Plasma antithrombin III deficiency in ischaemic stroke in the young.
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A deficiency of plasma antithrombin III has been identified as a potential risk factor for thrombosis. In a pilot study of 56 patients aged less than 40 years who presented with ischaemic stroke of unknown etiology, we detected only one case of plasma antithrombin III deficiency. Antithrombin III activity was estimated by a chromogenic assay. Hence, antithrombin III deficiency, though rare, should be considered while evaluating young patients with stroke of unknown etiology. (+info)
Neuronal nitric oxide synthase activation and peroxynitrite formation in ischemic stroke linked to neural damage.
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Nitric oxide (NO) is a new intercellular messenger that occurs naturally in the brain without causing overt toxicity. Yet, NO has been implicated as a mediator of cell death in cell death. One explanation is that ischemia causes overproduction of NO, allowing it to react with superoxide to form the potent oxidant peroxynitrite. To address this question, we used immunohistochemistry for citrulline, a marker for NO synthase activity, and 3-nitrotyrosine, a marker for peroxynitrite formation, in mice subjected to reversible middle cerebral artery occlusion. We show that ischemia triggers a marked augmentation in citrulline immunoreactivity but more so in the peri-infarct than the infarcted tissue. This increase is attributable to the activation of a large population (approximately 80%) of the neuronal isoform of NO synthase (nNOS) that is catalytically inactive during basal conditions, indicating a tight regulation of physiological NO production in the brain. In contrast, 3-nitrotyrosine immunoreactivity is restricted to the infarcted tissue and is not present in the peri-infarct tissue. In nNOS(Delta/Delta) mice, known to be protected against ischemia, no 3-nitrotyrosine immunoreactivity is detected. Our findings provide a cellular localization for nNOS activation in association with ischemic stroke and establish that NO is not likely a direct neurotoxin, whereas its conversion to peroxynitrite is associated with cell death. (+info)
Caspase-8 and caspase-3 are expressed by different populations of cortical neurons undergoing delayed cell death after focal stroke in the rat.
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A number of studies have provided evidence that neuronal cell loss after stroke involves programmed cell death or apoptosis. In particular, recent biochemical and immunohistochemical studies have demonstrated the expression and activation of intracellular proteases, notably caspase-3, which act as both initiators and executors of the apoptotic process. To further elucidate the involvement of caspases in neuronal cell death induced by focal stroke we developed a panel of antibodies and investigated the spatial and temporal pattern of both caspase-8 and caspase-3 expression. Our efforts focused on caspase-8 because its "apical" position within the enzymatic cascade of caspases makes it a potentially important therapeutic target. Constitutive expression of procaspase-8 was detectable in most cortical neurons, and proteolytic processing yielding the active form of caspase-8 was found as early as 6 hr after focal stroke induced in rats by permanent middle cerebral artery occlusion. This active form of caspase-8 was predominantly seen in the large pyramidal neurons of lamina V. Active caspase-3 was evident only in neurons located within lamina II/III starting at 24 hr after injury and in microglia throughout the core infarct at all times examined. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, gel electrophoresis of DNA, and neuronal cell quantitation indicated that there was an early nonapoptotic loss of cortical neurons followed by a progressive elimination of neurons with features of apoptosis. These data indicate that the pattern of caspase expression occurring during delayed neuronal cell death after focal stroke will vary depending on the neuronal phenotype. (+info)
Ubiquitin stress response in postischemic hippocampal neurons under nontolerant and tolerant conditions.
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Ubiquitin, an essential protein in nonlysosomal proteolytic system, is expressed after metabolic stress to the cell. The authors investigated stress response of ubiquitin in the hippocampus of the Mongolian gerbil after forebrain ischemia. The level of hippocampal ubiquitin was compared with that under ischemic tolerance induced by ischemic preconditioning. The authors also studied ubiquitin gene expression using in situ hybridization method. Transient ischemia resulted in consumption of free ubiquitin and an increase of multiubiquitin chains. These changes were transient in the hippocampus outside of the CA1 region where neurons survived, whereas it was persistent in the CA1 region where neurons were destined to die after ischemia. Under tolerant condition, subsequent ischemia provoked rapid recovery and further increase of free ubiquitin. The signal of ubiquitin messenger ribonucleic acid was continuously detected after ischemia, not only under tolerant conditions, but without tolerance induced by preconditioning. Thus, ubiquitin stress response takes place, at least at a transcriptional level, in dying CA1 neurons. Under tolerant conditions, however, subsequent ischemia in the CA1 region induces the stress response of ubiquitin up to the translational level, leading to the rapid restoration of protein synthesis and to eventual neuronal survival. (+info)
Neuroprotective effects of a novel nitrone, NXY-059, after transient focal cerebral ischemia in the rat.
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Recent results have demonstrated that the spin trapping agent alpha-phenyl-N-tert-butyl nitrone (PBN) reduces infarct volume in rats subjected to 2 hours of middle cerebral artery occlusion, even when given 1 to 3 hours after the start of recirculation. In the current study, the authors assessed the effect of NXY-059, a novel nitrone that is more soluble than PBN. Loading doses were given of 0.30, 3.0, or 30 mg x kg(-1) followed by 0.30, 3.0, or 30 mg x kg(-1) x h(-1) for 24 or 48 hours. Dose-response studies showed that when treatment was begun 1 hour after recirculation, 0.30 mg x kg(-1) had a small and 30 mg x kg(-1) a marked effect on infarct volume. At equimolar doses (3.0 mg x kg(-1) for NXY-059 and 1.4 mg x kg(-1) for PBN), NXY-059 was more efficacious than PBN. Similar results were obtained when a recovery period of 7 days was allowed. The window of therapeutic opportunity for NXY-059 was 3 to 6 hours after the start of recirculation. Studies of the transfer constant of [14C]NXY-059 showed that, in contrast to PBN, this more soluble nitrone penetrates the blood-brain barrier less extensively. This fact, and the pronounced antiischemic effect of NXY-059, suggest that the delayed events leading to infarction may be influenced by reactions occurring at the blood-endothelial interface. (+info)
Neuronal protection in stroke by an sLex-glycosylated complement inhibitory protein.
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Glycoprotein adhesion receptors such as selectins contribute to tissue injury in stroke. Ischemic neurons strongly expressed C1q, which may target them for complement-mediated attack or C1qRp-mediated clearance. A hybrid molecule was used to simultaneously inhibit both complement activation and selectin-mediated adhesion. The extracellular domain of soluble complement receptor-1 (sCR1) was sialyl Lewis x glycosylated (sCR1sLex) to inhibit complement activation and endothelial-platelet-leukocyte interactions. sCR1 and sCR1sLex colocalized to ischemic cerebral microvessels and C1q-expressing neurons, inhibited neutrophil and platelet accumulation, and reduced cerebral infarct volumes. Additional benefit was conferred by sialyl Lewis x glycosylation of the unmodified parent sCR1 molecule. (+info)
Targeted inhibition of intrinsic coagulation limits cerebral injury in stroke without increasing intracerebral hemorrhage.
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Agents that restore vascular patency in stroke also increase the risk of intracerebral hemorrhage (ICH). As Factor IXa is a key intermediary in the intrinsic pathway of coagulation, targeted inhibition of Factor IXa-dependent coagulation might inhibit microvascular thrombosis in stroke without impairing extrinsic hemostatic mechanisms that limit ICH. A competitive inhibitor of native Factor IXa for assembly into the intrinsic Factor X activation complex, Factor IXai, was prepared by covalent modification of the Factor IXa active site. In a modified cephalin clotting time assay, in vivo administration of Factor IXai caused a dose-dependent increase in time to clot formation (3.6-fold increase at the 300 micrograms/kg dose compared with vehicle-treated control animals, P < 0.05). Mice given Factor IXai and subjected to middle cerebral artery occlusion and reperfusion demonstrated reduced microvascular fibrin accumulation by immunoblotting and immunostaining, reduced 111In-labeled platelet deposition (42% decrease, P < 0.05), increased cerebral perfusion (2.6-fold increase in ipsilateral blood flow by laser doppler, P < 0.05), and smaller cerebral infarcts than vehicle-treated controls (70% reduction, P < 0.05) based on triphenyl tetrazolium chloride staining of serial cerebral sections. At therapeutically effective doses, Factor IXai was not associated with increased ICH, as opposed to tissue plasminogen activator (tPA) or heparin, both of which significantly increased ICH. Factor IXai was cerebroprotective even when given after the onset of stroke, indicating that microvascular thrombosis continues to evolve (and may be inhibited) even after primary occlusion of a major cerebrovascular tributary. (+info)
Block of rapid depolarization induced by in vitro energy depletion of rat dorsal vagal motoneurones.
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1. The ionic mechanisms contributing to the rapid depolarization (RD) induced by in vitro ischaemia have been studied in dorsal vagal motoneurones (DVMs) of brainstem slices. Compared with CA1 hippocampal neurones, RD of DVMs was slower, generally occurred from a more depolarized membrane potential and was accompanied by smaller increases in [K+]o. 2. RD was not induced by elevation of [K+]o to values measured around DVMs during in vitro ischaemia or by a combination of raised [K+]o and 2-5 microM ouabain. 3. Neither TTX (5-10 microM) nor TTX combined with bepridil (10-30 microM), a Na+-Ca2+ exchange inhibitor, slowed RD. Block of voltage-dependent Ca2+ channels with Cd2+ (0.2 mM) and Ni2+ (0.3 mM) led to an earlier onset of RD, possibly because [K+]o was higher than that measured during in vitro ischaemia in the absence of divalent ions. 4. When [Na+]o was reduced to 11.25-25 mM, RD did not occur, although a slow depolarization was observed. RD was slowed (i) by 10 mM Mg2+ and 0.5 mM Ca2+, (ii) by a combination of TTX (1.5-5 microM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) and D-2-amino-5-phosphonovalerate (AP5, 50 microM) and (iii) by TTX (1.5-5 microM) and AP5 (50 microM). 5. Ni2+ at concentrations of 0.6 or 1.33 mM blocked RD whereas 0.6 mM Cd2+ did not. A combination of Cd2+ (0.2 mM), Ni2+ (0.3 mM), AP5 (50 microM) and bepridil (10 microM) was largely able to mimic the effects of high concentrations of Ni2+. 6. It is concluded that RD is due to Na+ entry, predominantly through N-methyl-D-aspartate receptor ionophores, and to Ca2+ entry through voltage-dependent Ca2+ channels. These results are consistent with known changes in the concentrations of extracellular ions when ischaemia-induced rapid depolarization occurs. (+info)