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A proof of concept study has been conducted for the design of a porous biodegradable material containing nanocapsules and two actives with independent release-bimodal drug-eluting implants. Completely safe synthetic material free from risk of prion and virus contamination was tested in vivo, and a method for controlling the rate of biodegradation of poly-2-cyanoacrylic polymer was developed. Novel perfluorinated 2-cyanoacrylic esters have been applied for the chemical modification of polyethyl-2-cyanoacrlylate copolymers. Internal imide-cycle formation has been used to retard the rate of enzymatic hydrolysis of the 2-cyanoacrylic copolymer main chain.

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1. We have investigated the role of reactive oxygen species (ROS) in cell death induced by ischemia or application of the excitatory amino acid agonist, N-methyl-D-aspartate (NMDA) or kainate (KA), in acutely isolated rat cerebellar granule cell neurons, studied by flow cytometry. Various fluorescent dyes were used to monitor intracellular calcium concentration, ROS concentration, membrane potential, and viability in acutely dissociated neurons subjected to ischemia and reoxygenation alone, NMDA or kainate alone, and ischemia and reoxygenation plus NMDA or kainate. 2. With ischemia followed by reoxygenation, ROS concentrations rose slightly and there was only a modest increase in cell death after 60 min. 3. When NMDA or kainate alone was applied to the cells there was a large increase in ROS and in intracellular calcium concentration but only a small loss of cellular viability. However, when NMDA or kainate was applied during the reoxygenation period there was a large loss of viability, accompanied by membrane depolarization, but the elevations of ROS and intracellular calcium concentration were not greater than seen with the excitatory amino acids alone. 4. These observations indicate that other factors beyond ROS and intracellular calcium concentration contribute to cell death in cerebellar granule cell neurons.

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The effects of vitamin E on lipid peroxidation, intracellular free Ca2+ concentration ([Ca2+]i), and cell death were investigated in the postischemic immature cerebellum. Deprivation of oxygen and glucose for 10-min in a suspension of freshly dissociated granule cells from the cerebellum of 9-day-old male rat pups resulted in a recovery-induced consumption of cell nonenzymatic antioxidants (ascorbic acid, glutathione, and alpha-tocopherol) and development of membrane lipid peroxidation as measured by the thiobarbituric acid method. The rate of lipid peroxidation of the postischemic cells was stimulated, not reduced, by treatment of the cells with vitamin E (5-30 microM alpha-tocopherol phosphate). In flow-cytometric studies a 10-min period of ischemia resulted in a small increase in intracellular calcium concentration, lipid peroxidation products and cell death, but in the presence of alpha-tocopherol the same treatment caused a dramatic increase in cell death, accompanied by a large increase in [Ca2+]i and lipid peroxidation products. Pretreatment of the cells with a mixture of three antioxidants (vitamin C/rutin/ubiquinol-10, 10/5/1) or nickel (Ni2+) reduced the alpha-tocopherol-induced increases in [Ca2+]i, and cell death. Hydrogen peroxide (1 mM) and the water-soluble analogue of vitamin E, trolox (50 microM), mimicked the effect of vitamin E on lipid peroxidation in the postischemic cells. Pretreatment of the cells with the intracellular Ca2+ chelator BAPTA-AM, reduced both the alpha-tocopherol-induced increase in [Ca2+]i and cell death. The effect of vitamin E on [Ca2+]i was age dependent and decreased abruptly during maturation of the cerebellum between the first and second weeks of life. Results of in vitro treatment of the immature cerebellar cells with the water-soluble form of vitamin E (alpha-tocopherol phosphate) suggest that, after consumption of cellular co-antioxidants, vitamin E may be converted to an alpha-tocopheroxyl radical, which act as a toxic prooxidant as cellular bioenergetics deteriorate.

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We have measured the transendothelial electrical resistance across the blood-brain barrier (BBB) with a microelectrode technique and determined the effects of subcutaneous injections (five injections over ten days) of lipopolysaccharide (LPS, 100 ng/g), recombinant mouse interleukin-6 (IL-6, 5 ng/g), and/or inorganic lead (lead, 2.5 5 micrograms/g) on the ion permeability of arterioles in the temporoparietal cortex of anaesthetized mice between 10 and 40 days of age. In controls the electrical resistance increased with age. It was decreased in animals treated with IL-6, but unaffected by lead at the different ages studied. In IL-6 treated mice, repeated neonatal exposure to lead (five injections between 2 and 10 days after birth) caused a delay in the increase in arteriole resistance with age. LPS injections caused a 36% increase in ion permeability of the BBB in twenty-day-old mice, and lead potentiated this effect of LPS. Intra-arterial injections of glutamate did not alter vascular resistance, but topical applications of glutamate on the cerebrum caused a reversible decrease in the resistance in mice not treated with lead, and an irreversible decrease in mice treated with lead. Injections of glutamate in the lumen of arterial vessels in the parietal and temporoparietal brain areas of mice pretreated with lead and LPS, plus a topical application of glutamate, caused depolarization of neurons in the temporoparietal cortex. These results suggest that disruption of the BBB can allow serum glutamate to penetrate the brain, causing further disruption of the BBB, and that lead irreversibly potentiates this cascade of harmful events.

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The distribution of intracellular ionized lead (Pb) and calcium in dissociated cerebellar cells of ten-day-old mice was measured by flow cytometry. There are no fluorescent probes specific for lead, whereas commonly used fluorescent calcium indicators bind heavy metals with greater affinity than they do calcium, which impedes discrimination of lead- and calcium-induced fluorescence changes. Therefore, we developed a method to determine [Pb2+]i and [Ca2+]i by employing a combination of the calcium indicator fluo-3 and the heavy-metal chelator TPEN. Using these methods, we studied the effects of multiple in vivo exposure (five subcutaneous injections over 10 days) to lipopolysaccharide (LPS, 100 ng/g), recombinant mouse interleukin-6 (IL-6, 5 ng/g) and/or inorganic lead (lead, 2.5 micrograms/g) on lead and calcium concentrations. Control cells had [Cai] of 112 nM. Lead exposure alone had little effect on [Ca2+]i and resulted in a mean [Pb2+]i of about 7 pM, and did not alter cell volume. A significant fraction of cells (about 44% of living cells) from animals treated with lead plus LPS were swollen, as determined by analysis of the light scattering pattern, and there was a small increase in the number of dead cells, identified with the nucleic acid stain, 7-aminoactinomycin. While [Ca2+]i was not significantly increased in animals treated with either only LPS or IL-6, lead and calcium concentrations were increased in animals exposed to lead and LPS or IL-6 in both the non-swollen and swollen cells, with a mean value of (Pb2+)i of 32 pM and (Ca2+)i of 155 nM in cells not swollen. Electrophysiological analysis showed that LPS injections caused decreases in the membrane potential of endothelial cells of the blood-brain barrier (BBB) and lead potentiated the effect of LPS. IL-6 mimicked the effects of LPS, but was less potent. Thus these experiments indicate a synergistic interaction between lead and cytokines on biophysical properties of both neurons and endothelial cells of the BBB.

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The effect of kainate on the voltage-activated current of cultured embryonic chick telencephalic neurones was studied by whole-cell voltage clamp recording. In addition to opening non-NMDA receptor coupled ion channels, kainate produced additional effects, with a slower time course: it modulated voltage-activated currents. These effects were blocked by the non-NMDA receptor antagonists CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and GYKI 53784 ((-)-1-(4-aminophenyl)-3- methylcarbamoyl-4-methyl-7,8-methylenedioxy-3,4- dihydro-5H-2,3-benzodiazepine), indicating that non NMDA receptors are involved. It has been shown that the reversible inhibition of outward potassium currents was partly due to a decrease of current amplitude and partly to a marked shift of the inactivation curve towards more negative potentials.

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The effect of HgCl2 on kainate (KA)-induced depression of voltage-gated potassium (K+) current in chick embryo telencephalic cells was studied using conventional and nystatin-perforated whole-cell patch-clamp recordings, fluorescence imaging, and flow cytometry techniques. Hg2+ (1 microM) alone did not effect the 4-aminopyridine-(4-AP)-sensitive transient K+ current in immature cells (Embryonic Day 5), but irreversibly potentiated the depressant effect of KA on this K+ current. A 50% potentiation of KA-induced depression of the K+ current was produced by an application of 0.19 microM Hg2+. Application of ionomycin (5 microM) or calcium ionophore A23187 (2 microM) suppressed the K+ current. To test the possibility that the 4-AP-sensitive transient K+ current is a Ca-inactivated current, the effect of intracellular Ca2+ concentration ([Ca2+]i) in the range of 30 nM to 2 microM was determined. The amplitude of the K+ current was sensitive to [Ca2+]i with half-maximal inactivation at 370 nm at +60 mV. The concentration-response curve of the K+ current inhibition by [Ca2+]i was shifted to lower [Ca2+]i and the slope of the curve was reduced in the presence of KA. Hg2+ potentiated these effects of KA. The Ca-dependence of the K+ current was maximal at the 5th embryonic day, declined to the 9th embryonic day, and was absent at the 11th embryonic day. Application of Hg2+ (0.1-1 microM) had no effect on the basal [Ca2+]i of freshly dissociated cells (10th day in ovo) and cells in culture (the 4-day cultures from the telencephalon of 5-day-old embryos), but potentiated KA-induced increase of [Ca2+]i in a Ca-free-EGTA solution in a concentration-dependent manner. Moreover 1 microM Hg2+ delayed and reduced the recovery to basal [Ca2+]i after washout of KA. Exposure to 5-30 microM H2+ caused an irreversible decline of membrane resistance, an increased cell size, and reduced cell granularity and complexity. Intracellular recording of spontaneous neuronal activity and immunocytochemical identification showed that the KA/Hg2+-sensitive Ca-inactivated K+ current exists in early differentiating telencephalic neurons. Because depression of the K+ current by KA and Hg2+ decreases the interspike interval and irreversibly perturbs the frequency code of information in the nervous system, the expression of this current during early neuroembryogenesis may be one of the reasons for the developmental toxicity of inorganic mercury.

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Role of cytoplasmic concentration of free Ca2+ ([Ca]in) and arachidonic acid in potentiating the effect of vitamin E (DL-alpha-tocopherol) on acetylcholine receptor activity in Helix pomatia neurons was studied using a two-microelectrode intracellular recording, voltage clamp and fluorescent calcium probe fura-2 technique. Elevation of [Ca]in by intracellular injection from a microelectrode or by depolarizing pulses and application of 0.1 microM-0.1 mM vitamin E enhanced the acetylcholine-induced chloride current both in LP11 and RBc4 neurons. Application of 10 microM arachidonic acid to the same neurons decreased this current. The use of fluorescent probe showed that vitamin E did not essentially change [Ca]in, but an increase of [Ca]in intensified both the enhancing effect of vitamin E and the depressing effect of arachidonic acid. The enhancing effect of calcium influx was considerably decreased after vitamin E application. The antioxidant action of vitamin E was probably not involved in the mechanism of its enhancing effect on acetylcholine-induced current, since synthetic antioxidant, ionol, depressed acetylcholine responses. A spectrum analysis has shown the interaction between vitamin E and arachidonic acid in solution. This interaction may be considered as the molecular mechanism responsible for the prevention by vitamin E of steady arachidonic acid production from membrane phospholipids and its down-regulatory effect on acetylcholine receptor activity. Our results support this suggestion, since an inhibitor of phospholipase A2, 4-bromophenacyl bromide, mimicked the enhancing effect of vitamin E.

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The effect of ultrasound (2.64 MHz, 0.5 W/cm2) on acetylcholine-induced (ACh-induced) current and surface distribution of ACh receptors (AChRs) were studied in neurons of the mollusc Helix pomatia. Upon switching on the ultrasound a negligibly small transient two-phase transmembrane current appeared; prolonged (5-25 min) action of beamed ultrasound waves significantly depressed the ACh-induced chloride current and caused the disappearance of functional nicotinic AChRs on parts of the neuronal soma distant from the axon. Pharmacological studies showed that the disappeared AChRs were responsible for changes in membrane permeability for chloride ions (AChRsCl). The results obtained in the present study indicate that ultrasound may be used as a selective inhibitor of AChRsCl in molluscan neurons.

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1. Intracellular recordings were made from identified LP11, RBc4, D1 and E4 neurons in perioesophageal ganglionic ring with buccal ganglia of the mollusc Helix pomatia. 2. The modulations of acetylcholine (ACh)-induced current by vitamin E in these neurons were investigated using two-microelectrode intracellular recording and voltage-clamp techniques. 3. ACh receptors function on LP11 and RBc4 neurons was strongly regulated by intracellular calcium ions. For these ACh receptors application of 10(-6) to 10(-4) M vitamin E and calcium influx both induced an enhancement of the ACh-induced chloride current. Application of 10(-5) to 5.10(-5) M arachidonic acid on the same identified LP11 and RBc4 neurons was shown to evoke a decrease of the ACh-induced chloride current. 4. The elevation of calcium levels into D1 and E4 neurons induced a faint decrease of ACh-induced chloride current, but vitamin E and arachidonic acid were ineffective. 5. The calmodulin inhibitor, chloropromazine (6.10(-5) M), strongly inhibited the enhancing effect of calcium influx on ACh-induced chloride current in LP11 and RBc4 neurons, but it had a weak influence on the effect of vitamin E. 6. The effect of vitamin E on surface distribution of functional ACh receptors in LP11 and RBc4 neurons was found. 7. Application of 10(-4) to 10(-6) M vitamin E (DL-alpha-tocopherol) triggered mechanisms, which after a 5 to 45-min period lead to appearance of functional ACh receptors on the parts of neuronal soma, which were further from the axon. 8. Arachidonic acid (vitamin F) evoked a disappearance of functional ACh receptors, which were activated by vitamin E.

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1. Intracellular recordings were made from identified and non-identified neurons in perioesophageal ganglionic ring with buccal ganglia of the mollusc, Helix pomatia. The influence of oxytocin (OXT) on neuronal integration: space and temporal summations of postsynaptic potentials (PSPs) in various neurons was investigated. The obtained data indicated that these PSPs were cholinergic PSPs. 2. Ten minute exposure to 10(-8) M OXT had no effects on the resting membranes, but triggered secondary mechanisms, which lead to enhancement of the excitatory PSP (EPSR) amplitudes and the decrease of the decay time constant (tau EPSR) obtained from the falling phase of the EPSP. 3. The enhancement of the EPSP amplitude and the decrease of tau EPSP after OXT application evoked the appearance of action potential under space summation of two spontaneous EPSPs and made easier the appearance of action potential under temporal summation of EPSPs produced by paired afferent stimuli, when the corresponding interstimuli interval was smaller than tau EPSP in the presence of OXT. 4. Ten minute exposure to 10(-8) M OXT made the integrated amplitude of the excitatory acetylcholine response and the inhibitory dopamine response in the neuron E5 more positive only when the interval between applications of these mediators was smaller than the time constant of desensitization of acetylcholine receptors in the presence of OXT. 5. The pharmacological studies showed that drugs, which elevate intracellular cyclic AMP levels, mimicked the influence of OXT on integration of PSPs in the investigated neurons.

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It was found that 10(-7)-10(-8) mol/l oxytocin (OT) or arginine-vasopressin (AVP) applications produced effects on functional properties of three types of acetylcholine (ACh) receptors on various neurons identified in the ganglia of Helix pomatia under voltage clamp conditions. OT and AVP depressed ACh-induced sodium-potassium-calcium current in neuron RBc3 without shift of reversal potential. Our data show that there are two types (subtypes) of ACh receptors which are connected with chloride current in neurons of Helix pomatia. OT decreased ACh-induced chloride current in neuron D4 and enhanced ACh-induced chloride current in neuron D5. These effects of OT were mimicked by the intracellular injection of cyclic AMP or application of isobutylmethylxanthine and an active cyclic AMP analog. AVP as a rule mimicked the effects of OT on functional properties of ACh receptors, but in neuron F8 effects of OT and AVP were independent. The present results suggest that cyclic AMP may be the second messenger mediating the OT- and AVP-induced modulations of functional properties of three types of ACh-receptors.

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