Tomoya Kinouchi

Edaravone, a Free Radical Scavenger, Inhibits MMP-9–Related Brain Hemorrhage in Rats Treated With Tissue Plasminogen Activator

Background and Purpose— Intracerebral hemorrhage, induced by recombinant tissue plasminogen activator (rtPA) in ischemic stroke, is attributable to the increased activity of matrix metalloproteinase-9 (MMP-9). Patients with acute infarct benefit from the neuroprotective drug edaravone, a free radical scavenger. We examined the mechanisms by which edaravone may help to suppress rtPA-induced brain hemorrhage. Methods— Male Wistar rats weighing 250 to 280 g were subjected to 3-hour transient middle cerebral artery occlusion (MCAO) and divided randomly into 3 groups. Immediately after reperfusion, 1 group was intravenously injected with 10 mg/kg rtPA, another with rtPA plus 3 mg/kg edaravone, and the 3rd group received no treatment. We assessed the hemorrhage volume and the activity of MMP-9 in the brain 24 hours postischemia. We also studied the activity of MMP-9, its mRNA expression, and nuclear factor-kappa B (NF-&kgr;B) activity in rtPA-stimulated human microvascular endothelial cells (HBECs). Results— The degree of hemorrhage and the level of endothelial cell–derived MMP-9 were elevated in rats treated with rtPA alone and attenuated in rats treated with rtPA plus edaravone. In rtPA-stimulated HBECs, edaravone suppressed the activity and mRNA expression of MMP-9 in a dose-dependent manner. Edaravone also inhibited NF-&kgr;B activation. Conclusions— We demonstrate that edaravone inhibits rtPA-induced cerebral hemorrhage in the ischemic brain of rats via the inhibition of MMP-9 expression in vivo, which is substantiated by inhibition of MMP-9 expression and NF-&kgr;B activation in HBECs. Edaravone may render thrombolytic therapy safer for the administration of rtPA in patients with ischemic stroke.

Reduction of endothelial tight junction proteins is related to cerebral aneurysm formation in rats.

Objective The formation of cerebral aneurysms is associated with endothelial damage and macrophage migration. Hypothesizing that the opening of tight junctions due to the disappearance of the tight junction proteins occludin and zona occludens-1 (ZO-1) in damaged endothelia allows macrophage migration, leading to cerebral aneurysm formation, we investigated the role of tight junction proteins. Methods The vascular wall of female rats subjected to hypertension, oophorectomy (OVX), and hemodynamic stress to induce cerebral aneurysms was evaluated morphologically, immunohistochemically, and by quantitative RT-PCR. We also assessed the regulation of tight junction proteins in human brain endothelial cells (HBECs). Results In the very early stage before aneurysm formation, the expression of occludin and ZO-1 was reduced in injured endothelial cell junctions exhibiting gaps. In the course of aneurysmal progression their reduction progressed and was correlated with macrophage migration. In hypertension along with OVX rats we observed an increase in angiotensin II and the degradation molecules matrix metalloproteinase-9 (MMP-9), nicotinamide-adenine dinucleotide phosphate oxidases and monocyte chemoattractant protein-1. The mineralocorticoid receptor blocker eplerenone increased occludin and ZO-1 expression; this was associated with a reduction in angiotensin II and the degradation molecules and resulted in the inhibition of macrophage exudation and aneurysm formation. In HBECs, occludin and ZO-1 downregulation by angiotensin II and estrogen deficiency was reversed by eplerenone, the MMP inhibitor SB3CT, and apocynin. Our results suggest that macrophage migration is associated with the reduction in tight junction proteins induced by the degradation molecules. Conclusion In rats, the destruction of tight junctions may facilitate macrophage migration and cerebral-aneurysm formation.

Role of Mineralocorticoid Receptor on Experimental Cerebral Aneurysms in Rats

Activation of the renin-angiotensin (Ang)-aldosterone system is involved in the pathology of vascular diseases. Although the blockade of the mineralocorticoid receptor protects against vascular diseases, its role in cerebral aneurysms remains to be elucidated. We treated female rats subjected to renal hypertension, increased hemodynamic stress, and estrogen deficiency for 3 months with the mineralocorticoid receptor blocker eplerenone (30 or 100 mg/kg per day) or vehicle (vehicle control). Eplerenone reduced the incidence of cerebral aneurysms and saline intake without lowering of the blood pressure. In the aneurysmal wall, the production of Ang II and nitrotyrosine was increased. The mRNA levels of Ang-converting enzyme 1 and NADPH oxidase subunits NOX4, Rac1, monocyte chemoattractant protein 1, and matrix metalloproteinase 9 were increased. Eplerenone brought about a reduction in these molecules, suggesting that mineralocorticoid receptor blockade suppresses cerebral aneurysm formation by inhibiting oxidative stress, inflammatory factors, local renin-Ang system activation, and saline intake. Other female rats implanted with pellets of the mineralocorticoid receptor agonist deoxycorticosterone acetate manifested a high incidence of cerebral aneurysm formation and the upregulation of molecules related to oxidative stress, inflammatory factors, and the local renin-Ang system; their saline intake was increased. We demonstrate that mineralocorticoid receptor activation at least partly contributes to the pathogenesis of cerebral aneurysms.

Protective mechanisms of the angiotensin II type 1 receptor blocker candesartan against cerebral ischemia: in-vivo and in-vitro studies.

Background Angiotensin II type 1 (AT1) receptor blockers decrease ischemia by mechanisms dependent on and independent of arterial blood pressure in hypertensive rats and AT1-R knockout mice, respectively. However, the detailed mechanisms underlying the effects of AT1 receptor blockers remain unclear. Aims To elucidate the systemic and focal effects of AT1 receptor blockers against cerebral ischemia in in-vivo and in-vitro studies. Methods Normotensive Wistar rats were treated for 2 weeks with 0.5 or 1 mg/kg candesartan cilexetil and then subjected to 2-h middle cerebral artery occlusion–reperfusion. Human umbilical endothelial cells were stimulated with the active form of candesartan and angiotensin II in the absence and presence of an angiotensin II type 2 (AT2) receptor antagonist. Results In candesartan-pretreated hypotensive and nonhypotensive rats, blood pressure was moderately increased during middle cerebral artery occlusion and fell gradually to the baseline after the reperfusion; it remained elevated in the control even after the reperfusion occlusion. Candesartan treatment resulted in a decrease in the cortical infarct volume and oxidative damage, the hypoxic status was improved, and the expression of repair-associated and growth-associated proteins in the cortical penumbra was augmented. Candesartan also increased the eNOS mRNA level and the lumen size of the middle cerebral artery. In human umbilical endothelial cells, candesartan increased the eNOS protein level AT2-R dependently, inhibited the expression of nicotinamide adenine dinucleotide phosphate oxidase subunits and angiotensin II-induced intracellular reactive oxygen species and nitric oxide, and promoted the extracellular release of nitric oxide, suggesting that it augmented the bioavailability of nitric oxide. Conclusion Among the mechanisms candesartan exerts in its protection against cerebral ischemia, restoration of endothelial function may represent an attractive therapeutic goal to address cerebral ischemia.

Statins Promote the Growth of Experimentally Induced Cerebral Aneurysms in Estrogen-Deficient Rats

Background and Purpose— The pathogenesis of cerebral aneurysms is linked to inflammation, degradation of the extracellular matrix, and vascular wall apoptosis. Statins exert pleiotropic effects on the vasculature, independent of their cholesterol-lowering properties. To explore the detailed pathogenesis of cerebral aneurysms, we examined their progression in a rat model and studied whether statins prevent their initiation and growth. Methods— Cerebral aneurysms were induced in female rats subjected to hypertension, increased hemodynamic stress, and estrogen deficiency. The development of aneurysm was assessed morphologically on corrosion casts. The effects of pravastatin (5, 25, or 50 mg/kg per day) and of simvastatin (5 mg/kg per day) on their aneurysms were studied. Human brain endothelial cells were also used to determine the effects of pravastatin. Results— Pravastatin (5 mg/kg per day) reduced endothelial damage and inhibited aneurysm formation; there was an association with increased endothelial nitric oxide synthase (eNOS) levels and a decrease in human brain endothelial cell adhesion molecules. Unexpectedly, 25 mg/kg per day and 50 mg/kg per day pravastatin and 5 mg/kg per day simvastatin promoted aneurysmal growth, and high-dose pravastatin induced aneurysmal rupture. The deleterious effects exerted by these statins were associated with an increase in apoptotic caspase-3 levels and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, suggesting that statins exert bidirectional effects. Conclusions— Our results provide the first evidence that cerebral aneurysm growth is partly associated with apoptosis and issue a warning that statins exert bidirectional effects on cerebral aneurysms. Additional intensive research is necessary to understand better their mechanisms and to identify patients in whom the administration of statins may elicit deleterious effects.

Activation of estrogen receptor-α and of angiotensin-converting enzyme 2 suppresses ischemic brain damage in oophorectomized rats.

Like the angiotensin II type 1 receptor blocker, endogenous estrogen (17&bgr;-estradiol) is neuroprotective against cerebral ischemia; its effects are thought to be mediated by estrogen receptors (ERs). To verify the role of ERs and the brain renin-angiotensin system in estrogen-deficient rats with ischemia induced by middle cerebral artery occlusion, we compared rats subjected to oophorectomy (OVX+) with sham-oophorectomized rats (OVX−) and OVX+ rats treated with 0.3 or 3.0 mg/kg of olmesartan for 2 weeks before middle cerebral artery occlusion. Independent of the blood pressure, the cortical infarct volume was larger in OVX+ than in OVX− rats. It was smaller in olmesartan-pretreated OVX+ rats. The expression of ER&agr; in the peri-infarct region was correlated with the reduction of cortical infarct but not that of ER&bgr; or G protein–coupled estrogen receptor. Olmesartan prevented ER&agr; downregulation in the cortical peri-infarct area, without affecting ER&bgr; or G protein–coupled estrogen receptor. Olmesartan also increased mRNA expression of angiotensin-converting enzyme 2, Bcl-2, and Bcl-xL and reduced angiotensin II and cleaved caspase 3. These effects were augmented by olmesartan and abolished by the ER inhibitor. In OVX+ rats treated with the ER&agr; agonist alone, the infarct size was decreased, and the neuroprotective genes were upregulated. These findings suggest that the transactivation of neuroprotective genes and the reduction in brain angiotensin II are ER&agr; dependent and that this may augment neuroprotection together with an angiotensin II type 1 receptor blockade by olmesartan. We present the new insight that the activation of ER&agr; independent of estrogen contributes at least partly to limiting cerebral ischemic damage.

Activation of Signal Transducer and Activator of Transcription-3 by a Peroxisome Proliferator-Activated Receptor Gamma Agonist Contributes to Neuroprotection in the Peri-Infarct Region After Ischemia in Oophorectomized Rats

Background and Purpose— The role of the phosphorylated signal transducer and activator of transcription-3 (p-STAT3) after cerebral ischemia by the peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;) agonist pioglitazone (PGZ) remains controversial. Whether the increase in p-STAT3 by estrogen is mediated by the estrogen receptor &agr; is also obscure. We examined the role of p-STAT3, PPAR&ggr;, and estrogen receptor &agr; against ischemic brain damage after PGZ treatment. Methods— Female Wistar rats subjected or not subjected to bilateral oophorectomy were injected with 1.0 or 2.5 mg/kg PGZ 2 days, 1 day, and 1 hour before 90-minute middle cerebral artery occlusion–reperfusion and compared with vehicle-control rats. Results— The cortical infarct size was larger in ovariectomized than in nonovarietomized rats; it was reduced by PGZ treatment. Inversely with the reduction of the infarct size, PPAR&ggr;, and p-STAT3 but not estrogen receptor &agr; in the peri-infarct area were increased in PGZ-treated compared with vehicle-control rats. The increase in PPAR&ggr; and p-STAT3 was associated with the transactivation of antiapoptotic and survival genes and the reduction of caspase-3 in this area. Inhibitors of PPAR&ggr; or STAT3 abolished the PGZ-induced neuroprotection and the increase in p-STAT3. More importantly, p-STAT3 increased by PGZ was bound to PPAR&ggr; and the complex translocated to the nucleus to dock to the response element through p-STAT3. Conclusions— Our findings suggest that the activation in the peri-infarct region of p-STAT3 and PPAR&ggr; by PGZ is essential for neuroprotection after ischemia and that PGZ may be of benefit even in postmenopausal stroke patients.

Acquired pial and dural arteriovenous fistulae following superior sagittal sinus thrombosis in patients with protein S deficiency: a report of two cases.

Two patients with protein S deficiency with acquired multiple pial and dural arteriovenous fistulae (AVFs) following superior sagittal sinus (SSS) thrombosis are reported. Case 1 is a 38-year-old male with protein S deficiency who developed generalized seizure due to SSS thrombosis. Local fibrinolysis was achieved in the acute stage. His 10-month follow-up angiogram revealed an asymptomatic acquired dural AVF arising from the middle meningeal artery and the anterior cerebral artery with drainage to the thrombosed cortical vein in the right frontal lobe. Furthermore, his 2-year follow-up angiogram revealed a de novo pial AVF from the middle cerebral artery in the Sylvian fissure with drainage to the cortical vein initially thrombosed. However, this asymptomatic pial AVF caused bleeding in the ipsilateral cerebral hemisphere 12 years after onset, whereas the dural AVF spontaneously disappeared. Surgical disconnection was successfully performed to eliminate the source of hemorrhage. Case 2 is a 50-year-old male with a past history of SSS thrombosis with protein S deficiency who developed pulsatile tinnitus and generalized seizure. His angiogram showed a cortical dural AVF in the left parietal lobe and a sporadic dural AVF involving the right sigmoid sinus. The parietal lesion was eliminated by transarterial embolization followed by craniotomy. However, a de novo pial AVF emerged from the middle cerebral artery adjacent to the previously treated lesion. Of four cortical AVFs in two patients, thrombosis of cortical veins caused by protein S deficiency might play an important role in their formation. Long-term follow-up is required because this peculiar disorder has an unusual clinical course.

Site-specific elevation of interleukin-1β and matrix metalloproteinase-9 in the Willis circle by hemodynamic changes is associated with rupture in a novel rat cerebral aneurysm model

The pathogenesis of subarachnoid hemorrhage remains unclear. No models of cerebral aneurysms elicited solely by surgical procedures and diet have been established. Elsewhere we reported that only few rats in our original rat aneurysm model manifested rupture at the anterior and posterior Willis circle and that many harbored unruptured aneurysms at the anterior cerebral artery-olfactory artery bifurcation. This suggests that rupture was site-specific. To test our hypothesis that a site-specific response to hemodynamic changes is associated with aneurysmal rupture, we modified our original aneurysm model by altering the hemodynamics. During 90-day observation, the incidence of ruptured aneurysms at the anterior and posterior Willis circle was significantly increased and the high incidence of unruptured aneurysms at the anterior cerebral artery-olfactory artery persisted. This phenomenon was associated with an increase in the blood flow volume. Notably, the level of matrix metalloproteinase-9 associated with interleukin-1β was augmented by the increase in the blood flow volume, suggesting that these molecules exacerbated the vulnerability of the aneurysmal wall. The current study first demonstrates that a site-specific increase in interleukin-1β and matrix metalloproteinase-9 elicited by hemodynamic changes is associated with rupture. Our novel rat model of rupture may help to develop pharmaceutical approaches to prevent rupture.

The accumulation of brain water-free sodium is associated with ischemic damage independent of the blood pressure in female rats.

Estrogen deficiency worsens ischemic stroke outcomes. In ovariectomized (OVX(+)) rats fed a high-salt diet (HSD), an increase in the body Na(+)/water ratio, which characterizes water-free Na(+) accumulation, was associated with detrimental vascular effects independent of the blood pressure (BP). We hypothesized that an increase in brain water-free Na(+) accumulation is associated with ischemic brain damage in OVX(+)/HSD rats. To test our hypothesis we divided female Wistar rats into 4 groups, OVX(+) and OVX(-) rats fed HSD or a normal diet (ND), and subjected them to transient cerebral ischemia. The brain Na(+)/water ratio was increased even in OVX(+)/ND rats and augmented in OVX(+)/HSD rats. The increase in the brain Na(+)/water ratio was positively correlated with expansion of the cortical infarct volume without affecting the BP. Interestingly, OVX(+) was associated with the decreased expression of ATP1α3, a subtype of the Na(+) efflux pump. HSD increased the expression of brain Na(+) influx-related molecules and the mineralocorticoid receptor (MR). The pretreatment of OVX(+)/HSD rats with the MR antagonist eplerenone reduced brain water-free Na(+) accumulation, up-regulated ATP1α3, down-regulated MR, and reduced the cortical infarct volume. Our findings show that the increase in the brain Na(+)/water ratio elicited by estrogen deficiency or HSD is associated with ischemic brain damage BP-independently, suggesting the importance of regulating the accumulation of brain water-free Na(+). The up-regulation of ATP1α3 and the down-regulation of MR may provide a promising therapeutic strategy to attenuate ischemic brain damage in postmenopausal women.