Yuiko Morita-Fujimura

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

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 Evans 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.

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 vivooccurred 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 cin 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.

Cytosolic Redistribution of Cytochrome C after Transient Focal Cerebral Ischemia in Rats

Recent in vitro cell-free studies have shown that cytochrome c release from mitochondria is a critical step in the apoptotic process. The present study examined the expression of cytochrome c protein after transient focal cerebral ischemia in rats, in which apoptosis was assumed to contribute to the expansion of the ischemic lesion. In situ labeling of DNA breaks in frozen sections after 90 minutes of middle cerebral artery (MCA) occlusion showed a significant number of striatal and cortical neurons, which were maximized at 24 hours after ischemia, exhibiting chromatin condensation, nuclear segmentation, and apoptotic bodies. Cytosolic localization of cytochrome c was detected immunohistochemically in the ischemic area as early as 4 hours after 90 minutes of MCA occlusion. Western blot analysis of the cytosolic fraction revealed a strong single 15-kDa band, characteristic of cytochrome c, only in the samples from the ischemic hemisphere. Western blot analysis of the mitochondrial fraction showed a significant amount of mitochondrial cytochrome c in nonischemic brain, which was decreased in ischemic brain 24 hours after ischemia. These results provide the first evidence that cytochrome c is being released from mitochondria to the cytosol after transient focal ischemia. Although further evaluation is necessary to elucidate its correlation with DNA fragmentation, our results suggest the possibility that cytochrome c release may play a role in DNA-damaged neuronal cell death after transient focal cerebral ischemia in rats.

Early appearance of activated matrix metalloproteinase-9 and blood-brain barrier disruption in mice after focal cerebral ischemia and reperfusion.

Blood-brain barrier (BBB) disruption is thought to play a critical role in the pathophysiology of ischemia/reperfusion. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that can degrade all the components of the extracellular matrix when they are activated. Gelatinase A (MMP-2) and gelatinase B (MMP-9) are able to digest the endothelial basal lamina, which plays a major role in maintaining BBB impermeability. The present study examined the expression and activation of gelatinases before and after transient focal cerebral ischemia (FCI) in mice. Adult male CD1 mice were subjected to 60 min FCI and reperfusion. Zymography was performed from 1 to 23 h after reperfusion using the protein extraction method with detergent extraction and affinity-support purification. MMP-9 expression was also examined by both immunohistochemistry and Western blot analysis, and tissue inhibitors to metalloproteinase-1 was measured by reverse zymography. The BBB opening was evaluated by the Evans blue extravasation method. The 88-kDa activated MMP-9 was absent from the control specimens, while it appeared 3 h after transient ischemia by zymography. At this time point, the BBB permeability alteration was detected in the ischemic brain. Both pro-MMP-9 (96 kDa) and pro-MMP-2 (72 kDa) were seen in the control specimens, and were markedly increased after FCI. A significant induction of MMP-9 was confirmed by both immunohistochemistry and Western blot analysis. The early appearance of activated MMP-9, associated with evidence of BBB permeability alteration, suggests that activation of MMP-9 contributes to the early formation of vasogenic edema after transient FCI.

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

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 measurement of ethidium fluorescence for superoxide radicals, was increased in mutant mice 1 day after both 5 and 10 minutes of global ischemia, compared with wild-type mice. Hippocampal injury in the PcomA hypoplastic brains showed significant exacerbation in mutant mice compared with wild-type littermates 3 days after 5 minutes of global ischemia, although a marked difference was not observed at 1 day. CONCLUSIONS These data suggest that superoxide radicals play an important role in the pathogenesis of delayed injury in the vulnerable hippocampal CA1 subregion after transient global ischemia.

Mild Hypothermia Attenuates Cytochrome C Release but Does Not Alter Bcl-2 Expression or Caspase Activation After Experimental Stroke

Mild hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not well known. The authors previously found that hypothermia reduces the density of apoptotic cells, but it is not certain whether temperature alters associated biochemical events. Mitochondrial release of cytochrome c has recently been shown to be a key trigger in caspase activation and apoptosis via the intrinsic pathway. Using a model of transient focal cerebral ischemia, the authors determined whether mild hypothermia altered expression of Bcl-2 family proteins, mitochondrial release of cytochrome c, and caspase activation. Mild hypothermia significantly decreased the amount of cytochrome c release 5 hours after the onset of ischemia, but mitochondrial translocation of Bax was not observed until 24 hours. Mild hypothermia did not alter Bcl-2 and Bax expression, and caspase activation was not observed. The present study provides the first evidence that intraischemic mild hypothermia attenuates the release of cytochrome c in the brain, but does not appear to affect other biochemical aspects of the intrinsic apoptotic pathway. They conclude that necrotic processes may have been interrupted to prevent cytochrome c release, and that the ameliorative effect of mild hypothermia may be a result of maintaining mitochondrial integrity. Furthermore, the authors show it is unlikely that mild hypothermia alters the intrinsic apoptotic pathway.

Overexpression of Copper and Zinc Superoxide Dismutase in Transgenic Mice Prevents the Induction and Activation of Matrix Metalloproteinases after Cold Injury-Induced Brain Trauma

Matrix metalloproteinases (MMPs), a family of proteolytic enzymes which degrade the extracellular matrix, are implicated in blood—brain barrier disruption, which is a critical event leading to vasogenic edema. To investigate the role of reactive oxygen species (ROS) in the expression of MMPs in vasogenic edema, the authors measured gelatinase activities before and after cold injury (CI) using transgenic mice that overexpress superoxide dismutase-1. A marked induction of pro-gelatinase B (pro-MMP-9) was seen 2 hours after CI and was maximized at 12 hours in wild-type mice. The pro-MMP-9 level was significantly lower in transgenic mice 4 hours (P < 0.001) and 12 hours (P < 0.05) after CI compared to wild-type mice. The activated MMP-9 was detected from 6 to 24 hours after injury. A mild induction of pro-gelatinase A (pro-MMP-2) was seen at 6 hours and was sustained until 7 days. In contrast, the activated form of MMP-2 appeared at 24 hours, was maximized at 7 days, and was absent in transgenic mice. Western blot analysis showed that the tissue inhibitors of metalloproteinases were not modified after CI. The results suggest that ROS production after CI may contribute to the induction and/or activation of MMPs and could thereby exacerbate endothelial cell injury and the development of vasogenic edema after injury.

Manganese Superoxide Dismutase Affects Cytochrome c Release and Caspase-9 Activation after Transient Focal Cerebral Ischemia in Mice

Release of cytochrome c from mitochondria to cytosol is a critical step in the mitochondrial-dependent signaling pathways of apoptosis. The authors have reported that manganese superoxide dismutase (Mn-SOD) attenuated cytochrome c release and apoptotic cell death after focal cerebral ischemia (FCI). To investigate downstream to the cytochrome c-dependent pathway, the authors examined caspase-9 activation after transient FCI by immunohistochemistry and Western blotting in both wild-type and Sod2 −/+ mice. Mice were subjected to 60 minutes of middle cerebral artery occlusion followed by 1, 2, 4, or 24 hours of reperfusion. Two hours after reperfusion, cytochrome c and caspase-9 were observed in the cytosol and significantly increased in Sod2 −/+ mutants compared with wild-type mice as shown by Western blotting. Immunofluorescent double labeling for cytochrome c and caspase-9 showed cytosolic cytochrome c 1 hour after transient FCI. Cleaved caspase-9 first appeared in the cytosol at 2 hours and colocalized with cytochrome c. Terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick and labeling (TUNEL) showed significant increase of positive cells in Sod2 −/+ mice compared with the wild-type in the cortex, but not in the caudate putamen. The current study revealed Mn-SOD might affect cytochrome c translocation and downstream caspase activation in the mitochondrial-dependent cell death pathway after transient FCI.

Increased Cytochrome c–Mediated DNA Fragmentation and Cell Death in Manganese–Superoxide Dismutase–Deficient Mice After Exposure to Subarachnoid Hemolysate

Background and Purpose — We sought to investigate the mechanisms for oxidative injury caused by subarachnoid hemolysate, a pro-oxidant. Methods — Injection of 50 &mgr;L of subarachnoid hemolysate or saline was performed in CD1 mice (n=75), mutant mice deficient in Mn–superoxide dismutase (Sod2 +/−; n=23), and their wild-type littermates (n=23). Subcellular location of cytochrome c was studied by immunocytochemistry, immunofluorescence, and immunoblotting of cellular fractions. DNA fragmentation was assessed though DNA laddering and terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end-labeling (TUNEL). Cell death was examined through basic histology. Results — Cytochrome c immunoreactivity was present in the cytosol of neurons at 2 hours after hemolysate injection and increased by 4 hours compared with saline-injected animals (P <0.02). Cytosolic cytochrome c was more abundant in Sod2 +/− mutants. DNA fragmentation was evident at 24 hours, but not 4 hours, after hemolysate injection as determined by DNA laddering and TUNEL staining (P <0.02). DNA fragmentation colocalized to cells with cytosolic cytochrome c and iron. In Sod2 +/− mutants, the extent of fragmentation was increased as determined by TUNEL staining (52% increase;P <0.02) and DNA laddering (optical density=0.819 versus 0.391;P <0.01). Cell death was evident on basic histology as early as 4 hours after hemolysate injection. No cell death was evident in controls. In Sod2 +/− mutants, cell death was increased by 51% compared with wild-type littermates (P <0.05). Conclusions — These results demonstrate that subarachnoid blood products are associated with the presence of cytochrome c in the cytosol and subsequent cell death in neurons. It appears that Mn–superoxide dismutase plays a role in preventing cell death after exposure to subarachnoid blood products.

RELEASE OF MITOCHONDRIAL CYTOCHROME C AND DNA FRAGMENTATION AFTER COLD INJURY-INDUCED BRAIN TRAUMA IN MICE: POSSIBLE ROLE IN NEURONAL APOPTOSIS

Recent studies have shown that release of mitochondrial cytochrome c is a critical step in the apoptosis process. In this study, we examined the subcellular distribution of the cytochrome c protein after cold injury (CI), in which apoptosis is assumed to participate. Western blotting and immunohistochemistry showed cytosolic cytochrome c as early as 1 h after CI, and correspondingly, there was a reduction in mitochondrial cytochrome c after injury. Neuronal distribution of cytosolic cytochrome c was shown by double staining with a neuronal nuclear marker by immunohistochemistry. A significant amount of DNA laddering was detected 4 h after CI, and increased in a time-dependent manner. These data suggest that early cytochrome c release from mitochondria may contribute to apoptosis induction after traumatic brain injury.