Shobu Namura

Ischemic Brain Injury Is Mediated by the Activation of Poly(ADP-Ribose)Polymerase

Poly(ADP-ribose)polymerase (PARP, EC 2.4.2.30), an abundant nuclear protein activated by DNA nicks, mediates cell death in vitro by nicotinamide adenine dinucleotide (NAD) depletion after exposure to nitric oxide. The authors examined whether genetic deletion of PARP (PARP null mice) or its pharmacologic inhibition by 3-aminobenzamide (3-AB) attenuates tissue injury after transient cerebral ischemia. Twenty-two hours after reperfusion following 2 hours of filamentous middle cerebral artery occlusion, ischemic injury was decreased in PARP−/− and PARP+/− mice compared with PARP+/+ litter mates, and also was attenuated in 129/SV wild-type mice after 3-AB treatment compared with controls. Infarct sparing was accompanied by functional recovery in PARP−/− and 3-AB–treated mice. Increased poly(ADP-ribose) immunostaining observed in ischemic cell nuclei 5 minutes after reperfusion was reduced by 3-AB treatment. Levels of NAD—the substrate of PARP—were reduced 2 hours after reperfusion and were 35% of contralateral levels at 24 hours. The decreases were attenuated in PARP−/− mice and in 3-AB–treated animals. Poly(ADP-ribose)polymerase cleavage by caspase-3 (CPP-32) has been proposed as an important step in apoptotic cell death. Markers of apoptosis, such as oligonucleosomal DNA damage, total DNA fragmentation, and the density of terminal deoxynucleotidyl transferase dUTP nick-end–labelled (TUNEL +) cells, however, did not differ in ischemic brain tissue of PARP−/− mice or in 3-AB–treated animals versus controls, although there were differences in the number of TUNEL-stained cells reflecting the decrease in infarct size. Thus, ischemic brain injury activates PARP and contributes to cell death most likely by NAD depletion and energy failure, although the authors have not excluded a role for PARP in apoptotic cell death at earlier or later stages in ischemic cell death. Inhibitors of PARP activation could provide a potential therapy in acute stroke.

Attenuation of Delayed Neuronal Death After Mild Focal Ischemia in Mice by Inhibition of the Caspase Family

Inhibitors of apoptosis and of excitotoxic cell death reduce brain damage after transient and permanent middle cerebral artery occlusion. We compared the neuroprotective effects of two caspase family inhibitors with the N-methyl-d-aspartate receptor antagonist (+)-MK-801 hydrogen maleate (MK-801) in a newly characterized cycloheximidesensitive murine model of transient middle cerebral artery occlusion (30 minutes) in which apoptotic cell death is prominent. Ischemic infarction, undetected by 2,3,5-triphenyltetrazolium chloride staining at 24-hour reperfusion, featured prominently in the striatum at 72 hours and 7 days on hematoxylin-eosin—stained sections. Markers of apoptosis, such as oligonucleosomal DNA damage (laddering) and terminal deoxynucleotidyl transferase—mediated dUTP-biotin nick-end labeling (TUNEL)–positive cells first appeared at 24 hours and increased significantly at 72 hours and 7 days after reperfusion. The TUNEL-labeled cells were mostly neurons and stained negative for glial (GFAP, glial fibrillary acid protein) and leukocyte specific markers (CD-45). The caspase inhibitors, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD.FMK; 120 ng intracerebroventricularly) or N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (z-DEVD.FMK; 480 ng intracerebroventricularly) decreased infarct size and neurologic deficits when administered 6 hours after reperfusion. The extent of protection was greater than in models of more prolonged ischemia or after permanent occlusion, and the therapeutic window was extended from 0 to 1 hours after 2-hour middle cerebral artery occlusion to at least 6 hours after brief ischemia. Also, z-VAD.FMK and z-DEVD.FMK treatment decreased oligonucleosomal DNA damage (DNA laddering) as assessed by quantitative autoradiography after gel electrophoresis. By contrast, MK-801 protected brain tissue only when given before ischemia (3 mg/kg intraperitoneally), but not at 3 or 6 hours after reperfusion. Despite a decrease in infarct size after MK-801 pretreatment, the amount of DNA laddering did not decrease 72 hours after reperfusion, thereby suggesting a mechanism distinct from inhibition of apoptosis. Hence, 30 minutes of reversible ischemia augments apoptotic cell death, which can be attenuated by delayed z-VADPMK and z-DEVD.FMK administration with preservation of neurologic function. By contrast, the therapeutic window for MK-801 does not extend beyond the time of occlusion, probably because its primary mechanism of action does not block the development of apoptotic cell death.

Intravenous administration of MEK inhibitor U0126 affords brain protection against forebrain ischemia and focal cerebral ischemia

Brain subjected to acute ischemic attack caused by an arterial blockage needs immediate arterial recanalization. However, restoration of cerebral blood flow can cause tissue injury, which is termed reperfusion injury. It is important to inhibit reperfusion injury to achieve greater brain protection. Because oxidative stress has been shown to activate mitogen-activated protein kinases (MAPKs), and because oxidative stress contributes to reperfusion injury, MAPK may be a potential target to inhibit reperfusion injury after brain ischemia. Here, we demonstrate that reperfusion after forebrain ischemia dramatically increases phosphorylation level of extracellular signal-regulated kinase 2 (ERK2) in the gerbil hippocampus. In addition, i.v. administration of U0126 (100–200 mg/kg), a specific inhibitor of MEK (MAPK/ERK kinase), protects the hippocampus against forebrain ischemia. Moreover, treatment with U0126 at 3 h after ischemia significantly reduces infarct volume after transient (3 h) focal cerebral ischemia in mice. This protection is accompanied by reduced phosphorylation level of ERK2, substrates for MEK, in the damaged brain areas. Furthermore, U0126 protects mouse primary cultured cortical neurons against oxygen deprivation for 9 h as well as nitric oxide toxicity. These results provide further evidence for the role of MEK/ERK activation in brain injury resulting from ischemia/reperfusion, and indicate that MEK inhibition may increase the resistance of tissue to ischemic injury.

Prolonged Therapeutic Window for Ischemic Brain Damage Caused by Delayed Caspase Activation

Apoptotic cell death is prominent in neurodegenerative disorders, such as Alzheimers disease and Huntingtons disease, and is found in cerebral ischemia. Using a murine model of delayed cell death, we determined that cleavage of zDEVD-amino-4-trifluoromethyl coumarin (zDEVD-afc) in brain homogenate, a measure of caspase activation, increased initially 9 hours after brief (30 minutes) middle cerebral artery occlusion along with caspase-3p20 immunoreactive cleavage product as determined by immunoblotting. zDEVD-afc cleavage activity was blocked by pretreatment or posttreatment with the caspase-inhibitor N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl-ketone (zDEVD-fmk), and ischemic damage was reduced when the drug was injected up to 9 hours after reperfusion. The protection was long lasting (21 days). Hence, the period before caspase activation defined the therapeutic opportunity for this neuroprotective agent after mild ischemic brain injury. Prolonged protection after caspase inhibition plus the extended treatment window may be especially relevant to the treatment of neurodegenerative disorders.

Neuroprotection by MAPK/ERK kinase inhibition with U0126 against oxidative stress in a mouse neuronal cell line and rat primary cultured cortical neurons

Oxidative stress is implicated in the pathogenesis of neuronal degenerative diseases. Oxidative stress has been shown to activate extracellular signal-regulated kinases (ERK)1/2. We investigated the role of these mitogen-activated protein kinases (MAPKs) in oxidative neuronal injury by using a mouse hippocampal cell line (HT22) and rat primary cortical cultures. Here, we show that a novel MAPK/ERK kinase (MEK) specific inhibitor U0126 profoundly protected HT22 cells against oxidative stress induced by glutamate, which was accompanied by an inhibition of phosphorylation of ERK1/2. U0126 also protected rat primary cultured cortical neurons against glutamate or hypoxia. However, U0126 was not protective against death caused by tumor necrosis factor alpha (TNFalpha), A23187, or staurosporine. These results indicate that MEK plays a central role in the neuronal death caused by oxidative stress.

Role of peroxynitrite and neuronal nitric oxide synthase in the activation of poly(ADP-ribose) synthetase in a murine model of cerebral ischemia-reperfusion

Poly(ADP-ribose) synthetase (PARS) activation, a downstream event of nitric oxide (NO) neurotoxicity has been implicated in cerebral reperfusion injury. The aim of our study was to identify the trigger of PARS activation during stroke. Formation of poly(ADP-ribose) profoundly increased in the early phase of reperfusion. Poly(ADP-ribose) formation was attenuated in mice deficient for neuronal NO synthase (nNOS). We next tested in glioma cells whether NO, or peroxynitrite (a cytotoxic oxidant formed from NO and superoxide) is the actual trigger of PARS activation. Peroxynitrite, but not various NO donors, activated PARS and suppressed cellular viability in a PARS-dependent fashion. Thus, nNOS is responsible for PARS activation in stroke. PARS activation, however, is not a direct result of NO production, but it occurs via peroxynitrite formation.

Reduction of post-traumatic brain injury and free radical production by inhibition of the caspase-1 cascade

Necrotic and apoptotic cell death both play a role mediating tissue injury following brain trauma. Caspase-1 (interleukin-1beta converting enzyme) is activated and oligonucleosomal DNA fragmentation is detected in traumatized brain tissue. Reduction of tissue injury and free radical production following brain trauma was achieved in a transgenic mouse expressing a dominant negative inhibitor of caspase-1 in the brain. Neuroprotection was also conferred by pharmacological inhibition of caspase-1 by intracerebroventricular administration of the selective inhibitor of caspase-1, acetyl-Tyr-Val-Ala-Asp-chloromethyl-ketone or the non-selective caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. These results indicate that inhibition of caspase-1-like caspases reduces trauma-mediated brain tissue injury. In addition, we demonstrate an in vivo functional interaction between interleukin-1beta converting enyzme-like caspases and free radical production pathways, implicating free radical production as a downstream mediator of the caspase cell death cascade.

Ebselen Reduces Cytochrome c Release From Mitochondria and Subsequent DNA Fragmentation After Transient Focal Cerebral Ischemia in Mice

Background and Purpose— The seleno-organic compound ebselen has both antioxidant and anti-inflammatory properties. Although ebselen has been shown to protect the brain against stroke, it is unclear how ebselen provides neuroprotection. In the present study the authors examined whether ebselen inhibits neuronal apoptosis resulting from transient focal cerebral ischemia in mice. The cytochrome c release and DNA fragmentation, both of which are biochemical markers of apoptosis, were compared between vehicle- and ebselen-treated mice. Methods— Cerebral ischemia was induced by transient middle cerebral artery occlusion for 30 minutes in ICR mice under halothane anesthesia. Ebselen (10 mg/kg) was given orally twice, 30 minutes before ischemia and 12 hours after reperfusion. By Western blot analysis, we examined release of mitochondrial cytochrome c. To evaluate brain damage, the brain sections were treated for terminal deoxynucleotidyl transferase–mediated DNA nick-end labeling (TUNEL) and Nissl staining. Prolonged neuroprotective efficacy of ebselen was determined by counting neuronal nuclei (NeuN) immunopositive cells at 21 days after ischemia. Results— Cytochrome c release was detected in the ischemic hemisphere at 3 to 24 hours after ischemia. Ebselen treatment diminished the cytochrome c release at 12 and 24 hours. In addition, ebselen decreased both DNA fragmentation determined by TUNEL and brain damage volume at 3 days after ischemia. Furthermore, ebselen increased the number of NeuN immunopositive cells at 21 days after ischemia. Conclusions— These results indicate that ebselen attenuates ischemic neuronal apoptosis by inhibiting cytochrome c release. Ebselen may be a potential compound in stroke therapy.

Matrix metalloproteinase inhibitor KB-R7785 attenuates brain damage resulting from permanent focal cerebral ischemia in mice.

Matrix metalloproteinases (MMPs) are proteolytic enzymes that can degrade the extracellular matrix. MMP-9 and MMP-2 have been implicated in brain injury formation. The authors examined the effect of MMP inhibitor KB-R7785 on brain infarct formation resulting from permanent focal cerebral ischemia in mice. Ischemia was induced by intraluminal middle cerebral artery occlusion (MCAO) in mice under halothane anesthesia. Zymography was conducted to measure the MMPs activity in ischemic brain tissues. Injection of KB-R7785 (100 mg/kg) 30 min before MCAO significantly decreased both MMP-9 activity and infarct volume determined at 24 h. In addition, KB-R7785 injected twice at 1 and 4.5 h after MCAO significantly decreased infarct volume. These results indicate that KB-R7785 has a protective efficacy against focal cerebral ischemia, and our data provide further evidence that MMP-9 contributes to brain infarct formation.

Chemokine receptor antagonist peptide, viral MIP-II, protects the brain against focal cerebral ischemia in mice.

The authors previously reported that mRNA for macrophage inflammatory protein-1α (MIP-1 α), a member of the CC chemokines, was expressed in glial cells after focal cerebral ischemia in rats. However, the function of chemokines in the ischemic brain remains unclear. Recently, viral macrophage inflammatory protein-II (vMIP-II), a chemokine analogue encoded by human herpesvirus-8 DNA, has been demonstrated to have antagonistic activity at several chemokine receptors. In the present study, the effects of vMIP-II and MIP-1α on ischemic brain injury were examined in mice to elucidate the roles of chemokines endogenously produced in the ischemic brain. Intracerebroventricular injection of vMIP-II (0.01–1 μg) reduced infarct volume in a dose-dependent manner when examined 48 hours after 1-hour middle cerebral artery occlusion followed by reperfusion. However, 1 μg MIP-1α increased infarct volume in the cortical region. These results supported the possibility that chemokines endogenously produced in the brain are involved in ischemic injury, and that chemokine receptors are potential targets for therapeutic intervention of stroke.