Tamiji Tsubokawa

Anti-apoptotic effect of granulocyte-colony stimulating factor after focal cerebral ischemia in the rat.

We investigated the molecular mechanisms of the anti-apoptotic properties of granulocyte-colony stimulating factor (G-CSF) on neurons and whether G-CSF affects glial cell survival following focal cerebral ischemia in rats. Sprague-Dawley rats were subjected to a transient 90 min middle cerebral artery occlusion (MCAO) by the intraluminal occlusion technique. Rats were treated with either a single dose of G-CSF (50 microg/kg, s.c.) at the onset of reperfusion or G-CSF (50 microg/kg body weight, s.c.) was administered starting at the onset of reperfusion and followed by the administration of the same dose per day for an additional 2 days. Brains were harvested either 24 h, 72 h or 2 weeks after reperfusion for assays of infarct volume, immunohistological studies and Western blot analysis for phosphorylated signal transducer and activator of transcription 3 (pSTAT3), Pim-1, bcl-2, Bax, cytochrome c, cellular inhibitor of apoptosis protein 2 (cIAP2), and cleaved caspase-3 levels. G-CSF significantly reduced infarct volume and ameliorated the early neurological outcome. G-CSF treatment significantly up-regulated pSTAT3, Pim-1, bcl-2 expression, and down-regulated cytochrome c release to the cytosol, Bax translocation to the mitochondria, and cleaved caspase-3 levels in neurons. The activation of the STAT3 pathway was accompanied by increased cIAP2 expression in glial cells. After MCAO, G-CSF treatment increased both neuronal and glial survival by effecting different anti-apoptotic pathways which reflects the multifactorial actions of this drug. These changes were associated with remarkable improvement in tissue preservation and behavioral outcome.

Role of c-Jun N-terminal kinase in early brain injury after subarachnoid hemorrhage

The c‐Jun N‐terminal kinase (JNK) is induced by cerebral ischemia and injurious blood components acutely after subarachnoid hemorrhage (SAH). We hypothesized that inhibition of JNK will prevent damage to the neurovascular unit in the early brain injury period after SAH. Ninety‐nine male SD rats (300–350 g) were randomly assigned to sham, SAH, and SAH treated with JNK inhibitor groups. SAH was induced by endovascular perforation. The JNK inhibitor SP600125 was administered intraperitoneally at 1 hr before and 6 hr after SAH. At 24 hr after SAH, we observed increased phosphorylation of JNK and c‐Jun. Signs of neurovascular damage were observed in the hemorrhagic brains; these included the increases of aquaporin (AQP)‐1 expression and brain water content as well as enhanced matrix metalloproteinase (MMP)‐9 activity, vascular collagen IV loss, increased VEGF tissue level, and Evans blue extravasation. The appearances of cleaved caspase‐3 expression, TUNEL‐positive cells, and apoptotic morphology in cerebral tissues were associated with neurological deficit after SAH. JNK inhibition prevented c‐Jun phosphorylation and suppressed AQP1, MMP‐9, VEGF, and caspase‐3 activation, with concomitant diminution of neuronal injury, blood–brain barrier preservation, reduced brain swelling, and improved neurological deficit in rats after SAH. This study demonstrates a multitude of beneficial effects of JNK inhibition, including protection of the neurovascular unit in early brain injury after SAH.

Lecithinized Superoxide Dismutase Improves Outcomes and Attenuates Focal Cerebral Ischemic Injury via Antiapoptotic Mechanisms in Rats

Background and Purpose— Recent studies have shown the antiapoptotic neuroprotective effects of lecithinized superoxide dismutase (PC-SOD) in different forms of brain injury. We tested the effects of PC-SOD in focal cerebral ischemia in the rat middle cerebral artery occlusion model (MCAO). Methods— Adult male Sprague-Dawley rats were treated with PC-SOD (0.3, 1.0, and 3.0 mg/kg) administered intravenously after 90 minutes of occlusion (beginning of reperfusion). Physiological parameters, neurological score, and infarct volume were assessed at 24 and 72 hours in 3 groups of animals: sham-operated (n=18), MCAO treated with vehicle (n=26), and MCAO treated with PC-SOD (n=37). Oxidative stress was evaluated by malondialdehyde assay, and the apoptotic mechanisms were studied by Western blotting. Results— PC-SOD treatment significantly reduced infarct volume and improved neurological scores at different time points compared with the vehicle-treated group. PC-SOD treatment decreased malondialdehyde levels, cytochrome c, and cleaved caspase 3 expression and increased mitochondrial Bcl-2 expression. Conclusions— Inhibition of oxidative stress with PC-SOD treatment improves outcomes after focal cerebral ischemia. This neuroprotective effect is likely exerted by antiapoptotic mechanisms.

Cathepsin and Calpain Inhibitor E64d Attenuates Matrix Metalloproteinase-9 Activity After Focal Cerebral Ischemia in Rats

Background and Purpose— Matrix metalloproteinases (MMPs) and cysteine proteases (calpain and cathepsin B) play an important role in cell death and are upregulated after focal cerebral ischemia. Because there is a significant interaction between MMP-9 with calpain and cathepsin B, we investigated the role of E64d (a calpain and cathepsin B inhibitor) on MMP-9 activation in the rat focal ischemia model. Methods— Male Sprague-Dawley rats were subjected to 2 hours of middle cerebral artery occlusion by using the suture insertion method followed by 22 hours of reperfusion. In the treatment group, a single dose of E64d (5 mg/kg IP) was administrated 30 minutes before the induction of focal ischemia, whereas the nontreatment group received dimethyl sulfoxide only. The neurological deficits, infarct volumes, Evans blue extravasation, brain edema, and MMP-9 activation in the brain were determined. Results— Pretreatment with E64d produced a significant reduction in the cerebral infarction volume (353.1±19.8 versus 210.3±23.7 mm3) and the neurological deficits. Immunofluorescence studies showed MMP-9, calpain, and cathepsin B activation colocalized to both neurons and the neurovascular endothelial cells after ischemia, which was reduced by E64d. Conclusion— These results suggest that E64d treatment provides a neuroprotective effect to rats after transient focal cerebral ischemia by inhibiting the upregulation of MMP-9.

Simvastatin Attenuation of Cerebral Vasospasm After Subarachnoid Hemorrhage in Rats Via Increased Phosphorylation of Akt and Endothelial Nitric Oxide Synthase

The mechanisms involved in simvastatin‐mediated attenuation of cerebral vasospasm after subarachnoid hemorrhage (SAH) are unclear. We investigated the role of the phosphatidylinositol 3‐kinase/Akt (PI3K/Akt) pathway and endothelial nitric oxide synthase (eNOS) in the cerebral vasculature in statin‐mediated attenuation of cerebral vasospasm using wortmannin, an irreversible pharmacological PI3K inhibitor, and a rat SAH endovascular perforation model. Simvastatin was administered intraperitoneally in two dosages (1 mg/kg and 20 mg/kg) at 0.5, 24, and 48 hr after SAH and histological parameters of ipsilateral intracranial carotid artery (ICA) were assessed at 24 and 72 hr. SAH significantly decreased ICA diameter and perimeter while increasing wall thickness at both 24 and 72 hr. High‐dosage simvastatin prevented the reduction of ICA diameter and perimeter following SAH, whereas both high and low dosages reduced wall thickness significantly at 24 and 72 hr. The effects of simvastatin were significantly reversed by wortmannin. High‐dosage simvastatin increased pAkt and peNOS (phosphorylated forms) levels without increasing Akt and eNOS expression compared with the SAH group and also improved neurological deficits at 24 and 72 hr. Simvastatin did not affect protein levels by itself compared with untreated sham group. The present study elucidates the critical role of the PI3K activation leading to phosphorylation of Akt and eNOS in simvastatin‐mediated attenuation of cerebral vasospasm after SAH.

Granulocyte colony-stimulating factor protects the brain against experimental stroke via inhibition of apoptosis and inflammation

Abstract Objectives: The molecular mechanisms of the anti-apoptotic and anti-inflammatory properties of granulocyte-colony stimulating factor (G-CSF) following focal cerebral ischemia in rats were examined in this study. Methods: Sprague–Dawley rats were randomly divided into three groups: sham, middle cerebral artery occlusion (MCAO) non-treatment and MCAO with G-CSF treatment. Focal ischemia was induced with the suture occlusion method for 90 minutes, and treatment was given at the onset of reperfusion. All animals were killed 24 hours after reperfusion. Assessment included neurological scores, infarction volumes, histology, immunofluorescent staining and Western blotting. Results: G-CSF significantly reduced the infarct volume and ameliorated the early neurological outcome scores. Western blot analysis showed that G-CSF treatment significantly elevated the cIAP2 levels and decreased the activation of caspase 3 in the ischemic cortex compared with the non-treated rats. Immunofluorescent works also showed that G-CSF treatment inhibited both neuronal and glial tumor necrosis factor α and interleukin 1β expressions. Discussion: The neuronal anti-apoptotic action of G-CSF may be mediated in part by the anti-apoptotic protein cIAP2. G-CSF also exerts anti-inflammatory actions after focal cerebral ischemia by preventing both neuronal and glial pro-inflammatory cytokine expressions.

Comparison of silicon-coated nylon suture to plain nylon suture in the rat middle cerebral artery occlusion model

UNLABELLED A variety of intraluminal sutures have been used in the middle cerebral artery occlusion model (MCAO) of focal ischemia. In the present study we tested commercially available silicon-coated nylon suture in the MCAO model and compared the results to traditional monofilament nylon suture occlusion. Twelve Sprague-Dawley male rats were randomly divided two groups, MCAO with 4-0 nylon suture (Group N, n=6) and MCAO with silicone-coated 4-0 nylon suture (Group S, n=6). Rats were sacrificed 24 h after reperfusion. Assessment included mortality rates, neurological evaluation, and infarct volume. One rat died in each group from subarachanoid hemorrhage. Neurological evaluation demonstrated that Group S tended to have worse neurological outcomes than Group N, although this difference was not statistically significant. On TTC stain Group S had significantly larger infarct volumes than Group N. We conclude that the commercially available silicone-coated occlusion suture provides better occlusion of the middle cerebral artery than the traditional uncoated nylon suture. CLASSIFICATION Disease-related neuroscience (Section 6).

Granulocyte-colony stimulating factor inhibits apoptotic neuron loss after neonatal hypoxia-ischemia in rats.

Neonatal hypoxia-ischemia (HI) is an important clinical problem with few effective treatments. Granulocyte-colony stimulating factor (G-CSF) is an endogenous peptide hormone of the hematopoietic system that has been shown to be neuroprotective in focal ischemia in vivo and is currently in phase I/II clinical trials for ischemic stroke in humans. We tested G-CSF in a rat model of neonatal hypoxia-ischemia in postnatal day 7 unsexed rat pups. Three groups of animals were used: hypoxia-ischemia (HI, n=67), hypoxia-ischemia with G-CSF treatment (HI+G, n=65), and healthy control (C, n=53). G-CSF (50 microg/kg, subcutaneous) was administered 1 h after HI and given on four subsequent days (five total injections). Animals were euthanized 24 h, 1, 2, and 3 weeks after HI. Assessment included brain weight, histology, immunohistochemistry, and Western blotting. G-CSF treatment was associated with improved quantitative brain weight and qualitative Nissl histology after hypoxia-ischemia. TUNEL demonstrated reduced apoptosis in group HI+G. Western blot demonstrated decreased expression of Bax and cleaved caspase-3 in group HI+G. G-CSF treatment was also associated with increased expression of STAT3, Bcl-2, and Pim-1, all of which may have participated in the anti-apoptotic effect of the drug. We conclude that G-CSF ameliorates hypoxic-ischemic brain injury and that this may occur in part by an inhibition of apoptotic cell death.

Neurovascular and neuronal protection by E64d after focal cerebral ischemia in rats.

Calpains and cathepsins are two families of proteases that play an important role in ischemic cell death. In this study, we investigated the effect of E64d, a μ‐calpain and cathepsin B inhibitor, in the prevention of neuronal and endothelial apoptotic cell death after focal cerebral ischemia in rats. Rats underwent 2 hr of transient focal ischemia from middle cerebral artery occlusion (MCAO) and were sacrificed 24 hr later. E64d (5 mg/ kg intraperitoneally) was administered 30 min before MCAO. Assessment included neurological function, infarction volume, brain water content, blood–brain barrier permeability, histology, and immunohistochemistry. The E64d‐treated rats had significant brain protection against ischemic damage. We observed a reduction of infarction volume, brain edema, and improved neurological scores in E64d‐treated rats compared with the nontreated control. Furthermore, there was a remarkable reduction in both proteases and caspase‐3 activation and apoptotic changes in both neurons and endothelial cells in E64d‐treated rats. These results suggest that E64d protects the brain against ischemic/reperfusion injury by attenuating neuronal and endothelial apoptosis.

Inhibition of integrin αvβ3 reduces blood-brain barrier breakdown in focal ischemia in rats

Ischemic stroke is a major cause of morbidity and mortality in industrialized nations. We tested the effect of postischemic treatment of cyclo‐RGDfV (cRGDfV), a selective inhibitor of integrin αvβ3, in the middle cerebral artery occlusion (MCAO) model of ischemic stroke in rats. Rats were randomly divided into three groups: sham operation (n = 13), MCAO with no treatment (n = 18), and MCAO with cRGDfV treatment (n = 28). Focal ischemia was induced with the suture occlusion method for 2 hr, and treatment was given 1 hr after reperfusion (3 hr after ischemia). All animals were sacrificed 24 hr after reperfusion. Assessment included neurological scores, infarction volumes, brain water content, Evans blue exudation, IgG exudation, histology, immunohistochemistry, and Western blotting. Treatment with cRGDfV ameliorated neurological deficits, reduced brain edema, and reduced exudation of Evans blue dye and IgG, but failed to reduce infarction volumes. Western blotting showed a reduction in phosphorylation of one subset of vascular endothelial growth factor (VEGF) receptors in the cRGDfV treatment group. Western blotting also demonstrated a significant reduction of fibrinogen in the cRGDfV treatment group. We conclude that poststroke treatment with cRGDfV reduces blood–brain barrier breakdown in focal ischemia, possibly through inhibition of VEGF‐mediated vascular breakdown.