Aag-initiated base excision repair promotes ischemia reperfusion injury in liver, brain, and kidney.

Inflammation is accompanied by the release of highly reactive oxygen and nitrogen species (RONS) that damage DNA, among other cellular molecules. Base excision repair (BER) is initiated by DNA glycosylases and is crucial in repairing RONS-induced DNA damage; the alkyladenine DNA glycosylase (Aag/Mpg) excises several DNA base lesions induced by the inflammation-associated RONS release that accompanies ischemia reperfusion (I/R). Using mouse I/R models we demonstrate that Aag(-/-) mice are significantly protected against, rather than sensitized to, I/R injury, and that such protection is observed across three different organs. Following I/R in liver, kidney, and brain, Aag(-/-) mice display decreased hepatocyte death, cerebral infarction, and renal injury relative to wild-type. We infer that in wild-type mice, Aag excises damaged DNA bases to generate potentially toxic abasic sites that in turn generate highly toxic DNA strand breaks that trigger poly(ADP-ribose) polymerase (Parp) hyperactivation, cellular bioenergetics failure, and necrosis; indeed, steady-state levels of abasic sites and nuclear PAR polymers were significantly more elevated in wild-type vs. Aag(-/-) liver after I/R. This increase in PAR polymers was accompanied by depletion of intracellular NAD and ATP levels plus the translocation and extracellular release of the high-mobility group box 1 (Hmgb1) nuclear protein, activating the sterile inflammatory response. We thus demonstrate the detrimental effects of Aag-initiated BER during I/R and sterile inflammation, and present a novel target for controlling I/R-induced injury.

Role of matrix metalloproteinases in delayed cortical responses after stroke.

Matrix metalloproteinases (MMPs) are zinc-endopeptidases with multifactorial actions in central nervous system (CNS) physiology and pathology. Accumulating data suggest that MMPs have a deleterious role in stroke. By degrading neurovascular matrix, MMPs promote injury of the blood-brain barrier, edema and hemorrhage. By disrupting cell-matrix signaling and homeostasis, MMPs trigger brain cell death. Hence, there is a movement toward the development of MMP inhibitors for acute stroke therapy. But MMPs may have a different role during delayed phases after stroke. Because MMPs modulate brain matrix, they may mediate beneficial plasticity and remodeling during stroke recovery. Here, we show that MMPs participate in delayed cortical responses after focal cerebral ischemia in rats. MMP-9 is upregulated in peri-infarct cortex at 7-14 days after stroke and is colocalized with markers of neurovascular remodeling. Treatment with MMP inhibitors at 7 days after stroke suppresses neurovascular remodeling, increases ischemic brain injury and impairs functional recovery at 14 days. MMP processing of bioavailable VEGF may be involved because inhibition of MMPs reduces endogenous VEGF signals, whereas additional treatment with exogenous VEGF prevents MMP inhibitor-induced worsening of infarction. These data suggest that, contrary to MMP inhibitor therapies for acute stroke, strategies that modulate MMPs may be needed for promoting stroke recovery.

Serine racemase deletion protects against cerebral ischemia and excitotoxicity.

D-Serine, formed from L-serine by serine racemase (SR), is a physiologic coagonist at NMDA receptors. Using mice with targeted deletion of SR, we demonstrate a role for D-serine in NMDA receptor-mediated neurotoxicity and stroke. Brain cultures of SR-deleted mice display markedly diminished nitric oxide (NO) formation and neurotoxicity. In intact SR knock-out mice, NO formation and nitrosylation of NO targets are substantially reduced. Infarct volume following middle cerebral artery occlusion is dramatically diminished in several regions of the brains of SR mutant mice despite evidence of increased NMDA receptor number and sensitivity.

PTEN deletion prevents ischemic brain injury by activating the mTOR signaling pathway.

It is increasingly clear that the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a negative regulator of neuronal cell survival. However, its molecular mechanisms remain poorly understood. Here we found that PTEN/mTOR is critical for controlling neuronal cell death after ischemic brain injury. Male rats were subjected to MCAO (middle cerebral artery occlusion) followed by pretreating with bpv (pic), a potent inhibitor for PTEN, or by intra-cerebroventricular infusion of PTEN siRNA. bpv (pic) significantly decreased infarct volume and reduced the number of TUNEL-positive cells. We further demonstrated that although bpv (pic) did not affect brain injury-induced mTOR protein expression, bpv (pic) prevented decrease in phosphorylation of mTOR, and the subsequent decrease in S6. Similarly, down-regulation of PTEN expression also reduced the number of TUNEL-positive cells, and increased phospho-mTOR. These data suggest that PTEN deletion prevents neuronal cell death resulting from ischemic brain injury and that its neuroprotective effects are mediated by increasing the injury-induced mTOR phosphorylation.

The cyclooxygenase site, but not the peroxidase site of cyclooxygenase-2 is required for neurotoxicity in hypoxic and ischemic injury.

Cyclooxygenase-2 (COX-2) activity has been implicated in the pathogenesis of ischemic injury, but the exact mechanisms responsible for its toxicity remain unclear. Infection of primary neurons with an adenovirus expressing wild type (WT) COX-2 increased the susceptibility of neurons to hypoxia. Infection with an adenoviral vector expressing COX-2 with a mutation at the cyclooxygenase site did not increase susceptibility to hypoxia, whereas over-expression of COX-2 with a mutation in the peroxidase site produced similar susceptibility to hypoxia as WT COX-2. Primary neuronal cultures obtained from transgenic mice bearing a mutation in the COX-2 cylooxygenase site were protected from hypoxia. Mice with a mutation in the cyclooxygenase site had smaller infarctions 24 h after 70 min of middle cerebral artery occlusion than WT control mice. COX-2 activity had no effect on the formation of protein carbonyls. Ascorbate radicals were detected by electron paramagnetic resonance as a product of recombinant COX-2 activity and were blocked by COX-2 inhibitors. Similarly, formation of ascorbate radicals was inhibited in the presence of COX-2 inhibitors and in homogenates obtained from COX-2 null mice. Taken together, these results indicate that the cyclooxygenase activity of COX-2 is necessary to exacerbate neuronal hypoxia/ischemia injury rather than the peroxidase activity of the enzyme.

Neuroprotection by baicalein in ischemic brain injury involves PTEN/AKT pathway.

Recently more evidences support baicalein (Bai) is neuroprotective in models of ischemic stroke. This study was conducted to determine the molecular mechanisms involved in this effect. Either permanent or transient (2 h) middle cerebral artery occlusion (MCAO) was induced in rats in this study. Permanent MCAO led to larger infarct volumes in contrast to transient MCAO. Only in transient MCAO, Bai administration significantly reduced infarct size. Baicalein also markedly reduced apoptosis in the penumbra of transient MCAO rats. Additionally, oxygen and glucose deprivation (OGD) was used to mimic ischemic insult in primary cultured cortical neurons. A rapid increase in the intracellular reactive oxygen species level and nitrotyrosine formation induced by OGD was counteracted by Bai, which is parallel with attenuated cell injury. The reduction of phosphorylation Akt and glycogen synthase kinase-3beta (GSK3beta) induced by OGD was restored by Bai, which was associated with preserved levels of phosphorylation of PTEN, the phophatase that negatively regulates Akt. As a consequence, Bcl-2/Bcl-xL-associated death protein phosphorylation was increased and the protein level of Bcl-2 in motochondria was maintained, which subsequently antagonize cytochrome c released in cytosol. LY294002 blocked the increase in phospho-AKT evoked by Bai and abolished the associated protective effect. Together, these findings provide evidence that Bai protects neurons against ischemia injury and this neuroprotective effect involves PI3K/Akt and PTEN pathway.

NADPH oxidase Nox1 contributes to ischemic injury in experimental stroke in mice.

Reactive oxygen species (ROS) are mediators of brain injury in ischemia/reperfusion. An involvement of the NADPH oxidase Nox2 has been demonstrated. In contrast, only little is known about the contribution of the Nox1 homologue in this context. Thus, we studied the role of Nox1 in early cerebral reperfusion injury in the middle cerebral artery filament occlusion model using Nox1 knockout mice. Genetic deletion of a functional Nox1 lead to a 55% attenuation in lesion size at 24h after induction of 1h ischemia (p<0.05). This result was paralleled by a significant improvement of neurological outcome, preservation of blood-brain barrier integrity and reduced cerebral edema in Nox1(y/)(-) compared to WT mice. Interestingly, no difference in infarct size between WT and Nox1(y/)(-) was observed with an occlusion time of 2h and longer. Apoptosis rate as measured by TUNEL staining was similar between the groups. Moreover, infusion of the antioxidant TEMPOL as well as of the unspecific NO-synthase inhibitor l-NAME elicited similar changes with respect to ischemic tissue damage between WT and Nox1-deficient mice. In conclusion, Nox1 is involved in the pathophysiology of cerebral ischemia. Our data however indicate that ROS-mediated direct cellular injury is unlikely to explain the protective effect achieved by genetic deletion of the enzyme.

Identification and preliminary characterization of ubiquitin C terminal hydrolase 1 (UCHL1) as a biomarker of neuronal loss in aneurysmal subarachnoid hemorrhage.

By using two different approaches, ubiquitin C-terminal hydrolase 1 (UCHL1) was identified as a potential cerebrospinal fluid (CSF) biomarker of neuronal loss in aneurysmal subarachnoid hemorrhage (ASAH) and presumably other CNS damage and disease states. Appropriate antibodies and a sensitive ELISA were generated, and the release of UCHL1 into CSF was compared with that of pNF-H and S100beta in a cohort of 30 ASAH patients. Both UCHL1 and pNF-H showed persistent release into CSF in almost all patients in the second week postaneurysmal rupture (AR), and S100beta levels rapidly declined to baseline levels in 23 of 30 patients. Seven of thirty patients showed persistently elevated S100beta levels over the first 5 days post-AR and also had relatively higher levels of pNF-H and UCHL1 higher compared with the rest. These patients proved to have very poor outcomes, with 6 of 7 expiring. Patients who did reduce S100beta levels tended to have a better outcome if pNF-H and UCHL1 levels were also lower, and elevated UCHL1 levels in the second week post-AR were particularly predictive of poor outcome. Acute coordinated releases of large amounts of UCHL1, pNF-H, and S100beta in 16 of 30 patients were observed, suggesting sudden loss of brain tissues associated with secondary events. We conclude that measurement of the CSF levels of these proteins reveals details of ASAH progression and recovery and predicts patient outcome.

Pre-ischemic treadmill training induces tolerance to brain ischemia: involvement of glutamate and ERK1/2.

Physical exercise has been shown to be beneficial in stroke patients and animal stroke models. However, the exact mechanisms underlying this effect are not yet very clear. The present study investigated whether pre-ischemic treadmill training could induce brain ischemic tolerance (BIT) by inhibiting the excessive glutamate release and event-related kinase 1/2 (ERK1/2) activation observed in rats exposed to middle cerebral artery occlusion (MCAO). Sprague-Dawley rats were divided into three groups (n = 12/group): sham surgery without prior exercise, MCAO without prior exercise and MCAO following three weeks of exercise. Pre-MCAO exercise significantly reduced brain infarct size (103.1 +/- 6.7 mm3) relative to MCAO without prior exercise (175.9 +/- 13.5 mm3). Similarly, pre-MCAO exercise significantly reduced neurological defects (1.83 +/- 0.75) relative to MCAO without exercise (3.00 +/- 0.63). As expected, MCAO increased levels of phospho-ERK1/2 (69 +/- 5%) relative to sham surgery (40 +/- 5%), and phospho-ERK1/2 levels were normalized in rats exposed to pre-ischemic treadmill training (52 +/- 6%) relative to MCAO without exercise (69% +/- 5%). Parallel effects were observed on striatal glutamate overflow. This study suggests that pre-ischemic treadmill training might induce neuroprotection by inhibiting the phospho-ERK1/2 over-activation and reducing excessive glutamate release.

Activated protein C promotes neovascularization and neurogenesis in postischemic brain via protease-activated receptor 1.

Activated protein C (APC) is a serine protease with anticoagulant and direct cytoprotective activities. Early postischemic APC application activates the cellular protein C pathway in brain endothelium and neurons, which is neuroprotective. Whether late APC administration after a transient ischemic attack is neuroprotective and whether APC influences brain repair is not known. Here, we determined safety and efficacy of late APC and tissue-plasminogen activator (tPA) administrations in a mouse model of transient brain ischemia. tPA given at 6 h after onset of ischemia killed all mice within 2 d, whereas APC given at 6 or 24 h after ischemia onset improved significantly functional outcome and reduced spread of the ischemic lesion. At 7 d postischemia, APC multiple dosing (0.8 mg/kg, i.p.) at 6-72 or 72-144 h enhanced comparably cerebral perfusion in the ischemic border by approximately 40% as shown by in vivo lectin-FITC angiography, blocked blood-brain barrier leakage of serum proteins, and increased the number of endothelial replicating cells by 4.5- to 4.7-fold. APC multidosing at 6-72 h or 72-144 h increased proliferation of neuronal progenitor cells in the subventricular zone (SVZ) by 40-50% and migration of newly formed neuroblasts from the SVZ toward the ischemic border by approximately twofold. The effects of APC on neovascularization and neurogenesis were mediated by protease-activated receptor 1 and were independent of the reduction by APC of infarction volume. Our data show that delayed APC administration is neuroprotective and mediates brain repair (i.e., neovascularization and neurogenesis), suggesting a significant extension of the therapeutic window for APC intervention in postischemic brain.

The HDAC inhibitor, sodium butyrate, stimulates neurogenesis in the ischemic brain.

In the healthy adult brain, neurogenesis normally occurs in the subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Cerebral ischemia enhances neurogenesis in neurogenic and non-neurogenic regions of the ischemic brain of adult rodents. This study demonstrated that post-insult treatment with a histone deacetylase inhibitor, sodium butyrate (SB), stimulated the incorporation of bromo-2'-deoxyuridine (BrdU) in the SVZ, DG, striatum, and frontal cortex in the ischemic brain of rats subjected to permanent cerebral ischemia. SB treatment also increased the number of cells expressing polysialic acid-neural cell adhesion molecule, nestin, glial fibrillary acidic protein, phospho-cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF) in various brain regions after cerebral ischemia. Furthermore, extensive co-localization of BrdU and polysialic acid-neural cell adhesion molecule was observed in multiple regions after ischemia, and SB treatment up-regulated protein levels of BDNF, phospho-CREB, and glial fibrillary acidic protein. Intraventricular injection of K252a, a tyrosine kinase B receptor antagonist, markedly reduced SB-induced cell proliferation detected by BrdU and Ki67 in the ipsilateral SVZ, DG, and other brain regions, blocked SB-induced nestin expression and CREB activation, and attenuated the long-lasting behavioral benefits of SB. Together, these results suggest that histone deacetylase inhibitor-induced cell proliferation, migration and differentiation require BDNF-tyrosine kinase B signaling and may contribute to long-term beneficial effects of SB after ischemic injury.

Reduction of lipoxidative load by secretory phospholipase A2 inhibition protects against neurovascular injury following experimental stroke in rat.

BACKGROUND: In animal models, ischemia reperfusion (IR) injury triggers membrane lipid degradation and accumulation of lipoxidative exacerbations in neurovascular unit, leading to blood brain barrier (BBB) damage and neurologic deficits. In this study, we investigated whether impeding membrane lipid breakdown by inhibiting secretory phospholipase A2 (sPLA2) activity reduces BBB leakage, leading to neuroprotection and functional recovery. METHODS: Focal cerebral IR injury was induced by middle cerebral artery occlusion (MCAO) in adult male rats. A sPLA2 inhibitor, 7,7-dimethyleicosadienoic acid (DEDA), was administered following IR injury. DEDA-treated animals were compared with vehicle-treated in terms of BBB leakage, edema, infarct volume, and neurological deficit. Membrane lipid degradation and the expression/activity of sPLA2 were also assessed. The role of one of the sPLA2 products, arachidonic acid (AA), on the morphology of the differentiated neuronal cell PC12 was examined by light microscopy. RESULTS: Treatment with DEDA after IR injury not only reduced BBB leakage but also decreased infarct volume and improved neurologic function. The treatment attenuated both the activity of sPLA2 and the levels of sPLA2-derived oxidized products. The metabolites of lipid oxidation/peroxidation, including the protein carbonyl, were reduced as well. The treatment also restored the levels of glutathione, indicating attenuation of oxidative stress. In vitro treatment of PC12 cells with DEDA did not restore the AA-mediated inhibition of neurite formation and the levels of glutathione, indicating that effect of DEDA is up stream to AA release. CONCLUSION: sPLA2-derived oxidative products contribute to significant neurovascular damage, and treatment with sPLA2 inhibitor DEDA ameliorates secondary injury by reducing exacerbations from lipoxidative stress.

Early diffusion-weighted MRI as a predictor of caspase-3 activation after hypoxic-ischemic insult in neonatal rodents.

BACKGROUND AND PURPOSE: Neonatal encephalopathy in human babies is a serious condition associated with permanent neurological deficits. Diffusion-weighted MRI (DWI) is increasingly used for early diagnosis of brain injury in human babies. The relationship between the presence of DWI abnormalities and cellular injury, including apoptosis, during the neonatal period are not well understood. We asked whether the extent of injury depicted on DWI can predict the presence of caspase-3 activation, a quantitative marker of apoptotic injury, after hypoxia-ischemia (H-I) in postnatal day 7 rats. METHODS: Injury volume was determined by DWI at 2 hours, 24 hours, and 7 days after H-I and compared with histology. Caspase-3 activation and microgliosis were determined at 24 hours post-H-I. RESULTS: DWI-defined lesions (eg, decreased apparent diffusion coefficient) at 24 hours post-H-I correlated with a major increase in caspase-3 activity in the injured hemisphere and predicted injury. A modest but significant increase in caspase-3 activity occurred in the cortex of rats that had no apparent diffusion coefficient decrease in the injured hemisphere but had unilaterally enlarged regions of high apparent diffusion coefficient at the ipsilateral ventricle/white matter interface. Caspase-3 activity was similar in both hemispheres in pups with unchanged DWI. CONCLUSIONS: Abnormal DWI signal at 24 hours post-H-I is predictive of caspase-3 activation and can be used as an indicator that injury involving an apoptotic-like mechanism is present. Our data also suggest that the presence of an enlarged unilateral region with high apparent diffusion coefficient at the ventricle/white matter interface without significant apparent diffusion coefficient decrease in the cortex is a sign of modest caspase-3 activation after H-I.

Protecting against cerebrovascular injury: contributions of 12/15-lipoxygenase to edema formation after transient focal ischemia.

BACKGROUND AND PURPOSE: The concept of the neurovascular unit suggests that effects on brain vasculature must be considered if neuroprotection is to be achieved in stroke. We previously reported that 12/15-lipoxygenase (12/15-LOX) is upregulated in the peri-infarct area after middle cerebral artery occlusion in mice, and 12/15-LOX contributes to brain damage after ischemia-reperfusion. The current study was designed to investigate 12/15-LOX involvement in vascular injury in the ischemic brain. METHODS: In cell culture, a human brain microvascular endothelial cell line was subjected to either hypoxia or H(2)O(2)-induced oxidative stress with or without lipoxygenase inhibitors. For in vivo studies, mice were subjected to 90 minutes middle cerebral artery occlusion, and the effects of either 12/15-LOX gene knockout or treatment with lipoxygenase inhibitors were compared. Expression of 12/15-LOX and claudin-5 as well as extravasation of immunoglobulin G were detected by immunohistochemistry. Edema was measured as water content of brain hemispheres according to the wet-dry weight method. RESULTS: Brain endothelial cells were protected against hypoxia and H(2)O(2) by the lipoxygenase inhibitor baicalein. After focal ischemia, 12/15-LOX was increased in neurons and endothelial cells. The vascular tight junction protein claudin-5 underwent extensive degradation in the peri-infarct area, which was partially prevented by the lipoxygenase inhibitor baicalein. Leakage of immunoglobulin G into the brain parenchyma was significantly reduced in 12/15-LOX knockout mice as well as wild-type mice treated with baicalein. Likewise, brain edema was significantly ameliorated. CONCLUSIONS: 12/15-LOX may contribute to ischemic brain damage not just by causing neuronal cell death, but also by detrimental effects on the brain microvasculature. 12/15-LOX inhibitors may thus be effective as both neuroprotectants and vasculoprotectants.

The obligatory role of COX-2 expression for induction of HO-1 in ischemic preconditioned rat brain.

The molecular mechanisms of preconditioning-induced ischemic tolerance (PCIT) have yet to be elucidated. We investigated whether minimal expression levels of COX-2 induced by preconditioning trigger HO-1, thereby inducing the synthesis of cytoprotective proteins. We show that both COX-2 and HO-1 are induced in rat brains subjected to preconditioning by middle cerebral artery (MCA) occlusion for 10 min followed by different amounts of reperfusion time (1-24 h). Although preconditioning significantly reduced the brain infarct size against severe ischemia (24 h MCA occlusion), pretreatment with the COX-2-selective inhibitor rofecoxib increased infarct size and abolished PCIT-induced COX-2 and HO-1 expression in vivo. We also found that PGE(2) increased the phosphorylation of Akt, which was significantly inhibited by the PI3 kinase inhibitor LY294002. Taken together, we conclude that the kinetic changes in COX-2 induction during the reperfusion period following preconditioning may be important for ischemic tolerance.

Uridine protects cortical neurons from glucose deprivation-induced death: possible role of uridine phosphorylase.

We previously reported that uridine blocked glucose deprivation-induced death of immunostimulated astrocytes by preserving ATP levels. Uridine phosphorylase (UPase), an enzyme catalyzing the reversible phosphorylation of uridine, was involved in this effect. Here, we tried to expand our previous findings by investigating the uridine effect on the brain and neurons using in vivo and in vitro ischemic injury models. Orally administrated uridine (50-200 mg/kg) reduced middle cerebral artery occlusion (1.5 h)/reperfusion (22 h)-induced infarct in mouse brain. Additionally, in the rat brain subjected to the same ischemic condition, UPase mRNA and protein levels were up-regulated. Next, we employed glucose deprivation-induced hypoglycemia in mixed cortical cultures of neurons and astrocytes as an in vitro model. Cells were deprived of glucose and, two hours later, supplemented with 20 mM glucose. Under this condition, a significant ATP loss followed by death was observed in neurons but not in astrocytes, which were blocked by treatment with uridine in a concentration-dependent manner. Inhibition of cellular uptake of uridine by S-(4-nitrobenzyl)-6-thioinosine blocked the uridine effect. Similar to our in vivo data, UPase expression was up-regulated by glucose deprivation in mRNA as well as protein levels. Additionally, 5-(phenylthio)acyclouridine, a specific inhibitor of UPase, prevented the uridine effect. Finally, the uridine effect was shown only in the presence of astrocytes. Taken together, the present study provides the first evidence that uridine protects neurons against ischemic insult-induced neuronal death, possibly through the action of UPase.

Caspase-3 is related to infarct growth after human ischemic stroke.

Our aim was to investigate caspase-3 plasma levels after stroke, its correlation with infarct expansion and neurological outcome. Caspase-3 plasma levels were determined by ELISA at different time points after stroke in 116 t-PA-treated patients and a control group of 40 healthy controls. Neurological status was evaluated by NIHSS scores and functional outcome by modified Rankin Scale. To assess brain infarct growth, serial brain magnetic resonance imaging scans including diffusion- (DWI) and perfusion-weighted (PWI) images were performed in a subgroup of 58 patients. Plasma caspase-3 levels were higher in stroke patients versus the control group throughout the acute phase of stroke. Furthermore, caspase-3 level at 24h was associated with poorer short- and long-term neurological outcome and positively correlated with infarct growth assessed by diffusion-weighted images. Our data suggest that caspase-3 could be involved in recruitment of ischemic brain tissue being a marker of infarct growth.

Endonuclease G expression in thalamic reticular nucleus after global cerebral ischemia.

Endonuclease G is a mitochondrial enzyme, known to be translocated to the nucleus after transient focal cerebral ischemia and contribute to DNA degradation. After global cerebral ischemia, delayed cell death is observed in the thalamic reticular nucleus but the mechanisms involved are not well described. The purpose of this study was to identify if Endonuclease G was expressed in the cell nucleus of parvalbumin(+) GABA'ergic neurons in relation to cell death after global cerebral ischemia in the thalamic reticular nucleus. The cell death in male Wister rats were studied from 6 h until 4 days after global cerebral ischemia induced by transient 2-vessel carotid occlusion with hypotension for 15 min. Hematoxylin-eosin staining and immunohistochemistry for Endonuclease G, Parvalbumin and Glial fibrillary acidic protein was performed after the ischemic insult. Eosinophilic neurons and vacuolization of the cytoplasm in parvalbumin(+) neurons were observed 2 days after ischemia. Endonuclease G immunoreactivity increased in the cytoplasm 12 h after ischemia and was translocated to the nucleus of parvalbumin(+) neurons after 24 h. In the nucleus of astroglia, Endonuclease G was expressed after 2 days with an apoptotic-like morphology and the number of Endonuclease G-expressing astroglia increased during the later time points. During the same period the number of parvalbumin(+) neurons decreased. In conclusion, this study has identified that Endonuclease G is translocated from the cytoplasm to the nucleus of neurons and expressed with apoptotic-like morphology in the nucleus of astroglia in the thalamic reticular nucleus after global cerebral ischemia.

ACE I/D polymorphism in Korean patients with ischemic stroke and silent brain infarction.

OBJECTIVES: Angiotensin-converting enzyme (ACE) polymorphism may play a role in stroke and silent brain infarction (SBI) susceptibility, but the results among the populations studied to date have not been consistent. Thus, we investigated the association between ACE genotypes and ischemic stroke and SBI in Korean patients. SUBJECTS AND METHODS: DNA samples from 237 stroke patients, 264 SBI patients and 234 age-matched controls were amplified using polymerase chain reaction to detect the ACE ins/del (I/D) polymorphism. Genotype was determined by the presence of a 490-bp band (I allele) or a 190-bp band (D allele) in agarose gel electrophoresis. RESULTS: Odds ratios of the I/D and D/D genotypes and the overall (I/D + D/D) for the I/I genotype were significantly different between stroke patients and normal controls. However, there was no significant difference between patients with SBI and controls. CONCLUSIONS: This study is the first report of a significant association between ACE polymorphism and ischemic stroke in the Asian population. Although no consistent associations have been found between ACE polymorphism and stroke in the populations studied to date, the ACE polymorphism may be a genetic determinant of ischemic stroke, at least in Korean patients.