Naoki Oyama

SIK2 Is a Key Regulator for Neuronal Survival after Ischemia via TORC1-CREB

The cAMP responsive element-binding protein (CREB) functions in a broad array of biological and pathophysiological processes. We found that salt-inducible kinase 2 (SIK2) was abundantly expressed in neurons and suppressed CREB-mediated gene expression after oxygen-glucose deprivation (OGD). OGD induced the degradation of SIK2 protein concomitantly with the dephosphorylation of the CREB-specific coactivator transducer of regulated CREB activity 1 (TORC1), resulting in the activation of CREB and its downstream gene targets. Ca(2+)/calmodulin-dependent protein kinase I/IV are capable of phosphorylating SIK2 at Thr484, resulting in SIK2 degradation in cortical neurons. Neuronal survival after OGD was significantly increased in neurons isolated from sik2(-/-) mice, and ischemic neuronal injury was significantly reduced in the brains of sik2(-)(/-) mice subjected to transient focal ischemia. These findings suggest that SIK2 plays critical roles in neuronal survival, is modulated by CaMK I/IV, and regulates CREB via TORC1.

Granulocyte Colony-Stimulating Factor Enhances Arteriogenesis and Ameliorates Cerebral Damage in a Mouse Model of Ischemic Stroke

Background and Purpose— Enhancing collateral artery growth is a potent therapeutic approach to treat cardiovascular ischemic disease from occlusive artery. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has gained attention for its ability to promote arteriogenesis, ameliorating brain damage, by the mechanisms involving monocyte upregulation. However, the recent clinical study testing its efficacy in myocardial ischemia has raised the question about its safety. We tested alternative colony-stimulating factors for their effects on collateral artery growth and brain protection. Methods— Brain hypoperfusion was produced by occluding the left common carotid artery in C57/BL6 mice. After the surgery, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, or GM-CSF (100 &mgr;g/kg/day) was administered daily for 5 days. The angioarchitecture for leptomeningeal anastomoses and the circle of Willis were visualized after the colony-stimulating factor treatment. Circulating blood monocytes and Mac-2-positive cells in the dorsal surface of the brain were determined. A set of animals underwent subsequent ipsilateral middle cerebral artery occlusion and infarct volume was assessed. Results— Granulocyte colony-stimulating factor as well as GM-CSF promoted leptomeningeal collateral growth after common carotid artery occlusion. Both granulocyte colony-stimulating factor and GM-CSF increased circulating blood monocytes and Mac-2-positive cells in the dorsal brain surface, suggesting the mechanisms coupled to monocyte upregulation might be shared. Infarct volume after middle cerebral artery occlusion was reduced by granulocyte colony-stimulating factor, similarly to GM-CSF. Macrophage colony-stimulating factor showed none of theses effects. Conclusions— Granulocyte colony-stimulating factor enhances collateral artery growth and reduces infarct volume in a mouse model of brain ischemia, similarly to GM-CSF.

Activation of NR2A Receptors Induces Ischemic Tolerance through CREB Signaling

Previous exposure to a nonlethal ischemic insult protects the brain against subsequent harmful ischemia. N-methyl-D-aspartate (NMDA) receptors are a highly studied target of neuroprotection after ischemia. Recently, NMDA receptor subtypes were implicated in neuronal survival and death. We focused on the contribution of NR2A and cyclic-AMP response element (CRE)-binding protein (CREB) signaling to ischemic tolerance using primary cortical neurons. Ischemia in vitro was modeled by oxygen–glucose deprivation (OGD). Ischemic tolerance was induced by applying 45-mins OGD 24 h before 180-mins OGD. Sublethal OGD also induced cross-tolerance against lethal glutamate and hydrogen peroxide. After sublethal OGD, expression of phosphorylated CREB and CRE transcriptional activity were significantly increased. When CRE activity was inhibited by CREB-S133A, a mutant CREB, ischemic tolerance was abolished. Inhibiting NR2A using NVP-AAM077 attenuated preconditioning-induced neuroprotection and correlated with decreased CRE activity levels. Activating NR2A using bicuculline and 4-aminopiridine induced resistance to lethal ischemia accompanied by elevated CRE activity levels, and this effect was abolished by NVP-AAM077. Elevated brain-derived neurotrophic factor (BDNF) transcriptional activities were observed after sublethal OGD and administration of bicuculline and 4-aminopiridine. NR2A-containing NMDA receptors and CREB signaling have important functions in the induction of ischemic tolerance. This may provide potential novel therapeutic strategies to treat ischemic stroke.

Granulocyte-Macrophage Colony-Stimulating Factor Enhances Leptomeningeal Collateral Growth Induced by Common Carotid Artery Occlusion

Background and Purpose— Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to accelerate collateral growth (arteriogenesis) at the circle of Willis in rat brain. However, the effect of GM-CSF on leptomeningeal collateral growth has not been established. We examined the effect of unilateral common carotid artery (CCA) occlusion and GM-CSF treatment on leptomeningeal collateral growth in mice. Methods— Adult mice were subjected to unilateral CCA occlusion or sham surgery followed by an alternate-day regimen of GM-CSF (20 &mgr;g/kg) or saline injection. On day 7, latex perfusion was performed in 1 set of mice to visualize the leptomeningeal vessels, and the number of Mac-2+ monocytes/macrophages on the dorsal surface of the brain was counted. In another set of mice, on day 7, permanent ipsilateral middle cerebral artery (MCA) occlusion was performed, and infarct volume was measured. Results— Leptomeningeal collateral growth was observed after CCA occlusion, and that was enhanced by GM-CSF treatment. An increase in the number of Mac-2+ cells on the surface of the brain occurred after CCA occlusion and was enhanced by GM-CSF treatment. Seven days after CCA occlusion, GM-CSF treatment decreased the infarct size attributable to subsequent MCA occlusion. Conclusion— After CCA occlusion, GM-CSF treatment enhanced leptomeningeal collateral growth and decreased the infarct size after MCA occlusion in mice.

Cilostazol, Not Aspirin, Reduces Ischemic Brain Injury via Endothelial Protection in Spontaneously Hypertensive Rats

Background and Purpose— It is well-established that hypertension leads to endothelial dysfunction in the cerebral artery. Recently, cilostazol has been used for the secondary prevention of ischemic stroke. Among antiplatelet drugs, phosphodiesterase inhibitors including cilostazol have been shown to have protective effects on endothelial cells. The aim of the present study is to investigate the effects of cilostazol and aspirin on endothelial nitric oxide synthase (eNOS) phosphorylation in the cerebral cortex, endothelial function, and infarct size after brain ischemia in spontaneously hypertensive rats (SHR). Methods— Five-week-old male SHR received a 5-week regimen of chow containing 0.1% aspirin, 0.1% cilostazol, 0.3% cilostazol, or the vehicle control. The levels of total and Ser1177-phosphorylated eNOS protein in the cerebral cortex were evaluated by Western blot. To assess the contribution of eNOS in maintaining cerebral blood flow, we monitored cerebral blood flow by laser-Doppler flowmetry after L-N5-(1-iminoethyl)ornithine infusion. Additionally, we evaluated residual microperfusion using fluorescence-labeled serum protein and infarct size after transient focal brain ischemia. Results— In SHR, the blood pressure and heart rate were similar among the groups. Cilostazol-treated SHR had a significantly higher ratio of phospho-eNOS/total eNOS protein than vehicle-treated and aspirin-treated SHR. Treating with cilostazol, but not aspirin, significantly improved cerebral blood flow response to L-N5-(1-iminoethyl)ornithine. Cilostazol also increased residual perfusion of the microcirculation and reduced brain damage after ischemia compared to vehicle control and aspirin. Conclusions— These findings indicate that cilostazol, but not aspirin, can attenuate ischemic brain injury by maintaining endothelial function in the cerebral cortex of SHR.

Depletion of Cultivatable Gut Microbiota by Broad-Spectrum Antibiotic Pretreatment Worsens Outcome After Murine Stroke

Background and Purpose— Antibiotics disturbing microbiota are often used in treatment of poststroke infections. A bidirectional brain–gut microbiota axis was recently suggested as a modulator of nervous system diseases. We hypothesized that gut microbiota may be an important player in the course of stroke. Methods— We investigated the outcome of focal cerebral ischemia in C57BL/6J mice after an 8-week decontamination with quintuple broad-spectrum antibiotic cocktail. These microbiota-depleted animals were subjected to 60 minutes middle cerebral artery occlusion or sham operation. Infarct volume was measured using magnetic resonance imaging, and mice were monitored clinically throughout the whole experiment. At the end point, tissues were preserved for further analysis, comprising histology and immunologic investigations using flow cytometry. Results— We found significantly decreased survival in the middle cerebral artery occlusion microbiota-depleted mice when the antibiotic cocktail was stopped 3 days before surgery (compared with middle cerebral artery occlusion specific pathogen-free and sham-operated microbiota-depleted mice). Moreover, all microbiota-depleted animals in which antibiotic treatment was terminated developed severe acute colitis. This phenotype was rescued by continuous antibiotic treatment or colonization with specific pathogen-free microbiota before surgery. Further, infarct volumes on day one did not differ between any of the experimental groups. Conclusions— Conventional microbiota ensures intestinal protection in the mouse model of experimental stroke and prevents development of acute and severe colitis in microbiota-depleted mice not given antibiotic protection after cerebral ischemia. Our experiments raise the clinically important question as to whether microbial colonization or specific microbiota are crucial for stroke outcome.

An angiotensin II type 1 receptor blocker can preserve endothelial function and attenuate brain ischemic damage in spontaneously hypertensive rats

Hypertension reduces endothelial nitric oxide synthase (eNOS) expression and leads to endothelial dysfunction. However, few studies have demonstrated the influences of hypertension on eNOS function in the cerebral cortex. The present study investigates the influences of hypertension on endothelial function in the cerebral cortex and the protective effects of antihypertensive agents against brain ischemia through the preservation of endothelial function. Five‐ and ten‐week‐old male Wistar rats and spontaneously hypertensive rats (SHR) were used for experiments. Five‐week‐old SHR received olmesartan, hydralazine, or vehicle for 5 weeks in drinking water. eNOS activation in the cerebral cortex was evaluated by analyzing levels of total and Ser1177‐phosphorylated eNOS protein by Western blot. Blood pressure of 10‐week‐old SHR without treatment was clearly high, and the ratio of phospho‐eNOS/total eNOS protein was significantly low. Five‐week treatment with olmesartan or hydralazine suppressed the elevation of blood pressure and the reduction of phosphorylated eNOS‐Ser1177 in SHR, and olmesartan was more effective in maintaining phosphorylation of eNOS‐Ser1177 than hydralazine. To assess the contribution of eNOS to maintaining cerebral blood flow (CBF), we monitored CBF by laser‐Doppler flowmetry after L‐N5‐(1‐iminoethyl)ornithine (L‐NIO) infusion. CBF response to L‐NIO was preserved in olmesartan‐treated SHR but not in hydralazine‐treated SHR. Furthermore, infarct volume 48 hr after transient focal brain ischemia in olmesartan‐treated SHR was significantly reduced compared with vehicle‐treated SHR. These findings indicate that chronic prehypertensive treatment with olmesartan could attenuate brain ischemic injury through the maintenance of endothelial function in the cerebral cortex in SHR.

Hypertension impairs leptomeningeal collateral growth after common carotid artery occlusion: Restoration by antihypertensive treatment

Chronic mild hypoperfusion has been shown to enlarge pial collateral vessels in normal mouse brains. The purpose of this study was to clarify the effect of hypertension on pial collateral vessel development after chronic hypoperfusion using spontaneously hypertensive rats (SHR). In normotensive rats, unilateral common carotid artery (CCA) occlusion enlarged leptomeningeal collateral vessels. CCA occlusion also preserved residual cerebral blood flow (CBF) and attenuated infarct size after middle cerebral artery (MCA) occlusion 14 days later. In contrast, in SHR, CCA occlusion neither enlarged the leptomeningeal anastomosis nor showed protective effects after MCA occlusion. However, decreasing blood pressure using an angiotensin II AT1 receptor blocker restored the beneficial effect of CCA occlusion on collateral growth as well as on residual CBF and infarct size after MCA occlusion. Adaptive responses in CBF autoregulation curves observed 14 days after CCA occlusion in normotensive rats were impaired in untreated SHR, but were restored after antihypertensive treatment. In conclusion, SHR have impaired leptomeningeal collateral growth after CCA occlusion, but antihypertensive treatment restores the beneficial effect of CCA occlusion on collateral circulation.

Postischemic administration of angiotensin II type 1 receptor blocker reduces cerebral infarction size in hypertensive rats

Lowering the blood pressure (BP) during the acute period following ischemic stroke is still a controversial treatment. In this study, we investigated the effect of postischemic treatment using the angiotensin II type 1 receptor blocker, candesartan, on brain damage in focal cerebral ischemia. Spontaneously hypertensive rats underwent transient occlusion of the middle cerebral artery for 1 h. Candesartan (0.1, 1 and 10 mg kg−1) or vehicle was administered orally 3 and 24 h after ischemia. Blood pressure and neurological function were monitored, and infarct volume was evaluated 48 h after occlusion. Cerebral blood flow was measured using laser Doppler flowmetry before and after treatment with candesartan. Activation of Rho-kinase in cerebral microvessels was evaluated by immunohistochemistry. Systolic blood pressure was markedly lowered with both moderate and high doses, but it did not fall with a low dose of candesartan. The infarct volume was reduced in rats treated with the low dose of candesartan but not in those treated with the moderate or high doses. Cerebral blood flow decreased in parallel with the reduction in BP 3 h after treatment using the moderate dose, but it did not change after treatment with the low dose of candesartan, compared with vehicle. Rho-kinase was activated in the brain vessels of the ischemic cortex, but treatment with candesartan suppressed it. Our results show that oral administration of candesartan after transient focal ischemia reduced infarct volume at doses that showed little effect on BP. The neurovascular protective effects of candesartan may be caused by the inhibition of Rho-kinase in brain microvessels.

High Plasma D-Dimer is a Marker of Deep Vein Thrombosis in Acute Stroke

This study investigated whether plasma D-dimer level is useful for detection of deep vein thrombosis (DVT) in patients with acute stroke. A total of 133 patients hospitalized within 3 days after stroke onset underwent duplex venous ultrasonographic examination of the lower limbs and repeated measurements of plasma D-dimer level. DVT was detected in 36 of 100 patients with ischemic stroke and in 25 of 33 patients with intracerebral hemorrhage (ICH) (76%; P < .001). Plasma D-dimer level on admission (7.5 ± 10.7 μg/mL vs 3.7 ± 10.1 μg/mL; P = .040) and its maximum level before the ultrasonographic examination (29.1 ± 48.7 μg/mL vs 5.5 ± 11.0 μg/mL; P < .001) were higher in the patients with DVT compared with those without DVT. On multivariate logistic regression analysis, the maximum D-dimer level was independently related to the identification of DVT (odds ratio [OR] 1.05; 95% confidence interval [CI], 1.00-1.09 per 1-μg/mL increase; P = .045), but the admission D-dimer level was not when it was included instead of the maximum D-dimer level. In addition, female sex (OR, 4.99), ICH (OR, 5.20), high Wells clinical score (OR, 2.40 per 1-point increase), and low protein level (OR, 0.21 per 1-g/dL increase) were independently related to the identification of DVT. The optimum cutoff value of the maximum D-dimer level for positive DVT was 5.5 μg/mL (sensitivity, 89%; specificity, 82%). Our findings suggest that high plasma D-dimer level during the course of acute stroke can help detect DVT on duplex venous ultrasonography.