Direct effects of audio-visual stimulation on EEG.

In the course of 2 months, 25 repetitions of a 20 min audio-visual stimulation (AVS) program with stimulations at 17, 9, 4, and 2 Hz were applied to 6 volunteers. EEG data were recorded from 6 scalp locations prior, during and after AVS. In order to identify direct and transient changes in EEG under influence of AVS, total power, relative frequency band powers and magnitude-squared coherences were estimated. Intense brain wave entrainment as a direct reaction to AVS was significant through increase of spectral powers and coherences around the stimulating frequency bands in the occipital areas, spreading also to the central and frontal regions. However, these excitations were 'short-lived'. On the other hand some signs of interhemispheric cooperation (coherences in the narrow bands around 2, 4, and 17 Hz at parieto-occipital areas) remained increased during the investigated 3 min after AVS. As going through further AVS sessions the driving response progressively enhanced for 2 and 4 Hz stimulation in centro-parietal locations. Progress was also found in the left and right hemisphere synchronization examined by coherences. In perspective, the results contribute to deeper comprehension of photic stimulation approaches as a technique of guided entrainment of the brain waves or intermediate increase of hemispheres' synchronization.

Pycnogenol efficiency on glycaemia, motor nerve conduction velocity and markers of oxidative stress in mild type diabetes in rats.

The aim of this study was to describe the effects of Pycnogenol at various doses on preprandial and postprandial glucose levels, the levels of thiobarbituric acid reactive substances (TBARs) and N-acetyl-beta-d-glucosaminidase (NAGA) and on motor nerve conduction velocity (MNCV) in streptozotocin (STZ)-induced diabetic rats. Pycnogenol treatment (10, 20, 50 mg/kg body weight (b.w.)/day) lasted for 8 weeks after induction of diabetes. Pycnogenol significantly decreased elevated levels of preprandial glycaemia in treated animals at all doses. At doses of 10 mg/kg b.w./day and 20 mg/kg b.w./day it significantly decreased elevated levels of postprandial glycaemia compared with diabetic non-treated animals. Pycnogenol failed to induce a significant decrease of postprandial glycaemia at a dose of 50 mg/kg b.w./day. Pycnogenol improved significantly the impaired MNCV at doses of 10 and 20 mg/kg b.w./day compared with non-treated animals. The levels of TBARs were elevated in diabetic rats. The levels of NAGA increased gradually despite the treatment. Pycnogenol failed to affect the increased levels of TBARs and NAGA. Pycnogenollowered the elevated levels of glycaemia and reduced the decline in motor nerve conduction velocity in STZ-induced diabetic rats. The effect of Pycnogenol on postprandial glycaemic levels and MNCV was not dose-dependent.

Protective effect of stobadine on NCV in streptozotocin-diabetic rats: augmentation by vitamin E.

Hyperglycaemia-induced oxidative stress makes an important contribution to the aetiology of diabetic neuropathy. Elevated reactive oxygen species (ROS) cause cumulative damage to neurons and Schwan cells, however, they also have a deleterious effect on nerve blood flow causing endoneurial hypoxia, which is responsible for early nerve conduction velocity (NCV) deficits and contributes to an increase in resistance to ischaemic conduction failure (RICF). We tested whether antioxidants - stobadine, vitamin E or the combination of these drugs, could prevent the early signs of neural dysfunction in animal model of diabetes in 8-9 weeks old male Wistar rats, made diabetic by streptozotocin (55 mg/kg i.v.) 4 months prior to testing. Neuropathy was evaluated electrophysiologically by measuring motor NCV and RICF of sciatic nerve in vitro. We observed that treatment with the combination of stobadine and vitamin E significantly (p < 0.001) reduced the NCV slowing in diabetic rats, although it did not fully prevent the NCV impairment. Significant effect (p < 0.05) was observed also in stobadine monotherapy. The RICF elevated in diabetic animals was not affected by any drug applied. This study confirmed that treatment with appropriate antioxidants, especially their combination could partially prevented the decrease in NCV in diabetic rats.

Stobadine-induced hastening of sensorimotor recovery after focal ischemia/reperfusion is associated with cerebrovascular protection.

In a model of 1 hour-intraluminal occlusion of rat middle cerebral artery (MCA), we investigated the spontaneous recovery of vascular functions and functional deficit together with ischemia volume evolution at 24 h, 3 days and 7 days of reperfusion. Infarct cerebral volumes and edema were quantified with histological methods. Endothelium-dependent and smooth muscle potassium inward rectifier current (Kir2.x)-dependent relaxing responses of MCA were tested using Halpern arteriograph and Kir2.x current density evaluated on MCA myocytes with whole-cell patch-clamp technique. Sensorimotor recovery was estimated according to performances obtained with adhesive removal test and prehensile traction test. A time-dependent improvement of smooth muscle K(+)-dependent vasorelaxation and Kir2.x current density is observed at 7 days of reperfusion while endothelium-dependent relaxation is still impaired. In parallel a significant reduction of functional deficit is observed at 7 days of reperfusion together with a time-matched reduction of striatal infarct and edema volumes. Administration of an antioxidant agent, stobadine, at time of reperfusion and 5 h later allowed: (i) a neuroprotective effect with a significant reduction of infarct size compared to vehicle-treated rats; (ii) a prevention of endothelial-dependent relaxation and Kir2.x current density reductions of MCA ipsilateral to occlusion; (iii) a hastening of the functional recovery. The beneficial effect of stobadine underlines a link between vascular protection, neuronal protection and sensorimotor recovery that could become a promising pharmacological target in the treatment of cerebral ischemia.

Neurogliovascular unit after cerebral ischemia: is the vascular wall a pharmacological target.

Ischemic stroke induces drastic alterations of the functions of the neurogliovascular unit with dramatic consequences on the well-being of the patients in terms of cognitive and motor handicap. Nowadays, only very few therapeutics are available as a treatment of ischemic stroke. Ischemia is a multifactorial pathology involving different cerebral cellular components such as neurons, astrocytes and vessels working as a functional unit. Recent experimental strategy investigation involving different agents with antioxidant properties (dt-BC, stobadine) or pleiotropic effects (lipopolysaccharide, LPS) has been developed to evaluate whether the vascular wall could be considered as a potential target in neuroprotection concept.

Improvement of band segmentation in Epo images via column shift transformation with cost functions.

In recent years, the development of methodology and laboratory techniques for doping control (DC) of recombinant erythropoietin (rEpo) has become one of the most important topics pursued by doping control laboratories accredited by World Anti-Doping Agency (WADA). The software system GASepo has been developed within the international WADA project as a support for Epo doping control. Although a great number of functions for automatic image processing have been involved in this software, for Epo images with considerably distorted bands additional effort is required from the user to interactively correct the results of improper band segmentation. In this paper a problem of geometrically distorted bands is addressed from the viewpoint of how to transform the lanes in distorted Epo images in order to reach better band segmentation. A method of band straightening via column shift transformation has been proposed that is formulated as an optimization procedure with cost functions. The method involves several novel approaches: two-stage optimization procedure, four cost functions and selection of relevant columns. The developed band straightening algorithm (BSA) has been tested on real Epo images with distorted bands. Based on the evaluation scheme involving the GASepo software itself a recommendation is made for implementation of the method with the cost function based on correlation matrix. Estimates of computational complexity of the individual steps of BSA are also given.

Development of the new group of indole-derived neuroprotective drugs affecting oxidative stress.

1. The role of oxidative stress, and accordingly uncontrolled reactive oxygen species generation/action, have been widely documented in a number of different neuronal pathologies. However, the concept of pharmacological interventions in prevention and therapy of oxidative stress-related diseases has not found adequate application in clinical practice. This may be due to the insufficient efficacy of drugs available, their unsuitable pharmacokinetics, side effects, toxicity, etc. 2. Based on stobadine, (--)-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido[4,3-b]indole, a well-known antioxidant, free radical scavenger, and neuroprotectant, it was attempted to develop new stobadine derivatives with improved pharmacodynamic and toxicity profiles, on applying molecular design, synthesis and adequate tests. Stobadine molecule was modified mostly by electron donating substitution on the benzene ring and by alkoxycarbonyl substitution at N-2 position. A total of >70 derivatives were prepared. 3. In a mice model of head trauma, some of the new stobadine derivatives administered i.v. immediately after the trauma, significantly improved sensomotoric outcome in the animals assessed 1 h later. Accordingly, decrease in brain edema was proved histologically as well as by brain wet weight assessment. 4. Putative neuroprotective action of the compounds was confirmed on rat hippocampal slices exposed to reversible 6 min hypoxia/low glucose by analysis of synaptic transmission in CA1 region neurons. Irreversible impairment of neurotransmission resulting from the hypoxia was significantly reduced by the presence of SMe1EC2, one of the new compounds, in concentration range 0.03-10.0x10(-6) mol l(-1). Both the neuroprotective and antioxidant effect of the compound closely resembled those of stobadine, melatonin, 21-aminosteroids, alpha-phenyl-tert-butylnitrone and others, all well-established antioxidants, except the range of effective concentrations was by 1-2 orders lower in SMe1EC2. 5. A remarkable antioxidant efficacy was observed in the new compounds in rat brain homogenates exposed to iron/ascorbate system by protection of lipids and creatine kinase against the oxidative impairment. A link between the neuroprotective and antioxidant/ scavenger properties in the compounds can be assumed. 6. Acute toxicity of some of the new pyridoindoles was diminished compared to stobadine. That might be due to the virtually full elimination of stobadine's undesired alpha (1)-adrenolytic activity attained by appropriate modifications of its molecule. 7. The new pyridoindoles extend the range of available neuroprotectants interfering with oxidative stress in neuronal tissue.

EEG responses to long-term audio-visual stimulation.

In this study, linear and nonlinear electroencephalogram (EEG) changes due to long-term audio-visual stimulation (AVS) were investigated. In the course of 2 months, 25 repetitions of a 20-min AVS program with stimulation frequencies in the range 2-18 Hz were applied to six healthy volunteers. EEG data were recorded from six head locations during relaxed wakefulness prior to AVS. Then linear spectral measures (total power, frequency band powers, spectral edge frequency, and spectral entropy), nonlinear measures of complexity (histogram-based entropy and correlation dimension), interdependency measures (linear correlation coefficient, mutual information, and coherence), and measures of subjective assessment were estimated. Evolution of these measures during the whole experiment period was analyzed with respect to the significance of their linear regression. Our results confirm that repetitive training with audio-visual stimulation does induce changes in the electro-cortical activity of the brain. Long-term AVS significantly increased power in theta-1, theta-2, and alpha-1 bands in the frontal and central cortex locations. Total power increased in the right central region. Interhemispheric coherence in alpha-1 band displayed a significant increase between frontal parts in contrast to the decrease of both linear correlation and mutual information. Correlation dimension significantly decreased in some locations while entropy displayed an ascending trend.

In vitro physiological evidence of enhanced antioxidant and neuroprotective action of 2,3-dihydromelatonin, a melatonin analogue.

As the capacity of the endogenous antioxidative system is limited, pharmacological treatment with antioxidants may help to protect neuronal tissue against increased amount of reactive oxygen species produced during oxidative stress. We attempted to improve resistance of rat hippocampal slices exposed to ischaemia in vitro (hypoxia (HYP) accompanied with decreased glucose concentration followed by reoxygenation (ROX)) and thus to diminish the impairment of synaptic transmission after HYP/ROX. We compared the protective features of the melatonin analogue 2,3-dihydromelatonin (2,3-DHM) with melatonin itself. In preliminary experiments, the compound 2,3-DHM compared to melatonin revealed enhanced antilipoperoxidation action in rat brain homogenates exposed to Fe/ascorbate system (-logIC(50) = 4.76 +/- 0.01 versus -logIC(50) = 2.51 +/- 0.02, respectively). In this study, 2,3-DHM (from 0.3 to 10 micromol l(-1)) applied at 30 min before the beginning of HYP and remaining all over the 6-min HYP as well as 20-min ROX, exerted a protective effect demonstrated by improvement of the population spike amplitude (PoS) recovery during ROX, with the maximum effect at 3 micromol l(-1). In accordance with this, the ratio of irreversibly damaged slices after HYP/ROX was decreased in the groups treated with 2,3-DHM. Moreover, a significant delay of PoS decay during HYP (expressed as half time, t(0.5)) was revealed at 2,3-DHM concentration 1 and 3 micromol l(-1)). An equipotent effect of melatonin and 2,3-DHM was achieved by a 100-times lower concentration of the latter (0.3 and 1 micromol l(-1)) compared to that of melatonin (30 and 100 micromol l(-1)). Further, compared to the highest effect of 2,3-DHM in the concentration 3 micromol l(-1) on the percentage of irreversibly damaged slices (only 20%), melatonin did not exert such pronounced effect, either in the concentration 30 or 100 micromol l(-1) (67 and 50%, respectively). We conclude that hydrogenation of the melatonin molecule significantly improved its antihypoxic effect in our model of rat hippocampal slices exposed to ischaemic conditions in vitro, similarly as it enhanced the antilipoperoxidation action of 2,3-DHM in our previous studies. These findings suggest that 2,3-DHM deserves more attention concerning its neuroprotective effect in oxidative stress-associated tissue damage.

Effect of antioxidants and NMDA antagonists on the density of NMDA binding sites in rat hippocampal slices exposed to hypoxia/reoxygenation.

Major interest is currently focused on the development and evaluation of effective strategies for the pharmacological therapy of human stroke and cerebral ischemia, as well as some neurodegenerative disorders in which increased production of free oxygen radicals and the neurotoxic effect of excitatory amino acids may take place. Selected N-methyl-D-aspartate (NMDA) antagonists and antioxidants in the model of experimental oxidative stress induced by hypoxia and reoxygenation in rat hippocampal slices were tested. The putative antiglutamatergic effect of the antioxidant stobadine and its neuroprotective effect during oxidative stress was studied. NMDA antaonists 2-amino-5-phosphonovaleric acid (APV) and Mg2+, as well as the antioxidants stobadine and trolox, prevented the decrease of NMDA binding site number induced by hypoxia/reoxygenation in rat hippocampal slices. Moreover, stobadine, APV and Mg2+ prevented the decrease of NMDA binding site number due to glutamic acid incubation. Stobadine does not inhibit [3H]-glutamate binding and therefore does not seem to interact directly with glutamate binding sites. Thus, its neuroprotective effect in rat hippocampus exposed to hypoxia/reoxygenation does not seem to be based on a direct antiglutamatergic effect. The protective action of stobadine against the decrease of NMDA binding site number elicited by hypoxia/reoxygenation in rat hippocampus could rather be due to its antioxidant and antiradical effect.

Postischemic reperfusion of the spinal cord: prolonged reperfusion alleviates the metabolic alterations induced by 25 min ischemia in the cervical and thoracolumbal segments.

In recent years, increasing amount of information has indicated that in some tissues the main damage due to oxidative stress does not occur during reperfusion but during the ischemic episode of the ischemia/reperfusion event. In this respect, serious doubts were also expressed about the origin of the increased amounts of free radicals which were believed to form and reported to appear in the perfusate during the first minutes of reperfusion. Moreover, speculative explanations were only available for a second increase in lipid peroxidation which was reported to occur after postischemic reperfusions exceeding 60 min. For this reasons, the present paper reports the results of investigation of ischemia/reperfusion injury to the cervical (CE) and thoracolumbal (ThL) segments of the spinal cord (SP) after an acute 25 min occlusion of the abdominal aorta, followed by 60-120 min reperfusion of the ischemic areas in rabbits. In CE and ThL segments of the SP, the ischemia induced: 1) a decrease in activities of superoxide dismutase (SOD), from 57.35+/-6.36 to 45.27+/-5.45 U x mg(-1) x min(-1) (S.E.M., 20.92%), p < 0.01, and from 58.36+/-5.45 to 33.00+/-4.55 U x mg(-1) x min(-1) (S.E.M., 43.46%), p < 0.001; 2) a significant decrease in gamma-glutamyl transpeptidase (gamma-GTP), from 114.66+/-1.45 to 99.88+/-4.4 micromol p-nitroaniline x mg(-1) x h(-1) (S.E.M. 12.89 %), p < 0.05 and from 112.24+/-1.20 to 95.09+/-2.40 micromol p-nitroaniline x mg(-1) x h(-1) (S.E.M., 16.26%), p < 0.05; 3) a considerable depression in Na,K-ATPase activity, from 7.14+/-0.58 to 5.08+/-0.32 micromol Pi x mg(-1) x h(-1) (S.E.M., 28.86%), p < 0.01, and from 7.23+/-0.11 to 5.09+/-0.31 micromol Pi x mg(-1) x h(-1) (S.E.M., 30.00%), p < 0.01. The Na,K-ATPase activity became decreased by ischemia and remained depressed significantly (all p < 0.01) throughout the experiment. After 60 min of reperfusion, SOD activity in the CE segment and that of gamma-GTP in the CE as well as ThL segments recovered, even slightly surpassing the control values, wheras SOD activity in the ThL segment became stabilized again close to its post-ischemic value. Prolonged, reperfusion for 120 min resulted in a further increase in gamma-GTP activity in the CE and ThL segments (to 132.79 and 132.30%, p < 0.01), and this was accompanied by a slight (p > 0.05) elevation in the content of conjugated dienes as well as by a new wave of depression of the SOD activity (p < 0.05) in both the CE and the ThL segment. From our results it could be concluded that all considerable damage to the spinal cord occurred during the ischemic period. In the period of reperfusion reparative changes started to predominate. This is in accordance with the recent discoveries indicating that, when coupled with an increase in tissue gamma-GTP activity, the post-ischemic reparative changes comprise a replenishment of the cell glutathione pool. This process is accompanied with a gradual increase in H2O2 production that results in repeatead inhibition of the SOD activity and a tendency to conjugated dienes formation.

4-Hydroxynonenal as a second messenger of free radicals and growth modifying factor.

Immunohistochemical analysis of the distribution of the lipid peroxidation product 4-hydroxynonenal (HNE) in the brain of baboons exposed to experimental hemorrhagic traumatic shock or sepsis showed that systemic oxidative stress and the thereby generated HNE affect the blood:brain barrier and the regulation of cerebral blood flow determining secondary brain damage. Similarly, HNE was determined during ischemia in the brain blood vessels of rats exposed to ischemia/reperfusion injury of the brain. After reperfusion, HNE disappeared from the blood vessels but remained in neurones and in glial cells. Since HNE modulates cell proliferation and differentiation (including proto-oncogene expression), it is postulated that HNE might have prominent local and systemic effects that are not only harmful but beneficial, too, determining the outcome of various pathophysiological conditions based on oxidative stress.

Indole derivatives as neuroprotectants.

It seems to be satisfactorily proved that reactive oxygen species (ROS) participate in numerous pathological processes in the nervous system (NS). Compounds able to interfere with the action of ROS might be useful in prevention and treatment of these pathologies. The search is focused on compounds with a suitable spectrum of pharmacological and pharmacokinetic properties, among which indole derivatives are distinct group with great potential to be further developed. The paper presents an overview of indole derived compounds in which protective action has been demonstrated in the NS in situations in which ROS are excessively generated, such as chemically induced oxidative stress, hypoxia/reoxygenation, ischemia/reperfusion. These compounds include indoleamines (melatonin), carbazoles (carvedilol), carbolines (tetrahydrocarbolines, pyrimidoindoles, vinpocetine). Special attention is paid to the gamma-carboline stobadine. A range of effects which seem to be associated with its neuroprotective actions (antioxidant and ROS scavenging effects, capability to pass the hematoencephalic barrier, pharmacokinetic properties, etc.) are considered. A novel compound with pyrimidoindole structure (U-101033E) is mentioned. Attention is drawn also to the neurotoxic potential demonstrated in some carbolines (2-amino-alpha-carboline, halogenated tetrahydro-beta-carboline "TaClo", harmane, norharmane). The indole nucleus seems to be a promising basis for design and synthesis of new derivatives able to protect the NS against oxidative stress in a variety of acute and chronic NS pathologies.

Effect of stobadine on lipid peroxidation in brain and heart after ischemia and reperfusion of the brain.

Stobadine (ST), a novel drug with pyridoindol structure, was recently found to prevent reperfusion injury in rat brain. The aim of the present study was to reveal whether ST may prevent peroxidative changes in the heart and brain that were triggered by postischemic reperfusion of the brain. In the brain, reperfusion significantly increased the contents of malondialdehyde (MDA) by 43.8% and conjugated diens (CD) by 24.5% when compared with the end of ischemia. In the heart, contents of MDA and CD in reperfusion became elevated three fold and by 41.7%, respectively, when comparing to the values at the end of ischemia. In the heart, no significant changes in activities of the superoxide dismutase (SOD) and glutathione peroxidase (GPx) induced by ischemia or reperfusion were detected. In contrast, reperfusion induced a slight decrease in GPx activity in the brain. In accordance with our previous results, an application of ST (2 mg/kg) to the femoral artery shortly prior to reperfusion of the ischemic brain, prevented significantly MDA and CD accumulation in brain. Nevertheless, ST was not able to prevent the brain-ischemia/reperfusion-induced elevation of MDA and CD contents in the heart.

Effect of stobadine, U-74389G, trolox and melatonin on resistance of rat hippocampal slices to oxidative stress.

Reactive oxygen species have been suggested to participate in the impairment of nervous tissue by oxidative stress, induced by hypoxia (HYP) followed by reoxygenation (ROX). Although the mechanisms of such injury are rather complex, antioxidants might exert some protective action under such circumstances. This study tested the effect of a series of compounds interfering with the generation and action of reactive oxygen species on impairment of synaptic transmission in the CA1 region of rat hippocampal slices exposed to HYP followed by ROX in vitro. Shortlasting HYP (typically 4.5-7.5 min under the conditions used) resulted in fast decay of the amplitude of population spikes evoked in the CA1 neurons by stimulation of Schäffer collaterals. The impairment was mostly irreversible. However, in the presence of the antioxidants stobadine, 21-aminosteroid U-74389G, melatonin and trolox (with optimal concentrations of 10-30 micromol/l, 10 micromol/l, 30-100 micromol/l and 200 micromol/l, respectively), the irreversible damage of the transmission was significantly diminished. The decay of the synaptic transmission failure during HYP was also delayed by stobadine, U-74389G and melatonin. The results demonstrated that compounds with antioxidant activity may effectively protect nervous tissue during HYP and ROX.

Effects of stobadine, melatonin, and other antioxidants on hypoxia/reoxygenation-induced synaptic transmission failure in rat hippocampal slices.

In vitro reversible ischemia was simulated with rat hippocampal slices in order to test the neuroprotective activity of selected antioxidants with emphasis on the pyridoindole stobadine. Slices were exposed to hypoxia (HYP) combined with lowered D-glucose concentration to induce synaptic transmission (ST) failure, which turned out to be irreversible in approximately 80%-100% of slices during reoxygenation (ROX). The amplitude of population spikes (PoS) evoked trans-synaptically by electrical stimulation of Schäffer collaterals and recorded in CA1 neurons was the parameter of ST. Pretreatment of slices with stobadine dissolved in slice superfusion media (1 to 100 microM) improved ST recovery after 20-min tissue ROX. Stobadine decreased the number of irreversibly damaged slices and increased the average amplitude of PoS during tissue ROX. The concentration-response relationship of protective activity was bell-shaped, with maximum at 3-30 microM. Moreover, the half-time of PoS decay (t1/2) during HYP was significantly delayed in stobadine treated groups (10 to 100 microM). The neurohormone melatonin (30 to 100 microM) and 21-aminosteroid U-74389G (10 microM) revealed similar protective activity on ST recovery and on t1/2 during HYP. Trolox (200 microM) improved the PoS recovery, yet it had no effect on t1/2. The iron chelator deferoxamine (250 and 500 microM) had no protective effects at all. alpha-Tocopherol administered to animals orally (200 mg/kg for 10 days) only marginally improved the PoS recovery. Comparing the protective effect of compounds tested on PoS recovery, we assume the following rank order of potency: U-74389G > stobadine > melatonin >> trolox. Our findings suggest that stobadine as well as trolox, U-74389G and melatonin, antioxidants with remarkably different chemical structures, exerted neuroprotective activity, probably determined by antioxidative properties of these compounds. Moreover, stobadine, U-74389G, and melatonin were able to delay the early ST decay during HYP, which might indicate improved energetic state of neurons in the treated tissue. The study supports the notion about the neuroprotective activity of certain antioxidants.

Streptozotocin diabetes-induced changes in aorta, peripheral nerves and stomach of Wistar rats.

In rats with diabetes induced by streptozotocin (STZ), we studied the reactivity of the aorta in response to vasoconstrictor and vasorelaxant agents, changes in conduction velocity in the sciatic nerve, and glutathion (GSH) content in the gastric mucosa as well as the occurrence of spontaneous gastric lesions. STZ-induced diabetes was found to be accompanied by endothelial injury, exhibited by diminished endothelium-dependent relaxation and by increased noradrenaline- and H2O2-induced contraction. Conduction velocity in the nerves from STZ-treated animals was significantly lower compared to that in nerves from control animals. Moreover, gastric hyperaemia, occasional gastric lesions, and a significant depletion of GSH in the gastric mucosa were observed in STZ-treated rats. Our experiments confirmed the suitability of Wistar rats for the model of STZ-induced diabetes.

Free radicals in rabbit spinal cord ischemia: electron spin resonance spectroscopy and correlation with SOD activity.

1. In nonanesthetized rabbits temporal occlusion of the abdominal aorta was used to induce oxidative stress in the lower part of the body including distal segments of the spinal cord. 2. Spinal cord samples were taken from the animals exposed to 25-min aortic occlusion (AO) or to occlusion followed by 1- or 2-hr reperfusion (AO/R1 or AO/R2, respectively) or from sham-operated animals (C). The presence of free radicals (FR) in the spinal cord samples frozen in liquid N2 was assessed by ESR spectroscopy without spin trapping. Moreover, superoxide dismutase (SOD) activity and conjugated diene (CD) levels were measured in the samples. 3. In the AO group FR were detected in the spinal cord regions close to the occlusion (lower thoracic and distal segments) along with a decrease in SOD activity. The calculated g value (g = 2.0291) indicated that the paramagnetic signal recorded might be attributed to superoxide radicals. FR were absent in the AO/R1 group. Concurrently, the SOD activity revealed a significant tendency to return to the control level. FR appeared again in the AO/R2 group, mostly in the upper and middle lumbar regions, along with a decrease in SOD activity. No sample from the C group revealed FR. A significant increase in CD levels was observed in the thoracolumbar region only in the AO/R2 group. The temporary absence of FR in the AO/R1 group suggests activation of defense antioxidant mechanisms (e.g., specific enzymatic systems such as SOD), which might have been exhausted later. 4. Changes in SOD activity similar to those observed in the thoracolumbar region, though less noticeable, occurred in the obviously noncompromised tissue (upper cervical region). This points to a kind of generalized response of the animal to aortic occlusion. 5. Direct ESR spectroscopy revealed the presence of FR as well as their time course in the spinal cord during the early phase of ischemia/reperfusion injury and the inverse relationship between FR and SOD activity.

Mechanisms of hippocampal reoxygenation injury.

Mechanisms of 12 min of hypoxia and subsequent reoxygenation were studied in rat hippocampal slices. General cell injury in reoxygenation was indicated by increased lactate dehydrogenase (LDH). Increase in conjugated dienes (CD) showed that oxygen radical burst induced lipid peroxidation (LPO). ATP increase was also involved in reoxygenation injury, since cyanide, an inhibitor of ATP synthesis, decreased this damage. The results obtained on using inhibitors of oxygen radicals generation, i.e., allopurinol, indomethacin, rotenone, and antimycin A, strongly suggest that the sources of oxygen radicals were the xanthine/xanthine oxidase system, prostaglandin synthesis, and mitochondrial respiratory chain. The involvement of oxygen radicals in oxidative stress was confirmed also by using chain-breaking antioxidants, trolox alpha-tocopherol and stobadine, [(-)-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido (4,3b)indole]. Stobadine added at the onset of reoxygenation was most effective, acting in a dose-dependent manner and found to be without effect when applied in hypoxia. Cytochrome-c oxidase was decreased in reoxygenated hippocampal slices treated with stobadine.

Antioxidant and pharmacodynamic effects of pyridoindole stobadine.

1. The review summarizes the most important data known so far on chemistry, pharmacodynamics, toxicology and clinics of the investigational agent, pyridoindole stobadine. 2. Stobadine was shown to be able to scavenge hydroxyl, peroxyl and alkoxyl radicals, to quench singlet oxygen, to repair oxidized amino acids and to preserve oxidation of SH groups by one-electron donation. These effects originated from its ability to form a stable nitrogen-centered radical on indole nitrogen. Consequently, it was able to diminish lipid peroxidation and protein impairment under oxidative stress. 3. In various in vitro and in vivo animal models, stobadine was shown to diminish the impairment of the myocardium induced by mechanisms involving reactive oxygen species (e.g., myocardial infarction, hypoxia/ reoxygenation, catecholamine overexposure). 4. The neuroprotective effect of stobadine was demonstrated in a series of in vivo and in vitro models (brain in situ, brain slices, spinal cord, autonomic ganglia, etc.) during ischemia/reperfusion and hypoxia/ reoxygenation or in the presence of chemical systems generating free oxygen radicals, and so forth. Stobadine improved animal survival rate and synaptic transmission recovery, maintained SH tissue level and diminished lipid peroxidation as well as impairment of Ca-sequestering intracellular systems. 5. Oxidation of low-density lipoproteins (LDLs), which plays a major role in the development of atherosclerosis, was decreased by stobadine in vitro. Both lipid and protein (apo B) components of LDL were protected against Cu(2+)-induced oxidation by this agent. 6. Stobadine proved to be an effective protectant in models of free radical pathology in vivo, such as cyclophosphamide-, MNNG- or 60Co-induced mutagenesis and alloxan-induced hyperglycemia. 7. Besides other remarkable pharmacodynamic effects, stobadine exerts antidysrhythmic, local anesthetic, alpha-adrenolytic, antihistaminic, myorelaxant and antiulcerogenic actions. 8. Pharmacokinetic analyses demonstrated that stobadine was readily absorbed from the gastrointestinal tract. Thanks to its balanced lipo-hydrophilic properties, it was distributed over both water and lipid phases in biological tissues. It was shown to easily penetrate the blood-brain barrier. 9. Acute, subchronic and chronic toxicity studies in several animal species, as well as numerous analyses of embryotoxicity, teratogenicity, mutagenicity and genotoxicity, revealed only a negligible toxic potential of this agent. 10. Phase-one clinical study demonstrated safety of the compound. Only slight side effects--namely, a slight hypotension and a slight sedative effect--were observed subsequent to the highest dose used. In phase-two clinical study, the patients with angina pectoris treated for 4 weeks with stobadine showed a significant decrease in the frequency of anginal attacks, in the number of self-administrations of sublingual nitroglycerine and in plasma lipoprotein, cholesterol and triglyceride levels. A slight decrease in systolic and diastolic blood pressure also was observed. 11. It is suggested that stobadine may be considered a contribution to the search for new effective cardio- and neuroprotectants based on antioxidant or free radical scavenging mechanisms of action.