Tsuyoshi Kihara

Role of Neutrophils in Radical Production During Ischemia and Reperfusion of the Rat Brain: Effect of Neutrophil Depletion on Extracellular Ascorbyl Radical Formation

A growing body of experimental data indicate that oxygen radicals may mediate the brain injury during ischemia–reperfusion. One potential source of oxygen radicals is activated neutrophils. To study the role of neutrophils in radical production during cerebral ischemia–reperfusion, we evaluated the effects of depletion of circulating neutrophils by administration of an anti-neutrophil monoclonal antibody (RP3) on radical formation in rats with 1-h middle cerebral artery (MCA) occlusion. In the present study, we employed a new electron spin resonance method coupled with brain microdialysis. The method uses the endogenous ascorbyl radical (AR) concentration as a marker of oxygen radicals and requires no spin-trapping agents. In the vehicle controls, extracellular AR decreased during MCA occlusion. After reperfusion, AR significantly increased at 30 min and 1 h, returned to near basal level until 2 h, and increased again at 24 h after reperfusion. In the rats treated with RP3, AR decreased during MCA occlusion to the same extent as in the vehicle control. However, RP3 treatment completely inhibited the increase in extracellular AR after reperfusion. RP3 treatment exerted no effect on the changes in extracellular ascorbate or tissue Po2 throughout the experimental period. In conclusion, neutrophils are a major source of oxygen radicals during reperfusion after focal cerebral ischemia.

Role of cell adhesion molecules in brain injury after transient middle cerebral artery occlusion in the rat

Activated neutrophils appear to be directly involved in tissue injury after focal cerebral ischemia and reperfusion. Intercellular adhesion molecules-1 (ICAM-1) and CD11/CD18 integrins have been implicated in ischemia-reperfusion induced neutrophil endothelial adhesion and transmigration. We therefore investigated the roles of CD11a/CD18 (LFA-1) and ICAM-1 in cerebral ischemia-reperfusion injury by using monoclonal antibodies, WT1 (anti-CD11a), WT3 (anti-CD18), and 1A29 (anti-ICAM-1). Rats were subjected to 1 h of middle cerebral artery occlusion (MCAO). Individual antibodies were administered at a dose of 5 mg/kg intraperitoneally at 15 min before ischemia and immediately after reperfusion. Rats were killed at 24 h after reperfusion, and brain edema, neutrophil infiltration and infarct size were measured. Sustained enhancement of ICAM-1 expression on capillaries was observed up to 24 h (beginning between 1 and 3 h after reperfusion). While, leukocytes began to infiltrate into the ischemic hemisphere between 6 and 12 h after reperfusion. Treatment with individual antibodies against cell adhesion molecules reduced edema formation and infarct size in addition to neutrophil accumulation 24 h after reperfusion. These results strongly implicate the invasion of neutrophils in the development of post-ischemic brain injury, and suggest that interactions between CD11a/CD18 and ICAM-1 contribute to neutrophil infiltration into the ischemic brain.

Role of platelet-activating factor and thromboxane A2 in radical production during ischemia and reperfusion of the rat brain

Oxygen radicals produced by activated neutrophils have been involved in brain injury during ischemia-reperfusion. Platelet-activating factor (PAF) is a candidate as one of the mediators of neutrophil activation during cerebral ischemia-reperfusion. Recent evidence indicates that PAF-induced neutrophil activation is mediated by thromboxane A2 (TXA2). To study the role of PAF and TXA2 in radical production during cerebral ischemia-reperfusion, we evaluated the effects of a PAF antagonist, Y-24180, and a TXA2 antagonist, S-1452, on radical formation in rats with 1 h middle cerebral artery (MCA) occlusion. In the present study, we employed a new electron spin resonance (ESR) method coupled with brain microdialysis. The method uses the endogenous ascorbyl radical (AR) concentration as a marker of oxygen radicals and requires no spin-trapping agents. In the vehicle controls, extracellular AR from the ischemic brain cortex decreased during MCA occlusion. Following reperfusion, AR significantly increased at 30 mm and 1 h, returned to near the basal levels at 2 h, and increased again at 24 h after reperfusion. In the rats treated with S-1452 or Y-24180, AR decreased during MCA occlusion to the same extent as in the vehicle control. However, pretreatment with Y-24180 or S-1452 significantly attenuated the increase in extracellular AR after reperfusion, while it exerted no effect on the changes in extracellular ascorbate or tissue pO2 throughout the experimental period. In conclusion, PAF and TXA2 might contribute to cerebral ischemia-reperfusion injury by increasing the generation of oxygen radicals.

A novel benzodiazepine inverse agonist, S-8510, as a cognitive enhancer

1. Pharmacological actions of a novel benzodiazepine receptor ligand, S-8510 (2-(3-isoxazolyl)-3,6,7,9-tetrahydroimidazo[4,5-d]pyrano+ ++[4,3-b] pyridine monophosphate monohydrate), were examined in in vitro and in vivo studies. 2. S-8510 was characterized as a partial inverse agonist with a modest GABA ratio and low efficacy. 3. S-8510 ameliorated memory impairment induced by cholinergic deficit in the water maze paradigm of Wistar rats. 4. S-8510 augmented LTP of the Schaffer collateral/commissural fiber-CA1 synapses in the hippocampal slice preparations of SD rat. 5. S-8510 increased the extracellular levels of acetylcholine and noradrenaline in the hippocampus of Wistar rat. 6. S-8510 selectively potentiated pentylenetetrazol-induced convulsion without affecting minimal electroconvulsive shock- or strychnine-induced convulsion in ddY mice. 7. S-8510 failed to induce any sign of anxiety in the Wistar rat pro-conflict test. 8. S-8510 showed antidepressant-like pharmacological actions in ddY mice. 9. These results suggest that S-8510 can be used as a therapeutic drug for senile dementia, including Alzheimers disease with little risk for inducing anxiety or convulsion.

Direct Detection of Ascorbyl Radical in Experimental Brain Injury: Microdialysis and an Electron Spin Resonance Spectroscopic Study

Abstract: To examine the role played by free radicals in brain injury, we performed experiments to detect radicals in the frontal cortex of rats, using electron spin resonance (ESR) and microdialysis. A dialysis probe was inserted into the frontal cortex, and spin adducts in perfusates were immediately detected by ESR. We obtained a relatively stable doublet signal, with parameters of g = 2.0057 and aH = 0.17 mT. This signal corresponded with that of the ascorbyl radical. Ascorbyl radical in the perfusate collected from the frontal cortex was augmented by microinjection of H2O2 and FeCl2 adjacent to the dialysis probe. When the rats were challenged with cold‐induced brain injury, ascorbyl radical and lactate dehydrogenase (LDH) level in the perfusate increased significantly. Pretreatment with superoxide dismutase and catalase attenuated the increase in ascorbyl radical and LDH level induced by the cold injury. Infusion of FeCl2 dissolved in perfusate caused a pronounced increase in ascorbyl radical and LDH level after the cold injury. We conclude that the direct detection of free radical formation further supports the hypothesis that free radicals play an important role in traumatic brain injury. Our findings also indicate that combined microdialysis with ESR spectroscopy is a useful in vivo method for monitoring free radical production in the brain.

Differential effects of ceruletide on amphetamine-induced behaviors and regional dopamine release in the rat.

This study concerned the effects of ceruletide, a cholecystokinin (CCK)-related peptide, on amphetamine-stimulated behaviors (hyperlocomotion and stereotypy) and amphetamine-induced dopamine (DA) release from the striatum and the nucleus accumbens of the rat. Also, behavioral alterations due to ceruletide administration were compared with the change in DA release from these areas. Ceruletide 160 micrograms/kg s.c., attenuated hyperlocomotion induced by amphetamine, 1 mg/kg and 3 mg/kg s.c., but had no effect on amphetamine-induced stereotypy. Results from in vivo microdialysis experiments showed that s.c. administration of ceruletide caused a significant inhibition of the amphetamine-induced increase in DA release in the nucleus accumbens but not in the striatum. These neurochemical inhibitory effects of ceruletide disappeared completely with bilateral subdiaphragmatic vagotomy. However, infusion of 1 microM of ceruletide into the nucleus accumbens through the dialysis probe had no effect on amphetamine-induced DA release. These results suggest that the inhibitory effect of peripheral administration of ceruletide on amphetamine-induced hyperlocomotion is closely related to the change in DA release from the nucleus accumbens. In the nucleus accumbens, systemically administered ceruletide acts initially on the peripheral organs and influences the activity of DA terminals via an unknown path related to the vagus. Ceruletide had different actions on the dopaminergic system in the striatum and that in the nucleus accumbens.

Ceruletide, a cholecystokinin-related peptide, attenuates haloperidol-induced increase in dopamine release from the rat striatum: an in vivo microdialysis study

The effects of ceruletide diethylamine (CLT), a cholecystokinin (CCK)-related peptide, on the spontaneous and haloperidol (HPD)-induced release of striatal dopamine (DA) were investigated with the in vivo microdialysis method. The striatum was perfused with Ringer solution containing different concentrations of K+. (1) When the dialysis tube was perfused with 4 mM K(+)-containing Ringer solution, CLT exerted no influence on the spontaneous and HPD-induced release of DA. (2) Increasing the K+ concentration in the perfusate from 4 to 15 mM failed to change the spontaneous and HPD-induced DA release. In this perfusion condition, the HPD-induced increase in DA release was significantly attenuated by CLT. (3) Perfusion of the striatum with the 20 mM K+ significantly reduced both the spontaneous and HPD-induced output of DA. (4) Even under the condition of perfusing the dialysis tube with the 4 mM K+, CLT significantly decreased the HPD-stimulated DA release in rats given HPD alone for the first 7 days and with CLT for the last 3 days. (5) Sixty consecutive daily administrations of HPD alone markedly reduced HPD-induced DA release from the striatum perfused with the Ringer solution containing 4 mM K+. From these results, we suggest that CLT, under the appropriate depolarization, can facilitate or induce depolarization inactivation of the A9 DA cells and/or nigrostriatal DA terminals, and consequently, produce significant inhibition of HPD-induced DA release from the rat striatum.

Ceruletide, a CCK-like peptide, attenuates dopamine release from the rat striatum via a central site of action

Ceruletide (CLT), a cholecystokinin-like peptide was given subcutaneously or via the perfusate to rats to clarify the site of action (peripheral vs. central location) of CLT, using in vivo microdialysis techniques. Striatal dopamine (DA) release induced by haloperidol (HPD) was significantly inhibited by subcutaneously administered CLT (160 micrograms/kg) when given with a perfusate containing 15 mM K+. Subdiaphragmatic vagotomies failed to block the inhibitory effect of CLT. CLT (10(-15)-10(-11) M) locally applied, via a dialysis tube, produced an inhibitory effect on HPD-induced DA release in the striatum in a dose-dependent manner. The inhibitory effect of CLT given subcutaneously on DA release was antagonized by both locally applied proglumide and systemically administered L-365,260. These findings suggest that systemically administered CLT can directly act on the striatal neurons via CCK-B receptors and produce an inhibitory effect on DA release in the striatum under appropriate depolarization.

ω-Agatoxin IVA-sensitive Ca2+ channel blocker, α-eudesmol, protects against brain injury after focal ischemia in rats.

Abstract ω-Agatoxin IVA-sensitive Ca 2+ channels have been thought to be involved in physiological excitatory amino acid glutamate release and these channels may also contribute to the development of ischemic brain injury. Recently, we demonstrated that α-eudesmol from Juniperus virginiana Linn. (Cupressaceae) inhibits potently the presynaptic ω-agatoxin IVA-sensitive Ca 2+ channels. In the present study, we investigated the effects of α-eudesmol on brain edema formation and infarct size determined after 24 h of reperfusion following 1 h of middle cerebral artery occlusion in rats. We first found that α-eudesmol concentration-dependently inhibited glutamate release from rat brain synaptosomes and that its inhibitory effect was Ca 2+ -dependent. In the middle cerebral artery occlusion study, intracerebroventricular (i.c.v.) treatment with α-eudesmol significantly attenuated the post-ischemic increase in brain water content. α-Eudesmol also significantly reduced the size of the infarct area determined by triphenyltetrazolium chloride staining after 24 h of reperfusion. Using a microdialysis technique, we further demonstrated that α-eudesmol inhibits the elevation of the extracellular concentration of glutamate during ischemia. From these results, we suggest that α-eudesmol displays an ability to inhibit exocytotic glutamate release and to attenuate post-ischemic brain injury.

Influence of Potassium Concentration in Microdialysis Perfusate on Basal and Stimulated Striatal Dopamine Release: Effect of Ceruletide, a Cholecystokinin-Related Peptide

Abstract: The in vivo microdialysis method was used to study the effect of the cholecystokinin‐related peptide, ceruletide, on extracellular levels of dopamine (DA) in the striatum following perfusion with various K+ concentrations. Increasing the K+ concentration in the perfusate from 4 to 15 or 17.5 mM did not change basal DA release or release evoked by electrical stimulation of the medial forebrain bundle (MFB). However, when the perfusing solution contained 20 or 30 mM K+, dose‐dependent reductions of both basal and MFB‐stimulated DA release occurred. Subcutaneous administration of ceruletide at 160 μg/kg had no influence on the basal or MFB‐stimulated DA release with 4 or 15 mM K+ in the perfusate. However, after perfusion with 17.5 mM K+, ceruletide significantly attenuated the basal and MFB‐stimulated DA release. Carbachol (10 μM) locally applied via the dialysis probe also attenuated MFB‐stimulated DA release after perfusion with 17.5 mM K+. From these results, we conclude that under appropriate depolarization of striatal DA terminals, ceruletide induces further depolarization and inactivation of nigrostriatal DA terminals. The present data suggest that this effect may be mediated via intrinsic cholinergic neurons in the striatum.