RÉSUMÉ
Matrix metalloproteinases (MMPs) are a family of more than twenty secreted and cell-surface endopeptidases. Among them, MMP2, MMP3 and MMP9 are involved in blood-brain barrier injury and neuronal death after cerebral ischaemia. On the other hand, very little is known about the expression of the other secreted MMPs. Herein, we compared the global changes in MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP12 and MMP13, and their endogenous inhibitors TIMP1 and TIMP2, both at the mRNA and protein levels, during the hyperacute (6 h), acute (24 h) and subacute (72 h) stages following transient focal cerebral ischaemia and treatment with recombinant tissue plasminogen activator (rtPA). We observed a significant increase in MMP1, MMP2, MMP9, MMP10, MMP13 and TIMP1 levels during the acute stage of reperfusion, which was further amplified during the subacute stage for MMP1, MMP2, MMP10 and TIMP1. In general, no change of MMP3, MMP7, MMP8, MMP12 and TIMP2 was observed. However, rtPA treatment induced a rapid increase in MMP1/TIMP2, MMP2/TIMP2, MMP8/TIMP2 and MMP9/TIMP2 ratios during the hyperacute stage of reperfusion compared to saline treatment, which may have potential implications in the early disruption of the blood-brain barrier after rtPA treatment.
Sujet(s)
Encéphalopathie ischémique/enzymologie , Encéphale/enzymologie , Secreted matrix metalloproteinases/métabolisme , Accident vasculaire cérébral/enzymologie , Inhibiteur tissulaire des métalloprotéinases/métabolisme , Animaux , Encéphale/effets des médicaments et des substances chimiques , Encéphalopathie ischémique/traitement médicamenteux , Encéphalopathie ischémique/génétique , Modèles animaux de maladie humaine , Fibrinolytiques/pharmacologie , Régulation de l'expression des gènes , Régulation de l'expression des gènes codant pour des enzymes , Mâle , Secreted matrix metalloproteinases/génétique , Souris de souche-129 , ARN messager/métabolisme , Accident vasculaire cérébral/traitement médicamenteux , Accident vasculaire cérébral/génétique , Traitement thrombolytique , Facteurs temps , Inhibiteur tissulaire des métalloprotéinases/génétique , Activateur tissulaire du plasminogène/pharmacologie , Régulation positiveRÉSUMÉ
Recent in vitro evidence suggests that T-type Ca(2+) channels are implicated in the mechanisms of ischemia-induced delayed neuronal cell death. The aim of this work was to study the neuroprotective potential of mibefradil and pimozide, both T-type Ca(2+) channel inhibitors, in an in vivo rat model of global ischemia. We performed blinded and randomized placebo vs. treatment experiments using 57 animals to test mibefradil and fourteen animals to test pimozide. Each treated animal received a single stereotactic intraventricular injection of mibefradil or intraperitoneal injection of pimozide prior to transient global cerebral ischemia. The primary endpoint was the number of neurons surviving in the CA1 region 72 h after insult as evaluated by NeuN-labeled cell counts. All physiological variables monitored immediately before and after ischemic insult were equivalent between all groups. Surviving neurons in the CA1 region were significantly more frequent in the treated groups compared to the placebo group (mibefradil: 36.8 ± 2.8 cells in a 200 × 100 µm counting area vs. placebo: 25.2 ± 3.2 [P < 0.01]; pimozide: 39.4 ± 1.12 vs. placebo: 27.8 ± 0.7 [P < 0.0001]). Thus, administration of mibefradil or pimozide effectively prevents neuronal death after ischemia in a rat model of global ischemia. This study provides further support for a neuroprotective effect of T-type Ca(2+) current inhibition during ischemia.
Sujet(s)
Région CA1 de l'hippocampe/effets des médicaments et des substances chimiques , Inhibiteurs des canaux calciques/usage thérapeutique , Accident ischémique transitoire/traitement médicamenteux , Mibéfradil/usage thérapeutique , Neurones/effets des médicaments et des substances chimiques , Neuroprotecteurs/usage thérapeutique , Pimozide/usage thérapeutique , Animaux , Encéphale/effets des médicaments et des substances chimiques , Encéphale/métabolisme , Région CA1 de l'hippocampe/anatomopathologie , Inhibiteurs des canaux calciques/métabolisme , Inhibiteurs des canaux calciques/pharmacocinétique , Inhibiteurs des canaux calciques/pharmacologie , Canaux calciques de type T/effets des médicaments et des substances chimiques , Canaux calciques de type T/génétique , Canaux calciques de type T/métabolisme , Mort cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/effets des médicaments et des substances chimiques , Phénomènes électrophysiologiques/effets des médicaments et des substances chimiques , Phénomènes électrophysiologiques/physiologie , Glucose/déficit , Cellules HEK293 , Humains , Hypoxie , Accident ischémique transitoire/sang , Accident ischémique transitoire/mortalité , Accident ischémique transitoire/anatomopathologie , Acide lactique/sang , Mâle , Potentiels de membrane/physiologie , Mibéfradil/métabolisme , Mibéfradil/pharmacocinétique , Mibéfradil/pharmacologie , Neurones/anatomopathologie , Neuroprotecteurs/métabolisme , Neuroprotecteurs/pharmacocinétique , Neuroprotecteurs/pharmacologie , Pimozide/pharmacologie , Rats , Rat Sprague-Dawley , Analyse de survie , Techniques de culture de tissus , TransfectionRÉSUMÉ
Cerebral oedema is a potentially lethal complication of brain infarction. Ischemia, by altering membrane ionic pump function, induces cell swelling and cytotoxic oedema. It also initiates early oxidative and inflammatory cascades leading to blood-brain barrier disruption, vasogenic oedema and haemorrhagic transformation. The mechanisms of blood-brain barrier disruption involve endothelial cell activation and endothelial basal membrane degradation by matrix metalloproteinases. Reperfusion by tissue plasminogen activators is the only treatment improving stroke prognosis. This treatment also increases vasogenic oedema and the risk of symptomatic haemorrhagic transformation, reducing the benefit of reperfusion. Experimental studies suggest that the inhibition of blood-brain barrier proteolysis reduces vasogenic oedema and the risk of haemorrhage. This recent progress in the understanding of blood-brain barrier disruption during ischaemia brings forward new therapeutic strategies using agents capable of interfering with the ischaemic cascade in order to increase the therapeutic window between the onset of ischaemia and thrombolytic reperfusion.
Sujet(s)
Barrière hémato-encéphalique/physiologie , Encéphalopathie ischémique/physiopathologie , Oedème cérébral/étiologie , Oedème cérébral/physiopathologie , Encéphalopathie ischémique/étiologie , Infarctus cérébral/complications , Humains , Reperfusion , Activateur tissulaire du plasminogène/usage thérapeutiqueRÉSUMÉ
The blood-brain barrier (BBB) is a complex biological system that consists of endothelial cells, pericytes and astrocytes, which are involved in the induction and maintenance of its physiological and ultrastructural characteristics. The BBB plays a primordial role in isolating the cerebral parenchyma as well as in controlling brain homeostasis by its selective permeability to nutriments and other molecules flowing through the cerebral microcapillaries. A better knowledge of this system is crucial in order to improve the efficiency of brain penetration by drugs, and in order to prevent BBB opening, leading to brain edema, in physiopathological situations such as brain ischemia, trauma or inflammatory processes.
Sujet(s)
Barrière hémato-encéphalique/physiologie , Encéphale/anatomie et histologie , Encéphale/physiologie , Animaux , Astrocytes/physiologie , Encéphale/cytologie , Endothélium vasculaire/cytologie , Endothélium vasculaire/physiologie , Humains , Péricytes/physiologie , Jonctions serrées/physiologieRÉSUMÉ
Oxidative stress generated during stroke is a critical event leading to blood-brain barrier (BBB) disruption with secondary vasogenic edema and hemorrhagic transformation of infarcted brain tissue, restricting the benefit of thrombolytic reperfusion. In this study, the authors demonstrate that ischemia-reperfusion-induced BBB disruption in mice deficient in copper/zinc-superoxide dismutase (SOD1) was reduced by 88% ( P < 0.0001) and 73% ( P < 0.01), respectively, after 3 and 7 hours of reperfusion occurring after 1 hour of ischemia by the inhibition of matrix metalloproteinases. Accordingly, the authors show that local metalloproteinase-generated proteolytic imbalance is more intense in ischemic regions of SOD1 mice than in wild-type litter mates. Moreover, active in situ proteolysis is, for the first time, demonstrated in ischemic leaking capillaries that produce reactive oxygen species. By showing that oxidative stress mediates BBB disruption through metalloproteinase activation in experimental ischemic stroke, this study provides a new target for future therapeutic strategies to prevent BBB disruption and potentially reperfusion-triggered intracerebral hemorrhage.
Sujet(s)
Barrière hémato-encéphalique/physiologie , Accident ischémique transitoire/métabolisme , Inhibiteurs de métalloprotéinases matricielles , Stress oxydatif/physiologie , Animaux , Oedème cérébral/métabolisme , Collagenases/métabolisme , Antienzymes/pharmacologie , Proenzymes/antagonistes et inhibiteurs , Proenzymes/métabolisme , Gelatinases/antagonistes et inhibiteurs , Gelatinases/métabolisme , Matrix metalloproteinase 2/métabolisme , Matrix metalloproteinase 9/métabolisme , Matrix metalloproteinases/métabolisme , Metalloendopeptidases/antagonistes et inhibiteurs , Metalloendopeptidases/métabolisme , Souris , Lignées consanguines de souris , Souris knockout , Accident vasculaire cérébral/métabolisme , Superoxide dismutase/génétique , Superoxydes/métabolismeRÉSUMÉ
Mitochondrial cytochrome c translocation to the cytosol initiates the mitochondrial-dependent apoptotic pathway. This event has not been previously reported in traumatic brain injury (TBI). The authors determined the expression of cytochrome c in cytosolic and mitochondrial fractions after severe TBI produced by the controlled cortical impact model in the mouse. One hour after trauma there was an increase in cytosolic cytochrome c immunoreactivity. The increases in cytosolic cytochrome c preceded DNA fragmentation, which started at 4 hours. Western blots of mitochondrial and cytosolic fractions confirmed that there was a translocation of cytochrome c from the mitochondria after TBI. Mice deficient in manganese superoxide dismutase (MnSOD) showed an increased loss of mitochondrial cytochrome c after trauma, but less apoptotic cell death 4 and 24 hours after injury compared with wild-type control mice. However, the overall cell death was increased in MnSOD mice, as illustrated by a larger cortical lesion in these animals. The results show that cytochrome c is released from the mitochondria after severe TBI partly by a free radical-dependent mechanism, and that massive mitochondrial cytochrome c release is a predictor of necrotic cell death rather than apoptosis.
Sujet(s)
Lésions encéphaliques/métabolisme , Cytochromes de type c/métabolisme , Mitochondries/métabolisme , Animaux , Lésions encéphaliques/anatomopathologie , Mâle , Souris , Mitochondries/anatomopathologie , Stress oxydatifRÉSUMÉ
Mouse astrocytes deficient in the mitochondrial form of superoxide dismutase do not grow in culture under 20% atmospheric O2 levels. By flow cytometry, immunocytochemistry, and enzymatic analysis we have shown that the oxygen block of cell division is due to a decrease in the number of cells entering the S phase of the cell cycle and is concomitant with higher DNA oxidation and impairment of mitochondrial functions. Seeding the cells under 5% O2 until the cultures become confluent can circumvent this problem. An initial hypoxic environment increases the resistance of manganese superoxide dismutase-deficient astrocytes to superoxide radicals artificially produced by paraquat treatment, preserves respiratory activity, and allows normoxic division during a subsequent passage. DNA oxidation is then not higher than in wild-type control cells. However, the adaptation of the cells is not due to compensation by other enzymes of the antioxidant defense system and is specific to cells totally lacking manganese superoxide dismutase. Alteration of the phenotype by prior hypoxia exposure in the SOD2-deficient mutant provide a unique model to study adaptative mechanisms of cellular resistance to oxygen toxicity.
Sujet(s)
Astrocytes/physiologie , Hypoxie cellulaire/physiologie , Mitochondries/métabolisme , Stress oxydatif/physiologie , Superoxide dismutase/métabolisme , Animaux , Astrocytes/cytologie , Astrocytes/enzymologie , Catalase/métabolisme , Division cellulaire/physiologie , Taille de la cellule , Cellules cultivées , Croisements génétiques , ADN/biosynthèse , Glutathione peroxidase/métabolisme , Homozygote , Isoenzymes/déficit , Isoenzymes/génétique , Isoenzymes/métabolisme , Souris , Souris knockout , Superoxide dismutase/déficit , Superoxide dismutase/génétiqueRÉSUMÉ
BACKGROUND AND PURPOSE: Subarachnoid hemolysate (SAH) has been associated with oxidative brain injury, cell death, and apoptosis. We hypothesized that over-expression of CuZn-superoxide dismutase (CuZn-SOD) would protect against injury after SAH, whereas reduction of its expression would exacerbate injury. METHODS: Saline (n=16) or hemolysate (n=50) was injected into transgenic mice overexpressing CuZn-SOD (SOD1-Tg), CuZn-SOD heterozygous knockout mutants (SOD1+/-), and wild-type littermates (Wt). Mice were killed at 24 hours. Stress gene induction was evaluated by immunocytochemistry and Western blotting for hemeoxygenase-1 and heat shock protein 70. Apoptosis was evaluated by 3'-OH nick end-labeling and DNA gel electrophoresis. Cell death was quantified through histological assessment after cresyl violet staining. RESULTS: Histological assessment demonstrated neocortical cell death in regions adjacent to the blood injection. Overall cell death was reduced 43% in SOD1-Tg mutants (n=6) compared with Wt littermates (n=6; P<0.02). In contrast, cell death was increased >40% in SOD1+/- mutants (n=6; P<0.05). Both hemeoxygenase-1 and heat shock protein 70 were induced after SAH. Apoptosis was also present after SAH, as evidenced by 3'-OH end-labeling and DNA laddering. However, the degree of stress gene induction and apoptosis did not vary between Wt, SOD1-Tg, and SOD1+/- mice. CONCLUSIONS: The extent of CuZn-SOD expression in the cytosol correlates with cell death after exposure to SAH in a manner separate from apoptosis. Overexpression of CuZn-SOD may potentially be an avenue for therapeutic intervention.
Sujet(s)
Hémoglobines , Hémolyse , Hémorragie meningée/enzymologie , Superoxide dismutase/biosynthèse , Animaux , Apoptose/génétique , Technique de Western , Encéphale/métabolisme , Encéphale/anatomopathologie , Mort cellulaire/génétique , Fragmentation de l'ADN/génétique , Modèles animaux de maladie humaine , Régulation de l'expression des gènes , Protéines du choc thermique HSP70/métabolisme , Protéines du choc thermique/génétique , Heme oxygenase (decyclizing)/métabolisme , Heme oxygenase-1 , Hémolyse/génétique , Hétérozygote , Humains , Immunohistochimie , Méthode TUNEL , Mâle , Protéines membranaires , Souris , Souris knockout , Souris transgéniques , Stress oxydatif/génétique , Hémorragie meningée/génétique , Superoxide dismutase/déficit , Superoxide dismutase/génétique , Activation de la transcriptionRÉSUMÉ
Spreading depression (SD) is a wave of sustained depolarization challenging the energy metabolism of cells without causing irreversible damage. SD is a major mechanism of gene induction that takes place in cortical injury, including ischemia. We studied the role of oxygen radicals in SD-induced c-fos and cyclooxygenase-2 (COX-2) induction using transgenic (Tg) mice that overexpress copper/zinc-superoxide dismutase (SOD1). The frequency, amplitude and duration of SD waves were similar in the Tg mice and wild-type littermates. c-fos and COX-2 mRNAs were strongly induced 1 and 4 h after SD. The induction of both genes was slightly but significantly less at 4 h in the Tg mice. The results indicate that even a mild, noninjurious metabolic stimulation increases the concentration of oxygen radicals to the level that contributes to gene expression.
Sujet(s)
Cortex cérébral/métabolisme , Dépression corticale envahissante/physiologie , Expression des gènes/physiologie , Gènes fos/physiologie , Prostaglandin-endoperoxide synthases/métabolisme , Superoxide dismutase/génétique , Animaux , Cyclooxygenase 2 , Humains , Isoenzymes/métabolisme , Protéines membranaires , Souris , Souris transgéniques , ARN messager/métabolisme , Superoxide dismutase-1RÉSUMÉ
There are two types of intracellular superoxide dismutases: the mitochondrial manganese SOD (MnSOD) and the cytoplasmic copper/zinc SOD (CuZnSOD). Mutant mice that lack MnSOD die shortly after birth because of cardiomyopathy and mitochondrial injury. In order to verify if CuZnSOD could compensate for MnSOD deficiency, a new mutant mouse that overexpresses CuZnSOD but is deficient in MnSOD was generated by crossing MnSOD knockout mice with CuZnSOD transgenic mice. CuZnSOD activity was significantly increased in the blood, brain, liver, and heart of MnSOD knockout, CuZnSOD transgenic mice when compared with nontransgenic mice. However, overexpression of CuZnSOD did not prevent neonatal lethality in mice that lack MnSOD, nor did it prevent oxidative aconitase inactivation, nor did it rescue MnSOD-deficient astrocytes in culture. Based on our findings, which emphasize the strong enzymatic compartmentalization of CuZnSOD and MnSOD, therapeutic antioxidant strategies should consider the final intracellular localization of the antioxidant used, especially when those strategies are directed against mitochondrial diseases.
Sujet(s)
Superoxide dismutase/génétique , Superoxide dismutase/métabolisme , Animaux , Animaux nouveau-nés , Astrocytes/cytologie , Astrocytes/enzymologie , Séquence nucléotidique , Division cellulaire , Cellules cultivées , Amorces ADN/génétique , Femelle , Expression des gènes , Mâle , Souris , Souris knockout , Souris transgéniques , Stress oxydatif , Superoxide dismutase/déficit , Distribution tissulaireRÉSUMÉ
Glutamate neurotoxicity in brain is normally prevented by rapid uptake of glutamate by astrocytes. Increased expression of Cu,Zn superoxide dismutase (SOD1) can increase resistance to cerebral ischemia and other oxidative insults, but the cellular mechanisms by which this occurs are not well established. Here we examine whether increased SOD1 expression can attenuate inhibition of astrocyte glutamate uptake by reactive oxygen species. Primary cortical astrocyte cultures were prepared from transgenic mice that overexpress human SOD1 and from nontransgenic littermate controls. Glutamate uptake was assessed after exposure of these cultures to xanthine oxidase plus hypoxanthine, an extracellular superoxide generating system, or to menadione, which generates superoxide in the cytosol. These treatments produced dose-dependent reductions in astrocyte glutamate uptake, and the reductions were significantly attenuated in the SOD1 transgenic astrocytes. A specific effect of reactive oxygen species on glutamate transporters was suggested by the much smaller inhibitory effects of xanthine oxidase/hypoxanthine and menadione on GABA uptake than on glutamate uptake. These findings suggest that the cerebroprotective effects of increased SOD1 expression during cerebral ischemia-reperfusion could be mediated in part by astrocyte glutamate transport.
Sujet(s)
Astrocytes/métabolisme , Cortex cérébral/métabolisme , Acide glutamique/métabolisme , Superoxide dismutase/métabolisme , Animaux , Astrocytes/effets des médicaments et des substances chimiques , Transport biologique , Cortex cérébral/cytologie , Humains , Hypoxanthine/pharmacologie , L-Lactate dehydrogenase/analyse , Souris , Souris transgéniques , Espèces réactives de l'oxygène/métabolisme , Superoxide dismutase/génétique , Superoxide dismutase-1 , Superoxydes/métabolisme , Vitamine K/pharmacologie , Xanthine oxidase/pharmacologieRÉSUMÉ
Overexpression of Cu,Zn superoxide dismutase (SOD1) reduces ischemic injury in some stroke models but exacerbates injury in a neonatal stroke model and in other settings. The current study used a SOD1 transgenic (SOD1-Tg) murine cortical culture system, derived from the same mouse strain previously used for the stroke models, to identify conditions that determine whether SOD1 overexpression in neurons is protective or detrimental. The nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine, spermine-NONOate, and diethylamine-NONOate produced less death in SOD1-Tg neurons than in wild-type neurons (p < 0.01). Also, NO produced markedly less 3-nitrotyosine in SOD1-Tg cells. In contrast, the superoxide generator menadione produced significantly greater death and nearly twice as much 2'7'-dichlorofluorescein fluorescence in SOD1-Tg neurons than in wild-type neurons, suggesting increased peroxide formation in the SOD1-Tg cells. No significant difference was observed in the vulnerability of the two cell types to H2O2, the product of the SOD reaction. Overexpression of SOD1 also had no effect on neuronal vulnerability to glutamate, N-methyl-D-aspartate, or kainate. These observations suggest that SOD1 overexpression can reduce neuronal death under conditions where peroxynitrite formation is a significant factor, but may exacerbate neuronal death under conditions of rapid intracellular superoxide formation or impaired H2O2 disposal.
Sujet(s)
Neurotoxines/métabolisme , Monoxyde d'azote/métabolisme , Espèces réactives de l'oxygène/métabolisme , Superoxide dismutase/génétique , Animaux , Astrocytes/cytologie , Mort cellulaire/physiologie , Cellules cultivées , Cortex cérébral/cytologie , Agonistes des acides aminés excitateurs/pharmacologie , Régulation de l'expression des gènes codant pour des enzymes/effets des médicaments et des substances chimiques , Régulation de l'expression des gènes codant pour des enzymes/physiologie , Acide glutamique/métabolisme , Acide glutamique/pharmacologie , Humains , Hydrazines/pharmacologie , Acide kaïnique/pharmacologie , Souris , Souris transgéniques , N-Méthyl-aspartate/pharmacologie , Neurones/composition chimique , Neurones/cytologie , Neurones/enzymologie , Donneur d'oxyde nitrique/pharmacologie , Oxydes d'azote , Pénicillamine/analogues et dérivés , Pénicillamine/pharmacologie , Spermine/analogues et dérivés , Spermine/pharmacologie , Tyrosine/analogues et dérivés , Tyrosine/analyse , Vitamine K/pharmacologieRÉSUMÉ
Excitotoxicity is implicated in the pathogenesis of several neurologic diseases, such as chronic neurodegenerative diseases and stroke. Recently, it was reported that excitotoxicity has a relationship to apoptotic neuronal death, and that the mitochondrial toxin, 3-nitropropionic acid (3-NP), could induce apoptosis in the striatum. Although striatal lesions produced by 3-NP could develop through an excitotoxic mechanism, the exact relationship between apoptosis induction and excitotoxicity after 3-NP treatment is still not clear. The authors investigated the role of excitotoxicity and oxidative stress on apoptosis induction within the striatum after intraperitoneal injection of 3-NP. The authors demonstrated that removal of the corticostriatal glutamate pathway reduced superoxide production and apoptosis induction in the denervated striatum of decorticated mice after 3-NP treatment. Also, the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, prevented apoptosis in the striatum after 3-NP treatment for 5 days, whereas the non-NMDA receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline, was ineffective. The authors also evaluated the initial type of neuronal death by 3-NP treatment for different durations from 1 to 5 days. In early striatal damage, apoptotic neuronal death initially occurred after 3-NP treatment. Our data show that excitotoxicity related to oxidative stress initially induces apoptotic neuronal death in mouse striatum after treatment with 3-NP.
Sujet(s)
Apoptose/physiologie , Corps strié/physiopathologie , Neurotoxines/métabolisme , Stress oxydatif/physiologie , Propionates/pharmacologie , Animaux , Caspases/métabolisme , Corps strié/métabolisme , Corps strié/anatomopathologie , Décérébration/métabolisme , Maléate de dizocilpine/pharmacologie , Antagonistes des acides aminés excitateurs/pharmacologie , Femelle , Méthode TUNEL , Souris , Souris de lignée C57BL , Mitochondries/effets des médicaments et des substances chimiques , Système nerveux/effets des médicaments et des substances chimiques , Système nerveux/physiopathologie , Composés nitrés , Propionates/intoxication , Quinoxalines/pharmacologie , Récepteurs du N-méthyl-D-aspartate/antagonistes et inhibiteurs , Superoxydes/métabolismeRÉSUMÉ
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-l. 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. Key Words: Metalloproteinases-Brain-Vasogenic edema-Reactive oxygen species-Superoxide dismutase.
Sujet(s)
Lésions encéphaliques/enzymologie , Basse température , Matrix metalloproteinase 2/métabolisme , Matrix metalloproteinase 9/métabolisme , Superoxide dismutase/métabolisme , Animaux , Activation enzymatique , Induction enzymatique , Proenzymes/métabolisme , Gelatinases/métabolisme , Mâle , Matrix metalloproteinase 1/métabolisme , Metalloendopeptidases/métabolisme , Souris , Lignées consanguines de souris , Souris transgéniques/génétique , Souris transgéniques/métabolisme , Superoxide dismutase/génétique , Inhibiteur tissulaire de métalloprotéinase-1/métabolisme , Régulation positiveRÉSUMÉ
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'-triphosphate-biotin nick end-labeling (TUNEL). RESULTS: A similar level of APE/Ref-1 was detected in the control brains from both groups. APE/Ref-1 was significantly reduced 1 hour after transient FCI in both groups, whereas the transgenic mice had less reduction than that seen in wild-type mice 1 and 4 hours after FCI. DNA laddering was detected 24 hours after FCI and was decreased in transgenic mice. Double staining with APE/Ref-1 and TUNEL showed that the neurons that lost APE/Ref-1 immunoreactivity became TUNEL positive. CONCLUSIONS: These results suggest that reactive oxygen species contribute to the early decrease of APE/Ref-1 and thereby exacerbate DNA fragmentation after transient FCI in mice.
Sujet(s)
Carbon-oxygen lyases/physiologie , Fragmentation de l'ADN/physiologie , Accident ischémique transitoire/enzymologie , Superoxide dismutase/physiologie , Animaux , Technique de Western , Agents colorants , Altération de l'ADN/génétique , Altération de l'ADN/physiologie , Fragmentation de l'ADN/génétique , Réparation de l'ADN/génétique , Réparation de l'ADN/physiologie , DNA-(apurinic or apyrimidinic site) lyase , Deoxyribonuclease IV (phage T4-induced) , Électrophorèse sur gel d'agar , Régulation de l'expression des gènes codant pour des enzymes , Immunohistochimie , Méthode TUNEL , Mâle , Souris , Souris transgéniques , Artère cérébrale moyenne/physiopathologie , Neurones/enzymologie , Neurones/anatomopathologie , Espèces réactives de l'oxygène/physiologie , Superoxide dismutase/génétique , Facteurs tempsRÉSUMÉ
Copper zinc superoxide dismutase (CuZnSOD) is an important enzyme for the detoxification of reactive oxygen species. Particularly in the central nervous system (CNS), reactive oxygen species are often associated with acute brain injuries and chronic neurodegeneration. It has been demonstrated in vivo that there is an inverse correlation between CuZnSOD activity and neuronal death after acute brain injury. To further understand the protective role of CuZnSOD upon neurons, we have generated transgenic mouse lines with targeted expression of the human CuZnSOD gene (SOD1) that is driven by a rat neuron-specific enolase gene promoter in neurons of the CNS. The transgenic SOD1 expression was restricted to the CNS identified by reverse transcriptase polymerase chain reaction and SOD gel electrophoresis assays. The CuZnSOD activity was significantly increased in the brain stem of the transgenic mice. Immunostaining of human CuZnSOD activity showed that Purkinje cells in the cerebellar cortex were the most intensely stained neurons in the CNS of the transgenic mice.
Sujet(s)
Encéphale/enzymologie , Neurones/enzymologie , Superoxide dismutase/génétique , Animaux , Tronc cérébral/enzymologie , Cervelet/enzymologie , Régulation de l'expression des gènes codant pour des enzymes , Humains , Isoenzymes/biosynthèse , Isoenzymes/génétique , Mâle , Souris , Souris transgéniques , Enolase/biosynthèse , Enolase/génétique , Régions promotrices (génétique) , Cellules de Purkinje/enzymologie , Rats , Protéines de fusion recombinantes/biosynthèse , Cartographie de restriction , RT-PCR/méthodes , Superoxide dismutase/biosynthèseRÉSUMÉ
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.
Sujet(s)
Barrière hémato-encéphalique , Encéphalopathie ischémique/enzymologie , Collagenases/métabolisme , Animaux , Activation enzymatique , Immunohistochimie , Mâle , Matrix metalloproteinase 9 , SourisRÉSUMÉ
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.
Sujet(s)
Encéphalopathie ischémique/métabolisme , Encéphale/métabolisme , Cytochromes de type c/métabolisme , Fragmentation de l'ADN , Accident ischémique transitoire/métabolisme , Mitochondries/métabolisme , Superoxide dismutase/métabolisme , Chlorométhyl cétones d'acides aminés/pharmacologie , Animaux , Apoptose , Pression sanguine , Encéphalopathie ischémique/génétique , Encéphalopathie ischémique/physiopathologie , Cardiomyopathie dilatée/génétique , Cortex cérébral/métabolisme , Cortex cérébral/anatomopathologie , Infarctus cérébral/métabolisme , Inhibiteurs de la cystéine protéinase/pharmacologie , Cytosol/métabolisme , Hétérozygote , Accident ischémique transitoire/génétique , Accident ischémique transitoire/physiopathologie , Mâle , Souris , Souris knockout , Stress oxydatif , Rats , Superoxide dismutase/déficit , Superoxide dismutase/génétique , Superoxydes/métabolismeRÉSUMÉ
We examined the effects of the free radical scavenger, 21-aminosteroid, on apurinic/apyrimidinic endonuclease (APE/Ref-1) protein expression and subsequent infarction volume after photothrombotic cortical cerebral ischemia in mice. Immunohistochemistry and Western blot analysis showed a significant reduction in APE/Ref-1 expression 6 and 24 h after ischemia in untreated animals, whereas in drug-treated animals the reduction was much less at the same time points. The administration of 21-aminosteroid significantly decreased subsequent infarction volume 3 days after ischemia. These data suggest that 21-aminosteroid prevents the early decrease of APE/Ref-1 expression, thereby reducing cortical infarction after photothrombotic cerebral ischemia.
Sujet(s)
Antioxydants/pharmacologie , Encéphalopathie ischémique/traitement médicamenteux , Carbon-oxygen lyases/effets des médicaments et des substances chimiques , Cortex cérébral/effets des médicaments et des substances chimiques , DNA-(apurinic or apyrimidinic site) lyase , Neuroprotecteurs/pharmacologie , Animaux , Encéphalopathie ischémique/physiopathologie , Carbon-oxygen lyases/métabolisme , Cortex cérébral/physiopathologie , Mâle , Souris , Lignées consanguines de souris , Prégnatriènes/pharmacologieRÉSUMÉ
Studies of neuronal injury and death after cerebral ischemia and various neurodegenerative diseases have increasingly focused on the interactions between mitochondrial function, reactive oxygen species (ROS) production and glutamate neurotoxicity. Recent findings suggest that increased mitochondrial ROS production precedes neuronal death after glutamate treatment. It is hypothesized that under pathological conditions when mitochondrial function is compromised, extracellular glutamate may exacerbate neuronal injury. In the present study, we focus on the relationship between mitochondrial superoxide production and glutamate neurotoxicity in cultured cortical neurons with normal or reduced levels of manganese-superoxide dismutase (MnSOD) activity. Our results demonstrate that neurons with reduced MnSOD activity are significantly more sensitive to transient exposure to extracellular glutamate. The increased sensitivity of cultured cortical neurons with reduced MnSOD activity is characteristically subject only to treatment by glutamate but not to other glutamate receptor agonists, such as N-methyl-d-aspartate, kainate and quisqualate. We suggest that the reduced MnSOD activity in neurons may exacerbate glutamate neurotoxicity via a mechanism independent of receptor activation.