Hideyuki Uchimura

Ovariectomy Exacerbates and Estrogen Replacement Attenuates Photothrombotic Focal Ischemic Brain Injury in Rats

BACKGROUND AND PURPOSE We previously reported the infarct volumes in female spontaneously hypertensive rats (SHR) to be significantly smaller than those in male SHR. The purpose of the present study was to determine whether estrogen is responsible for the sex difference in ischemic vulnerability in SHR. METHODS In experiment 1, 1 week (short-term) or 4 weeks (long-term) after the ovariectomy (OVX), female SHR (5 months old) were randomly subjected to photothrombotic occlusion of the middle cerebral artery, and the infarct volumes were determined. In experiment 2, the rats were randomly assigned to 3 groups (ie, the sham-ovariectomized, ovariectomized, and estrogen replacement groups). In the replacement group, estradiol valerate (200 microgram/kg) was subcutaneously injected once a week after the OVX. Four weeks after the OVX or sham-OVX, all rats were subjected to middle cerebral artery occlusion. Changes in regional cerebral blood flow were determined by laser-Doppler flowmetry. RESULTS In experiment 1, the infarct volume produced 1 week after the OVX was not different from that of the sham-ovariectomized group. In contrast, the infarct volume produced 4 weeks after the OVX was significantly larger than that of the sham-ovariectomized group (82.4+/-11.6 versus 54.5+/-16.0 mm(3), P=0.0058). In experiment 2, estradiol replacement after the OVX was observed to attenuate the infarct volume compared with the ovariectomized group (55.6+/-18.8 versus 78.5+/-21.0 mm(3), P=0.0321). The degrees of regional cerebral blood flow reduction did not differ among the sham-ovariectomized, ovariectomized, and estrogen replacement groups. CONCLUSIONS Chronic estrogen depletion was thus found to increase the infarct size, which was attenuated by estradiol replacement. These findings indicate that estrogen contributes to the sex difference in ischemic vulnerability and that endogenous estrogen also has a neuroprotective effect against ischemic brain damage.

Regional differences in cofactor saturation of glutamate decarboxylase (GAD) in discrete brain nuclei of the rat. Effect of repeated administration of haloperidol on GAD activity in the substantia nigra.

Glutamate decarboxylase (GAD) activities with and without added pyridoxal-5′-phosphate were determined in discrete brain nuclei of freeze-dried samples. The distribution of GAD holoenzyme activity as well as the cofactor saturation, was found to be uneven in the discrete nuclei. In addition, it was found that repeated haloperidol treatment reduced GAD holoenzyme activity in the substantia nigra pars reticulata.

Monoamine oxidase activities for serotonin and tyramine in individual limbic and lower brain stem nuclei of the rat.

MONOAMINE oxidase (MAO; EC 1.4.3.4.) is a major enzyme in the catabolism of the putative neurotransmitter monoamines. During the past decade, it has been suggested that M A 0 in the brain exists in multiple forms (YOUDIM, 1972) and has been classified into two forms, type A and type B, by studies with substrate-specific inhibitors (JOHNSTON, 1968; YANG & NEFF, 1974). Some workers have reported the discriminative action of several hormones on dimerent forms of M A 0 (TONG & D~ORIO, 1976; YOUDIM & HOLZBAUER, 1976). the differential maturation of the two types of M A 0 (JOURDIKIAN et al., 1975) and different effects of selective M A 0 inhibitors on some behaviours (FUENTES & NEW, 1975) and drugs (Rom & GILLIS, 1975). However, little information referring to the physiological significance of this multiplicity in the discrete brain regions was reported. The limbic system consists of many nuclei and areas where catecholaminergic and serotonergic pathways are projected from the cell bodies located in the lower brain stem (DAHLSTR~M & FUXE, 1964; UNGERSTEDT. 1971). The biogenic amines in the brain are known to play different roles in various autonomic, neuroendocrine and behavioural functions. The present study describes the distribution of M A 0 activities towards serotonin which is a specific substrate for MAOA and tyramine which is a substrate for both MAO-A and MAO-B in individual limbic regions, main monoamine-containing cell groups and some hypothalamic areas, using the micromethod with freeze-dried sections (UCHImRA et a/., 1974; HIRANO et a/., 1975).

Cerebral blood flow and ischemia-induced neurotransmitter release in the striatum of aged spontaneously hypertensive rats.

Background and Purpose: We found age‐related vulnerability to cerebral ischemia in the hippocampus and striatum in spontaneously hypertensive rats. Further study revealed that ischemia‐induced release of hippocampal taurine, an inhibitory amino acid, was reduced by 40% in aged rats compared with adult rats, which suggested an impaired inhibitory function against excitotoxicity in aged rats. The purpose of this study was to examine whether ischemia‐induced neurotransmitter release is altered in the striatum of aged spontaneously hypertensive rats. Methods: Five adult (5‐6 months) and five aged (18‐22 months) female spontaneously hypertensive rats were subjected to 20 minutes of cerebral ischemia induced by bilateral carotid artery occlusions and 120 minutes of recirculation under amobarbital anesthesia (100 mg/kg i.p.). Cerebral blood flow was determined using the hydrogen clearance method, and extracellular concentrations of neurotransmitters were determined with the brain dialysis technique in the striatum. Results: During ischemia, cerebral blood flow in aged rats decreased to 8.7±1.2 (mean±SEM) mL/100 g per minute (11% of the resting), which was not different from 5.2±1.7 mL/100 g per minute (8% of the resting) in adult rats, and extracellular dopamine and amino acids (glutamate, aspartate, and taurine) increased by approximately 170‐ and 10‐30‐fold, respectively, and returned to baseline after 20‐40 minutes of recirculation. These values of neurotransmitters, however, were not different between aged and adult rats during ischemia and reperfusion. Conclusions: It is unlikely that a presynaptic mechanism is involved in age‐related vulnerability in the striatum of hypertensive rats. (Stroke 1993;24:577‐580)

Choline acetyltransferase and acetylcholinesterase activities in limbic nuclei of the rat brain

THE HISTOCHEMICAL distribution of acetylcholinesterase (AChE, acetylcholine acetyl-hydrolase, EC 3.1.1.7) in the limbic system has been illustrated by SHUTE & LEWIS (1967). LEWIS & SHUTE (1967) and JACOBOWITZ & PALKOvm (1974). However, choline acetyltransferase (ChAT, acetyl-CoA-choline 0-acetyltransferase, EC 2.3.1.6) is a more spenfic marker for cholinergic neurons than AChE (SILVER, 1967; FONNUM, 1970; CHUPPMELLI et ol., 1976). In addition, the histochemical staining method has been used to obtain relative AChE values (STORM-MATHISEN, 1970). For study of physiological functions of the limbic system, therefore, i t is very important to determine O A T and AchE activities in limbic nuclei and areas. This paper describes the quantitative distribution of ChAT and AChE in limbic regions, using the microdissection technique of individual nuclei with freeze-dried sections (UCHIMURA et a!., 1974, 1975) and a radiochemical micromethod to assay the enzymes.

Angiotensin II Increases Norepinephrine Turnover in the Anteroventral Third Ventricle of Spontaneously Hypertensive Rats

We evaluated the effect of angiotensin II (Ang II) administered by intracerebroventricular injection on norepinephrine turnover in the anteroventral third ventricle in adult spontaneously hypertensive rats (SHR, n = 35) and age-matched Wistar-Kyoto rats (WKY, n = 38). Ang II (100 ng) or saline (vehicle control) was administered into the cerebral ventricle 30 minutes after injection of alpha-methyl-p-tyrosine (250 mg/kg IP). Norepinephrine turnover was assessed by evaluation of the norepinephrine concentration before and 1 hour after such administration. The pressor response to Ang II administration was significantly greater in SHR than in WKY (+43 +/- 3 versus +23 +/- 2 mm Hg, P < .01). Baseline norepinephrine turnover (response to saline) was reduced in the ventral median preoptic nucleus of SHR. Ang II significantly increased norepinephrine turnover in the organum vasculosum lamina terminalis and ventral median preoptic nucleus of SHR (organum vasculosum lamina terminalis: 40 +/- 5% by Ang II versus 18 +/- 6% by saline, P < .05; ventral median preoptic nucleus: 32 +/- 3% by Ang II versus 21 +/- 2% by saline, P < .05) but not of WKY (37 +/- 5% versus 29 +/- 5%, P = NS, and 30 +/- 2% versus 32 +/- 3%, P = NS, respectively). Thus, norepinephrine turnover in the anteroventral third ventricle region induced by intracerebroventricular administration of Ang II was increased in SHR. This effect may contribute to the enhanced pressor response to central Ang II seen in this model.

Preparation of tyrosine-O-[35S]sulfated cholecystokinin octapeptide from a nonsulfated precursor peptide

A rapid and simple one-pot method for O-sulfation of nonsulfated cholecystokinin octapeptide (CCK-8) was developed using sulfuric acid and dicyclohexylcarbodiimide (DCC) without protection of the amino acid side chains. The extent of sulfation was increased with increasing the amount of reactants, sulfuric acid, and DCC, and reached maximum (40%) with fourfold molar excess of sulfuric acid and 40-fold molar excess of DCC. The excess of nonsulfated peptide inhibited the sulfation. The sulfation product was purified by HPLC or TLC to give a pure sulfated substance which showed exactly the same behavior as that of an authentic O-sulfated CCK-8 on HPLC or TLC. The purified sulfated peptide was active in stimulating amylase secretion from rat pancreatic fragments, and amino acid analysis showed that the tyrosine residue in the peptide existed in O-sulfated form. Sulfation with [35S]sulfuric acid-DCC produced a radioactive substance, from which O-[35S]sulfated CCK-8 could be easily purified by two-dimensional TLC.

Analysis of Exploding Plasma Behavior in a Dipole Magnetic Field.

Numerical analyses on plasma behaviors in a dipole magnetic field are performed using a three-dimensional (3D) hybrid code. Results are compared with the experimental data and magnetohydrodynamics (MHD) analysis. Dependence of plasma expansion on initial plasma energy and location are discussed by temporal evolutions of plasma position and magnetic field strength. An overall good agreement in the expansion behavior of plasmas among these results is found. The asymmetrical shape of the expanding plasma in the cross-field direction is also noticed, and the reason for this is discussed. For future engineering applications, these results will be useful in designing an optimal configuration of the magnetic thrust chamber for laser fusion rockets, and for studying the effective explosive methods for protecting the earth from collisions by asteroids or comets.

Mass Fragmentographic Determination of Homovanillic Acid in Individual Dopaminergic Neuron Systems of Rat Brain: Application of Reaction Gas Chromatography

Abstract: Homovanillic acid (HVA) levels of 12 discrete rat brain areas were determined by a mass fragmentographic method using the reaction gas chromatographic technique. The use of reaction gas chromatography increased the sensitivity for determination of HVA. The sensitivity of this method allows measurement of HVA in small amounts of brain tissue. The HVA levels in polar, medial, and lateral fields of prefrontal cortex, anterior cingulate cortex, septum, amygdala, A12, A13, and A14 dopaminergic neurons were 0.417 ± 0.018 ng/mg protein, 0.689 ± 0.004, 0.753 ± 0.024, 0.496 ± 0.029, 1.311 ± 0.046, 0.555 ± 0.008, 1.949 ± 0.077, 1.109 ± 0.112, and 0.489 ± 0.019, respectively. The HVA levels in these areas are first reported in the present paper.

Serotonin and 5-hydroxyindoleacetic acid concentrations in individual hypothalamic nuclei and other brain areas of rat

Serotonin and 5-hydroxyindoleacetic acid (5-HIAA) were measured in individual nuclei of rat hypothalamus and other brain areas using HPLC with electrochemical detection. 5-HIAA levels were first demonstrated in hypothalamic and some discrete brain areas. The 5-HIAA/5-HT ratio was highest in the n. caudatus putamen, high in the n. ventromedialis and lowest in the n. suprachiasmaticus.