Showing papers by "Miki Fujimura published in 1999"

Early appearance of activated matrix metalloproteinase-9 after focal cerebral ischemia in mice: a possible role in blood-brain barrier dysfunction.

Abstract: During cerebral ischemia blood-brain barrier (BBB) disruption is a critical event leading to vasogenic edema and secondary brain injury. Gelatinases A and B are matrix metalloproteinases (MMP) able to open the BBB. The current study analyzes by zymography the early gelatinases expression and activation during permanent ischemia in mice (n = 15). ProMMP-9 expression was significantly (P < 0.001) increased in ischemic regions compared with corresponding contralateral regions after 2 hours of ischemia (mean 694.7 arbitrary units [AU], SD ± 238.4 versus mean 107.6 AU, SD ± 15.6) and remained elevated until 24 hours (mean 745.7 AU, SD ± 157.4). Moreover, activated MMP-9 was observed 4 hours after the initiation of ischemia. At the same time as the appearance of activated MMP-9, we detected by the Evan's blue extravasation method a clear increase of BBB permeability. Tissue inhibitor of metalloproteinase-1 was not modified during permanent ischemia at any time. The ProMMP-2 was significantly (P < 0.05) increased only after 24 hours of permanent ischemia (mean 213.2 AU, SD ± 60.6 versus mean 94.6 AU, SD ± 13.3), and no activated form was observed. The appearance of activated MMP-9 after 4 hours of ischemia in correlation with BBB permeability alterations suggests that MMP-9 may play an active role in early vasogenic edema development after stroke.

Manganese superoxide dismutase mediates the early release of mitochondrial cytochrome C and subsequent DNA fragmentation after permanent focal cerebral ischemia in mice.

Abstract: Recent studies have shown that release of mitochondrial cytochrome c is a critical step in the apoptosis process. We have reported that cytosolic redistribution of cytochrome c in vivo occurred after transient focal cerebral ischemia (FCI) in rats and preceded the peak of DNA fragmentation. Although the involvement of reactive oxygen species in the cytosolic redistribution of cytochrome c in vitro has been suggested, the detailed mechanism by which cytochrome c release is mediated in vivo has not yet been established. Also, the role of mitochondrial oxidative stress in cytochrome c release is unknown. These issues can be addressed using knock-out mutants that are deficient in the level of the mitochondrial antioxidant manganese superoxide dismutase (Mn-SOD). In this study we examined the subcellular distribution of the cytochrome c protein in both wild-type mice and heterozygous knock-outs of the Mn-SOD gene (Sod2 −/+) after permanent FCI, in which apoptosis is assumed to participate. Cytosolic cytochrome c was detected as early as 1 hr after ischemia, and correspondingly, mitochondrial cytochrome c showed a significant reduction 2 hr after ischemia ( p < 0.01). Cytosolic accumulation of cytochrome c was significantly higher in Sod2 −/+ mice compared with wild-type animals ( p < 0.05). N -benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone (z-VAD.FMK), a nonselective caspase inhibitor, did not affect cytochrome c release after ischemia. A significant amount of DNA laddering was detected 24 hr after ischemia and increased in Sod2 −/+ mice. These data suggest that Mn-SOD blocks cytosolic release of cytochrome c and could thereby reduce apoptosis after permanent FCI.

Mitochondrial release of cytochrome c corresponds to the selective vulnerability of hippocampal CA1 neurons in rats after transient global cerebral ischemia.

Abstract: Release of cytochrome c from mitochondria to the cytosol is a critical step in apoptotic cell death after focal cerebral ischemia. The relationship among cytochrome c release, selective vulnerability, and delayed death of hippocampal CA1 neurons after transient global ischemia was examined. Global ischemia was induced by 10 min of bilateral common carotid artery occlusion and hypotension in rats. Cytosolic expression of cytochrome c was evaluated by immunohistochemistry and Western blotting. Apoptosis after global ischemia was also characterized by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL) staining and DNA gel electrophoresis. Immunohistochemistry showed cytosolic cytochrome c-positive cells exclusively in the CA1 subregion of the hippocampus as early as 2 hr after ischemia. Double fluorescent immunostaining confirmed that CA1 neurons and a small number of astrocytes expressed cytochrome c. Western blot analysis revealed a band (15 kDa) of cytochrome c in the cytosolic fraction and a corresponding decrease in the mitochondrial fraction. A significant number of TUNEL-positive cells appeared only in the CA1 pyramidal cell layer of the hippocampus, and DNA gel electrophoresis showed a significant amount of DNA fragmentation 3-5 d after ischemia. Our data provide the first evidence that cytochrome c was released to the cytosol from mitochondria in CA1 neurons after global ischemia and that the release preceded DNA fragmentation. These findings suggest cytochrome c involvement in the delayed death of hippocampal CA1 neurons in rats after transient global ischemia.

Exacerbation of Delayed Cell Injury After Transient Global Ischemia in Mutant Mice With CuZn Superoxide Dismutase Deficiency

Abstract: Background and Purpose—We have demonstrated that copper-zinc superoxide dismutase (CuZn-SOD), a cytosolic isoenzyme of SODs, has a protective role in the pathogenesis of superoxide radical–mediated brain injury. Using mice bearing a disruption of the CuZn-SOD gene (Sod1), the present study was designed to clarify the role of superoxide anion in the pathogenesis of selective vulnerability after transient global ischemia. Methods—Sod1 knockout homozygous mutant mice (Sod1 −/−) with a complete absence of endogenous CuZn-SOD activity, heterozygous mutant mice (Sod1 +/−) with a 50% decrease in the activity, and littermate wild-type mice (male, 35 to 45 g) were subjected to global ischemia. Since the plasticity of the posterior communicating artery (PcomA) has been reported to influence the outcome of hippocampal injury, we assessed the relation between the plasticity of PcomAs and the decrease of regional cerebral blood flow in global ischemia. Results—The fluorescence intensity of hydroethidine oxidation, a m...

Copper-Zinc Superoxide Dismutase Prevents the Early Decrease of Apurinic/Apyrimidinic Endonuclease and Subsequent DNA Fragmentation After Transient Focal Cerebral Ischemia in Mice

Abstract: Background and Purpose—DNA damage and its repair mechanism are thought to be involved in ischemia/reperfusion injury in the brain. We have previously shown that apurinic/apyrimidinic endonuclease (APE/Ref-1), a multifunctional protein in the DNA base excision repair pathway, rapidly decreased after transient focal cerebral ischemia (FCI) before the peak of DNA fragmentation. To further investigate the role of reactive oxygen species in APE/Ref-1 expression in vivo, we examined the expression of APE/Ref-1 and DNA damage after FCI in wild-type and transgenic mice overexpressing copper-zinc superoxide dismutase. Methods—Transgenic mice overexpressing copper-zinc superoxide dismutase and wild-type littermates were subjected to 60 minutes of transient FCI by intraluminal blockade of the middle cerebral artery. APE/Ref-1 protein expression was analyzed by immunohistochemistry and Western blot analysis. DNA damage was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase–mediated uridine 5′-...

Reduction of Apurinic/Apyrimidinic Endonuclease Expression After Transient Global Cerebral Ischemia in Rats: Implication of the Failure of DNA Repair in Neuronal Apoptosis

Abstract: Background and Purpose—To clarify the relationship between apurinic/apyrimidinic endonuclease (APE/Ref-1), a multifunctional protein in the DNA base excision repair pathway, and delayed neuronal cell death associated with apoptosis, we examined the expression of APE/Ref-1 before and after transient global ischemia in rats Methods—Global ischemia was induced by bilateral common carotid artery occlusion and hypotension Expression of the APE/Ref-1 protein was evaluated by Western blot and immunohistochemical analyses Apoptosis after global ischemia was observed by DNA electrophoresis and terminal deoxynucleotidyl transferase–mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) staining Results—Immunohistochemistry showed the nuclear expression of APE/Ref-1 in the control brains Nuclear immunoreactivity of APE/Ref-1 was significantly decreased 2 days after 10 minutes of ischemia in the hippocampal CA1 subregion Western blot analysis of a sample from the normal brains showed a characteristic

Early decrease of apurinic/apyrimidinic endonuclease expression after transient focal cerebral ischemia in mice.

Abstract: The authors examined the protein expression of apurinic/apyrimidinic endonuclease (APE/Ref-1), a multifunctional protein in the DNA base excision repair pathway, before and after transient focal ischemia in mice. Immunohistochemistry showed the nuclear expression of APE/Ref-1 in the entire region of the control brains. Nuclear immunoreactivity was decreased as early as 5 minutes after 60 minutes of ischemia in the ischemic core, which was followed by a significant reduction of APE/Ref-1-positive cells in the entire middle cerebral artery territory. Western blot analysis of the sample from the nonischemic brain showed a characteristic 37-kDa band, which was reduced after ischemia. A significant amount of DNA fragmentation was observed at 24 hours, but not at 4 hours, after ischemia. The authors' data provide the first evidence that APE/Ref-1 rapidly decreases after transient focal ischemia, and that this reduction precedes the peak of DNA fragmentation in the brain regions that are destined to show necrosis and apoptosis. Although further examination is necessary to elucidate the direct relationship between the APE/Ref-1 decrease and ischemic necrosis and apoptosis, our results suggest the possibility that rapid decrease of APE/Ref-1 and the failure of the DNA repair mechanism may contribute to necrosis or apoptosis after transient focal ischemia.

Inhibition of Interleukin-1β Converting Enzyme Family Proteases (Caspases) Reduces Cold Injury-Induced Brain Trauma and DNA Fragmentation in Mice:

Abstract: The authors examined the effect of z-VAD.FMK, an inhibitor that blocks caspase family proteases, on cold injury-induced brain trauma, in which apoptosis as well as necrosis is assumed to play a role. A vehicle alone or with z-VAD.FMK was administered into the cerebral ventricles of mice 15 minutes before and 24 and 48 hours after cold injury. At 24 hours after cold injury, infarction volumes in the z-VAD.FMK-treated animals were significantly smaller than infarction volumes in the vehicle-treated animals, and were further decreased at 72 hours (0.92 ± 1.80 mm3, z-VAD.FMK-treated animals; 7.46 ± 3.53 mm3, vehicle-treated animals; mean ± SD, n = 7 to 8). The amount of DNA fragmentation was significantly decreased in the z-VAD.FMK-treated animals compared with the vehicle-treated animals, as shown by terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling staining and DNA gel electrophoresis. By Western blot analysis, both the proform and activated form of interleukin-1β converting enzyme (caspase 1) were detected in the control brain, and the activated form showed moderate reduction after cold injury-induced brain trauma. These results indicate that caspase inhibitors could reduce cold injury-induced brain trauma by preventing neuronal cell death by DNA damage. The caspase family proteases appear to contribute to the mechanisms of cell death in cold injury-induced brain trauma and to provide therapeutic targets for traumatic brain injury.

Early Decrease of XRCC1, a DNA Base Excision Repair Protein, May Contribute to DNA Fragmentation After Transient Focal Cerebral Ischemia in Mice

Abstract: Background and Purpose —DNA damage and the DNA repair mechanism are known to be involved in ischemia/reperfusion injury in the brain. The x-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in the DNA base excision repair pathway by interacting with DNA ligase III and DNA polymerase β. The present study examined the protein expression of XRCC1 and DNA fragmentation before and after transient focal cerebral ischemia (FCI). Methods —Adult male CD-1 mice were subjected to 60 minutes of FCI by intraluminal blockade of the middle cerebral artery. XRCC1 protein expression was analyzed by immunohistochemistry and Western blot analysis. DNA damage was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase–mediated uridine 5′-triphosphate-biotin nick end-labeling (TUNEL). The spatial relationship between XRCC1 expression and DNA damage was examined by double staining with XRCC1 and TUNEL after FCI. Results —Immunohistochemistry showed the nuclear expression of XRCC1 in all regions of the control brains and that it was predominant in the hippocampus. The XRCC1 level was markedly reduced in the caudate putamen at 10 minutes, further decreased in the entire middle cerebral artery territory at 1 hour, and remained reduced until 4 and 24 hours after FCI. Western blot analysis of the normal control brain showed a characteristic band of 70 kDa, which decreased after FCI. A significant amount of DNA fragmentation was detected by DNA gel electrophoresis 24 hours but not 4 hours after FCI. Double staining showed that the neurons that lost XRCC1 immunoreactivity became TUNEL positive. Conclusions —These results suggest that the early decrease of XRCC1 and the failure of the DNA repair mechanism may contribute, at least in part, to DNA fragmentation after FCI.

Studies of Neuronal Necrosis and Apoptosis after Global Cerebral Ischemia in Superoxide Dismutase Transgenic and Knockout Mutants

Abstract: We have demonstrated that copper zinc-superoxide dismutase (CuZnSOD), a cytosolic isoenzyme of SOD, has a protective role in the pathogenesis of superoxide-radical-mediated brain injury, including focal transient ischemia/reperfusion and excitotoxic neuronal injury in culture. The development of mice that overexpress and underexpress the CuZnSOD-deficient gene (Sod1) has provided a model for assessing the role of CuZnSOD in ischemic brain injury. Employing both Sod1 overexpressors and knockout mutant mice, the present study is designed to clarify whether CuZnSOD plays a protective role in the pathogenesis of hippocampal injury after transient global ischemia and reperfusion. To study the role of oxidative stress on the vulnerability of hippocampal CA1 neurons in Sod1 transgenic (Tg) or knockout mutant mice, a reliable and reproducible model of transient global cerebral ischemia has been developed that is based on plasticity of the posterior communicating artery. Our results indicate that neuronal injury in the hippocampus was markedly reduced in Sod1 Tg mice versus non-Tg mice 3 days after 5 min of ischemia. In contrast, significant exacerbation of neuronal injury was observed in Sod1 (-/-) knockout mice compared with wild-type littermates. However, no difference was observed at 1 day in either Sod1 Tg or knockout mutant mice. These data indicate that superoxide radicals play an important role in the pathogenesis of delayed neuronal death in the vulnerable hippocampus CA1 subregion following transient global cerebral ischemia.