Thrombin inhibitory activity of some polyphenolic compounds.

Thrombin, also known as an active plasma coagulation factor II, belongs to the family of serine proteases and plays a crucial role in blood coagulation process. The process of thrombin generation is the central event of the hemostatic process and regulates blood coagulant activity. For this reason, thrombin inhibition is key to successful novel antithrombotic pharmacotherapy. The aim of our present study was to examine the effects of the well-known polyphenolic compounds on the activity of thrombin, by characterization of its interaction with selected polyphenols using different biochemical methods and biosensor BIAcore analyses. Only six compounds, cyanidin, quercetin, silybin, cyanin, (+)-catechin and (-)-epicatechin, of all examined in this study polyphenols caused the inhibition of thrombin amidolytic activity. But only three of the six compounds (cyanidin, quercetin and silybin) changed thrombin proteolytic activity. BIAcore analyses demonstrated that cyanidin and quercetin caused a strong response in the interaction with immobilized thrombin, while cyanin and (-)-epicatechin induced a low response. Lineweaver-Burk curves show that used polyphenol aglycones act as competitive thrombin inhibitors. Our results suggest that polyphenolic compounds might be potential structural bases and source to find and project nature-based, safe, orally bioavailable direct thrombin inhibitors.

Recrystallization of plane strain compressed Al-1 wt.% Mn alloy single crystals of typical unstable orientations.

A systematic study of crystal lattice reorientation in early stages of recrystallization has been carried out to correlate the orientations of recrystallization nuclei with the deformation microtexture and with slip systems. Microstructure and texture of Al-1 wt.% Mn single crystals of unstable initial orientations of {112}111, {100}001 and {001}110 have been examined by high-resolution field-emission gun scanning electron microscope local orientation measurements. All single crystals were channel-die deformed at room temperature and then annealed for a short time. It was shown that often observed presence of the 112 directions as rotation axes in the formation of new nuclei orientation directly suggested a close link with the deformation process.

Atrophy, but not necrosis, in rabbit skeletal muscle denervated for periods up to one year.

Our understanding of the effects of long-term denervation on skeletal muscle is heavily influenced by an extensive literature based on the rat. We have studied physiological and morphological changes in an alternative model, the rabbit. In adult rabbits, tibialis anterior muscles were denervated unilaterally by selective section of motor branches of the common peroneal nerve and examined after 10, 36, or 51 wk. Denervation reduced muscle mass and cross-sectional area by 50-60% and tetanic force by 75%, with no apparent reduction in specific force (force per cross-sectional area of muscle fibers). The loss of mass was associated with atrophy of fast fibers and an increase in fibrous and adipose connective tissue; the diameter of slow fibers was preserved. Within fibers, electron microscopy revealed signs of ultrastructural disorganization of sarcomeres and tubular systems. This, rather than the observed transformation of fiber type from IIx to IIa, was probably responsible for the slow contractile speed of the muscles. The muscle groups denervated for 10, 36, or 51 wk showed no significant differences. At no stage was there any evidence of necrosis or regeneration, and the total number of fibers remained constant. These changes are in marked contrast to the necrotic degeneration and progressive decline in mass and force that have previously been found in long-term denervated rat muscles. The rabbit may be a better choice for a model of the effects of denervation in humans, at least up to 1 yr after lesion.

Scanning electron microscopy and transmission electron microscopy in situ studies of grain boundary migration in cold-deformed aluminium bicrystals.

The crystallography of recrystallization has been investigated in channel-die deformed pure aluminium bicrystals with {100}<011>/{110}<001> orientations. The microstructural and microtextural changes during the early stages of recrystallization were followed by systematic local orientation measurements using scanning and transmission electron microscopes. In particular, orientation mapping combined with in situ sample heating was used to investigate the formation and growth of new grains at very early stages of recrystallization. Grain boundary migration and 'consumption' of the as-deformed areas was always favoured along directions parallel to the traces of the {111} slip planes that had been most active during deformation.

Different receptor subtypes are involved in the serotonin-induced modulation of epileptiform activity in rat frontal cortex in vitro.

The frontal cortex is innervated by serotonergic terminals from the raphe nuclei and it expresses diverse 5-HT receptor subtypes. We investigated the effects of 5-HT and different 5-HT receptor subtype-selective agonists on spontaneous discharges which had developed in rat cortical slices perfused with a Mg2+-free medium and the GABA(A) receptor antagonist picrotoxin. The frequency of synchronous discharges, recorded extracellularly in superficial layers (II/III) of the frontal cortex, was dose-dependently enhanced by 5-HT (2.5-40 microM). That excitatory effect was blocked by the 5-HT2 receptor selective antagonist ketanserin. The 5-HT2A/2C receptor-selective agonist DOI and the 5-HT4 receptor agonist zacopride also increased the frequency of spontaneous discharges. In the presence of ketanserin, 5-HT decreased the discharge rate; a similar effect was observed when the 5-HT1A receptor agonist 8-OH-DPAT or the 5-HT1B receptor agonist CGS-12066B was applied. The 5-HT3 receptor agonist m-CPBG was ineffective. In conclusion, 5-HT produces multiple effects on epileptiform activity in the frontal cortex via activation of various 5-HT receptor subtypes. The excitatory action of 5-HT, which predominates, is mediated mainly by 5-HT2 receptors. The inhibitory effects can be attributed to activation of 5-HT1A and 5-HT1B receptors.

Functional and biological test of a 20 channel implantable stimulator in sheep in view of functional electrical stimulation walking for spinal cord injured persons.

A newly developed implantable stimulator with 20 output channels, mainly intended for the stimulation of lower extremities in paraplegics, was implanted in 6 sheep over a time period of 26 weeks. Five epineural electrodes each were used to contact various nerves at different locations to elicit hip and knee extension and flexion and to make carrousel and selective stimulation possible. Different electrode application strategies in view of paraplegic standing and walking were investigated. Additional implanted electrodes allowed M-wave monitoring for selectivity investigations in 3 sheep. Stimulator, electrode leads, and electrodes proved to be reliable. Selective stimulation with electrodes placed on the trunk of the sciatic nerve could be demonstrated but with bad reproducibility. Histological investigation of the tissues surrounding electrodes and leads showed the expected stable foreign body response. Strong hip and knee extension could be gained in all cases while only weak flexion forces could be elicited in most cases. Muscle biopsies showed that daily stimulation for 8 h at threshold level caused an increase in muscle Type I fibers and a decrease in Type IIc fibers. Implants and electrodes fulfill the most important functional and biological criteria for their clinical application for paraplegic walking. The intention to provide selective flexion functions via epineural stimulation could not be demonstrated sufficiently in this animal model.

Opposite effects of antidepressants and corticosterone on the sensitivity of hippocampal CA1 neurons to 5-HT1A and 5-HT4 receptor activation.

Using extracellular and intracellular ex vivo recording techniques we studied changes in the reactivity of hippocampal pyramidal CA1 neurons to serotonin (5-HT) and to the 5-HT1A- and 5-HT4 receptor agonists (+/-)-2-dipropylamino-8-hydroxy- 1,2,3 ,4-tetrahydronaphthalene hydrobromide (8-OH-DPAT) and zacopride, respectively, evoked by repeated electroconvulsive shock (ECS), imipramine and corticosterone treatments. Rats were subjected to ECS for 1 or 10 days, treated with imipramine for 1, 7, 14 or 21 days (10 mg/kg p.o., twice daily) and with corticosterone for 7 days (10 mg/kg s.c., twice daily). Hippocampal slices were prepared 2 days after the last treatment. Activation of 5-HT1A receptors decreased the amplitude of population spikes evoked by stimulation of the Schaffer/collateral-commissural pathway and hyperpolarized CA1 cells. Activation of 5-HT4 receptors increased the population spike amplitude and decreased the amplitude of slow afterhyperpolarization. Both repeated ECS and imipramine enhanced the effects related to 5-HT1A receptor activation and attenuated the effects of 5-HT4 receptor activation. The action of imipramine was significant after a 7-day treatment and reached a maximum after 14 daily applications, remaining at the same level in a group of animals treated for 21 days. Repeated corticosterone attenuated the inhibitory effect of 5-HT and 8-OH-DPAT on the population spike amplitude and enhanced the increase in population spike amplitude induced by zacopride. These findings indicate that antidepressant treatments and repeated corticosterone have opposite effects on hippocampal responsiveness to 5-HT1A and 5-HT4 receptor activation. In consequence, antidepressants enhance, whereas corticosterone reduces the 5-HT-mediated inhibition of hippocampal CA1 cells, which may be relevant to the antidepressant and pro-depressant effects of either treatment, respectively.

Pruritometer 2: portable recording system for the quantification of scratching as objective criterion for the pruritus.

Studies to evaluate therapies for itching (pruritus) related diseases often require the quantification of the itch sensation. Like all subjective symptoms the evaluation of itching is difficult and can only be done indirectly. With the Pruritometer 2 a measuring system is introduced that evaluates itching by detecting scratching movements. Based on the Pruritometer 1, that processes the signals of a piezoelectric vibration sensor, fixed on the midfinger of the patients dominant hand, and triggers a simple counter, the Pruritometer 2 allows to store the scratch activity during a 24 hours period. For each adjustable time slice of this time period, the amount of scratches and the scratch intensity are recorded. All data can be transferred to a PC via infrared link for further processing with a standard software package. An additional PC-software allows to set various parameters for optimal scratch detection and to test the patient attached system, also via the infrared link. All electronic components are shockproof encapsulated in a milled housing and are attached to a textile watchstrap that is worn by the patient like a wristwatch.

Basic design and construction of the Vienna FES implants: existing solutions and prospects for new generations of implants.

We can distinguish 3 generations of FES implants for activation of neural structures: 1. RF-powered implants with antenna displacement dependent stimulation amplitude; 2. RF-powered implants with stabilised stimulation amplitude; and 3. battery powered implants. In Vienna an 8-channel version of the second generation type has been applied clinically to mobilisation of paraplegics and phrenic pacing. A 20-channel implant of the second generation type for mobilisation of paraplegics and an 8-channel implant of the third generation type for cardiac assist have been tested in animal studies. A device of completely new design for direct stimulation of denervated muscles is being tested in animal studies. There is a limited choice of technologically suitable biocompatible and bioresistant materials for implants. The physical design has to be anatomically shaped without corners or edges. Electrical conductors carrying direct current (D.C.) have to be placed inside a hermetic metal case. The established sealing materials, silicone rubber and epoxy resin, do not provide hermeticity and should only embed DC-free components. For electrical connections outside the hermetic metal case welding is preferable to soldering; conductive adhesives should be avoided. It is advisable to use a hydrophobic oxide ceramic core for telemetry antenna coils embedded in sealing polymer. Cleaning of all components before sealing in resin is of the utmost importance as well as avoidance of rapid temperature changes during the curing process.

[Electrophysiologic tests for testing the effects of antidepressant drugs and corticosterone on reactivity of serotonin receptors in the hippocampus]. / Badania elektrofizjologiczne wplywu leków przeciwdepresyjnych i kortykosteronu na reaktywnosc receptorów serotoninowych w hipokampie.

Disturbances in the serotonin (5-HT) system and the limbic-hypothalamo-pituitary-adrenal axis (LHPA) have been implicated in the pathophysiology of depression. It is well established that hippocampus is a central component of limbic circuitry that participates in the modulation of cognition, mood and behavior, and is involved in the control of the LHPA axis. Therefore, the hippocampus provides a unique environment to study the interplay between serotonergic system, antidepressants and corticosteroids. Activity of hippocampal cells can be modulated by 5-HT via inhibitory 5-HT1A and excitatory 5-HT4 receptors. Repeated treatment with antidepressants increases the responsiveness of hippocampal pyramidal neurons to the 5-HT1A and attenuates the responsiveness to the 5-HT4 receptor agonists, with a time course which correlates with the delayed onset of the therapeutic effect of antidepressants in humans. Moreover, repeated corticosterone, which may constitute a model of a prolonged nonadaptable stress, has opposite effect on hippocampal responsiveness to the 5-HT1A and 5-HT4 receptor activation. Such an action results in an enhancement of the 5-HT-mediated inhibition by antidepressants and a reduction in the inhibitory effect of 5-HT by corticosterone which may be relevant to antidepressant/antiaxiety and proaxiety effects, respectively, of both treatments.

Repeated corticosterone administration increases excitatory effect of 5-HT4 receptor agonist in the rat hippocampus.

The objective of the present study was to investigate whether repeated exposure of rats to high level of corticosterone affects responses of CA1 hippocampal cells to the 5-HT4 receptor agonist zacopride. To assess responsiveness of CA1 neurons to zacopride we used extracellular recording of population spikes evoked in CA1 cells by the stimulation of the Schaffer/collateral-commissural pathway in hippocampal slices. Rats were treated with corticosterone for 7 days (10 mg/kg sc, twice daily), slices were prepared two days after the last treatment. Zacopride induced an increase in the amplitude of population spike and repeated corticosterone treatment enhanced this excitatory effect. It is concluded that repeated treatment with corticosterone increases the responsiveness of hippocampal CA1 neurons to the 5-HT4 receptor activation.

[EMG monitoring in functional electrostimulation]. / EMG-Monitoring bei funktioneller Elektrostimulation.

When using functional electrical stimulation (FES), correct adjustment of stimulation parameters, and monitoring of the stimulated muscle is mandatory if tissue damage is to be avoided. Although several FES systems are already in regular use, a method for direct muscle monitoring is still lacking. This paper investigates the suitability of the electromyogram (EMG) for such a purpose. In six sheep, the right latissimus dorsi muscle (LDM) and the associated thoracodorsal nerve were exposed. Stimulation was effected via electrodes placed on the nerve. Three electrodes were placed in the LDM for EMG recording, and the tendon was connected to a force transducer for isometric force measurement. Stimulation was applied for one second (burst), followed by a three-second pause. The stimulation current was increased in 0.2 mA steps, starting at 0 mA and ending at 4 mA. Throughout the investigation, the EMG signal was monitored with an oscilloscope. In addition, the EMG signal and the force transducer signal were recorded for subsequent analysis. An analysis of the data of all six sheep revealed an almost linear relationship between muscle force and m-wave amplitude (magnitude of r = 0.95, p < 0.001). M-wave monitoring during EMG recording with three intramuscular electrodes is a reliable method of monitoring FES-induced muscle activity, but the absolute force cannot be measured.

Neuropeptide Y reduces epileptiform discharges and excitatory synaptic transmission in rat frontal cortex in vitro.

Neuropeptide Y reduced spontaneous and stimulation-evoked epileptiform discharges in rat frontal cortex slices perfused with a magnesium-free solution and with the GABA(A) receptor antagonist picrotoxin. To investigate the mechanism of that action, effects of neuropeptide Y on intrinsic membrane properties and synaptic responses of layer II/III cortical neurons were studied using intracellular recording. Neuropeptide Y (1 microM) had no detectable effect on the membrane properties of neurons. The evoked synaptic potentials were attenuated by neuropeptide Y. Moreover, the pharmacologically isolated excitatory postsynaptic potentials, mediated by N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors, were reversibly depressed by neuropeptide Y. The most pronounced inhibitory effect of neuropeptide Y was observed on late polysynaptic excitatory postsynaptic potentials. To assess a putative postsynaptic action of neuropeptide Y, N-methyl-D-aspartate was locally applied in the presence of tetrodotoxin. The N-methyl-D-aspartate-evoked depolarizations were unaffected by neuropeptide Y, which suggests that the depression of excitatory postsynaptic potentials was due to an action at sites presynaptic to the recorded neurons. These data show that neuropeptide Y attenuates epileptiform discharges and the glutamate receptor-mediated synaptic transmission in the rat frontal cortex. The above results indicate that neuropeptide Y may regulate neuronal excitability within the cortex, and that neuropeptide Y receptors are potential targets for an anticonvulsant therapy.

Roles of group II metabotropic glutamate receptors in modulation of seizure activity.

Evidence suggests that metabotropic glutamate receptors (mGluR) are involved in mediating seizures and epileptogenesis. In the present experiments, the selective, group II mGluR agonist (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylic acid (LY354740, 0.1-1.0 microM) inhibited spontaneous epileptiform discharges which developed in rat cortical slices in Mg2+-free medium. LY354740 (4-16 mg/kg) administered prior to an injection of pentylenetetrazol (80 mg/kg) or picrotoxin (3.2 mg/kg) produced a dose-dependent decrease in the number of mice exhibiting clonic convulsions, but had no effect on N-methyl-D-aspartate (NMDA, 150 mg/kg)-induced convulsions. LY354740 (4-16 mg/kg) did not affect lethality induced in mice by pentylenetetrazol, picrotoxin or NMDA. LY354740 potentiated the anticonvulsant activity of the conventional antiepileptic drug diazepam, significantly decreasing the ED50 for that drug's effect on pentylenetetrazol-induced convulsions by 30%, but had no influence on anticonvulsant activity of ethosuximide and valproic acid. A pharmacokinetic interaction between LY354740 and diazepam, leading to the lowering of the plasma level of free diazepam, was also demonstrated. Our data suggest that the group II mGluR agonist LY354740 possesses anti-seizure activity and may modify the effects of some conventional antiepileptic drugs.

Long-term electromyogram recording from the posterior cricoarytenoid muscle as a potential biological trigger for phrenic pacing: results of an animal study.

Diaphragm pacing has been used to restore respiration in approximately 1,000 patients worldwide suffering from high quadriplegia or from central alveolar hypoventilation syndrome. Compared with conventional mechanical ventilation, electrophrenic respiration (EPR) reduces the risk of pulmonary infections and increases the mobility of patients. Voluntary activation of the pacemaker during speech would improve patients' quality of life and allow application of EPR in a more physiological way. An animal study was performed to investigate the electromyogram (EMG) of the posterior cricoarytenoid (PCA) muscle and the movement of the glottis via impedance measurement (electroglottography) with the aim to examine reproducibility and stability of the recordings from the PCA muscle as a potential biological trigger for a phrenic pacemaker. The EMG of the PCA muscle was recorded via implanted electrodes for a 200 day period. The EMG signal proved stable for that period, artifacts caused by movements can be suppressed, and swallowing can be detected. In contrast, impedance measurement to detect movement of the glottis proved not useful. Based on the results of this study, the use of the PCA EMG as a biological trigger for a phrenic pacemaker has to be considered a realistic option.

Neuropeptide Y suppresses epileptiform activity in rat frontal cortex and hippocampus in vitro via different NPY receptor subtypes.

Neuropeptide Y (NPY) and different NPY receptor (Y) subtype-selective agonists were tested for their effects on spontaneous epileptiform discharges which developed in rat cortical and hippocampal slices in Mg(2+)-free medium. Epileptiform activity, recorded extracellularly, was attenuated by NPY (0.5-1 microM) in both the frontal cortex and hippocampal CA3/CA1 pyramidal cell layers. In the cortex the Y1/5 selective agonist [Leu31 Pro34] NPY was more effective than the Y2 preferring agonist NPY13-36 and the Y2/5 preferring agonist NPY3-36. The suppression of epileptiform discharges induced by NPY in cortical slices was blocked by the selective Y1 receptor antagonist (R)-N2-(diphenylacetyl)-N-((4-hydroxyphenyl)methyl] argininamide (BIBP 3226). In the hippocampus, NPY13-36 and NPY3-36 were more effective than [Leu31 Pro34] NPY. In conclusion, the antiepileptic activity of NPY is mediated predominantly by the Y1 receptor subtype in the frontal cortex and by Y2 and probably Y5 receptors in the hippocampal CA3/CA1 areas.

Battery-powered implantable nerve stimulator for chronic activation of two skeletal muscles using multichannel techniques.

Chronic activation of skeletal muscle is used clinically in representative numbers for diaphragm pacing to restore breathing and for dynamic graciloplasty to achieve fecal continence. The 3 different stimulation techniques currently used for electrophrenic respiration (EPR) all apply high frequency powered implants. It was our goal to make these stimulation methods applicable for EPR by a battery-powered nerve stimulator that would maximize the patient's freedom of movement. Additionally, the system should allow the implementation of multichannel techniques and alternating stimulation of 2 skeletal muscles as a further improvement in graciloplasty. Generally, the developed implantable nerve stimulator can be used for simultaneous and alternating activation of 2 skeletal muscles. Stimulation of the motor nerve is achieved by either single channel or multichannel methods. Carousel stimulation and sequential stimulation can be used for graciloplasty as well as for EPR. For EPR we calculated an operating time of the implant battery of 4.1 years based on the clinically used stimulation parameters with carousel stimulation. The multichannel pulse generator is hermetically sealed in a titanium case sized 65 x 17 mm (diameter x height) and weighs 88 g.

Personal computer supported eight channel surface stimulator for paraplegic walking: first results.

Today functional electrical stimulation (FES) is used among other treatments to restore hand and arm function, to restore mobility of the lower extremities, for phrenic pacing, and in cardiomyoplasty. Common to all FES applications is that they require careful setup of stimulation parameters. To improve these tasks, personal computer (PC) based software for stimulation parameter evaluation and data acquisition was written. First, the described software was used to mobilize paraplegic patients in conjunction with an 12C bus controlled 8 channel surface stimulator. Electrodes were placed on each leg on the m. quadriceps and m. gluteus for hip and knee extension and the peroneal nerve to elicit flexion reflex. The fourth channel was used to correspond to subjects' individual needs. The stimulation patterns for standing up, walking, and sitting down easily could be set up and optimized by adjusting up to 128 stimulation parameters in a task-specific way.

MYOSTIM-FES to prevent muscle atrophy in microgravity and bed rest: preliminary report.

Long-term flights in microgravity cause atrophy and morphological changes of skeletal muscles. Training with mechanical devices is insufficient regarding the required time to exercise and space for devices. The objective of this project is to develop a passive training method based on functional electrostimulation (FES) to preserve muscle mass and fiber composition with minimal impairment to the cosmonaut. For a pilot experiment on the MIR space station, a suitable 8 channel FES device was developed. It consists of electrode trousers that carry surface electrodes and cables, 2 interconnected 4 channel stimulators, and a laptop personal computer (PC) for stimulator programming and processing compliance data. An automatic extensive training of 4 muscle groups of the lower extremities is performed for 6 h/day, with 1 s on and 2 s off tetanic contractions at 20-30% of maximum tetanic muscle force. The synchronous activation of antagonists of the thigh and lower leg prevents uncoordinated movements.