Hypoxic preconditioning enhances neural stem cell transplantation therapy after intracerebral hemorrhage in mice.

Previous studies have shown that intraparenchymal transplantation of neural stem cells ameliorates neurological deficits in animals with intracerebral hemorrhage. However, hemoglobin in the host brain environment causes massive grafted cell death and reduces the effectiveness of this approach. Several studies have shown that preconditioning induced by sublethal hypoxia can markedly improve the tolerance of treated subjects to more severe insults. Therefore, we investigated whether hypoxic preconditioning enhances neural stem cell resilience to the hemorrhagic stroke environment and improves therapeutic effects in mice. To assess whether hypoxic preconditioning enhances neural stem cell survival when exposed to hemoglobin, neural stem cells were exposed to 5% hypoxia for 24 hours before exposure to hemoglobin. To study the effectiveness of hypoxic preconditioning on grafted-neural stem cell recovery, neural stem cells subjected to hypoxic preconditioning were grafted into the parenchyma 3 days after intracerebral hemorrhage. Hypoxic preconditioning significantly enhanced viability of the neural stem cells exposed to hemoglobin and increased grafted-cell survival in the intracerebral hemorrhage brain. Hypoxic preconditioning also increased neural stem cell secretion of vascular endothelial growth factor. Finally, transplanted neural stem cells with hypoxic preconditioning exhibited enhanced tissue-protective capability that accelerated behavioral recovery. Our results suggest that hypoxic preconditioning in neural stem cells improves efficacy of stem cell therapy for intracerebral hemorrhage.

Postconditioning mitigates cell death following oxygen and glucose deprivation in PC12 cells and forebrain reperfusion injury in rats.

Postconditioning mitigates ischemia-induced cellular damage via a modified reperfusion procedure. Mitochondrial permeability transition (MPT) is an important pathophysiological change in reperfusion injury. This study explores the role of MPT modulation underlying hypoxic postconditioning (HPoC) in PC12 cells and studies the neuroprotective effects of ischemic postconditioning (IPoC) on rats. Oxygen-glucose deprivation (OGD) was performed for 10 hr on PC12 cells. HPoC was induced by three cycles of 10-min reoxygenation/10-min rehypoxia after OGD. The MPT inhibitor N-methyl-4-isoleucine cyclosporine (NIM811) and the MPT inducer carboxyatractyloside (CATR) were administered to selective groups before OGD. Cellular death was evaluated by flow cytometry and Western blot analysis. JC-1 fluorescence signal was used to estimate the mitochondrial membrane potential (△Ψm ). Transient global cerebral ischemia (tGCI) was induced via the two-vessel occlusion and hypotension method in male Sprague Dawley rats. IPoC was induced by three cycles of 10-sec reperfusion/10-sec reocclusion after index ischemia. HPoC and NIM811 administration attenuated cell death, cytochrome c release, and caspase-3 activity and maintained △Ψm of PC12 cells after OGD. The addition of CATR negated the protection conferred by HPoC. IPoC reduced neuronal degeneration and cytochrome c release and cleaved caspase-9 expression of hippocampal CA1 neurons in rats after tGCI. HPoC protected PC12 cells against OGD by modulating the MPT. IPoC attenuated degeneration of hippocampal neurons after cerebral ischemia.

Synergy of combined tPA-edaravone therapy in experimental thrombotic stroke.

Edaravone, a potent antioxidant, may improve thrombolytic therapy because it benefits ischemic stroke patients on its own and mitigates adverse effects of tissue plasminogen activator (tPA) in preclinical models. However, whether the combined tPA-edaravone therapy is more effective in reducing infarct size than singular treatment is uncertain. Here we investigated this issue using a transient hypoxia-ischemia (tHI)-induced thrombotic stroke model, in which adult C57BL/6 mice were subjected to reversible ligation of the unilateral common carotid artery plus inhalation of 7.5% oxygen for 30 min. While unilateral occlusion of the common carotid artery suppressed cerebral blood flow transiently, the addition of hypoxia triggered reperfusion deficits, endogenous thrombosis, and attenuated tPA activity, leading up to infarction. We compared the outcomes of vehicle-controls, edaravone treatment, tPA treatment at 0.5, 1, or 4 h post-tHI, and combined tPA-edaravone therapies with mortality rate and infarct size as the primary end-points. The best treatment was further compared with vehicle-controls in behavioral, biochemical, and diffusion tensor imaging (DTI) analyses. We found that application of tPA at 0.5 or 1 h--but not at 4 h post-tHI--significantly decreased infarct size and showed synergistic (p<0.05) or additive benefits with the adjuvant edaravone treatment, respectively. The acute tPA-edaravone treatment conferred >50% reduction of mortality, ∼ 80% decline in infarct size, and strong white-matter protection. It also improved vascular reperfusion and decreased oxidative stress, inflammatory cytokines, and matrix metalloproteinase activities. In conclusion, edaravone synergizes with acute tPA treatment in experimental thrombotic stroke, suggesting that clinical application of the combined tPA-edaravone therapy merits investigation.

Transplantation of neural stem cells that overexpress SOD1 enhances amelioration of intracerebral hemorrhage in mice.

Previous studies have shown that intraparenchymal transplantation of neural stem cells (NSCs) ameliorates neurologic deficits in animals with intracerebral hemorrhage (ICH). However, massive grafted cell death after transplantation, possibly caused by a hostile host brain environment, lessens the effectiveness of this approach. We focused on the effect of oxidative stress against grafted NSCs and hypothesized that conferring antioxidant properties to transplanted NSCs may overcome their death and enhance neuroprotection after ICH. Copper/zinc-superoxide dismutase (SOD1) is a specific antioxidant enzyme that counteracts superoxide anions. We investigated whether genetic manipulation to overexpress SOD1 enhances survival of grafted NSCs and accelerates amelioration of ICH. Neural stem cells that overexpress SOD1 were administered intracerebrally 3 days after ICH in a mouse model. Histologic and behavioral tests were examined after ICH. Copper/zinc-superoxide dismutase overexpression protected the grafted NSCs via a decrease in production of reactive oxygen species. This resulted in an increase in paracrine factors released by the NSCs, and an increase in surviving neurons in the striatum and a reduction in striatal atrophy. In addition, SOD1 overexpression showed progressive improvement in behavioral recovery. Our results suggest that enhanced antioxidative activity in NSCs improves efficacy of stem cell therapy for ICH.

The role of PARL and HtrA2 in striatal neuronal injury after transient global cerebral ischemia.

The presenilin-associated rhomboid-like (PARL) protein and high temperature requirement factor A2 (HtrA2) are key regulators of mitochondrial integrity and play pivotal roles in apoptosis. However, their roles after cerebral ischemia have not been thoroughly elucidated. To clarify these roles, mice were subjected to transient global cerebral ischemia, and striatal neuronal injury was assessed. Western blot and coimmunoprecipitation analyses revealed that PARL and processed HtrA2 localized to mitochondria, and that PARL was bound to HtrA2 in sham animals. Expression of PARL and processed HtrA2 in mitochondria significantly decreased 6 to 72 hours after ischemia, and the binding of PARL to HtrA2 disappeared after ischemia. In contrast, expression of processed HtrA2 increased 24 hours after ischemia in the cytosol, where HtrA2 was bound to X chromosome-linked inhibitor-of-apoptosis protein (XIAP). Administration of PARL small interfering RNA inhibited HtrA2 processing and worsened ischemic neuronal injury. Our results show that downregulation of PARL after ischemia is a key step in ischemic neuronal injury, and that it decreases HtrA2 processing and increases neuronal vulnerability. In addition, processed HtrA2 released into the cytosol after ischemia contributes to neuronal injury via inhibition of XIAP.

Crush, culotte, T and protrusion: which 2-stent technique for treatment of true bifurcation lesions? - insights from in vitro experiments and micro-computed tomography.

BACKGROUND: Percutaneous coronary intervention of complex true bifurcation lesions often fails to ensure continuous stent coverage and strut apposition in both the side branch and main vessel. Struts left unopposed floating in the lumen disturb blood flow and are increasingly recognized as increasing the risk of stent thrombosis. METHODS AND RESULTS: In this study, we compared the results of different bifurcation treatment strategies: Crush (n=5); Culotte (n=3); T-/T with Protrusion (TAP) (n=4) using drug-eluting stents deployed in-vitro in representative coronary bifurcation models. After final kissing balloon post-dilatation, the rate of malapposition within the bifurcation quantified from micro-computed tomography scanning was on average 41.5 ± 8.2% with the Crush technique, reduced to respectively 31.4 ± 5.2% with Culotte and 36.7 ± 8.0% with T-/TAP approach. Overlaying layers of struts in the Crush and Culotte techniques lead to a significantly higher rate of strut malapposition in the proximal vessel than with the T-/TAP technique (Crush: 39.1 ± 10.7%, Culotte: 26.1 ± 7.7%, TAP: 4.2 ± 7.2%, P<0.01). Maximal wall-malapposed strut distance was also found on average to be higher with the Crush (1.36 ± 0.4mm) and Culotte techniques (1.32 ± 0.1mm) than with T-/TAP (1.08 ± 0.1mm, P=0.04). CONCLUSIONS: In this model, the Crush technique resulted in a higher risk of malapposition than either the Culotte or T-/TAP technique.

Prevention of JNK phosphorylation as a mechanism for rosiglitazone in neuroprotection after transient cerebral ischemia: activation of dual specificity phosphatase.

Rosiglitazone, a synthetic peroxisome proliferator-activated receptor-γ (PPARγ) agonist, prevents cell death after cerebral ischemia in animal models, but the underlying mechanism has not been clarified. In this study, we examined how rosiglitazone protects neurons against ischemia. Mice treated with rosiglitazone were subjected to 60 minutes of focal ischemia followed by reperfusion. Rosiglitazone reduced infarct volume after ischemia and reperfusion. We show that this neuroprotective effect was reversed with a PPARγ antagonist. Western blot analysis showed a significant increase in expression of phosphorylated stress-activated protein kinases (c-Jun N-terminal kinase (JNK) and p38) in ischemic brain tissue. Rosiglitazone blocked this increase. Furthermore, we observed that rosiglitazone increased expression of the dual-specificity phosphatase 8 (DUSP8) protein and messenger RNA in ischemic brain tissue. Dual-specificity phosphatase 8 is a mitogen-activated protein kinase phosphatase that can dephosphorylate JNK and p38. Another key finding of the present study was that knockdown of DUSP8 in primary cultured cortical neurons that were subjected to oxygen-glucose deprivation diminished rosiglitazone's effect on downregulation of JNK phosphorylation. Thus, rosiglitazone's neuroprotective effect after ischemia is mediated by blocking JNK phosphorylation induced by ischemia via DUSP8 upregulation.

Complement component 3 inhibition by an antioxidant is neuroprotective after cerebral ischemia and reperfusion in mice.

Oxidative stress after stroke is associated with the inflammatory system activation in the brain. The complement cascade, especially the degradation products of complement component 3, is a key inflammatory mediator of cerebral ischemia. We have shown that pro-inflammatory complement component 3 is increased by oxidative stress after ischemic stroke in mice using DNA array. In this study, we investigated whether up-regulation of complement component 3 is directly related to oxidative stress after transient focal cerebral ischemia in mice and oxygen-glucose deprivation in brain cells. Persistent up-regulation of complement component 3 expression was reduced in copper/zinc-superoxide dismutase transgenic mice, and manganese-superoxide dismutase knock-out mice showed highly increased complement component 3 levels after transient focal cerebral ischemia. Antioxidant N-tert-butyl-α-phenylnitrone treatment suppressed complement component 3 expression after transient focal cerebral ischemia. Accumulation of complement component 3 in neurons and microglia was decreased by N-tert-butyl-α-phenylnitrone, which reduced infarct volume and impaired neurological deficiency after cerebral ischemia and reperfusion in mice. Small interfering RNA specific for complement component 3 transfection showed a significant increase in brain cells viability after oxygen-glucose deprivation. Our study suggests that the neuroprotective effect of antioxidants through complement component 3 suppression is a new strategy for potential therapeutic approaches in stroke.

Interleukin 6-preconditioned neural stem cells reduce ischaemic injury in stroke mice.

Transplantation of neural stem cells provides a promising therapy for stroke. Its efficacy, however, might be limited because of massive grafted-cell death after transplantation, and its insufficient capability for tissue repair. Interleukin 6 is a pro-inflammatory cytokine involved in the pathogenesis of various neurological disorders. Paradoxically, interleukin 6 promotes a pro-survival signalling pathway through activation of signal transducer and activator of transcription 3. In this study, we investigated whether cellular reprogramming of neural stem cells with interleukin 6 facilitates the effectiveness of cell transplantation therapy in ischaemic stroke. Neural stem cells harvested from the subventricular zone of foetal mice were preconditioned with interleukin 6 in vitro and transplanted into mouse brains 6 h or 7 days after transient middle cerebral artery occlusion. Interleukin 6 preconditioning protected the grafted neural stem cells from ischaemic reperfusion injury through signal transducer and activator of transcription 3-mediated upregulation of manganese superoxide dismutase, a primary mitochondrial antioxidant enzyme. In addition, interleukin 6 preconditioning induced secretion of vascular endothelial growth factor from the neural stem cells through activation of signal transducer and activator of transcription 3, resulting in promotion of angiogenesis in the ischaemic brain. Furthermore, transplantation of interleukin 6-preconditioned neural stem cells significantly attenuated infarct size and improved neurological performance compared with non-preconditioned neural stem cells. This interleukin 6-induced amelioration of ischaemic insults was abolished by transfecting the neural stem cells with signal transducer and activator of transcription 3 small interfering RNA before transplantation. These results indicate that preconditioning with interleukin 6, which reprograms neural stem cells to tolerate oxidative stress after ischaemic reperfusion injury and to induce angiogenesis through activation of signal transducer and activator of transcription 3, is a simple and beneficial approach for enhancing the effectiveness of cell transplantation therapy in ischaemic stroke.

Involvement of mitogen-activated protein kinase pathways in expression of the water channel protein aquaporin-4 after ischemia in rat cortical astrocytes.

Brain edema after ischemic brain injury is a key determinant of morbidity and mortality. Aquaporin-4 (AQP4) plays an important role in water transport in the central nervous system and is highly expressed in brain astrocytes. However, the AQP4 regulatory mechanisms are poorly understood. In this study, we investigated whether mitogen-activated protein kinases (MAPKs), which are involved in changes in osmolality, might mediate AQP4 expression in models of rat cortical astrocytes after ischemia. Increased levels of AQP4 in primary cultured astrocytes subjected to oxygen-glucose deprivation (OGD) and 2 h of reoxygenation were observed, after which they immediately decreased at 0 h of reoxygenation. Astrocytes exposed to OGD injury had significantly increased phosphorylation of three kinds of MAPKs. Treatment with SB203580, a selective p38 MAPK inhibitor, or SP600125, a selective c-Jun N-terminal kinase inhibitor, significantly attenuated the return of AQP4 to its normal level, and SB203580, but not SP600125, significantly decreased cell death. In an in vivo study, AQP4 expression was upregulated 1-3 days after reperfusion, which was consistent with the time course of p38 phosphorylation and activation, and decreased by the p38 inhibition after transient middle cerebral artery occlusion (MCAO). These results suggest that p38 MAPK may regulate AQP4 expression in cortical astrocytes after ischemic injury.

Neural stem cells genetically modified to overexpress cu/zn-superoxide dismutase enhance amelioration of ischemic stroke in mice.

BACKGROUND AND PURPOSE: The harsh host brain microenvironment caused by production of reactive oxygen species after ischemic reperfusion injury offers a significant challenge to survival of transplanted neural stem cells (NSCs) after ischemic stroke. Copper/zinc-superoxide dismutase (SOD1) is a specific antioxidant enzyme that counteracts superoxide anions. We have investigated whether genetic manipulation to overexpress SOD1 enhances survival of grafted stem cells and accelerates amelioration of ischemic stroke. METHODS: NSCs genetically modified to overexpress or downexpress SOD1 were administered intracerebrally 2 days after transient middle cerebral artery occlusion. Histological and behavioral tests were examined from Days 0 to 28 after stroke. RESULTS: Overexpression of SOD1 suppressed production of superoxide anions after ischemic reperfusion injury and reduced NSC death after transplantation. In contrast, downexpression of SOD1 promoted superoxide generation and increased oxidative stress-mediated NSC death. Transplantation of SOD1-overexpressing NSCs enhanced angiogenesis in the ischemic border zone through upregulation of vascular endothelial growth factor. Moreover, grafted SOD1-overexpressing NSCs reduced infarct size and improved behavioral performance compared with NSCs that were not genetically modified. CONCLUSIONS: Our findings reveal a strong involvement of SOD1 expression in NSC survival after ischemic reperfusion injury. We propose that conferring antioxidant properties on NSCs by genetic manipulation of SOD1 is a potential approach for enhancing the effectiveness of cell transplantation therapy in ischemic stroke.

Minocycline-preconditioned neural stem cells enhance neuroprotection after ischemic stroke in rats.

Transplantation of neural stem cells (NSCs) offers a novel therapeutic strategy for stroke; however, massive grafted cell death following transplantation, possibly due to a hostile host brain environment, lessens the effectiveness of this approach. Here, we have investigated whether reprogramming NSCs with minocycline, a broadly used antibiotic also known to possess cytoprotective properties, enhances survival of grafted cells and promotes neuroprotection in ischemic stroke. NSCs harvested from the subventricular zone of fetal rats were preconditioned with minocycline in vitro and transplanted into rat brains 6 h after transient middle cerebral artery occlusion. Histological and behavioral tests were examined from days 0-28 after stroke. For in vitro experiments, NSCs were subjected to oxygen-glucose deprivation and reoxygenation. Cell viability and antioxidant gene expression were analyzed. Minocycline preconditioning protected the grafted NSCs from ischemic reperfusion injury via upregulation of Nrf2 and Nrf2-regulated antioxidant genes. Additionally, preconditioning with minocycline induced the NSCs to release paracrine factors, including brain-derived neurotrophic factor, nerve growth factor, glial cell-derived neurotrophic factor, and vascular endothelial growth factor. Moreover, transplantation of the minocycline-preconditioned NSCs significantly attenuated infarct size and improved neurological performance, compared with non-preconditioned NSCs. Minocycline-induced neuroprotection was abolished by transfecting the NSCs with Nrf2-small interfering RNA before transplantation. Thus, preconditioning with minocycline, which reprograms NSCs to tolerate oxidative stress after ischemic reperfusion injury and express higher levels of paracrine factors through Nrf2 up-regulation, is a simple and safe approach to enhance the effectiveness of transplantation therapy in ischemic stroke.

Induction of thioredoxin-interacting protein is mediated by oxidative stress, calcium, and glucose after brain injury in mice.

Oxidative stress and glucose affect the expression of various genes that contribute to both reactive oxygen species generation and antioxidant systems. However, systemic alteration of oxidative stress-related gene expression in normal brains and in brains with a high-glucose status after ischemic-reperfusion has not been explored. Using a polymerase chain reaction array system, we demonstrate that thioredoxin-interacting protein (Txnip) is induced by both oxidative stress and glucose. We found that Txnip mRNA is induced by ischemic-reperfusion injury and that Txnip is located in the cytoplasm of neurons. Moreover, in vitro oxygen-glucose deprivation (OGD) and subsequent reoxygenation without glucose and in vivo administration of 3-nitropropionic acid also promoted an increase in Txnip in a time-dependent manner, indicating that oxidative stress without glucose can induce Txnip expression in the brain. However, calcium channel blockers inhibit induction of Txnip after OGD and reoxygenation. Using the polymerase chain reaction array with ischemic and hyperglycemic-ischemic samples, we confirmed that enhanced expression of Txnip was observed in hyperglycemic-ischemic brains after middle cerebral artery occlusion. Finally, transfection of Txnip small interfering RNA into primary neurons reduced lactate dehydrogenase release after OGD and reoxygenation. This is the first report showing that Txnip expression is induced in neurons after oxidative or glucose stress under either ischemic or hyperglycemic-ischemic conditions, and that Txnip is proapoptotic under these conditions.

Release of mitochondrial apoptogenic factors and cell death are mediated by CK2 and NADPH oxidase.

Activation of the NADPH oxidase subunit, NOX2, and increased oxidative stress are associated with neuronal death after cerebral ischemia and reperfusion. Inhibition of NOX2 by casein kinase 2 (CK2) leads to neuronal survival, but the mechanism is unknown. In this study, we show that in copper/zinc-superoxide dismutase transgenic (SOD1 Tg) mice, degradation of CK2α and CK2α' and dephosphorylation of CK2ß against oxidative stress were markedly reduced compared with wild-type (WT) mice that underwent middle cerebral artery occlusion. Inhibition of CK2 pharmacologically or by ischemic reperfusion facilitated accumulation of poly(ADP-ribose) polymers, the translocation of apoptosis-inducing factor (AIF), and cytochrome c release from mitochondria after ischemic injury. The eventual enhancement of CK2 inhibition under ischemic injury strongly increased 8-hydroxy-2'-deoxyguanosine and phosphorylation of H2A.X. Furthermore, CK2 inhibition by tetrabromocinnamic acid (TBCA) in SOD1 Tg and gp91 knockout (KO) mice after ischemia reperfusion induced less release of AIF and cytochrome c than in TBCA-treated WT mice. Inhibition of CK2 in gp91 KO mice subjected to ischemia reperfusion did not increase brain infarction compared with TBCA-treated WT mice. These results strongly suggest that NOX2 activation releases reactive oxygen species after CK2 inhibition, triggering release of apoptogenic factors from mitochondria and inducing DNA damage after ischemic brain injury.

Neuroprotection by interleukin-6 is mediated by signal transducer and activator of transcription 3 and antioxidative signaling in ischemic stroke.

BACKGROUND AND PURPOSE: Interleukin-6 (IL-6) has been shown to have a neuroprotective effect in brain ischemic injury. However, its molecular mechanisms are still poorly understood. In this study, we investigated the neuroprotective role of the IL-6 receptor (IL-6R) by IL-6 in the reactive oxygen species defense system after transient focal cerebral ischemia (tFCI). METHODS: IL-6 was injected in mice before and after middle cerebral artery occlusion. Coimmunoprecipitation assays were performed for analysis of an IL-6R association after tFCI. Primary mouse cerebral cortical neurons were transfected with small interfering RNA probes targeted to IL-6Rα or gp130 and were used for chromatin-immunoprecipitation assay, luciferase promoter assay, and cell viability assay. Reduction in infarct volumes by IL-6 was measured after tFCI. RESULTS: IL-6R was disrupted through a disassembly between IL-6Rα and gp130 associated by protein oxidation after reperfusion after tFCI. This suppressed phosphorylation of signal transducer and activator of transcription 3 (STAT3) and finally induced neuronal cell death through a decrease in manganese-superoxide dismutase. However, IL-6 injections prevented disruption of IL-6R against reperfusion after tFCI, consequently restoring activity of STAT3 through recovery of the binding of STAT3 to gp130. Moreover, IL-6 injections restored the transcriptional activity of the manganese-superoxide dismutase promoter through recovery of the recruitment of STAT3 to the manganese-superoxide dismutase promoter and reduced infarct volume after tFCI. CONCLUSIONS: This study demonstrates that IL-6 has a neuroprotective effect against cerebral ischemic injury through IL-6R-mediated STAT3 activation and manganese-superoxide dismutase expression.

Manganese superoxide dismutase deficiency exacerbates ischemic brain damage under hyperglycemic conditions by altering autophagy.

Both preischemic hyperglycemia and suppression of SOD2 activity aggravate ischemic brain damage. This study was undertaken to assess the effect of SOD2 mutation on ischemic brain damage and its relation to the factors involved in autophagy regulation in hyperglycemic wild-type (WT) and heterozygous SOD2 knockout (SOD2(-/+)) mice subjected to 30-min transient focal ischemia. The brain samples were analyzed at 5 and 24 h after recirculation for ischemic lesion volume, superoxide production, and oxidative DNA damage and protein levels of Beclin 1, damage-regulated autophagy modulator (DRAM), and microtubule-associated protein 1 light chain 3 (LC3). The results revealed a significant increase in infarct volume in hyperglycemic SOD2(-/+) mice, and this was accompanied with an early (5 h) significant rise in superoxide production and reduced SOD2 activity in SOD2(-/+) mice as compared to WT mice. The superoxide production is associated with oxidative DNA damage as indicated by colocalization of the dihydroethidium (DHE) signal with 8-OHdG fluorescence in SOD2(-/+) mice. In addition, while ischemia in WT hyperglycemics increased the levels of autophagy markers Beclin 1, DRAM, and LC3, ischemia in hyperglycemic, SOD2-deficient mice suppressed the levels of autophagy stimulators. These results suggest that SOD2 knockdown exacerbates ischemic brain damage under hyperglycemic conditions via increased oxidative stress and DNA oxidation. Such effect is associated with suppression of autophagy regulators.

Jailed side branches: fate of unapposed struts studied with 3D frequency-domain optical coherence tomography.

We report the case of a 64-year-old man treated for stable angina with two bare-metal stents in the proximal-mid segment of the left anterior descending artery at the bifurcation with the first diagonal and second septal branches without final kissing balloon dilatation. Seven months later he complained of recurrent angina. Frequency-domain optical coherence tomography (OCT) with three-dimensional (3D) reconstruction was performed, showing a thick rim of neointimal proliferation at the ostium of both branches, causing severe restenosis. Deployment of a stent in a bifurcation main branch without opening the struts at the side-branch ostium may facilitate focal restenosis.

NADPH oxidase is involved in post-ischemic brain inflammation.

Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is widely expressed in brain tissue including neurons, glia, and endothelia in neurovascular units. It is a major source of oxidants in the post-ischemic brain and significantly contributes to ischemic brain damage. Inflammation occurs after brain ischemia and is known to be associated with post-ischemic oxidative stress. Post-ischemic inflammation also causes progressive brain injury. In this study we investigated the role of NOX2 in post-ischemic cerebral inflammation using a transient middle cerebral artery occlusion model in mice. We demonstrate that mice with NOX2 subunit gp91(phox) knockout (gp91 KO) showed 35-44% less brain infarction at 1 and 3 days of reperfusion compared with wild-type (WT) mice. Minocycline further reduced brain damage in the gp91 KO mice at 3 days of reperfusion. The gp91 KO mice exhibited less severe post-ischemic inflammation in the brain, as evidenced by reduced microglial activation and decreased upregulation of inflammation mediators, including interleukin-1ß (IL-1ß), tumor necrosis factor-α, inducible nitric oxide synthases, CC-chemokine ligand 2, and CC-chemokine ligand 3. Finally, we demonstrated that an intraventricular injection of IL-1ß enhanced ischemia- and reperfusion-mediated brain damage in the WT mice (double the infarction volume), whereas, it failed to aggravate brain infarction in the gp91 KO mice. Taken together, these results demonstrate the involvement of NOX2 in post-ischemic neuroinflammation and that NOX2 inhibition provides neuroprotection against inflammatory cytokine-mediated brain damage.

Consistent injury to medium spiny neurons and white matter in the mouse striatum after prolonged transient global cerebral ischemia.

A reproducible transient global cerebral ischemia (tGCI) mouse model has not been fully established. Although striatal neurons and white matter are recognized to be vulnerable to ischemia, their injury after tGCI in mice has not been elucidated. The purpose of this study was to evaluate injuries to striatal neurons and white matter after tGCI in C57BL/6 mice, and to develop a reproducible tGCI model. Male C57BL/6 mice were subjected to tGCI by bilateral common carotid artery occlusion (BCCAO). Mice whose cortical cerebral blood flow after BCCAO decreased to less than 13% of the pre-ischemic value were used. Histological analysis showed that at 3 days after 22 min of BCCAO, striatal neurons were injured more consistently than those in other brain regions. Quantitative analysis of cytochrome c release into the cytosol and DNA fragmentation in the striatum showed consistent injury to the striatum. Immunohistochemistry and Western blot analysis revealed that DARPP-32-positive medium spiny neurons, the majority of striatal neurons, were the most vulnerable among the striatal neuronal subpopulations. The striatum (especially medium spiny neurons) was susceptible to oxidative stress after tGCI, which is probably one of the mechanisms of vulnerability. SMI-32 immunostaining showed that white matter in the striatum was also consistently injured 3 days after 22 min of BCCAO. We thus suggest that this is a tGCI model using C57BL/6 mice that consistently produces neuronal and white matter injury in the striatum by a simple technique. This model can be highly applicable for elucidating molecular mechanisms in the brain after global ischemia.

NADPH oxidase mediates striatal neuronal injury after transient global cerebral ischemia.

Medium spiny neurons (MSNs) constitute most of the striatal neurons and are known to be vulnerable to ischemia; however, the mechanisms of the vulnerability remain unclear. Activated forms of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase (NOX), which require interaction between cytosolic and membrane-bound subunits, are among the major sources of superoxide in the central nervous system. Although increasing evidence suggests that NOX has important roles in neurodegenerative diseases, its roles in MSN injury after transient global cerebral ischemia (tGCI) have not been elucidated. To clarify this issue, C57BL/6 mice were subjected to tGCI by bilateral common carotid artery occlusion for 22 minutes. Western blot analysis revealed upregulation of NOX subunits and recruitment of cytosolic subunits to the cell membrane at early (3 to 6 hours) and late (72 hours) phases after tGCI. Taken together with immunofluorescent studies, this activation arose in MSNs and endothelial cells at the early phase, and in reactive microglia at the late phase. Pharmacological and genetic inhibition of NOX attenuated oxidative injury, microglial activation, and MSN death after tGCI. These findings suggest that NOX has pivotal roles in MSN injury after tGCI and could be a therapeutic target for brain ischemia.