Yutaka Hirashima

Elevation of platelet activating factor, inflammatory cytokines, and coagulation factors in the internal jugular vein of patients with subarachnoid hemorrhage

The aim of the present study was to examine the changes of inflammatory and coagulation factors in blood of the internal jugular vein, not of peripheral vein, in patients with subarachnoid hemorrhage (SAH). The results show that while interleukin-6 (IL-6) and platelet activating factor (PAF) concentrations increased within first 4 days after SAH and remained elevated up to 14 days, interleukin-1 β (IL-1 β showed a transient increase between 5–9 days after SAH and tumor necrosis factor-α (TNF-α) remained unchanged. Also different coagulation factors were increased between 5–9 days after SAH. Moreover, patients with delayed ischemic neurological deficits (DIND) displayed the highest levels of PAF and the coagulation factors, von Willebrand factor (vWF) and thrombin-antithrombin III complex (TAT). These results suggest that elevation of PAF and other inflammatory cytokines following SAH may cause the hypercoagulation state that is associated with cerebral vasospasm and internal jugular vein may be more adequate vessel for sampling blood to examine these factors.

Growth Inhibition and Radiosensitization of Cultured Glioma Cells by Nitric Oxide Generating Agents

The authors examined the effect of nitric oxide (NO) generating agents on the growth and radiosensitivity of cultured glioma cells. Three glioma, rat C6, and human T98G and U87 cell lines were treated with the NO generating agents, S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP). These agents released NO in the cell culture media and inhibited the growth of the glioma cells. Growth-inhibition was attenuated by hemoglobin, a known inhibitor of NO, suggesting it is mediated by NO. When C6 and T98G cells were irradiated in the presence of SNAP or SNP at 100 µM, radiosensitization was observed. SNAP at 100 µM exhibited a sensitizer enhancement ratio (SER) of 1.4 for C6 cells and 1.8 for T98G cells. SNP at 100 µM only radiosensitized T98G cells with a SER of 1.9. The effect of SNP on radiosensitization of C6 cells was unclear. We conclude that NO generating agents are potential growth inhibitors and radiosensitizers for malignant glioma cells. NO mediated radiosensitization of glioma cells by NO generating agents may offer a new therapeutic approach for malignant glioma.

Extrinsic pathway of blood coagulation and thrombin in the cerebrospinal fluid after subarachnoid hemorrhage.

OBJECTIVE The involvement of thrombin in the pathophysiology of subarachnoid hemorrhage (SAH) was investigated by comparing thrombin expression and extrinsic pathway activation in the cerebrospinal fluid (CSF) and blood of patients with SAH with the neurological grades, outcome, and presence of delayed cerebral vasospasm. METHODS Blood and CSF samples were obtained from 38 patients with SAH on Days 3 through 5, 7 through 9, and 12 through 14 after the onset of SAH. CSF samples were also obtained from control patients. Thrombin-antithrombin III complex, prothrombin fragment F1 +2, tissue factor, and tissue factor pathway inhibitor were analyzed using enzyme-linked immunosorbent assay. RESULTS No markers in the blood or CSF were correlated with neurological grades and outcome. Thrombin-antithrombin III complex and prothrombin fragment F1 +2 levels were significantly higher in the CSF of patients with SAH than in the blood or the CSF of control patients and were significantly higher in patients with vasospasm than in patients without vasospasm on Days 7 through 9. Tissue factor levels were significantly higher in the CSF of patients with SAH than in the blood, but the levels were close to those in the CSF of control patients. Tissue factor pathway inhibitor levels in the CSF of patients with SAH and control patients were under the detection limit. CONCLUSION Thrombin in the blood may not reflect the pathophysiology of SAH. Imbalance between tissue factor and tissue factor pathway inhibitor in the CSF may tend to thrombin generation under normal physiological conditions and also after SAH. Thrombin in the CSF may be involved in the pathophysiology of vasospasm.

Inhibition of cellular proliferation and induction of apoptosis by curcumin in human malignant astrocytoma cell lines

SummaryNuclear factor (NF)-κB is known to control cellular proliferation and apoptosis. In malignant astrocytoma cells, it was reported that NF-κB was activated aberrantly and promoted their proliferation. Thus, inhibition of NF-κB activity is considered to be a promising therapeutic strategy for malignant astrocytoma. Recently, curcumin, the major constituent of turmeric, was reported to inhibit NF-κB activity. In this study, we investigated inhibitory effects of curcumin on NF-κB activity and cellular proliferation, and induction of apoptosis by curcumin in human malignant astrocytoma cell lines. Alteration of NF-κB activity in NP-2 human malignant astrocytoma cell line after treatment with curcumin was examined using electrophoretic mobility shift assay. Alterations of DNA synthesis and cellular growth in five human malignant astrocytoma cell lines after treatment with curcumin were examined using [3H]thymidine incorporation assay and the trypan blue dye exclusion method, respectively. Induction of apoptosis by curcumin in NP-2 and NP-3 human malignant astrocytoma cell lines was examined by DNA-fragmentation analysis and morphological observation. We found that the NF-κB activity in NP-2 was significantly reduced by curcumin. The DNA synthesis and the cellular growth were inhibited by curcumin in dose-dependent manner in all the five malignant astrocytoma cell lines. Nuclear condensation and fragmentation, and DNA fragmentation were observed in both NP-2 and NP-3 after the treatment with curcumin. These results indicate that curcumin inhibits the cellular proliferation and induces apoptosis in human malignant astrocytoma cell lines. These results are considered to be resulted from the inhibition of NF-κB activity by curcumin.

A case of delayed brain abscess due to a retained intracranial wooden foreign body: a case report and review of the last 20 years

SummaryA 13-year-old female is presented. When she was six years old, she had fallen, holding wooden chopsticks and got stuck with a chopstick in the right upper eyelid. She was brought to a physician immediately, but a residual foreign body was missed and no particular symptom had developed during 7 years. She visited our department with fever and headache, and a brain abscess and an intracranial foreign body were found on computed tomography (CT) and magnetic resonance image (MRI) 7 years after the penetrating injury. She underwent removal of the object and abscess by craniotomy and recovered without neurological abnormalities. Since intracranial retained wooden foreign bodies frequently cause delayed complications of severe central nervous system infection, surgical removal is necessary even in the absence of symptoms.

Independent Predictors of Late Hydrocephalus in Patients with Aneurysmal Subarachnoid Hemorrhage – Analysis by Multivariate Logistic Regression Model

Object: We determined independent variables contributing to the development of late hydrocephalus after subarachnoid hemorrhage (SAH). Methods: A total of 114 consecutive patients who underwent surgery for aneurysms within 72 h after SAH were studied. Thirty-nine patients underwent ventriculoperitoneal shunt (VPS) placement (14 patients within 30 days and 25 patients more than 30 days after onset). Univariate and multivariate analyses were performed to assess relationships among various variables and shunt placement. Results: Three variables were found to be independently associated with VPS patients: (1) the rate of SAH clearance; (2) the duration of external cerebrospinal fluid drainage, and (3) presence of neurological deficits 2 weeks after surgery, which indicates brain damage mainly caused by intraoperative manipulation and cerebral vasospasm. Conclusion: As in previous reports, intraoperative clot removal and duration of external CSF drainage were found to be closely related to the incidence of hydrocephalus. Brain damage due to intraoperative manipulation and cerebral vasospasm is seemed to be involved in the occurrence of late hydrocephalus in this study.

Involvement of platelet-activating factor (PAF) in glutamate neurotoxicity in rat neuronal cultures.

To clarify the role of platelet-activating factor (PAF) in glutamate neurotoxicity, in vitro experiments using primary neuronal cultures were performed. The anti-PAF immunoglobulin-G (aPAF-IgG) and the three PAF receptor antagonists (BN52021, CV6209, and E5880) were tested for their neuroprotective activity in primary neuronal cultures isolated from embryonic rat cerebral cortex. The cultured neurons were exposed to glutamate (1 mM) for 60 min. Twenty-four hours after this exposure, aPAF-IgG demonstrated evidence of protective effects against neuronal damage in a dose-dependent manner. Protective effects also were observed in cultures treated with the three PAF antagonists (P < 0.05 at 1 microg/ml aPAF-IgG, P < 0.01 at 100 microM BN52021, P < 0.05 at 10 nM CV6209 and P < 0.01 at 10 nM E5880). The Fura-2 assay was used to estimate whether low dosages of exogenous PAF affect cultured neurons. The cultured neurons were loaded with Fura-2/AM. After preincubation for 120 min, the Fura-2-loaded neurons were exposed to various concentrations of PAF for 60 min. By measuring the fluorescent intensity of the medium as representing the amount of Fura-2 released from damaged neurons, we detected an increased release of Fura-2, even at low doses of PAF (P < 0.01 at 10 nM PAF). We further studied PAF production by neurons in response to glutamate. The level of PAF measured in the medium exposed to glutamate was significantly higher than the level in the medium unexposed to glutamate (P < 0.05). Our results suggest an important role of PAF in glutamate neurotoxicity.

Neuroprotective Role of Transgenic PAF-Acetylhydrolase II in Mouse Models of Focal Cerebral Ischemia

Background and Purpose— Platelet-activating factor (PAF) and oxidized unsaturated free fatty acids have been postulated to aggravate neuronal damage in the postischemic brain. Type II PAF-acetylhydrolase (PAF-AH II) not only terminates signals by PAF by its PAF-hydrolyzing activity but also protects cells against oxidative stress. We examined whether PAF-AH II can rescue cerebral neurons against ischemic insults. Methods— Transgenic mice overexpressing human PAF-AH II in neurons were generated and enzyme expressions were examined biochemically and histochemically. The mice were subjected to 60 minutes of transient middle cerebral artery occlusion followed by reperfusion for 24 hours. The infarction and apoptosis were estimated by TTC staining and fluorescence TUNEL staining, respectively. Results— Overexpression of PAF-AH II was found in brains of transgenic mice by Western blot and enzymatic activity analyses. In immunohistochemistry, human PAF-AH II expression was found throughout the central nervous system, especially in neurons of neocortex, hippocampus, and basal ganglia. The neurological deficit scores, cerebral edema index, and relative infarction volume were all significantly (P<0.05) lower in transgenic mice (1.30±0.72, 1.12±0.04, and 14.0±7.7%, respectively) than in wild-type mice (2.56±0.93, 1.23±0.12, and 31.9±9.7%, respectively). Percentages of apoptotic cells were also significantly (P<0.001) lower in transgenic mice (cortex, 5.2±3.3%; hippocampus, 3.4±7.0%) than in wild-type mice (cortex, 41.1±16.9%; hippocampus, 58.9±15.3%). Conclusions— These results indicate that PAF-AH II exerts strong neuroprotective effects against ischemic injury and suggest a possibility for clinical use of this enzyme in cerebral ischemia.

Correlation of glutamate-induced apoptosis with caspase activities in cultured rat cerebral cortical neurons

In cultured rat cortical neurons lactate dehydrogenase (LDH) activity in the medium, a cell-death marker, increased gradually after exposure to glutamate (100 microM to 1 mM) for 60 min and reached a plateau at 24 to 30 h. Neuronal death was mainly apoptotic as suggested by typical electron microscopic findings, fluorescent double staining with membrane-permeating and nonpermeating chromatin dyes, nick end labeling, and assessment of DNA fragmentation by agarose gel electrophoresis. After 1 mM glutamate exposure, a rise of interleukin-1beta converting enzyme (ICE)-like protease activity in neurons was parallel to cysteine protease p32 (CPP32)-like protease activity and declined before CPP32-like protease activity reached the peak (at 6 h). LDH activity in the medium of glutamate-exposed neurons was decreased by specific ICE and/or CPP32 inhibitors, acetyl-L-tyrosyl-L-valyl-L-alanyl-L-aspart-1-al (Ac-YVAD-CHO) and acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspart-1-al (Ac-DEVD-CHO), respectively, in a dose-dependent manner. Fluorescent double staining of nuclei also demonstrated that at 100 microM each inhibitor prevented neuronal apoptosis and that this effect was additive. Among agonists corresponding to various glutamate receptor subtypes, N-methyl-D-aspartate (NMDA) and kainate induced apoptosis in cortical neuronal cultures while alpha-amino-3-hydroxy-5-methylisoxazole-4-propinate (AMPA) did not. The metabotropic glutamate receptor agonist, 1-aminocyclopentane-1S, 3R-dicarboxylate (ACPD) prevented apoptosis. Interestingly, apoptosis at 24 h after agonist or antagonist exposure correlated closely with caspase activity 6 h after exposure.

Tissue factor and cancer procoagulant expressed by glioma cells participate in their thrombin-mediated proliferation.

The relationship between coagulation cascade activation and glioma cell proliferation was examined. The human glioma cell lines T98G, TM-1 and normal human astrocyte cell strain (NHA) were examined. Using anti-tissue factor (TF) antibody, immunocytochemical detection of TF antigen was obtained in both cell lines and cell strain. TF antigen in cell lysates was also measured by enzyme linked immunosorbent assay (ELISA). In a one-stage clotting assay, T98G, TM-1 and NHA revealed procoagulant activity (PCA) in normal human plasma and factor VII deficient plasma. PCA in normal human plasma was significantly inhibited by both inhibitory anti-TF antibody and cysteine protease inhibitor HgCl2. This result indicates that T98G, TM-1 and NHA cells express not only TF but also cancer procoagulant (CP) at the same time.In a cell proliferation assay, thrombin induced proliferation in T98G and TM-1 cells in a dose-dependent fashion and in NHA cell in a bell-shaped fashion. This mitogenic stimulant was inhibited by the specific thrombin inhibitor hirudin. The combinations of coagulation factors II, V, and X with or without factor VII induced proliferation in T98G, TM-1, and NHA cells. The maximal mitogenic stimulatory effects were larger in glioma cells than in NHA. These mitogenic stimulatory effects were also inhibited by hirudin. Each coagulation factor on its own or in any other combination of coagulation factors had no proliferative effect. Thus, these mitogenic stimulatory effects were considered to be the effect of thrombin.In conclusion, T98G and TM-1 human glioma cells express two different types of procoagulants TF and CP. In the presence of coagulation factors, these glioma cells can generate thrombin and this thrombin generation is capable of inducing glioma cell proliferation in vitro.