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Extracellular concentrations of ascorbic acid, glutathione, cysteine, uric acid, tyrosine, and tryptophan were monitored using intracerebral microdialysis in the left frontoparietal cortex of spontaneous hypertensive rats before, and for 3 h after, either focal ischemia [left middle cerebral artery occlusion (MCAO)] or sham operation. The size of the ischemic area and the position of the microdialysis probe were checked using the enzyme histotopochemical acid phosphatase reaction. The probe was always located in the cortex inside the stained area. Ascorbic acid levels rose immediately after MCAO and remained at about 12-fold for 3 h. There was a transient release of glutathione during 1-1.5 h. Uric acid concentrations were also increased but the differences did not reach significance. The levels of the amino acids tyrosine and tryptophan increased steadily after MCAO. The increases in cysteine were variable but significant. In some experiments, the pH of the dialysate was measured online. The parameters ascorbic acid, glutathione, cysteine, and pH are suitable for the early detection of cortical ischemic events by microdialysis.

We describe an improved high-performance liquid chromatographic method for the determination of tyrosine, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, tryptophan and homovanillic acid in cerebrospinal fluid and nerve tissue, using the new microbore cartridges with 5 micron average particle size. The first four substances are quantified fluorometrically and the last two electrochemically. Both detectors are connected to the same integrator through a relay which can be switched as required. Data are collected in an on-line personal computer and evaluated statistically. An improvement in the method for extraction and separation of catecholamines is also reported.

Blood platelets and brain membranes of SIV and Lewis rats both exhibited two saturable binding sites for 5-hydroxytryptamine (5-HT) in the concentration range 1-100 nM. The Kd values for the high-affinity sites were 4-6 nM and for the low-affinity sites 20-40 nM in both tissues of both rat strains. Blood platelets had 100-200 times more binding sites per mg protein (Bmax) than brain membranes. Thus, 5-HT receptors of platelets may be used as models for those of cerebral 5-HT-neurons.

Experimental autoimmune encephalomyelitis influenced catecholaminergic and indoleaminergic neurotransmitter systems in the central nervous system of Lewis rats. During paralysis, serotonin and noradrenaline were significantly reduced compared to animals injected with complete Freund's adjuvant in the posterior dorsomedial brainstem and in lower spinal cord segments. These diminutions remained after recovery from neurological signs in T11-S1. The serotonin metabolite 5-hydroxyindoleacetic acid was greatly augmented during the attack in all segments but was depleted during recovery in the lumbar spinal cord, which may indicate a normalized turnover at a reduced serotonin level. These results suggest functional impairment of monoaminergic neurons of the brainstem and spinal cord followed by permanent damage to some monoaminergic fibers in the spinal cord.

Three aspects of the pathophysiology of experimental autoimmune encephalomyelitis (EAE) are discussed: firstly, the possible electrophysiological effects in the CNS of myelin basic protein, which is released during demyelination; secondly, the partial degeneration of monoaminergic and glutamatergic neurons which occurs during an attack of EAE in addition to demyelination; thirdly, the importance of ischemic events, accompanied by free radical release, in EAE. Especially the third aspect could have therapeutic implications. Treatment with radical scavengers, N-methyl-D-aspartate receptor blockers, or calcium blockers (as suggested for ischemia) might prove effective for EAE. Our present aim is to investigate whether these results are also relevant for MS, for which EAE is an animal model.

Myelin basic protein (MBP) isolated from bovine spinal cord caused a marked shape change reaction of human blood platelets which was not accompanied by the release reaction and not inhibited by methysergide and spiroperidol. Only those basic proteins, including MBP, which had previously shown to exert neuronal depolarisation also induced the shape change reactions. Therefore, these findings may extend the use of platelets as neuronal models.

Bath application of 10(-5) M myelin basic protein (MBP) to various types of cultured nerve cells resulted in a membrane depolarization amounting to a change of 41 +/- 15 mV. Excitability could be restored by repolarizing the membrane by means of current injection through the recording electrode. The action of MBP persisted in the presence of tetrodotoxin (TTX), Co2+ or D-600 as well as in low Na+ and low Cl- solutions, whereas it was abolished by increasing extracellular Ca2+ concentrations. The action MBP was mimicked by ouabain. We propose that the effect of the protein might be generated by blockade of an ion pump. It is speculated that MBP may exert a direct effect on neuronal membranes in demyelinating diseases.

The water-soluble scavengers ascorbic acid (Asc), cysteine (Cys), glutathione (GSH) and uric acid (UA) as well as DNA content were determined in 40 breast tissue samples (neoplastic and non-neoplastic tissues from 20 patients). To allow proper homogenization to take place, a fixed number of sections was cut from a tissue cylinder of known diameter. Adjacent sections were stained with hematoxylin-eosin and the fractions of epithelium, fat and connective tissue were estimated as a percentage of the section area. Protein-free extracts were injected into a reverse-phase high-pressure liquid chromatography system and scavengers quantified with 2 electrochemical detectors (gold and glassy carbon). DNA and all scavengers, except UA, were greatly increased in cancer tissues in nearly all cases. Amounts of Asc and GSH in neoplastic tissue correlated closely with DNA values and percentage of epithelium, those of Cys not so closely and those of UA not at all. We assume that Asc and GSH were located mainly in the epithelium, UA mainly in the extracellular space and Cys in both spaces. When values were expressed as mumol/g DNA, a parameter related to content per cell, values were higher in neoplastic than in non-neoplastic tissue for Asc (18/20 cases), GSH (17/20) and Cys (14/20) and lower in neoplastic tissue for UA (19/20). It is known that increased GSH protects cells against certain drugs in tissue cultures. For in vivo treatment the presence of increased Asc (and to a lesser extent Cys) in addition to GSH could be of importance.

Pretreatment with zinc aspartate increased the survival rate of mice poisoned with a lyophilisate from the mushroom Amanita phalloides (APL). In the livers of the zinc-treated animals, confluent necrosis was cleared earlier. Zinc also prevented the reduction of brain noradrenaline caused by the mushroom as well as the rise of brain dopamine and brain weight. D-Penicillamine also protected against the lethal effects of APL. The survival afforded by zinc pretreatment was marginally ameliorated by additional oxygenation.

Rat cerebral cortex slices were incubated with 1muM [3H]4-aminobutyrate or [3H]L-glutamine. The subcellular distribution of the accumulated labelled substances were determined by fractionating the nuclei-free homogenates on a 5-step discontinuous sucrose density gradient in a B XIV zonal rotor. The gradient was designed to separate the synaptosomes into 3 subpopulations of increasing density. The patterns of distribution of [3H]4-aminobutyrate and [3H-a1L-glutamate in the three synaptosomal peaks were distinctly different. This indicates the presence of separate types of nerve ending accumulating these two potential neurotransmitters, which are known to be metabolically closely linked through the enzyme glutamate decarboxylase. Recentrifugation of the 3 synaptosomal peaks on flat step gradients in a swingout rotor did not result in any further enrichment in transmitter-specific synaptosomes.

Diurnal cycles for 8 ligand receptor pairs and choline acetyltransferase (ChAT) activity in 3 brain regions differed markedly in two rat lines, both of Wistar origin. Statistically significant differences between diurnal cycles in the two rat lines were found in the following parameters: 24 h means in 6 of 11 measurements, magnitude of cycle amplitudes, and phase position in 6 of 11 measurements, up to complete reversal of the acrophases in the case of ChAT activity in hippocampus. The importance of these findings--such major differences in two closely related rat lines--is obvious in any attempt to compare receptor-binding studies per se between laboratories using the same strain but not line, in studies of receptor rhythm characteristics, and in particular, for analysing the effects of brain-reactive drugs. While there are some reports on strain-dependency of cyclic functions, we are not aware that line-dependency has previously been described.

We investigated whether the development of sympathetic innervation of the spleen is affected by lymphoid cells. Splenic noradrenaline (NA) levels of athymic nude mice (nu/nu) and normal thymus-bearing littermates (nu/+) were determined at different times during ontogeny. While no differences were detected at birth, higher splenic NA levels were found in 7-, 11-, and 21-day-old athymic mice. Thymus transplantation or thymocyte injection to newborn nude mice resulted in splenic NA levels comparable to those of normal nu/+ mice. Histochemical studies fully confirmed such differences. Taken together with previous studies, these results suggest that T lymphocytes or their products exert an inhibitory influence on sympathetic nerve fibers, thus leading to decreased NA content in the spleen. The data also illustrate the capacity of a nonneuronal cell in a peripheral organ to affect the process of autonomic innervation of this organ.

We investigated the effects of chronic relapsing experimental allergic encephalomyelitis (first attack and first recovery) on uric acid (UR) concentrations in Lewis rat spinal cord. Perchloric extracts of 9 regions between spinal cord segments C3 and Co1 were injected into a reversed-phase high-performance liquid chromatographic system, and UR was quantified electrochemically. In all regions UR, which was lower than 0.5 micrograms/g wet tissue in rats injected with adjuvant (controls), increased to a variable extent during the attack (5-12 micrograms/g, maximum in T11-L2) and returned during recovery to a residual level of 1-2 micrograms/g. These findings can be partly attributed to blood-brain barrier damage.

Interleukin-1 (IL-1) is a hormone that, apart from playing a key role in immune and inflammatory processes, can also affect mechanisms under brain control. To gain a better understanding of the action of this cytokine on the CNS, its effects on the contents of norepinephrine (NE), dopamine (DA) and serotonin (5-HT), and their main metabolites and precursors, were evaluated in different regions of the forebrain, brain stem, and spinal cord. Following administration of human recombinant IL-1 (beta form) to rats, a modest decrease in the content of NE was observed in the hypothalamus as well as in the dorsal posterior brain stem. However, the most relevant finding was that 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), the main NE metabolite, and the relation MHPG/NE were increased in all the regions studied, revealing a stimulatory effect of IL-1 on NE metabolism in the CNS. This effect seems to be specific for NE since no comparable changes in the brain content of DA, 5-HT, or its metabolite, 5-hydroxyindole acetic acid, were detected after administration of the cytokine. However, tryptophan was significantly increased in all brain regions and in the cervical spinal cord. The capacity of IL-1 to affect the metabolism of NE, a neurotransmitter involved in the control of a variety of brain functions, provides further proof for the relevance of this cytokine in brain-immune interactions.

The specificity of I125-MBP uptake and subcellular distribution on a discontinuous sucrose density gradient, compared to those of histone H 4 and cytochrome c, showed a high affinity of MBP for mitochondria and heavy synaptosomes, and of histone H 4 for lighter synaptosomes. One heavier synaptosomal subpopulation was almost equally labelled by both proteins. Cytochrome c showed only a low uptake into particular material. Receptor interaction studies of MBP with H3-labelled 5-hydroxytryptamine and naloxone gave negative results.

Both bovine and human myelin basic protein (MBP) have been shown to have electrophysiological activity. As MBP is susceptible to proteolytic degradation, our aim was to discover whether the resulting peptides retained this activity. Bovine MBP was completely cleaved by plasmin into at least nine peptides. The electrophysiological activities of this peptide mixture and of bovine MBP were directly compared on the hemisected frog spinal cord. The peptide mixture and intact bovine MBP had quantitatively and qualitatively similar effects (dose-dependent long-lasting depolarization, about 100 times more active than glutamate). Four peptides (molecular weights 14,000, 10,500, 8,000, 4,500) from thrombin or cathepsin D cleavage of bovine MBP also showed electrophysiological activity, positively correlated to their molecular weights. As MBP-like material occurs in increased concentrations in the cerebrospinal fluid during demyelinating diseases, peptides resulting from proteolytic degradation of MBP, e.g. in demyelinating foci of multiple sclerosis, might cause neuronal disturbances.

The effects of myelin basic protein (MBP) on the ventral root response in rat spinal cord in situ and on the spontaneous bioelectric activity of cultured rat Purkinje cells were investigated. Injection of 2 or 4 microl MBP (10(-4) M/l) into the motoneurone pool reduced the ventral root response to dorsal root stimulation. The depression was long-lasting (30-50 min) and partially reversible. At 5 x 10(-6) M/l MBP abolished the spontaneous activity of most cultured Purkinje cells, an effect which was not blocked by haloperidol, propranolol, phenoxybenzamine and bicuculline. The EC(50) for the depression was 4 + or - 1 x 10(-6) M/l.

Serum from patients with multiple sclerosis (MS) and other diseases was added to the medium perfusing isolated hemisected frog spinal cord, and the effect on ventral root responses (VRR) tested. As control, a standardized commercial human serum (Moni-trol I) was used. In 25 out of 40 spinal cord preparations Moni-trol gave inhibition of VRR ranging from 2.8 to 23.1%. 76% of the tests with confirmed MS sera showed inhibition of VRR, after deduction of the control serum effect, while 53% of the tests with sera from other diseases were inhibitory. Sera from cases of suspected but clinically unconfirmed MS gave strong inhibition (above 20%).

Ascorbic acid, cysteine, glutathione and uric acid were determined by reverse-phase high-pressure liquid chromatography (HPLC) in 46 breast tissue samples [neoplastic (C) and non-neoplastic (N) from the same patient]. Cholesterol, alpha-tocopherol and gamma-tocopherol were quantified in 64 similar samples by extraction into heptane followed by direct-phase HPLC. DNA was measured in all samples and the percentages of epithelium, fat and connective tissue were estimated in sections adjacent to the sample. Results confirm previous findings that ascorbic acid and glutathione, expressed as mumol/g DNA, were greatly increased in the epithelium of neoplastic tissue. Similar increases in cysteine could be accounted for by the presence of inflammatory cells. Although values of alpha- and gamma-tocopherol correlated with the percentage of fat in both types of tissue, these compounds were also present in the epithelium. Because of the varying amounts of fat in the samples, no significant difference could be found between N and C values. Cholesterol correlated with fat in N and epithelium in C. Consideration of 10 cases with equal amounts of fat in C and N tissue suggests that cholesterol is reduced in C in the epithelial cells.

Binding in 14 ligand membrane receptor pairs and choline acetyltransferase activity were studied at 4-hour intervals during a 24-hour cycle (12:12 light:dark). Cortex, hippocampus, cerebellum, striatum and brainstem were prepared from 3-, 12- and 24-month-old male rats. Cholinergic, alpha- and beta-adrenergic, dopaminergic, serotonergic, gabaergic and opiate binding were determined. In the aged animals, the times of the binding maxima are no longer locked to the same time of the light:dark cycle, and the cycle phases themselves are shifted in comparison to those of the young and adult group. Cycle amplitudes were smallest in the 12-month-old rats, increasing significantly in the 24-month group. The age changes in the overall (24-hour) means fall into 4 different patterns, none of which shows a linear age-related decrease. This points out the great importance of including an adult group (12 months) in all ageing studies on small mammals.

By combining tissue pO2 measurements with morphological visualization and counting of perfused capillaries, early microvascular changes were investigated in paralysis of skeletal muscle of the rat. The models included experimental allergic encephalomyelitis (EAE), experimental allergic neuritis (EAN), 6-aminonicotinamide intoxication, and peripheral denervation. The results showed that in all models tissue oxygenation was either normal or elevated. In most cases this was accompanied by a reduced perfusion of the capillary bed, supporting the hypothesis of a dysfunction in oxidative metabolism. The effects of paralysis on capillary perfusion were more marked in red than in white muscles, except in spastic paresis. Flaccid paralysis had more deleterious effects on muscle microcirculation than spastic paralysis. Our data suggest that: (1) microvascular changes occur very early after the onset of paralysis and (2) later developing changes in the skeletal muscle such as atrophy cannot be ascribed to a lack of oxygen supply but rather, at least in part, to a dysfunction in oxidative metabolism.

Binding of 3H-labeled agonists and antagonists to muscarinic-cholinergic, alpha- and beta-adrenergic, dopaminergic, serotoninergic, and opiate receptors was studied in four regions of the neocortex and in hippocampus, thalamus, putamen, and caudatus in autoptic material from patients with senile dementia of Alzheimer type and of mixed vascular-Alzheimer pathogenesis. Different patterns of changes in ligand binding were found for the two groups. Some of these changes were quantitatively correlated with the histological scores of plaques and neurofibrillary tangles.

An automated high-performance liquid chromatographic method with electrochemical and fluorimetric detection and on-line data evaluation is described for the simultaneous measurement of indoleaminergic and catecholaminergic neurotransmitters, some of their metabolites and precursors and ascorbic and uric acids. Deproteinized tissue extracts from the central nervous system or peripheral organs are injected without prior purification (recovery greater than 90%). A switching system enables the compounds to be passed as necessary through one, two or three reversed-phase columns, which are then eluted simultaneously (analysis time 25 min). Fifty samples per day can be analysed with a precision of 95% for neurotransmitters and about 90% for ascorbic and uric acids.

Antioxidants (ascorbic acid, glutathione, cysteine, alpha-tocopherol) and uric acid were measured using two high-pressure liquid chromatographic methods in 3 regions (cervical, thoracic, lumbar) of the spinal cord and in blood of Lewis rats during the attack and recovery of experimental autoimmune encephalomyelitis (EAE). Uric acid, which is thought to be a marker of free radical release, was greatly increased and glutathione correspondingly decreased in lumbar and thoracic regions. Cysteine and ascorbic acid were practically unchanged, whereas alpha-tocopherol was significantly increased during attack and recovery. Results, which could have therapeutic implications, generally support the hypothesis that free radicals are released during EAE.

Basic proteins and polypeptides (BPP) such as myelin basic protein (MBP), polyornithine (M.W. 40,000), polylysine and protamine, which are known to cause neuronal depolarization in the central nervous system, induced a shape change reaction in blood platelets of various species, including man. This reaction was not accompanied by platelet aggregation or marked alterations of 5-hydroxytryptamine release. Cyclic nucleotide levels were also unchanged. The shape change induced by polyornithine was inhibited by heparin but not by antagonists of 5HT, catecholamines or gamma-aminobutryic acid, substances which are known to have no effect on the MBP-induced neuronal depolarization. Other basic substances, e.g. low molecular weight polyornithine (M.W. 4,000), cytochrome c, spermine and spermidine, did not induce either platelet shape change or (as shown before) neuronal depolarization. It is concluded, that 1) the shape change reaction of platelets seems to be a sensitive and simple means of detecting those BPP which induce functional changes in mammalian cells and 2) the use of platelets as models for neurons can be extended to include the action of BPP on the plasma membranes.

The effects of experimental autoimmune encephalomyelitis (attack and recovery) on levels of six amino acids have been investigated in nine regions of the Lewis rat spinal cord between segments C3 and Co1 and in the brainstem. Amino acids were analyzed by separation of their 4'-dimethylaminoazobenzene-4-sulfonyl chloride derivatives on a reversed-phase column using a ternary gradient. Glutamate and gamma-aminobutyric acid were reduced by 10-30% in all segments during the attack, whereas taurine, lysine, glutamine, and glycine were all greatly increased (up to 300%). Most values except those of taurine, as well as glutamate in certain segments, returned to normal on recovery. Because some of these compounds have neurotransmitter function, these changes may contribute to the neurological symptoms of experimental autoimmune encephalomyelitis.

The effects of human myelin basic protein (HMBP) and its individual microheterogeneous components on the electrical activity of the frog spinal cord are compared with those of bovine MBP (BMBP) and glutamate (Glu). HMBP causes a dose-dependent depolarization of the ventral (VR) and dorsal (DR) roots with a reduction of the evoked root potentials. The amplitude of the depolarization induced by 0.01 mM HMBP corresponds to that observed with application of 1 mM Glu. A weaker depolarization persists during blockade of synaptic transmission by MgSO4 or tetrodotoxin, suggesting a direct depolarizing effect on motoneurones and primary afferents. HMBP causes a long-lasting tachyphylaxis. The individual microheterogeneous components of HMBP (peaks 1, 2 and 3), and a mixture of smaller myelin basic proteins elicit similar responses, whereas BMBP and its microheterogeneous components are slightly less active.

The uptake of [3H]glycine and [3H]L-glutamate into slices of the dorsal and ventral halves of rat spinal cord taken from the thoracal (T3-9) and lumbar (L1-6) regions was determined in the concentration range 2 - 1 000muM. For both amino acids and in all four tissue pieces (thoracal dorsal and ventral, lumbar dorsal and ventral) biphasic linear regressions were obtained from the Lineweaver-Burk plots, demonstrating saturable transport mechanisms both in the high-affinity and in the low-affinity range. No significant dorsal-ventral or lumbar-thoracal differences were found for the transport constants Km of both amino acids. For the maximum velocity V similar dorsal-ventral and lumbar-thoracal gradients were found for both glycine and L-glutamate. These results, which are not entirely in agreement with the distribution of the endogenous pools of these amino acids, are critically discussed in view of the proposed function of high affinity uptake at the synapse.

Dry weight, total basic protein and myelin basic protein (MBP) in human corpus callosum tissue were found to be significantly reduced with age, confirming other reports. The heterogeneous patterns of the MBP samples were investigated and could be classified into three groups: a normal one with peak 1 larger than peak 3; an old-age pattern with peak 1 smaller than peak 3; and a pathological pattern with peak 1 greatly reduced. Because peak 1 is the only one which is significantly reduced with age, it is suggested that future investigations could be simplified. The physiological and pathophysiological role of MBP heterogeneity is discussed.

Catecholamine and indoleamine neurotransmitters, together with some of their precursors and metabolites, were determined using HPLC in three brain and two spinal cord regions of Lewis rats with chronic relapsing allergic encephalomyelitis and of control rats injected with complete Freund's adjuvant. Three attacks and two recovery phases were investigated. Changes are found mainly in the spinal cord. In the lumbosacral region both 5-hydroxytryptamine and noradrenaline are reduced during the entire course of the disease, whereas in the craniothoracal region 5-hydroxytryptamine is unchanged and only noradrenaline is reduced during the attacks, returning to normal during the first recovery. The precursors tyrosine and tryptophan are greatly elevated during the first two attacks in both regions. The 5-hydroxytryptamine turnover marker 5-hydroxyindoleacetic acid is increased in the first attack in both regions, then it decreases in the later stages, indicating destruction of nerve fibers. On the fourth and seventh days after inoculation values are generally not significantly different from controls in all regions. The possible correlation of neurochemical results with neurological signs is discussed.