Nobuo Araki

A novel in vivo assay system for consecutive measurement of brain nitric oxide production combined with the microdialysis technique

A novel spectrophotometric nitrite (NO2-)/nitrate (NO3-) assay system for a small quantity (5 microliter) of dialysate sample obtained by in vivo brain microdialysis was developed based on the diazotization reaction. The system has the advantage of in vivo consecutive measurement, high precision, good reproducibility, technical simplicity, relatively short resolution time (2.5-20 min), and wide availability. The NO3- level in the rat striatum was found to be 3 times higher than the NO2- level. A nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester, reduced striatal NO2-/citrulline formation in a dose-related manner and increased arginine, indicating that the tissue NO2- level detected by this assay system adequately reflects the striatal NO synthase activity.

Presynaptic ionotropic glutamate receptors modulate in vivo release and metabolism of striatal dopamine, noradrenaline, and 5-hydroxytryptamine: involvement of both NMDA and AMPA/kainate subtypes

In order to explore further the presynaptic modulation of monoamine release by glutamatergic nerve fibers, we investigated the effects of selective agonists for ionotropic glutamate (GLU) receptors on striatal release of dopamine (DA), noradrenaline (NA) and 5-hydroxytryptamine (5-HT). In the striatum of anesthetized Sprague-Dawley rats, in vivo microdialysis was performed to measure the release of monoamines and metabolities, and also to administer GLU agonists locally in the tissue. L-GLU and its selective agonists (N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and kainate (KA)) evoked simultaneous release of striatal DA, NA and 5-HT in a dose-dependent manner. Pretreatment with MK-801 (5 mg/kg i.p.), a noncompetitive NMDA receptor antagonist, selectively suppressed NMDA-evoked monoamine release. The rank order of GLU agonist efficacy in releasing monoamines was different among DA, NA, and 5-HTergic terminals: AMPA = KA > NMDA for DA release, AMPA > NMDA = KA for NA release, and NMDA = AMPA = KA for 5-HT release. In conclusion, presynaptic ionotropic GLU receptors exist extensively on monoaminergic terminals including not only catecholaminergic (DA and NA) but also indoleaminergic (5-HT) terminals in the rat striatum. Their subtypes include both NMDA subtype and AMPA/KA subtype, and show a differential distribution among these three monoaminergic terminals and a differential contribution to facilitating monoamine release.

FK506 abrogates delayed neuronal death via suppression of nitric oxide production in rats

BACKGROUND AND PURPOSEnThe mechanism of the neuroprotective effect of FK506 in relation to nitric oxide (NO) production has not been clarified in vivo. We have investigated the effect of FK506 on ischemia-induced NO production in association with the pathogenesis of delayed neuronal death (DND) in rats.nnnMETHODSnIn vivo microdialysis was performed in the hippocampus of male Sprague-Dawley rats (250-350 g). Dialysate samples were collected every 3 min. In the ischemia group (n=16), global ischemia was induced for 21 min and reperfusion was achieved. In the FK506 treatment group (n=25), FK506 (1 mg/kg, i.v.) was administered 21 min prior to the onset of global ischemia. Sham operations were done (n=15). The levels of NO(2)(-) in the dialysate samples were determined by the Griess reaction. The animals were decapitated 7 days after ischemia. Coronal brain sections were stained with hematoxylin and eosin.nnnRESULTSnIn the ischemia group, the NO(2)(-) level significantly increased during ischemia. In the FK506 treatment group, there was no significant change in the NO(2)(-) level during ischemia. In histological examinations, FK506 treatment showed a neuroprotective effect against DND.nnnCONCLUSIONSnThe effect of FK506 inhibiting NO production contributes to the neuro-protective effect of FK506 on DND in the hippocampus.

Correlation of in vivo nitric oxide and cGMP with glutamate/glutamine metabolism in the rat striatum.

We have examined how the suppression of endogenous production of nitric oxide (NO) in the striatal tissue affects release of glutamate (GLU) and glutamine (GLN) in pentobarbital-anesthetized male Sprague-Dawley rats. For the quantitative measurement of tissue NO production and amino acid release, an in vivo assay system for extracellular nitrite (NO2-) and amino acids was employed using an in vivo microdialysis technique. An NO synthase inhibitor (NG-nitro-L-arginine methyl ester, L-NAME) in concentrations ranging between 4-40 mM was perfused into the rat striatum using the assay system. Tissue NO production was found to be inversely proportional to the L-NAME concentration. L-NAME likewise decreased striatal levels of GLU and GLN. Furthermore, tissue NO production showed a positive correlation with GLU (R = 0.62, P < 0.02) and GLN (R = 0.86, P < 0.001) concentrations. Exogenous application of NO and cGMP by intrastriatal perfusion with 0.1-2.5 mM hydroxylamine and 0.4-10 mM 8-bromo-cGMP, respectively, increased striatal GLU release in a dose-related manner. Hydroxylamine reduced GLN release, and 8-bromo-cGMP showed a tendency to decrease GLN. In conclusion, striatal GLU/GLN metabolism is a function of the tissue concentration of NO. Normal endogenous concentration of NO causes GLU to be released at a consistent basal level, and enhanced tissue NO production facilitates GLU release via pathways including cGMP formation. We hypothesize that NO may suppress GLN formation by astrocytes.

Presynaptic glutamate receptors facilitate release of norepinephrine and 5-hydroxytryptamine as well as dopamine in the normal and ischemic striatum

We investigated the effects of selective glutamate (Glu) agonists on the release of monoamine neurotransmitters and their implication in the enhanced monoamine release in cerebral ischemia. In the striatum of anesthetized Sprague-Dawley rats, in vivo microdialysis was performed and the release of excitatory amino acids (Glu and aspartate (Asp)) and monoamines (dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT)) was measured by high-performance liquid chromatography with an electrochemical detector. (1) Forebrain ischemia by 4-vessel occlusion generated significant correlations between the Glu and Asp levels and the DA, NE and 5-HT levels (r = 0.922-0.967, P < 0.01, n = 6). (2) L-Glu and its selective agonists (N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and kainate (KA)) evoked a simultaneous release of striatal DA, NE and 5-HT in a dose-dependent manner (P < 0.01, ANOVA, n = 8). The maximal monoamine release evoked by the Glu agonists showed different magnitudes in the order of DA >> NE > 5-HT (118-, 16- and 9-fold from the baseline levels by 62.5 mM L-Glu, respectively). Each Glu agonist exerted a different magnitude of transmitter release and the order of agonist efficacy was different among NE, 5-HT and DA release: AMPA = KA > L-Glu = NMDA for DA release, AMPA > L-Glu = NMDA = KA for NE release, and L-Glu = NMDA = KA = AMPA for 5-HT release.(ABSTRACT TRUNCATED AT 250 WORDS)

Sweating Function and Retinal Vasomotor Reactivity in Migraine

Sweating function and retinal arterial reactivity in patients with migraine were examined during headache-free intervals. The sweat glands were stimulated by intradermal injection of pilocarpine hydrochloride, and molds of sweat droplets were obtained using Silastic. The number of droplets in classic migraine was significantly lower than that in the controls. The retinal arterial response to changes in perfusion pressure was measured by means of a fundus camera. The retinal vasomotor index (-change in retinal arterial diameter (%)/change in effective MABP (mmHg)) was significantly lower in classic and in common migraine than in controls. The above results suggest that sweating function and retinal arterial reactivity are impaired in patients with migraine and that the impairment may play an important role in the pathophysiology of migraine attacks.

Prognostic value of cerebral blood flow autoregulation in the long-term prognosis of ischemic cerebrovascular disease.

The correlation between long-term prognosis, cerebral blood flow (CBF) and CBF autoregulation was studied in 34 patients with cerebral infarction (mean age, 64 years). CBF was measured by the nitrous oxide method 1-6 months (mean 87 days) after disease onset. CBF autoregulation was evaluated quantitatively from the Dysautoregulation Index (DI) (delta CBF/delta effective MABP). Reductions in effective MABP were induced with a tilt table. No significant correlation was noted among CBF, DI and activities of daily living at the time of measurement. The patients physical condition was reevaluated by questionnaire 2 years or more (mean 32 months) later. Better functional state at follow-up was related to higher CBF and lower DI values although the differences were not significant. The relationships among CBF, DI and changes in physical condition during the period were evaluated. The mean CBF values in patients with a better prognosis exceeded those of poor prognosis patients. The CBF values in the group who became independent significantly exceeded those in the group that deteriorated (P less than 0.05). The CBF values in the latter showed small but significant decreases during head-up tilting (P less than 0.05). The DI in this group was significantly higher than in the groups with a less severe outcome (P less than 0.01, P less than 0.05, respectively). In conclusion, determinations of CBF autoregulation, together with flow values, in the chronic state may have some value in predicting the long-term prognosis in cerebral infarction.

In Vivo Measurement of Superoxide in the Cerebral Cortex Utilizing Cypridina luciferin analog (MCLA) Chemiluminescence

Temproal profile of superoxide (O2 −) generation following cerebral hypoxia and ischemia has been obscure, although it has been implicated in the progression of reperfusion injury. We have examined the time course of O 2 − generation in the cat cortex following reversible hypoxia and forebrain incomplete ischomia. We used 20 cats anesthetized with halothane inhalation. A closed cranial window was placed over the exposed temporoparietal cortex, and Cypridina luciferin analog (MCLA), a chemiluminescence probe for the measurement of O 2 − , was dissolved in the artificial cerebrospinal fluid (ACSF) and superfused continuously throughout the experiments. We simultaneously monitored a reflectance (398 nm) from the cortex by means of an in vivo fluoromicroscope with two photomultiplier tubes. We subtracted a hemodynamic artifact from the MCLA chemiluminescence. Hypoxia was induced by pure N2 inhalation for 1 min, and forebrain incomplete ischemia was induced by 30-min ligation of both common carotid arteries combined with hypotension (= 50 mmHg). As a result, the MCLA chemiluminescence increased following reoxygenation and reperfusion, indicating an enhanced O 2 − generation. It was significantly reduced during hypoxia and severe ischemia, but fluctuated during mild ischemia. We speculate a breakdown of arachidonic acid and a biochemical interaction between endothelial cells and polymorphonuclear leukocytes as a source of O 2 − generation.

The role of noradrenergic nerve terminal in the autonomic action potentials of the pial arteries

脳軟膜動脈より導出した活動電位がいかなる種類の神経線維に由来するかを明らかにする目的で, ノルアドレナリン作動神経系の関与について検討した. [対象および方法] 成猫15匹を用い, ノルアドレナリン作動神経終末部に働き, dopamine-β-hydroxylaseを阻害する作用のあるフサリン酸を頚動脈内に投与した際の脳軟膜動脈活動電位の変化, および脳灌流圧変化に対する脳軟膜動脈活動電位の反応性を, 電気生理学的手法により検討した. [結果] (1) フサリン酸投与後, 全例において脳軟膜動脈活動電位の放電頻度は有意に減少した. (2) フサリン酸投与後の脳軟膜動脈活動電位の血圧変動に対する反応性は, フサリン酸4mg/kg以下の投与例では保たれていたが, フサリン酸8mg/kg以上の投与例では, 投与10～80分後に消失した. [結論] 脳軟膜動脈より導出した活動電位は, ノルアドレナリン作動神経, とくにその終末部と密接に関係していると考えられる.

Autonomic Nervous Action Potential of Cerebral Veins

1. Averaged action potential waves were recorded from cerebral arteries and from cerebral veins during cervical sympathetic stimulation. The averaged potential waves were suppressed after the administration of hexamet honium or tetrodotoxin.