NS398 on infarct volume in C57BL/6 mice

Before MCA occlusion, rectal temperature was 35.7 0.9 °C in vehicle-treated mice (n = 7) and 36.0 0.7 °C in mice treated with NS398 (n = 7). Treatment with NS398 did not alter rectal temperature during the 4-day survival period (P > .05; analysis of variance). For example, 24 hours after MCA occlusion, rectal temperature was 36.0 0.7° C before NS398 and 36.2 0.3 2 hours after. In vehicle-treated mice, MCA occlusion produced an infarct involving mostly the cerebral cortex (Table 1). Size and regional distribution of the infarct were similar to those previously reported (Iadecola et al., 1997; Zhang et al., 1997). Administration of NS398 reduced infarct volume (P < .05; t-test; Table 1) and ameliorated the motor deficits produced by MCA occlusion (P < .05 at 96 hours; Mann-Whitney U test) (Fig. 1).

Figure 1.

Effect of the cyclooxygenase-2 inhibitor NS398 on the motor deficits produced by middle cerebral artery occlusion. Administration of NS398 reduced the neurologic deficits both in C57BL/6 (A) and in 129/SVeV mice (B). The effect is delayed and reaches statistical significance 4 days after MCA occlusion (P < .05; Mann-Whitney U test).

Full figure and legend (111K)

Table 1 - Effect of NS398 on infarct volume after middle cerebral artery occlusion in wild-type mice.

Full table

Effect of NS398 or 7-NI on infarct volume in iNOS -/- mice

Before MCA occlusion, rectal temperature was 36.6 0.7 °C in vehicle-treated iNOS -/- mice (n = 5) and 36.1 0.8 °C in mice treated with NS398 (n = 5). Treatment with NS398 did not alter rectal temperature during the 4-day survival period (P > .05; analysis of variance). In agreement with previous observations, infarct volume in iNOS -/- mice was smaller than that of C57BL/6 mice (-32 14% in neocortex; P < .05) (cf. Iadecola et al., 1997). However, in contrast to C57BL/6 mice, treatment with NS398 did not reduce infarct volume (Table 2). Rather, NS398 tended to increase the volume of injury, although the effect did not reach statistical significance (P > .05). NS398 did not affect the motor deficits produced by MCA occlusion (P > .05; Fig. 2). To rule out the possibility that the lack of effectiveness of NS398 in iNOS -/- mice was due to the fact that infarct volume was already maximally reduced, we studied the effect of the nNOS inhibitor 7-NI on infarct volume in iNOS -/- mice. Treatment with 7-NI (n = 6) reduced neocortical infarct volume by 20 10% compared to vehicle-treated controls (P < .05; n = 5).

Figure 2.

Effect of the cyclooxygenase-2 inhibitor NS398 on the motor deficits produced by middle cerebral artery occlusion in iNOS -/- mice. At variance with C57BL/6 and 129/SVeV mice (cf. Fig. 1), administration of NS398 did not reduce the neurologic deficits in iNOS -/- mice (P < .05; Mann-Whitney U test).

Full figure and legend (70K)

Table 2 - Effect of NS398 on infarct volume after middle cerebral artery occlusion in iNOS -/- mice.

Full table

Effect of NS398 on infarct volume in 129/SVeV mice

iNOS -/- mice, as most mice generated by homologous recombination, have a mixed genetic background comprising both C57BL/6 and 129/SVeV strains (MacMicking et al., 1995). To rule out the possibility that the lack of protection by NS398 in iNOS -/- mice was related to strain differences, we tested the effect of NS398 on infarct volume in 129/SVeV mice. In vehicle-treated mice (n = 5), infarct volume did not differ from that of C57BL/6 mice (Table 1; P > .05). Treatment with NS398 (n = 6) reduced neocortical infarct volume by 22 7% (Table 1; P < .05) and ameliorated the motor deficits produced by MCA occlusion (P < .05 at 96 hours) (Fig. 1).

Effect of NS398 on resting CBF and on the reactivity to hypercapnia in iNOS -/- mice

In these experiments (n = 6), we sought to explore the possibility that the absence of protection by NS398 in iNOS -/- mice was related to cerebral hemodynamic effects of NS398 present only in iNOS -/-. Mean arterial pressure was 95 8 mm Hg before NS398 administration and 90 7 60 minutes after (P > .05). NS398 did not affect resting CBF or the CBF increase produced by hypercapnia (Fig. 3). The intensity of the hypercapnia challenge was comparable before and after NS398 (before: PCO₂ = 54.5 3 mm Hg; after: 55.3 2; P > .05).

Figure 3.

Effect of NS398 on resting CBF (A), and on the increases in CBF produced by hypercapnia (B) in anesthetized inducible nitric oxide synthase -/- mice (n = 6). CBF was monitored in the parietal cortex by laser-Doppler flowmetry. Data in panel A are expressed in arbitrary perfusion units obtained from the output of the laser-Doppler flowmeter. NS398 (20 mg/kg; intraperitoneally) did not influence resting CBF (P > .05; paired t-test) or the reactivity of CBF to hypercapnia.

Full figure and legend (70K)

DISCUSSION

We sought to investigate further the role of iNOS and COX-2 in focal cerebral ischemic damage. Previous investigations have indicated that NO produced by iNOS increases COX-2 catalytic output, raising the possibility that COX-2 reaction products contribute to the brain damage produced by iNOS-derived NO (Nogawa et al., 1998). In this study, we sought to provide evidence in support of this hypothesis. We reasoned that if iNOS-derived NO is required for the toxic effects of COX-2 to take place, then inhibition of COX-2 in iNOS -/- mice, which do not express iNOS after cerebral ischemia (Iadecola et al., 1997), should not provide added protection. Consistent with this prediction we found that the COX-2 inhibitor NS398 reduced the infarct produced by MCA occlusion in wild-type mice but not in iNOS -/- mice. This observation indicates that iNOS-derived NO is required for the toxicity of COX-2 reaction products to occur.

The lack of effectiveness of NS398 in iNOS -/- mice cannot be attributed to genetic differences in the susceptibility to this inhibitor in the parental strains because NS398 reduced ischemic damage both in C57BL/6 and in 129/SVeV mice, the two strains used to generate the null mice (MacMicking et al., 1995). Similarly, the absence of protection cannot result from the fact that iNOS -/- mice are maximally protected from cerebral ischemic damage because administration of the nNOS inhibitor 7-NI reduced infarct volume in iNOS -/- mice. Furthermore, the failure of NS398 to reduce infarct volume in the iNOS -/- mice cannot be attributed to confounding cerebrovascular effects of NS398. (1) NS398 was administered 6 hours after induction of cerebral ischemia, at a time when hemodynamic effects are no longer able to influence the outcome of permanent cerebral ischemia (e.g., Zhang and Iadecola, 1994). (2) Administration of NS398, at the same dose used in ischemia experiments, did not alter arterial pressure, resting CBF, or the reactivity of CBF to hypercapnia in iNOS null mice. While these observations are preliminary and need to be expanded to other aspects of cerebrovascular regulation, they suggest that NS398 is devoid of cerebrovascular effects that could potentially antagonize the protection exerted by the drug. Therefore, the lack of protection by NS398 in iNOS -/- mice is unlikely to be caused by factors other than deletion of iNOS gene.

The mechanisms by which NO and its derived chemical species exert their cytotoxic effect are multiple and are thought to include inhibition of ATP-producing enzymes (Kroncke et al., 1997), DNA damage (Szabo and Ohshima, 1997), and oxidative damage produced by peroxynitrite, a highly reactive chemical species formed by the reaction of NO with superoxide (Beckman et al., 1990). Peroxynitrite-induced DNA single-strand breaks activate the DNA repair enzyme poly(ADP)ribose synthetase which worsens energy depletion and contributes to ischemic brain injury (Eliasson et al., 1997; Szabo and Dawson, 1998). In addition, DNA strand breaks induce the expression of p53, a tumor-suppressor protein that induces cell cycle arrest and apoptosis (Forrester et al., 1996). The results of the present study suggest that iNOS-derived NO could produce tissue damage also by activating COX-2 and increasing the toxic output of this enzyme. Therefore, COX-2 reaction products could be an additional mechanism by which NO exerts its deleterious effects on the postischemic brain.

However, the mechanisms by which COX-2 contributes to cerebral ischemic injury have not been elucidated. One possibility is that pro-inflammatory prostanoids synthesized by COX-2 contribute to the inflammatory reaction that involves the ischemic brain and, by this mechanism, worsen damage (Seibert et al., 1995). In addition, COX-2 could act as a source of superoxide for peroxynitrite formation. The two catalytic steps of COX-2, cyclooxygenase and peroxidase, are associated with production of reactive oxygen species (Vane et al., 1998). Superoxide produced by COX-2 activity could react with NO to form peroxynitrite, a highly reactive oxidant thought to mediate many of the toxic effects of NO (Kroncke et al., 1997). Irrespective of its mechanisms, the present results suggest that iNOS-derived NO is required for the full expression of COX-2 neurotoxicity.

Another new finding of this study is that NS398 reduces ischemic brain damage also in mice. The reduction in infarct volume afforded by NS398 cannot be attributed to effects on body temperature, arterial pressure or blood gases. While NS398 does not influence rectal temperature (present study), this inhibitor does not affect arterial pressure in anesthetized mice (present study) or in awake rats (Nogawa et al., 1997). Because of their small blood volume, serial measurements of arterial blood gases were not made in the mice in which infarct volume was studied. However, experiments in rats indicate that NS398 does not affect blood gases (Nogawa et al., 1997). Selective COX-2 inhibitors have previously been reported to reduce the damage resulting from focal ischemia or global ischemia in rats (Nakayama et al., 1998; Nogawa et al., 1997). The observation that the COX-2 inhibitor NS398 reduces ischemic damage also in mice suggests that the participation of COX-2 in ischemic brain injury is preserved across species. However, it would be important to determine whether COX-2 inhibition reduces infarct volumes also in other stroke models, such as those with reperfusion. We have recently found that iNOS and COX-2 are expressed also in the human brain in the acute stages of focal cerebral ischemia (Forster et al., 1999; Iadecola et al., 1999). As in rodents, iNOS expression occurs in neutrophils and vascular cells whereas COX-2 expression occurs also in neurons at the infarct border. While it remains to be established whether iNOS-derived NO and COX-2 reaction products contribute to the pathogenesis of human stroke, the similarity in the timing and cellular localization of iNOS and COX-2 between rodents and humans suggests similar roles in the mechanisms of damage. nNOS inhibition reduces infarct volume in iNOS null mice. This observation confirms that both nNOS and iNOS contribute to ischemic brain injury (Iadecola, 1997). However, the fact that nNOS inhibition in iNOS null mice does not completely eliminate ischemic damage underscores the pathogenic importance of factors independent of the NO pathways.

In conclusion, we have shown that the COX-2 inhibitor NS398 reduces cerebral ischemic damage in wild-type mice but not in iNOS -/- mice. The effect cannot be attributed to variations in the genetic background of iNOS -/- mice, to cerebral hemodynamic effects of NS398, or to the fact that the maximal protection possible was achieved in the iNOS -/- mice. The data suggest that iNOS-derived NO is required for the expression of toxicity mediated by COX-2 reaction products. Thus, COX-2 reaction products may be an important factor in the deleterious effect exerted by NO derived from iNOS in the postischemic brain.

References

1. Beckman JS, Beckman TW, Chen J, Marshall PA & Freeman BA. (1990) Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 87: 1620−1624. | PubMed | ChemPort |
2. Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL & Bartkowski H. (1986) Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17: 472−476. | PubMed | ISI | ChemPort |
3. Collaco-Moraes Y, Aspey B, Harrison M & de Belleroche J. (1996) Cyclo-oxygenase-2 messenger RNA induction in focal cerebral ischemia. J Cereb Blood Flow Metab 16: 1366−1372. | PubMed | ChemPort |
4. Dawson VL, Brahmbhatt HP, Mong JA & Dawson TM. (1994) Expression of inducible nitric oxide synthase causes delayed neurotoxicity in primary mixed neuronal-glial cortical cultures. Neuropharmacology 33: 1425−1430. | Article | PubMed | ISI | ChemPort |
5. Eliasson MJ, Sampei K, Mandir AS, Hurn PD, Traystman RJ, Bao J, Pieper A, Wang ZQ, Dawson TM, Snyder SH & Dawson VL. (1997) Poly(ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia. Nat Med 3: 1089−1095. | Article | PubMed | ISI | ChemPort |
6. Forrester K, Ambs S, Lupold SE, Kapust RB, Spillare EA, Weinberg WC, E F-B, Wang XW, Geller DA, Tzeng E, Billiar TR & Harris CC. (1996) Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53. Proc Natl Acad Sci USA 93: 2442−2447. | Article | PubMed | ChemPort |
7. Forster C, Clark HB, Ross ME & Iadecola C. (1999) Inducible nitric oxide synthase expression in human cerebral infarcts. Acta Neuropathologica 97: 215−220. | Article | PubMed | ChemPort |
8. Hewett SJ, Csernansky CA & Choi DW. (1994) Selective potentiation of NMDA-induced neuronal injury following induction of astrocytic iNOS. Neuron 13: 487−494. | Article | PubMed | ISI | ChemPort |
9. Hewett SJ, Muir JK, Lobner D, Symons A & Choi DW. (1996) Potentiation of oxygen-glucose deprivation-induced neuronal death after induction of iNOS. Stroke 27: 1586−1591. | PubMed | ChemPort |
10. Iadecola C. (1997) Bright and dark sides of nitric oxide in ischemic brain damage. Trends Neurosci 20: 132−138. | Article | PubMed | ISI | ChemPort |
11. Iadecola C, Forster C, Shigeru N, Clark HB & Ross ME. (1999) Cyclooxygenase-2 immunoreactivity in the human brain following cerebral ischemia. Acta Neuropathol 98: 9−14. | Article | PubMed | ChemPort |
12. Iadecola C & Zhang F. (1996) Permissive and obligatory roles of NO in cerebrovascular responses to hypercapnia and acetylcholine. Am J Physiol 271 (Regulatory Integrative Comp Physiol 40): R990−R1001. | PubMed | ChemPort |
13. Iadecola C, Zhang F, Casey R, Clark HB & Ross ME. (1996) Inducible nitric oxide synthase gene expression in vascular cells after transient focal cerebral ischemia. Stroke 27: 1373−1380. | PubMed | ISI | ChemPort |
14. Iadecola C, Zhang F, Casey R, Nagayama M & Ross ME. (1997) Delayed reduction in ischemic brain injury and neurological deficits in mice lacking the inducible nitric oxide synthase gene. J Neurosci 17: 9157−9164. | PubMed | ISI | ChemPort |
15. Iadecola C, Zhang F & Xu X. (1995a) Inhibition of inducible nitric oxide synthase ameliorates cerebral ischemic damage. Am J Physiol 268: R286−R292. | PubMed | ChemPort |
16. Iadecola C, Zhang F, Xu X, Casey R & Ross ME. (1995b) Inducible nitric oxide synthase gene expression in brain following cerebral ischemia. J Cereb Blood Flow Metab 15: 378−384. | PubMed | ISI | ChemPort |
17. Kamii H, Mikawa S, Murakami K, Kinouchi H, Yoshimoto T, Reola L, Carlson E, Epstein CJ & Chan PH. (1996) Effects of nitric oxide synthase inhibition on brain infarction in SOD-1-transgenic mice following transient focal cerebral ischemia. J Cereb Blood Flow Metab 16: 1153−1157. | PubMed | ChemPort |
18. Kirk R. 1982 Experimental Design: Procedures for the Behavioral Sciences Monterey, CA, Brooks/Cole Publishing (p 911).
19. Kroncke KD, Fehsel K & Kolb-Bachofen V. (1997) Nitric oxide: cytotoxicity versus cytoprotection-how, why, when, and where? Nitric Oxide 1: 107−120. | Article | PubMed | ChemPort |
20. Lin T-N, He YY, Wu G, Khan M & Hsu CY. (1993) Effect of brain edema on infarct volume in a focal cerebral ischemia model in rats. Stroke 24: 117−121. | PubMed | ISI | ChemPort |
21. MacMicking J, Xie QW & Nathan C. (1997) Nitric oxide and macrophage function. Annu Rev Immunol 15: 323−350. | Article | PubMed | ISI | ChemPort |
22. MacMicking JD, Nathan C, Hom G, Chartrain N, Fletcher DS, Trumbauer M, Stevens K, Xie QW, Sokol K, Hutchinson N, Chen H & Mudgett JS. (1995) Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81: 641−650. | Article | PubMed | ISI | ChemPort |
23. Miettinen S, Fusco FR, Yrjanheikki J, Keinanen R, Hirvonen T, Roivainen R, Narhi M, Hokfelt T & Koistinaho J. (1997) Spreading depression and focal brain ischemia induce cyclooxygenase-2 in cortical neurons through N-methyl-D-aspartic acid-receptors and phospholipase A2. Proc Natl Acad Sci USA 94: 6500−6505. | Article | PubMed | ChemPort |
24. Nagayama M, Zhang F & Iadecola C. (1998) Delayed treatment with aminoguanidine decreases focal cerebral ischemic damage and enhances neurologic recovery in rats. J Cereb Blood Flow Metab 18: 1107−1113. | Article | PubMed | ChemPort |
25. Nakayama M, Uchimura K, Zhu RL, Nagayama T, Rose ME, Stetler RA, Isakson PC, Chen J & Graham SH. (1998) Cyclooxygenase-2 inhibition prevents delayed death of CA1 hippocampal neurons following global ischemia. Proc Natl Acad Sci USA 95: 10954−10959. | Article | PubMed | ChemPort |
26. Nogawa S, Forster C, Zhang F, Nagayama M, Ross ME & Iadecola C. (1998) Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia. Proc Natl Acad Sci USA 95: 10966−10971. | Article | PubMed | ChemPort |
27. Nogawa S, Zhang F, Ross ME & Iadecola C. (1997) Cyclo-oxygenase-2 gene expression in neurons contributes to ischemic brain damage. J Neurosci 17: 2746−2755. | PubMed | ChemPort |
28. Planas AM, Soriano MA, Rodriguez-Farre E & Ferrer I. (1995) Induction of cyclooxygenase-2 mRNA and protein following transient focal ischemia in the rat brain. Neurosci Lett 200: 187−190. | Article | PubMed | ChemPort |
29. Samdeni AF, Dawson TM & Dawson VL. (1997) Nitric oxide synthase in models of focal ischemia. Stroke 28: 1283−1288. | PubMed |
30. Seibert K, Masferrer J, Zhang Y, Gregory S, Olson G, Hauser S, Leahy K, Perkins W & Isakson P. (1995) Mediation of inflammation by cyclooxygenase-2. Agents Actions Suppl 46: 41−50. | PubMed | ChemPort |
31. Szabo C & Dawson VL. (1998) Role of poly(ADP-ribose) synthetase in inflammation and ischaemia-reperfusion. Trends Pharmacol Sci 19: 287−298. | Article | PubMed | ISI | ChemPort |
32. Szabo C & Ohshima H. (1997) DNA damage induced by peroxynitrite: subsequent biological effects. Nitric Oxide 1: 373−385. | Article | PubMed | ChemPort |
33. Vane JR, Bakhle YS & Botting RM. (1998) Cyclooxygenases 1 and 2. Annu Rev Pharmacol Toxicol 38: 97−120. | Article | PubMed | ISI | ChemPort |
34. Vodovotz Y, Kwon NS, Pospischil M, Manning J, Paik J & Nathan C. (1994) Inactivation of nitric oxide synthase after prolonged incubation of mouse macrophages with IFN-gamma and bacterial lipopolysaccharide. J Immunol 152: 4110−4118. | PubMed | ISI | ChemPort |
35. Wu KK. (1995) Inducible cyclooxygenase and nitric oxide synthase. Adv Pharmacol 33: 179−207. | PubMed | ChemPort |
36. Zhang F, Eckman C, Younkin S, Hsiao KK & Iadecola C. (1997) Increased susceptibility to ischemic brain damage in transgenic mice overexpressing the amyloid precursor protein. J Neurosci 17: 7655−7661. | PubMed | ISI | ChemPort |
37. Zhang F & Iadecola C. (1994) Reduction of focal cerebral ischemic damage by delayed treatment with nitric oxide donors. J Cereb Blood Flow Metab 14: 574−580. | PubMed | ISI | ChemPort |
38. Zingarelli B, Southan GJ, Gilad E, O'Connor M, Salzman AL & Szabo C. (1997) The inhibitory effects of mercaptoalkylguanidines on cyclo-oxygenase activity. Br J Pharmacol 120: 357−366. | PubMed | ChemPort |

Acknowledgements

The authors thank Ms. Deborah Kabes for editorial assistance and Ms. Tracy Aber for managing the mouse colony.