Haruhiko Kikuchi

Intravenous administration of MEK inhibitor U0126 affords brain protection against forebrain ischemia and focal cerebral ischemia

Brain subjected to acute ischemic attack caused by an arterial blockage needs immediate arterial recanalization. However, restoration of cerebral blood flow can cause tissue injury, which is termed reperfusion injury. It is important to inhibit reperfusion injury to achieve greater brain protection. Because oxidative stress has been shown to activate mitogen-activated protein kinases (MAPKs), and because oxidative stress contributes to reperfusion injury, MAPK may be a potential target to inhibit reperfusion injury after brain ischemia. Here, we demonstrate that reperfusion after forebrain ischemia dramatically increases phosphorylation level of extracellular signal-regulated kinase 2 (ERK2) in the gerbil hippocampus. In addition, i.v. administration of U0126 (100–200 mg/kg), a specific inhibitor of MEK (MAPK/ERK kinase), protects the hippocampus against forebrain ischemia. Moreover, treatment with U0126 at 3 h after ischemia significantly reduces infarct volume after transient (3 h) focal cerebral ischemia in mice. This protection is accompanied by reduced phosphorylation level of ERK2, substrates for MEK, in the damaged brain areas. Furthermore, U0126 protects mouse primary cultured cortical neurons against oxygen deprivation for 9 h as well as nitric oxide toxicity. These results provide further evidence for the role of MEK/ERK activation in brain injury resulting from ischemia/reperfusion, and indicate that MEK inhibition may increase the resistance of tissue to ischemic injury.

Neuroprotective effect of an antioxidant, ebselen, in patients with delayed neurological deficits after aneurysmal subarachnoid hemorrhage.

OBJECTIVE The effect of ebselen, a seleno-organic compound with antioxidant activity through a glutathione peroxidase-like action, on the outcome of subarachnoid hemorrhage was evaluated in a multicenter placebo-controlled double-blind clinical trial. METHODS Patients who suffered aneurysmal subarachnoid hemorrhages of Hunt and Kosnik Grades II through IV at admission and were able to start drug treatment within 96 hours of the ictus were enrolled. Early surgery was performed whenever possible. Oral administration of ebselen granules suspended in water (150 mg, twice a day) or placebo was started immediately after admission and continued for 2 weeks. The major end points were the Glasgow Outcome Scale at 2 weeks, 1 month, and 3 months after the start of treatment. The incidence of delayed ischemic neurological deficits clinically diagnosed as resulting from vasospasm and the incidence and extent of low-density areas on postoperative computed tomographic scans were also studied as secondary outcome measures. RESULTS Intent-to-treat analysis of the 286 patients enrolled in the trial (145 patients administered ebselen and 141 administered placebo) revealed that the incidence of clinically diagnosed delayed ischemic neurological deficits was unaltered. There were 52 (receiving ebselen) and 58 (receiving placebo) patients with delayed deficits; however, a significantly better outcome was observed after ebselen treatment than after placebo (P = 0.005, chi2 test). There was a corresponding decrease in the incidence and extent of low-density areas (P = 0.032, Wilcoxon rank sum test). CONCLUSION Ebselen reduced brain damage in patients with delayed neurological deficits after subarachnoid hemorrhage and may be a promising neuroprotective agent.

Small Unruptured Intracranial Aneurysm Verification Study: SUAVe Study, Japan

Background and Purpose— The natural history and optimal management of incidentally found small unruptured aneurysms <5 mm in size remain unclear. A prospective study was conducted to determine the optimal management for incidentally found small unruptured aneurysms. Methods— From September 2000 to January, 2004, 540 aneurysms (446 patients) were registered. Four hundred forty-eight unruptured aneurysms <5 mm in size (374 patients) have been followed up for a mean of 41.0 months (1306.5 person-years) to date. We calculated the average annual rupture rate of small unruptured aneurysms and also investigated risk factors that contribute to rupture and enlargement of these aneurysms. Results— The average annual risks of rupture associated with small unruptured aneurysms were 0.54% overall, 0.34% for single aneurysms, and 0.95% for multiple aneurysms. Patient <50 years of age (P=0.046; hazard ratio, 5.23; 95% CI, 1.03 to 26.52), aneurysm diameter of ≥4.0 mm (P=0.023; hazard ratio, 5.86; 95% CI, 1.27 to 26.95), hypertension (P=0.023; hazard ratio, 7.93; 95% CI, 1.33 to 47.42), and aneurysm multiplicity (P=0.0048; hazard ratio, 4.87; 95% CI, 1.62 to 14.65) were found to be significant predictive factors for rupture of small aneurysms. Conclusions— The annual rupture rate associated with small unruptured aneurysms is quite low. Careful attention should be paid to the treatment indications for single-type unruptured aneurysms <5 mm. If the patient is <50 years of age, has hypertension, and multiple aneurysms with diameters of ≥4 mm, treatment should be considered to prevent future aneurysmal rupture.

Neuroprotection by MAPK/ERK kinase inhibition with U0126 against oxidative stress in a mouse neuronal cell line and rat primary cultured cortical neurons

Oxidative stress is implicated in the pathogenesis of neuronal degenerative diseases. Oxidative stress has been shown to activate extracellular signal-regulated kinases (ERK)1/2. We investigated the role of these mitogen-activated protein kinases (MAPKs) in oxidative neuronal injury by using a mouse hippocampal cell line (HT22) and rat primary cortical cultures. Here, we show that a novel MAPK/ERK kinase (MEK) specific inhibitor U0126 profoundly protected HT22 cells against oxidative stress induced by glutamate, which was accompanied by an inhibition of phosphorylation of ERK1/2. U0126 also protected rat primary cultured cortical neurons against glutamate or hypoxia. However, U0126 was not protective against death caused by tumor necrosis factor alpha (TNFalpha), A23187, or staurosporine. These results indicate that MEK plays a central role in the neuronal death caused by oxidative stress.

Bone regeneration by basic fibroblast growth factor complexed with biodegradable hydrogels

The objective of this study is to enhance the bone induction activity of basic fibroblast growth factor (bFGF) for reconstruction of skull bone defects which has been clinically recognized as almost impossible. For this purpose, we prepared biodegradable hydrogels from gelatin with an isoelectric point of 4.9 which is capable of polyionic complexing with basic bFGF. When implanted in rabbit skull defects of 6 mm in diameter (6 defects per experimental group), the gelatin hydrogels incorporating 100 microg of bFGF promoted bone regeneration at the defect in marked contrast to free bFGF of the same dose, finally closing the bone defects after 12 weeks of implantation as is apparent from histological examination. In dual energy X-ray absorptometry analysis, the bone mineral density at the skull defects enhanced by the hydrogels was significantly higher than that by free bFGF at doses ranging from 2 to 200 microg/defect (P < 0.05). The extent of bone regeneration induced by gelatin hydrogels incorporating 100 microg of bFGF increased with a decrease in their water content. Histological examination indicated that more slowly degrading hydrogels of lower water content prolonged the retention period of osteoblasts in the bone defects. This led to enhanced bone regeneration compared with faster degrading hydrogels of higher water content. It was concluded that this biodegradable hydrogel system was a promising surgical tool to assist self-reconstruction of the skull bone.

Prevention of Rat Cerebral Aneurysm Formation by Inhibition of Nitric Oxide Synthase

BACKGROUND Cerebral saccular aneurysm is a major cause of subarachnoid hemorrhage, one of the cerebrovascular diseases with the highest mortality. The mechanisms underlying the development of aneurysms, however, still remain unclear. We have made a series of reports on an animal model of experimentally induced cerebral aneurysms that resemble human cerebral aneurysms in their location and morphology, suggesting that the arterial wall degeneration associated with aneurysm formation develops near the apex of arterial bifurcation as a result of an increase in wall shear stress. Using the animal model and human specimens, we examined the role of nitric oxide (NO) in the degenerative changes and cerebral aneurysm formation. METHODS AND RESULTS Inducible NO synthase (iNOS) was immunohistochemically located at the orifice of human and rat aneurysms. Nitrotyrosine distribution was also seen in the human aneurysm. Although no iNOS immunostaining was found in normal arteries, iNOS immunoreactivity was observed in parallel with the development of early aneurysmal changes in rats. In contrast, during the early development of aneurysm, endothelial NOS immunostaining in the endothelium was weakened compared with that in the control arteries. An NOS inhibitor, aminoguanidine, attenuated both early aneurysmal changes and the incidence of induced aneurysms. A defibrinogenic agent, batroxobin, which may diminish shear stress by reduction of blood viscosity, prevented iNOS induction as well as early aneurysmal changes. CONCLUSIONS The evidence suggests that NO, particularly that derived from iNOS, is a key requirement for the development of cerebral aneurysm. The iNOS induction may be caused by an increase in shear stress near the apex.

Expression of Interleukin-1β Converting Enzyme Gene Family and bcl-2 Gene Family in the Rat Brain following Permanent Occlusion of the Middle Cerebral Artery

Recent investigations have been suggesting that some neuronal subpopulations may die via programmed cell death after focal ischemic injury. To clarify the possible roles of the genes involved in the cell-death program, this study examined the expression of three members of the interleukin-1β converting enzyme (Ice) gene family (Ice, Nedd2, and Yama/CPP32) and two members of the bcl-2 gene family (bcl-2 and bcl-x) in the rat brain after permanent occlusion of the middle cerebral artery. Northern blot analysis revealed a transient induction of Nedd2 mRNA 8 h after the ischemic insult (3.8-fold) and an increase in Yama/CPP32 mRNA 16 to 24 h after the insult (5.8-fold at 24 h), whereas the expression of Ice remained constant. The expression of bcl-2 and bcl-x remained constant after the ischemic insult. Taking into account the key role of the Ice gene family in the execution of programmed cell death, the induction of Ice gene family might play a causative role in apoptotic cell death.

The introduction of microvascular surgery to hepatic artery reconstruction in living-donor liver transplantation : its surgical advantages compared with conventional procedures

&NA; Microvascular surgery for the reconstruction of the graft artery has been used since the 8th case in our series of 14 liver transplantations using living-related donors, and the clinical results have been compared between the first seven cases (the Loupe group) and the last seven cases (the Micro group). Seven arteries in 7 grafts were reconstructed with the use of loupe magnification in the Loupe group, while 8 arteries in 7 grafts were anastomosed with microscopic techniques in the Micro group. Statistically, there was no difference between the two groups in general background, including age, body weight and primary disease of the recipient, and in medical and surgical factors possibly relating to postoperative thrombosis of the hepatic artery.In two cases in the Loupe group, one or two additional reconstructions were necessary to obtain sufficient blood flow, while 8 arteries were anastomosed in the Micro group without any arterial complication in the postoperative period. There was no difference in time required for completing the arterial reconstruction (45.1±18.1 min in the Loupe versus 44.4±6.9 min in the Micro [mean±SEM]). Postoperative ultrasonic Doppler duplex study demonstrated a temporary decrease in the arterial flow in 2 cases of the Loupe group, and partial thrombosis of the artery was suspected. Additionally there were two episodes of hepatic artery thrombosis in 1 case of the Loupe group, in which emergent revision for thrombectomy and reanastomosis was performed at the first episode. This illustrated the higher incidence of arterial complications in the Loupe group compared with the Micro group (4 episodes/7 arteries in the Loupe versus 0/8 arteries in the Micro, P<0.05). In the present series there were no graft failures or arterial complications in the three deaths in the series. The clinical improvements achieved by micro-vascular surgery in living-donor liver transplantation suggest an alternative technical strategy for dealing with problematic arterial reconstruction in adult liver transplantation.

Ischemic tolerance due to the induction of HSP70 in a rat ischemic recirculation model

Various studies have demonstrated an increase in heat shock protein 70 (HSP70) synthesis in the brain following transiently induced ischemia, suggesting a protective role for HSP70 against ischemic insult. In this study, we determined the time course of HSP70 mRNA and protein induction in rat hippocampus following ischemia using Pulsinellis four-vessel occlusion model, and suggested a protective role for HSP70 induction in limiting ischemic damage to neurons and delayed neuronal death. In Northern blotting analysis using human HSP70 DNA as a probe, the accumulation of HSP70 mRNA after 5 min ischemia became evident at 4 h, and continued until 16 h, while after 30 min ischemia, HSP70 mRNA appeared at 2 h, and continued above control level until 24 h after treatment. In immunoblot analysis using anti-HSP70 antibody, induction of HSP70 protein appeared 24 h and reached a maximum 48 h after 5 min ischemia. In immunohistochemical analysis using anti-HSP70 antibody, staining was not detected in CA1 neurons until 16 h after 5 min ischemia, but staining in CA1 gradually increased 1 day after ischemia and reached a maximum level 2 days after ischemia. Similar time profiles in the staining pattern of HSP70 protein were observed in CA3 and CA4 neuronal cells following 30 min ischemia. When rats pretreated with 5 min ischemia (non-lethal for CA1 pyramidal neurons) were exposed to a 30 min, lethal period of ischemia, 2 days after pretreatment, considerable staining of HSP70 was observed. Pretreated rats had much less neuronal damage in the CA1 sector than did rats subjected to lethal, 30 min ischemia alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous Recording of Epileptiform Discharges by MEG and Subdural Electrodes in Temporal Lobe Epilepsy

Spontaneous epileptiform discharges were recorded by whole head magnetoencephalography (MEG) and subdural electrodes simultaneously from two patients with medically intractable temporal lobe epilepsy. In one patient whose epileptiform discharges emerged from the lateral temporal lobe, simultaneously recorded MEG could estimate equivalent current dipole reliably near the tumor. The amplitude of the dipole was in proportion not only to the amplitude of epileptiform discharge but also to the number of subdural electrodes involved. In the other patient, MEG detected only a small proportion of epileptiform discharges, even when they were recorded by subdural electrodes from the mesial temporal lobe. It is concluded that the amplitude and the depth of epileptiform discharges would largely affect the sensitivity of dipole localization by MEG.