Hiroyuki Sakata

Oxidative Stress in Ischemic Brain Damage: Mechanisms of Cell Death and Potential Molecular Targets for Neuroprotection

Significant amounts of oxygen free radicals (oxidants) are generated during cerebral ischemia/reperfusion, and oxidative stress plays an important role in brain damage after stroke. In addition to oxidizing macromolecules, leading to cell injury, oxidants are also involved in cell death/survival signal pathways and cause mitochondrial dysfunction. Experimental data from laboratory animals that either overexpress (transgenic) or are deficient in (knock-out) antioxidant proteins, mainly superoxide dismutase, have provided strong evidence of the role of oxidative stress in ischemic brain damage. In addition to mitochondria, recent reports demonstrate that NADPH oxidase (NOX), an important pro-oxidant enzyme, is also involved in the generation of oxidants in the brain after stroke. Inhibition of NOX is neuroprotective against cerebral ischemia. We propose that superoxide dismutase and NOX activity in the brain is a major determinant for ischemic damage/repair and that these major anti- and pro-oxidant enzymes are potential endogenous molecular targets for stroke therapy.

Reperfusion and Neurovascular Dysfunction in Stroke: from Basic Mechanisms to Potential Strategies for Neuroprotection

Effective stroke therapies require recanalization of occluded cerebral blood vessels. However, reperfusion can cause neurovascular injury, leading to cerebral edema, brain hemorrhage, and neuronal death by apoptosis/necrosis. These complications, which result from excess production of reactive oxygen species in mitochondria, significantly limit the benefits of stroke therapies. We have developed a focal stroke model using mice deficient in mitochondrial manganese-superoxide dismutase (SOD2−/+) to investigate neurovascular endothelial damage that occurs during reperfusion. Following focal stroke and reperfusion, SOD2−/+ mice had delayed blood-brain barrier breakdown, associated with activation of matrix metalloproteinase and high brain hemorrhage rates, whereas a decrease in apoptosis and hemorrhage was observed in SOD2 overexpressors. Thus, induction and activation of SOD2 is a novel strategy for neurovascular protection after ischemia/reperfusion. Our recent study identified the signal transducer and activator of transcription 3 (STAT3) as a transcription factor of the mouse SOD2 gene. During reperfusion, activation of STAT3 and its recruitment into the SOD2 gene were blocked, resulting in increased oxidative stress and neuronal apoptosis. In contrast, pharmacological activation of STAT3 induced SOD2 expression, which limits ischemic neuronal death. Our studies point to antioxidant-based neurovascular protective strategies as potential treatments to expand the therapeutic window of currently approved therapies.

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.

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.

Hemoglobin-Induced Oxidative Stress Contributes to Matrix Metalloproteinase Activation and Blood–Brain Barrier Dysfunction in vivo

Hemoglobin (Hb) released from extravasated erythrocytes is implicated in brain edema after intracerebral hemorrhage (ICH). Hemoglobin is a major component of blood and a potent mediator of oxidative stress after ICH. Oxidative stress and matrix metalloproteinases (MMPs) are associated with blood–brain barrier (BBB) dysfunction. This study was designed to elucidate whether Hb-induced oxidative stress contributes to MMP-9 activation and BBB dysfunction in vivo. An intracerebral injection of Hb into rat striata induced increased hydroethidine (HEt) signals in parallel with MMP-9 levels. In situ gelatinolytic activity colocalized with oxidized HEt signals in vessel walls, accompanied by immunoglobulin G leakage and a decrease in immunoactivity of endothelial barrier antigen, a marker of endothelial integrity. Administration of a nonselective MMP inhibitor prevented MMP-9 levels and albumin leakage in injured striata. Moreover, reduction in oxidative stress by copper/zinc-superoxide dismutase (SOD1) overexpression reduced oxidative stress, MMP-9 levels, albumin leakage, and subsequent apoptosis compared with wild-type littermates. We speculate that Hb-induced oxidative stress may contribute to early BBB dysfunction and subsequent apoptosis, partly through MMP activation, and that SOD1 overexpression may reduce Hb-induced oxidative stress, BBB dysfunction, and apoptotic cell death.

Genetics and Biomarkers of Moyamoya Disease: Significance of RNF213 as a Susceptibility Gene

Moyamoya disease is characterized by a progressive stenosis at the terminal portion of the internal carotid artery and an abnormal vascular network at the base of the brain. Although its etiology is still unknown, recent genome-wide and locus-specific association studies identified RNF213 as an important susceptibility gene of moyamoya disease among East Asian population. A polymorphism in c.14576G>A in RNF213 was identified in 95% of familial patients with moyamoya disease and 79% of sporadic cases, and patients having this polymorphism were found to have significantly earlier disease onset and a more severe form of moyamoya disease, such as the presentation of cerebral infarction and posterior cerebral artery stenosis. The exact mechanism by which the RNF213 abnormality relates to moyamoya disease remains unknown, while recent reports using genetically engineered mice lacking RNF213 by homologous recombination provide new insight for the pathogenesis of this rare entity. Regarding biomarkers of moyamoya disease, moyamoya disease is characterized by an increased expression of angiogenic factors and pro-inflammatory molecules such as vascular endothelial growth factors and matrix metalloproteinase-9, which may partly explain its clinical manifestations of the pathologic angiogenesis, spontaneous hemorrhage, and higher incidence of cerebral hyperperfusion after revascularization surgery. More recently, blockade of these pro-inflammatory molecules during perioperative period is attempted to reduce the potential risk of surgical complication including cerebral hyperperfusion syndrome. In this review article, we focus on the genetics and biomarkers of moyamoya disease, and sought to discuss their clinical implication.

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.

Enhanced post-ischemic angiogenesis in mice lacking RNF213; a susceptibility gene for moyamoya disease.

Moyamoya disease (MMD) is a chronic occlusive cerebrovascular disease with unknown etiology that is characterized by the development of abnormal vascular networks at the base of the brain. Recent genome-wide studies identified RNF213 as an important MMD susceptibility gene. However, the exact mechanism by which the RNF213 abnormality leads to MMD remains unknown. Thus, we sought to clarify the role of RNF213 in angiogenesis under ischemic conditions using conventional RNF213 knockout mice. We assessed the infarction volume, cerebral edema, and vascular density in the ischemic brain after transient middle cerebral artery occlusion (tMCAO). To further evaluate systemic angiogenesis following chronic ischemia, we investigated blood flow recovery using laser speckle flowmetry, the severity of ambulatory impairments, and vascular density in the hind-limb after permanent femoral artery ligation. Results were compared between homozygous RNF213 knockout mice (RNF213 -/-) and wild-type littermates (Wt). No significant differences were observed in infarction volume or the formation of edema following tMCAO, or in vascular density 28 days after tMCAO between RNF213 -/- and Wt. Blood flow recovery was significantly improved in RNF213 -/- from 3 to 28 days after femoral artery ligation, and angiogenesis as shown by vascular density in the hind-limb was significantly enhanced in RNF213 -/- at 28 days. The amelioration of ambulatory impairments was also evident in RNF213 -/-. Angiogenesis was enhanced in mice lacking RNF213 after chronic hind-limb ischemia, which suggested the potential role of the RNF213 abnormality in the development of pathological vascular networks in chronic ischemia.

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.

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.