Published online before print April 8, 2004, doi: 10.1161/01.STR.0000126044.83777.f4
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Stroke. 2004;35:1449.)
© 2004 American Heart Association, Inc.
Original Contributions |
Reperfusion-Induced Oxidative/Nitrative Injury to Neurovascular Unit After Focal Cerebral Ischemia
From the Department of Neurology (Y.G.-Ö., T.D.), Faculty of Medicine, Institute of Neurological Sciences & Psychiatry, Hacettepe University, Ankara, Turkey; and Department of Histology-Embryology (A.C.), Ankara University School of Medicine, Ankara, Turkey.
Correspondence to Dr Turgay Dalkara MD, PhD, Department of Neurology, Hacettepe University Hospitals, Ankara 06100, Turkey. E-mail tdalkara{at}hacettepe.edu.tr
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background and Purpose— Use of thrombolysis in stroke is limited by a short therapeutic window because delayed reperfusion may cause brain hemorrhage and edema. Available evidence suggests a role for superoxide, NO, and peroxynitrite in reperfusion-induced injury. However, depending on their cellular origin and interactions between them, these molecules may exert protective or deleterious actions, neither of which is characterized in the intact brain.
Methods— Using fluorescent probes, we determined superoxide and peroxynitrite formation within neurons, astrocytes, and endothelium, and the association between oxidative/nitrative stress and vascular injury in mice brains subjected to 2-hour middle cerebral artery occlusion and 3 or 5 hours of reperfusion.
Results— Both signals were colocalized, suggesting that the main source of peroxynitrite in the reperfused brain was a reaction between superoxide and NO. Superoxide and peroxynitrite formation was particularly intense in microvessels and astrocytic end-feet surrounding them, and overlapped with dense mitochondrial labeling. Sites of oxidative/nitrative stress on microvessels were colocalized with markers of vascular injury such as Evans blue (EB) leakage and matrix metalloproteinase-9 (MMP-9) expression, suggesting an association between peroxynitrite and microvascular injury. Supporting this idea, partial inhibition of endothelial NO synthesis at reperfusion with a low dose of L-nitroarginine (1 mg/kg IP) reduced 3-nitrotyrosine formation in microvessels and EB extravasation.
Conclusion— During reperfusion, intense superoxide, NO, and peroxynitrite formation on microvessels and surrounding end-feet may lead to cerebral hemorrhage and edema by disrupting microvascular integrity. Combination of thrombolysis with agents diminishing oxidative/nitrative stress may reduce reperfusion-induced injury and extend the therapeutic window for thrombolysis.
Key Words: blood-brain barrier • matrix metalloproteinases • nitric oxide • peroxynitrite • reactive oxygen species • reperfusion injury • thrombolysis
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Thrombolytic therapy cannot be administered to most stroke patients arriving at the hospital several hours after stroke because delayed recirculation bears risk of brain hemorrhage and edema.1 An increased production of superoxide and NO in the brain has been demonstrated after reperfusion.2,3 A concomitant surge in production of these radicals can lead to formation of peroxynitrite, a powerful oxidant.4,5 Overproduction of these radicals may play important roles in reperfusion-induced injury. Supporting this view, scavengers of free radicals or inhibitors of lipid peroxidation protect the brain after ischemia/reperfusion.6,7 Mice overexpressing superoxide dismutase (SOD) are more resistant to ischemia associated with reperfusion,8 and conversely, larger infarcts develop in SOD knockout mice.9,10 NO’s role is more complex since it has both beneficial and deleterious actions during ischemia/reperfusion depending on the cell type in which it is generated.11 Excessive NO generation by nNOS is cytotoxic and, accordingly, nNOS knockouts are resistant to ischemia.12 On the contrary, eNOS knockouts develop larger infarcts because NO of endothelial origin promotes survival by improving residual blood flow during ischemia.13 However, the role of eNOS is more complicated because endothelial NO surge during reperfusion may contribute to brain injury via peroxynitrite formation, on one hand,14 and may improve microcirculation by preventing platelet and neutrophil aggregation, on the other.15
Consequently, there is controversy about the roles of superoxide, NO, and peroxynitrite in reperfusion injury because in vivo interactions between them are not characterized, and they may exert opposing actions depending on their cellular origin, distance between their sources, and in vivo diffusibility.5,11 It has been proposed that superoxide is not a powerful oxidant but gains significant toxicity by being converted to peroxynitrite after interacting with NO.5 Conversely, peroxynitrite is thought by some authors not to reach toxic concentrations during reperfusion but exert a favorable action by reducing endothelial dysfunction and leukocyte adhesion.16 Therefore, we have investigated the cellular origins of these radicals and the spatial relationship between them in reperfused brain in vivo.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Middle Cerebral Artery Occlusion
Animal housing, care, and application of experimental procedures were done in accordance with institutional guidelines. Swiss albino mice (28 to 34g) were fasted overnight with free access to water before the experiment, and were anesthetized with chloral hydrate (400 mg/kg, IP). Body temperature was maintained at 37.0±0.1°C during the experiment and only animals with arterial blood pressure in physiological ranges were included in the study. Proximal occlusion of the right middle cerebral artery (MCA) was performed with a filament as described before.14 The regional cerebral blood flow (rCBF) was monitored by laser-Doppler flowmetry with a probe placed over the skull to ensure that a desired level of ischemia and reperfusion was achieved. Forty mice were subjected to 2 hours of proximal MCA occlusion and 3 (n=34) or 5 (n=6) hours of reperfusion.
Detection of Superoxide
Dihydroethidium (HEt, Molecular Probes, 2 mg/200 µL PBS) was given intravenously just before reperfusion to monitor superoxide production.9,17 Hydroethidine (HEt) is oxidized by superoxide to ethidium and an unidentified product (Et+X), which can be detected with fluorescent microscopy (Ex=543 nm and Em>570 nm).9,17,18 This red fluorescence has been repeatedly shown to be inhibited by SOD and superoxide scavengers in heart and brain, and is used for semiquantitative intracellular superoxide detection.18 After 3 or 5 hours of reperfusion, mice were perfused transcardially with heparinized saline followed by 10% formaldehyde. Brains were postfixed in 10% formaldehyde for 24 hours and then were cryoprotected in 20% and 30% sucrose. Fifteen-µm–thick sections passing through the anterior commissure were kept frozen at –20°C until use. Sections were washed 3 times in a blocking and aldehyde-reducing solution.
Immunohistochemistry
We monitored peroxynitrite formation by detecting nitrosylated tyrosine residues on proteins with anti-3–nitrotyrosine (3-NT) antibody (Upstate Biotechnology). Neurons, astrocytes, and endothelial cells were labeled with antibodies against microtubule-associated protein-2 (MAP-2; Sigma), glial fibrillary acidic protein (GFAP; Sigma), and endothelium (CD-34; Chemicon), respectively. Matrix metalloproteinase-9 (MMP-9) expression was detected with anti-MMP-9 antibody (Chemicon). Mitochondria were labeled with antiprohibitin antibody (Research Diagnostics) or Mitotracker (Molecular Probes). Sections were incubated first with primary antibodies (1:100) at 37°C for 90 minutes and then with secondary antibodies (1:200) for 90 minutes. Fluorescein isothiocyanate (FITC) goat anti-mouse IgG, Cy2 goat anti-rabbit IgG, Cy3 goat anti-rabbit IgG, and Cy3 goat anti-mouse IgG (all from Jackson ImmunoResearch) were used as appropriate. Primary antibody omission incubations with either blocking solution or PBS were performed to test the specificity of immunoreactivity.
Sections were mounted in glycerol/PBS medium containing 25 mg/mL sodium azide. Specimens were examined by a Zeiss LSM-510 confocal laser-scanning microscope. Single optical sections (2048x2048 pixel) were collected. Digitized images were pseudocolored according to their original fluorochromes.
Colocalization of 3-Nitrotyrosine With Evans Blue Extravasation and MMP-9
Breakdown of blood-brain barrier (BBB) was assessed by Evans blue (EB) extravasation, which fluoresces red when excited. EB (0.3 mL of 4% solution; Sigma) was injected through the tail vein before reperfusion. Three or 5 hours after reperfusion, mice were transcardially perfused and frozen sections were obtained as described under Detection of Superoxide. The sections were double-labeled with MMP-9 or 3-NT antibody and colocalization of signals was assessed with confocal microscopy.
Fluorometric Measurement of Evans Blue Leakage
EB extravasation was quantified in L-nitroarginine (L-NA) and vehicle-treated mice 3 hours after reperfusion (n=6 per group). Transcardially perfused brains were removed and each hemisphere was homogenized in 1 mL 50% trichloroacetic acid and then centrifuged at 14 000g for 20 minutes at 4°C.14 The supernatant was diluted 4-fold with ethanol. Fluorescence intensity was measured by a microplate reader (620/680 nm; Wallac; Perkin–Elmer). Calculations were based on external standard readings and extravasated EB dye was expressed as micrograms/per hemisphere.
Cell Count and Statistics
Superoxide or 3-NT–positive cells were manually counted over the ischemic hemispheres in 5 selected core regions (frontal, parietal, insular cortices, preoptic area, and lateral striatum) under x400 magnification. Ratios of those positively-labeled to the total number of neurons, astrocytes, or endothelia in each microscopic field were individually calculated. Cell counts were also obtained from another group of mice subjected to 2 hours ischemia and 3 hours reperfusion, but treated with L-NA (1 mg/kg, IP) or 7-nitroindazole (7-NI) (50 mg/kg, IP) before reperfusion. Mean values are given with their standard deviations, and the number of animals and brain sections studied are indicated in parentheses under Results. Groups were compared by analysis of variance (ANOVA); P<0.05 was considered to be significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Superoxide and Peroxynitrite Formation After Reperfusion
In the nonischemic hemisphere, there were only occasional 3-NT–positive cells, whereas cells generating superoxide were more common in agreement with previous reports.9,17 On the other hand, numerous cells in the ischemic MCA territory were strongly labeled with 3-NT (n=4 mice, 16 sections) and Et+X (n=4,12), 3 hours after reperfusion (Figure 1). The size of the Et+X (n=2.8) or 3-NT (n=2.6) positive granules increased when reperfusion was extended to 5 hours. Both signals were more prominent in ischemic core. Double-labeling studies with cell-specific markers showed that 3-NT and Et+X fluorescence were scattered in neurons, astrocytes, and endothelial cells (Figure 1). Most neurons (79±18% of all MAP-2-positive cells; n=4,12) were labeled with anti-3–NT antibody (Figures 1 and 2
) and displayed Et+X fluorescence (78±8%; n=4,12). Only half of the astrocytes labeled with GFAP displayed 3-NT immunolabeling (51±10%; n=4,12) and Et+X fluorescence (47±10%; n=4,12). 3-NT and Et+X positivity were dispersed in cell bodies as well as processes of neurons and astrocytes. All Et+X signals were colocalized with 3-NT signals (weighted colocalization coefficient; 0.89±0.03), except the small 3-NT granular staining scattered in neuropil (Figure 1). Accordingly, the number of Et+X- and 3-NT–labeled cells were similar for all 3 cell types (Figure 2). Consistent with our previous report showing a striking 3-NT staining (diaminobenzidine-labeled) of microvessels relative to parenchyma,14 fluorescent markers disclosed that astrocytic end-feet ensheathing microvessels and endothelia were intensely positive for both signals (Figure 1). 48±17% of CD-34–positive endothelial cells were labeled with anti-3–NT antibody (n=4,12) and displayed Et+X fluorescence (51±13%, n=4,12) (Figure 2). The area of intense Et+X as well as 3-NT fluorescence on microvessels overlapped with dense mitochondrial labeling observed with 2 different markers of mitochondria (n=3,6) (Figure 3B and 3C).
|
|
|
Colocalization of 3-Nitrotyrosine With Evans Blue Extravasation and MMP-9
Three (n=2) and 5 (n=2) hours after reperfusion, EB leakage was detectable around several microvessels, especially in the ischemic core (Figure 3E). Microvessels that became permeable to EB were almost all labeled with 3-NT (n=4,8) (Figure 3G). Since EB leakage had just started to emerge at these time points,19 this colocalization suggests a causal relationship between peroxynitrite formation and microvascular injury. Supporting this view, 3-NT labeling was also colocalized with MMP-9 expression on the vessel wall 5 hours after reperfusion (n=2,4) (Figure 3H). MMP-9 expression was not at detectable levels 3 hours after reperfusion.
NOS Inhibition and Blood-Brain Barrier
A low dose of L-NA administered 15 minutes before reperfusion significantly reduced EB leakage 3 hours after reperfusion [0.33±0.06 versus 0.12±0.03 microgram/hemisphere in L-NA (n=6) and vehicle-treated (n=6) mice, respectively] (Figure 4). Contrary to the nonselective NOS inhibitor L-NA, 7-nitroindazole (7-NI) (in vivo selective inhibitor of nNOS) was not protective when given before reperfusion in our previous study and, hence, we proposed that NO of endothelial origin might be a major player in reperfusion-induced injury.14 In the present study, taking advantage of double-labeling methods, we were able to investigate the inhibition of neuronal and endothelial peroxynitrite formation individually. L-NA (n=3,12) significantly decreased the number of 3-NT–labeled endothelia (17±4 versus 48±17%) and astrocytes (28±8 versus 51±10%) by 64 and 45%, respectively, compared with untreated mice (n=4,12) (Figure 2), but not the number of 3-NT–labeled neurons (72±17 versus 79±18%). On the other hand, 7-NI (n=3,12) given 30 minutes before reperfusion had no effect on 3-NT labeling in endothelial cells (45±9 versus 48±17%). Prereperfusion administration of both L-NA (n=3,9) and 7-NI (n=3,9) did not alter the number of Et+X-positive cells, suggesting that observed fluorescence reflected superoxide formation and was not altered by peroxynitrite. Details of distribution of both signals are given in Figure 2; however, it should be noted that counting of 3-NT or Et+X-labeled cells did not take into account the changes in signal intensity in individual cells, and hence, is only a semiquantitative measure.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
We detected superoxide and nitrotyrosine formation in neurons, astrocytes, and endothelial cells soon after reperfusion in intact brains. Double-labeling with several fluorescent probes gave us the opportunity to study cellular distribution of these molecules and interactions among them under in vivo conditions. Both signals were highly colocalized; since superoxide has a relatively low diffusibility, this finding suggests that NO, diffusing freely across membranes, reached to superoxide-generating foci and led to peroxynitrite formation.4,5 Since there is no significant leukocyte infiltration and no inducible NOS activity in the mouse brain 3 hours after reperfusion,20,21 we think that the major sources of NO were nNOS expressing neurons and the endothelium, whereas superoxide was generated within all 3 cell types.
High constitutive SOD activity may account for the relatively less numerous superoxide and peroxynitrite signals detected in the soma and processes of astrocytes.22 Notably, however, astrocytic end-feet and endothelia were intensely labeled for both anions. This area of intense radical formation overlapped with dense populations of mitochondria, which may be a significant source of superoxide on recirculation.23 Endothelial enzymes can also contribute to superoxide generation.24 eNOS has been reported to be a major source of NO during reperfusion, although it accounts for less than 10% of NO generation in normal mouse brain.25 Therefore, NO produced by eNOS can diffuse and react with superoxide generated in end-feet and endothelia to form peroxynitrite, which may account for the intense 3-NT labeling in and around microvessels.2,14,25
The reaction between NO and superoxide was argued by some authors to promote protection by reducing superoxide toxicity, although this reaction occurs at the expense of generation of a stronger oxidant, peroxynitrite.16 Moreover, peroxynitrite was reported to have beneficial effects in coronary circulation.16,26 Therefore, we searched for evidence, whether or not peroxynitrite was indeed toxic to the cerebral microvasculature. We found that 3-NT signal was always colocalized with EB leakage or MMP-9 expression in microvessels after reperfusion, suggesting an association between peroxynitrite and vascular injury. Further supporting this idea, inhibition of NO synthesis by L-NA just before reperfusion decreased EB extravasation, consistent with suppression of MMP-9 expression with NOS inhibition in our previous report.14 Additionally, by using double labeling methods, we have obtained direct evidence showing that the low-dose L-NA decreased 3-NT formation in endothelia and astrocytes, supporting our idea that partial inhibition of the eNOS may contribute to preservation of BBB14 by protecting vascular and end-feet integrity. These findings are in contrast to studies reporting solely a protective role for NO in coronary ischemia/reperfusion.27 The discrepancy, in addition to differences in tissue biology (eg, urate generation in the heart may efficiently detoxify peroxynitrite),28 may also be related to differences in vasculature (eg, tight endothelial junctions and astrocytic end-feet in the brain may be particularly vulnerable to oxidative/nitrative injury).29–31 Moreover, loss of the selective permeability of BBB may jeopardize recovery of neural tissue after reperfusion.32 Hence, the deleterious effects of NO during brain reperfusion may surpass its positive effects such as inhibition of platelet and leukocyte adhesion to endothelium.15 Possibly because of this dual action of NO, high doses of NOS inhibitors increase infarct size, whereas low doses are protective.33 Inhibition of peroxynitrite formation is also feasible by suppressing superoxide levels. In parallel to our results, Asahi et al found a decrease in reperfusion-induced vascular injury and reduction in hemorrhagic complications when a free radical scavenger was administered at reperfusion.34 Similarly, SOD-overexpressing–transgenic mice were resistant to reperfusion injury, and studies with these mice suggested that the neurotoxic role of NO in ischemia/reperfusion depended on the reaction with superoxide to form peroxynitrite.6,35
Taken together, these findings suggest that superoxide, NO, and peroxynitrite formation in and around microvessels contribute to loss of selective BBB permeability and activation of MMPs. Supporting this idea, activation of MMP-9 via S-nitrosylation, and reduced MMP activation in mice overexpressing SOD have recently been reported.36,37 MMP-mediated degradation of collagen and laminins in basal lamina disrupts the integrity of the vascular wall.38,39 Oxidative/nitrative injury triggers recruitment and migration of white cells, which release enzymes that increase basal lamina degradation and vascular permeability.20 These events eventually lead to parenchymal hemorrhage and vasogenic brain edema. Moreover, disruption of the BBB, and, hence, loss of regulation of selective supply of nutrients to the brain, may jeopardize recovery of already damaged cells. Therefore, coadministration of thrombolytic agents with free radical34 or peroxynitrite scavengers,40 or low doses of nonselective NO synthase inhibitors14 may reduce reperfusion-induced injury and extend the therapeutic window for thrombolysis.
|
Acknowledgments |
|---|
The authors are grateful to Dr Kamil Topalkara for his permission to use panels E and F in Figure 2. This study was supported by The Turkish Academy of Sciences (T.D.), Brain Research Association (Y.G.-Ö.), and Ankara Biotechnology Institute (A.C.).
Received July 2, 2003; accepted February 2, 2004.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Hacke W, Brott T, Caplan L, Meier D, Fieschi C, von Kummer R, Donnan G, Heiss WD, Wahlgren NG, Spranger M, Boysen G, Marler JR. Thrombolysis in acute ischemic stroke: controlled trials and clinical experience. Neurology. 1999; 53: S3–S14.[Medline] [Order article via Infotrieve]
2. Peters O, Back T, Lindauer U, Busch C, Megow D, Dreier J, Dirnagl U. Increased formation of reactive oxygen species after permanent and reversible middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab. 1998; 18: 196–205.[CrossRef][Medline] [Order article via Infotrieve]
3. Kumura E, Yoshimine T, Iwatsuki KI, Yamanaka K, Tanaka S, Hayakawa T, Shiga T, Kosaka H. Generation of nitric oxide and superoxide during reperfusion after focal cerebral ischemia in rats. Am J Physiol. 1996; 270: C748–C52.[Medline] [Order article via Infotrieve]
4. Beckman JS. The double-edged role of nitric oxide in brain function and superoxide-mediated injury. J Dev Physiol. 1991; 15: 53–59.[Medline] [Order article via Infotrieve]
5. Beckman JS. Interactions of oxidants, nitric oxide, and antioxidant defenses in cerebral ischemia and injury. In: Ginsberg MD, Bogousslavsky J, eds. Cerebrovascular Disease. Boston, Mass: Blackwell Science; 1998: 455–470.
6. Chan PH. Reactive oxygen radicals in signaling and damage in the ischemic brain. J Cereb Blood Flow Metab. 2001; 21: 2–14.[CrossRef][Medline] [Order article via Infotrieve]
7. Kontos HA. Oxygen radicals in cerebral ischemia: the 2001 Willis lecture. Stroke. 2001; 32: 2712–2716.
8. Yang G, Chan PH, Chen J, Carlson E, Chen SF, Weinstein P, Epstein CJ, Kamii H. Human copper-zinc superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal cerebral ischemia. Stroke. 1994; 25: 165–170.[Abstract]
9. Kim GW, Kondo T, Noshita N, Chan PH. Manganese superoxide dismutase deficiency exacerbates cerebral infarction after focal cerebral ischemia/reperfusion in mice: implications for the production and role of superoxide radicals. Stroke. 2002; 33: 809–815.
10. Kondo T, Reaume AG, Huang TT, Murakami K, Carlson E, Chen S, Scott RW, Epstein CJ, Chan PH. Edema formation exacerbates neurological and histological outcomes after focal cerebral ischemia in CuZn-superoxide dismutase gene knockout mutant mice. Acta Neurochir Suppl (Wien). 1997; 70: 62–64.[Medline] [Order article via Infotrieve]
11. Dalkara T, Endres M, Moskowitz MA. Mechanisms of NO neurotoxicity. Prog Brain Res. 1998; 118: 231–239.[Medline] [Order article via Infotrieve]
12. Huang Z, Huang PL, Panahian N, Dalkara T, Fishman MC, Moskowitz MA. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science. 1994; 265: 1883–1885.
13. Huang Z, Huang PL, Ma J, Meng W, Ayata C, Fishman MC, Moskowitz MA. Enlarged infarcts in endothelial nitric oxide synthase knockout mice are attenuated by nitro-L–arginine. J Cereb Blood Flow Metab. 1996; 16: 981–987.[CrossRef][Medline] [Order article via Infotrieve]
14. Gursoy-Ozdemir Y, Bolay H, Saribas O, Dalkara T. Role of endothelial nitric oxide generation and peroxynitrite formation in reperfusion injury after focal cerebral ischemia. Stroke. 2000; 31: 1974–1980.
15. Gidday JM, Park TS, Shah AR, Gonzales ER. Modulation of basal and postischemic leukocyte-endothelial adherence by nitric oxide. Stroke. 1998; 29: 1423–1429.
16. Lefer AM. Nitric oxide: a critical determinant in ischemia-reperfusion. In: Ignarro LJ, ed. Nitric oxide: Biology and Pathobiology. San Diego, Calif: Academic Press; 2000; 649–660.
17. Murakami K, Kondo T, Kawase M, Li Y, Sato S, Chen SF, Chan PH. Mitochondrial susceptibility to oxidative stress exacerbates cerebral infarction that follows permanent focal cerebral ischemia in mutant mice with manganese superoxide dismutase deficiency. J Neurosci. 1998; 18: 205–213.
18. Zhao H, Kalivendi S, Zhang H, Joseph J, Nithipatikom K, Vasquez-Vivar J, Kalyanaraman B. Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic Biol Med. 2003; 34: 1359–1368.[CrossRef][Medline] [Order article via Infotrieve]
19. Belayev L, Busto R, Ikeda M, Rubin LL, Kajiwara A, Morgan L, Ginsberg MD. Protection against blood-brain barrier disruption in focal cerebral ischemia by the type IV phosphodiesterase inhibitor BBB022: a quantitative study. Brain Res. 1998; 787: 277–285.[CrossRef][Medline] [Order article via Infotrieve]
20. del Zoppo GJ, von Kummer R, Hamann GF. Ischaemic damage of brain microvessels: inherent risks for thrombolytic treatment in stroke. J Neurol Neurosurg Psychiatry. 1998; 65: 1–9.
21. Yoshida T, Waeber C, Huang Z, Moskowitz MA. Induction of nitric oxide synthase activity in rodent brain following middle cerebral artery occlusion. Neurosci Lett. 1995; 194: 214–218.[CrossRef][Medline] [Order article via Infotrieve]
22. Lindenau J, Noack H, Possel H, Asayama K, Wolf G. Cellular distribution of superoxide dismutases in the rat CNS. Glia. 2000; 29: 25–34.[CrossRef][Medline] [Order article via Infotrieve]
23. Oldendorf WH, Cornford ME, Brown WJ. The large apparent work capability of the blood-brain barrier: a study of the mitochondrial content of capillary endothelial cells in brain and other tissues of the rat. Ann Neurol. 1977; 1: 409–417.[CrossRef][Medline] [Order article via Infotrieve]
24. Beetsch JW, Park TS, Dugan LL, Shah AR, Gidday JM. Xanthine oxidase-derived superoxide causes reoxygenation injury of ischemic cerebral endothelial cells. Brain Res. 1998; 786: 89–95.[CrossRef][Medline] [Order article via Infotrieve]
25. Wei G, Dawson VL, Zweier JL. Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia. Biochim Biophys Acta. 1999; 1455: 23–34.[Medline] [Order article via Infotrieve]
26. Lefer DJ, Scalia R, Campbell B, Nossuli T, Hayward R, Salamon M, Grayson J, Lefer AM. Peroxynitrite inhibits leukocyte-endothelial cell interactions and protects against ischemia-reperfusion injury in rats. J Clin Invest. 1997; 99: 684–691.[Medline] [Order article via Infotrieve]
27. Jones SP, Lefer DJ. Using gene-targeted mice to investigate the pathophysiology of myocardial reperfusion injury. Basic Res Cardiol. 2000; 95: 499–502.[CrossRef][Medline] [Order article via Infotrieve]
28. Teng RJ, Ye YZ, Parks DA, Beckman JS. Urate produced during hypoxia protects heart proteins from peroxynitrite-mediated protein nitration. Free Radic Biol Med. 2002; 33: 1243–1249.[CrossRef][Medline] [Order article via Infotrieve]
29. Gobbel GT, Chan TY, Chan PH. Nitric oxide- and superoxide-mediated toxicity in cerebral endothelial cells. J Pharmacol Exp Ther. 1997; 282: 1600–1607.
30. Imaizumi S, Kondo T, Deli MA, Gobbel G, Joo F, Epstein CJ, Yoshimoto T, Chan PH. The influence of oxygen free radicals on the permeability of the monolayer of cultured brain endothelial cells. Neurochem Int. 1996; 29: 205–211.[CrossRef][Medline] [Order article via Infotrieve]
31. Greenacre S, Ridger V, Wilsoncroft P, Brain SD. Peroxynitrite: a mediator of increased microvascular permeability? Clin Exp Pharmacol Physiol. 1997; 24: 880–882.[Medline] [Order article via Infotrieve]
32. Betz AL, Dietrich WD. Blood-brain barrier dysfunction in cerebral ischemia. In: Ginsberg MD, Bogousslavsky J, eds. Cerebrovascular Disease. Boston, Mass: Blackwell Science; 1998: 358–370.
33. Margaill I, Allix M, Boulu RG, Plotkine M. Dose- and time-dependence of L-NAME neuroprotection in transient focal cerebral ischaemia in rats. Br J Pharmacol. 1997; 120: 160–163.[CrossRef][Medline] [Order article via Infotrieve]
34. Asahi M, Asahi K, Wang X, Lo EH. Reduction of tissue plasminogen activator-induced hemorrhage and brain injury by free radical spin trapping after embolic focal cerebral ischemia in rats. J Cereb Blood Flow Metab. 2000; 20: 452–457.[Medline] [Order article via Infotrieve]
35. Kamii H, Mikawa S, Murakami K, Kinouchi H, Yoshimoto T, Reola L, Carlson E, Epstein CJ, Chan PH. Effects of nitric oxide synthase inhibition on brain infarction in SOD-1-transgenic mice following transient focal cerebral ischemia. J Cereb Blood Flow Metab. 1996; 16: 1153–1157.[CrossRef][Medline] [Order article via Infotrieve]
36. Kim GW, Gasche Y, Grzeschik S, Copin JC, Maier CM, Chan PH. Neurodegeneration in striatum induced by the mitochondrial toxin 3-nitropropionic acid: role of matrix metalloproteinase-9 in early blood-brain barrier disruption? J Neurosci. 2003; 23: 8733–8742.
37. Gu Z, Kaul M, Yan B, Kridel SJ, Cui J, Strongin A, Smith JW, Liddington RC, Lipton SA. S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death. Science. 2002; 297: 1186–1190.
38. Rosenberg GA, Navratil M. Metalloproteinase inhibition blocks edema in intracerebral hemorrhage in the rat. Neurology. 1997; 48: 921–926.
39. Asahi M, Wang X, Mori T, Sumii T, Jung JC, Moskowitz MA, Fini ME, Lo EH. Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood-brain barrier and white matter components after cerebral ischemia. J Neurosci. 2001; 21: 7724–7732.
40. Yu ZF, Bruce-Keller AJ, Goodman Y, Mattson MP. Uric acid protects neurons against excitotoxic and metabolic insults in cell culture, and against focal ischemic brain injury in vivo. J Neurosci Res. 1998; 53: 613–625.[CrossRef][Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  P. Li and T. H. Murphy Two-Photon Imaging during Prolonged Middle Cerebral Artery Occlusion in Mice Reveals Recovery of Dendritic Structure after Reperfusion J. Neurosci., November 12, 2008; 28(46): 11970 - 11979. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M C Pustovrh, A Jawerbaum, V White, E Capobianco, R Higa, N Martinez, J J Lopez-Costa, and E Gonzalez The role of nitric oxide on matrix metalloproteinase 2 (MMP2) and MMP9 in placenta and fetus from diabetic rats Reproduction, October 1, 2007; 134(4): 605 - 613. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Pacher, J. S. Beckman, and L. Liaudet Nitric Oxide and Peroxynitrite in Health and Disease Physiol Rev, January 1, 2007; 87(1): 315 - 424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Millan, T. Sobrino, M. Castellanos, F. Nombela, J. F. Arenillas, E. Riva, I. Cristobo, M. M. Garcia, J. Vivancos, J. Serena, et al. Increased Body Iron Stores Are Associated With Poor Outcome After Thrombolytic Treatment in Acute Stroke Stroke, January 1, 2007; 38(1): 90 - 95. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-H. Jung, K. Chu, S.-Y. Ko, S.-T. Lee, D.-I. Sinn, D.-K. Park, J.-M. Kim, E.-C. Song, M. Kim, and J.-K. Roh Early Intravenous Infusion of Sodium Nitrite Protects Brain Against In Vivo Ischemia-Reperfusion Injury Stroke, November 1, 2006; 37(11): 2744 - 2750. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Xie, P. E. Ray, and B. L. Short NF-{kappa}B Activation Plays a Role in Superoxide-Mediated Cerebral Endothelial Dysfunction After Hypoxia/Reoxygenation Stroke, May 1, 2005; 36(5): 1047 - 1052. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. M. Faraci Oxidative Stress: The Curse That Underlies Cerebral Vascular Dysfunction? Stroke, February 1, 2005; 36(2): 186 - 188. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||