The effects of various antiepileptic drugs on E-shock-induced amnesia in mice: dissociability of effects on convulsions and effects on memory.

Antiepileptic drugs prevent tonic convulsions induced by cerebral electroshocks. The present study investigated whether these drugs can also modulate the amnestic effect of the cerebral electroshock. It could be shown that phenytoin, ethosuximide, valproinate sodium, phenobarbitone, and clonazepam dose-dependently worsened the amnestic effect of electroshock (despite prevention of the convulsion). Carbamazepine, in contrast, significantly reduced the amnestic effect of the cerebral electroshock even at doses below the threshold for anticonvulsant activity.

In vitro techniques for the assessment of neurotoxicity.

Risk assessment is a process often divided into the following steps: a) hazard identification, b) dose-response assessment, c) exposure assessment, and d) risk characterization. Regulatory toxicity studies usually are aimed at providing data for the first two steps. Human case reports, environmental research, and in vitro studies may also be used to identify or to further characterize a toxic hazard. In this report the strengths and limitations of in vitro techniques are discussed in light of their usefulness to identify neurotoxic hazards, as well as for the subsequent dose-response assessment. Because of the complexity of the nervous system, multiple functions of individual cells, and our limited knowledge of biochemical processes involved in neurotoxicity, it is not known how well any in vitro system would recapitulate the in vivo system. Thus, it would be difficult to design an in vitro test battery to replace in vivo test systems. In vitro systems are well suited to the study of biological processes in a more isolated context and have been most successfully used to elucidate mechanisms of toxicity, identify target cells of neurotoxicity, and delineate the development and intricate cellular changes induced by neurotoxicants. Both biochemical and morphological end points can be used, but many of the end points used can be altered by pharmacological actions as well as toxicity. Therefore, for many of these end points it is difficult or impossible to set a criterion that allows one to differentiate between a pharmacological and a neurotoxic effect. For the process of risk assessment such a discrimination is central. Therefore, end points used to determine potential neurotoxicity of a compound have to be carefully selected and evaluated with respect to their potential to discriminate between an adverse neurotoxic effect and a pharmacologic effect. It is obvious that for in vitro neurotoxicity studies the primary end points that can be used are those affected through specific mechanisms of neurotoxicity. For example, in vitro systems may be useful for certain structurally defined compounds and mechanisms of toxicity, such as organophosphorus compounds and delayed neuropathy, for which target cells and the biochemical processes involved in the neurotoxicity are well known. For other compounds and the different types of neurotoxicity, a mechanism of toxicity needs to be identified first. Once identified, by either in vivo or in vitro methods, a system can be developed to detect and to evaluate predictive ability for the type of in vivo neurotoxicity produced. Therefore, in vitro tests have their greatest potential in providing information on basic mechanistic processes in order to refine specific experimental questions to be addressed in the whole animal.

Psychometric alterations in treatment with the MAO-A-inhibitor moclobemide.

Two groups of depressed patients were treated either with a selective MAO-A-inhibitor, moclobemide (n = 13) or a tetracyclic antide-pressant, maprotiline (n = 18), in a 28 days lasting double blind investigation. Before and after treatment psychopathologic symptoms were rated, motor performance was proven, and acoustic and visual sensomotoric performance were investigated. Deterioration of psychomotor performance were seen in patients without amelioration of their psychopathologic symptoms, especially when treated with moclobemide. These findings were regarded as a hint that possibly the therapeutic agent interacts with the wrong transmitter system and perhaps this is the reason for the deterioration of psychomotor functions.

Moclobemide and maprotiline in the treatment of inpatients with major depressive disorder.

A double-blind study with the substituted benzamide moclobemide, a novel reversible, short acting MAOI with predominant inhibition of MAO-A, and maprotiline, the most selective noradrenaline reuptake inhibitor available at present has been conducted in n = 40 severely depressed inpatients suffering from predominantly endogenous depressions. No significant differences between the two drugs were found using global HRSD, HAMA and self rating scales. Regarding the clinical profile moclobemide seemed to be more effective in retarded depressives, maprotiline was superior in alleviating depressive agitation and sleep disturbances. The latter symptoms were responsible for three cases of treatment withdrawal in the moclobemide group. No case of hypertensive crisis could be registered, though patients were not subject to food restrictions. Maximal concentrations of moclobemide in CSF were reached two hours after oral application, compared to tricyclic antidepressants a high CSF/plasma ratio could be detected.

Clinical, biochemical and psychometric findings with the new MAO-A-inhibitors moclobemide and brofaromine in patients with major depressive disorder.

N = 53 inpatients with major depressive disorder have been treated with the reversible, selective MAO-A-inhibitors moclobemide (double-blind versus maprotiline) and brofaromine (open study), respectively. Clinically, significant improvement of depression and an activating profile of action could be observed, typical side effects were sleep disturbances, agitation and weight loss. The neurobiochemical data showed an increase of noradrenaline plasma concentrations under treatment with moclobemide. Visual reaction times improved with antidepressant treatment. MAO-A inhibitors proved to be effective antidepressants in the treatment of hospitalized patients with predominantly endogenous depressions.

The use of an unbaited radial maze in neurotoxicology: II. Sensory inputs, general malaise and locomotor activity.

An unbaited 6-arm radial tunnel maze (6-arm RTM) is used in our laboratory to screen for working- and reference memory deficits in rats in the course of neurotoxicological studies. In the 6-arm RTM animals are minimally stressed, and do not need food reward as a reinforcer. Maze behavior is assessed using mean error score of arm repetitions as a measure of 'working memory', left-right discrimination within each arm (expressed as percent "blind-alley" visits) as a measure of 'reference memory' and trial time (time to complete 12 arm entries) as a measure of locomotor activity. As shown for other mazes sensory deficits may affect spatial orientation. Sensory dysfunction are likely to be induced by neurotoxic compounds, while at toxic doses reduced locomotor activity is a common finding. When using the 6-arm RTM in the course of neurotoxicity studies to assess cognitive functions such potentially confounding effects have to be excluded to allow the conclusion of a cognitive impairment. The aim of the following experiments was to assess if visual, vibrissal or olfactory dysfunction, or hypoactivity may affect memory parameters in the 6-arm RTM. Loss of visual, olfactory or vibrissal input alone did not affect any of the maze parameters. Combined loss of visual and vibrissal or olfactory inputs increased mean error score. Loss of olfaction prolonged trial time under both lighting conditions. Acrylamide- and vitamin B6-induced sensorymotor dysfunctions and hypoactivity did not affect mean error score or percent blind-alley visits in the 6-arm RTM. Similarly, gastrointestinal distress and hypoactivity induced by lithium chloride injections did not affect parameters of working or reference memory. Results indicate that in the 6-arm RTM (1) olfactory, visual and, to a minor extent, vibrissal input contribute to the sensory information necessary for spatial orientation and (2) reduced locomotor activity secondary to impaired sensorymotor abilities or drug-induced illness do not influence working or reference memory parameter.

Impaired tunnel-maze behavior in rats with sensory lesions: vestibular and auditory systems.

Maze behavior of rodents provides insight into processes of learning and memory and also serves to assess cognitive functions in neurotoxicity tests. Neurotoxic agents may impair maze behavior by acting upon different parts of the nervous system. To assess the dependence of maze learning upon vestibular and/or auditory input, the two systems were lesioned. Daily treatment of rat pups with streptomycin (400 mg/kg sc) on postnatal day 11 to 22 caused irreversible impairment of vestibular and auditory functions, whereas, 20 injections of neomycin in adult rats (100 mg/kg sc, postnatal weeks 8 to 11) led to hearing loss only. Hearing loss was assessed by absence of Preyer's reflex and impaired vestibular function by loss or shortened duration of postrotatory nystagmus. Learning in the unbaited 6-arm radial maze was tested at the age of 2 to 3 mon using a maze configuration that allowed to assess order of arm entries ("working memory") and left-right discrimination within each arm ("reference memory"). Treatment with streptomycin but not with neomycin led to impaired order of arm entries. Since treatment with streptomycin failed to induce any signs of brain lesions, impaired maze learning is considered to result from destruction of vestibular hair cell receptors with subsequent vestibular impairment and not from hearing loss or cognitive impairment.

Susceptibility of various areas of the nervous system of hens to TOCP-induced delayed neuropathy.

Sensitivity of in-life parameters, biochemical endpoints, and susceptibility of various areas of the chicken nervous system to delayed neuropathy induced by tri-orthocresyl phosphate (TOCP) was assessed. Groups of hens were exposed to a single oral dose of TOCP of 0, 50, 200 or 500 mg/kg and the animals observed for 21 days. Perfusion fixed, paraffin embedded tissue sections were stained with Bodian's silver and Luxol blue and semi-thin epoxy sections with toluidine blue. Sciatic and tibial nerves, lumbosacral, midthoracic, and upper cervical spinal cord, medulla oblongata and cerebellum were examined using a semiquantitative scoring system. In pair-dosed hens inhibition of brain and spinal cord neurotoxic esterase (NTE) and cholinesterase and of plasma and erythrocyte cholinesterases was determined 24 hr and 48 hr after administration. At all dose levels NTE in brain and spinal cord and plasma cholinesterase was inhibited markedly. Quantitative inhibition of NTE was seen also in absence of neuropathy. Ataxia and body weight loss occurred in high-dose animals only, while dose-related neuropathy was seen in the distal tibial nerve, medulla oblongata and cerebellum. Ataxia was correlated best with neuropathy in peripheral nerves while degeneration of nerve fibers in the cerebellum, seen best in mid-longitudinal sections, was the most sensitive histological indicator of TOCP-induced delayed neuropathy. The particular susceptibility of spinocerebellar neurons was recognized long ago, but often has been neglected in delayed neurotoxicity studies and respective guidelines. Optimal sensitivity of toxicity tests is a prerequisite for risk assessment, can be cost efficient, and nowadays should be a main interest of animal welfare in order to reduce animals' suffering. Based on these data, determination of NTE inhibition together with histopathological examination of longitudinal sections of distal tibial nerves, mid-longitudinal sections of rostral cerebellum and cross sections of upper cervical spinal cord represents an optimally sensitive and cost efficient test requirement.

The use of an unbaited tunnel maze in neurotoxicology: I. Trimethyltin-induced brain lesions.

In our laboratory we use an unbaited 6-arm radial tunnel maze (6-arm RTM) to assess working and reference memory in the course of neurotoxicity studies. The 6-arm RTM is believed to measure parameters comparable to those assessed in radial-arm mazes, but without the need of food deprivation and rewarding of animals. This is especially useful in the course of neurotoxicity studies as interferences of e.g. food deprivation with drug pharmacokinetics can be avoided. Since the 6-arm RTM is less evaluated than conventional mazes the aim of this study was to further confirm mean error score as measure of 'working memory', left-right discrimination within each radial arm (expressed as percent "blind-alley" visits) as measure of 'reference memory', and number of arm entries/min as a measure of motor activity. Therefore, hippocampal lesions were induced by injecting animals with the neurotoxicant trimethyltin (TMT). TMT at a dose of 5 mg/kg slightly lesioned hippocampal CA3 pyramidal cells in 3 of 8 animals, but did not affect behavioral measures in the 6-arm RTM. In all surviving animals treated with 7 or 9 mg/kg TMT moderate to marked loss of CA3 pyramidal cells was observed, while in 4 of these 7 rats CA4 pyramidal cells were also affected. Other brain lesions were not observed. TMT-induced brain lesions led to increased mean error score and number of arm visits during the retention phase and after changing maze configuration, whereas percent "blind-alley" visits were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of oxiracetam on learning and memory in animals: comparison with piracetam.

The effects of oxiracetam and piracetam were compared in learning and memory tests in rats and mice. In the dose range examined, the two nootropics were equally active in reducing the amnesia induced by cerebral electroshock in the mouse. Step-down retention performance, however, was distinctly improved by oxiracetam but unaffected by piracetam, no matter whether it was given before or immediately after the learning trial. Oxiracetam also improved acquisition performance in aged (24- to 27-month-old) rats in an active-avoidance situation at doses of 30 and 100 mg/kg i.p. whereas piracetam showed no effect at 100 mg/kg i.p.

Nicotine and amphetamine: differential tolerance and no cross-tolerance for ingestive effects.

Rats chronically treated twice daily with nicotine (0.4 mg/kg, SC) or d,1-amphetamine (1.5 mg/kg, SC) exhibited different patterns of anorexia, hypodipsia, and body weight loss. Amphetamine-treated rats developed tolerance to these ingestive effects and to weight loss, whereas nicotine-injected rats did not. There was little, if any, evidence for cross-tolerance when the drugs were switched between the two groups. These results indicate that different mechanisms underlie the ingestive effects of nicotine and amphetamine.

Evaluation of the neurotoxic potential of chemicals in animals.

This review provides a scientific view on how to evaluate effectively the neurotoxic potential of chemicals in order to provide adequate safeguards for human health. Detection of compounds that may cause direct, persistent, adverse effects on the nervous system should be given the most critical attention. Evaluation of the neurotoxic potential of a chemical should include descriptions of functional and morphological effects as well as the determination of the dose response, no-observed-effect level, time course and reversibility of effects. Evaluation of the nervous system in the context of standard toxicity studies that use a variety of species and study durations are appropriate screening tests (Tier 1) for the detection of potential neurotoxicity. Studies specifically designed to assess neurotoxicity (Tier 2) should be performed with chemicals for which there is an indication of neurotoxic potential and where the available data are inadequate for risk assessment. Tier 2 studies should evaluate the function and structure of the nervous system by comprehensive clinical examinations and neuropathological assessment. These studies may be conducted in conjunction with standard toxicity studies so that any potential neurotoxicity can be interpreted in the context of other systemic toxicity. More specific neurotoxicity tests (Tier 3) may be necessary for advanced characterization of discovered neurotoxicants.

Spongiform neuropathy induced in dogs by prolonged, low-level administration of 6-aminonicotinamide (6-ANA).

The objective of this study was to demonstrate the effects of prolonged exposure to 6-ANA at low dose-levels in dogs. A male and a female Beagle dog received daily oral repetitive doses of 1 mg/kg or less for 20 weeks. Both dogs showed lacrimation, conjunctivitis, reduced motility and anemia since the second week of treatment. The female dog was more affected than the male and at the end of treatment period it had tremor, hanging lower jaw, stepping gait of the hind limbs, hunched posture, and general debilitation. Post-mortem examination of the female dog revealed prominent brain edema with pressure atrophy of the dorsal cranial bones. Microscopic examination of the nervous system revealed spongiform neuropathy in both animals mainly affecting the telencephalic cortex and hippocampal fascia dentata, the substantia gelatinosa in the spinal cord and the dorsal root and autonomic ganglia. The changes were produced by vacuolation of astrocytes in the central nervous system and perineuronal satellite cells in the ganglia. Examination of the other organs revealed thymic atrophy and high hematopoietic activity of the bone marrow in both dogs. The male had severe interstitial edema and vacuolar degeneration of the testicular seminiferous tubules and the female had marked chronic pyelonephritis. This chemically induced spongiform neuropathy in dogs obviously represents a subchronic form of the "energy deprivation syndrome" induced by impaired glucose utilization. Vacuolar degeneration of the testicular seminiferous epithelium may have the same pathogenesis.

Optimal conduct of the neuropathology evaluation of organophosphorus induced delayed neuropathy in hens.

Susceptibility of various areas of the nervous system to TOCP (triorthocresyl phosphate) induced delayed neuropathy was assessed in groups of seven hens respectively, intoxicated with a single oral does of 500 or 1000 mg/kg body weight. 18 hens were used as negative controls. About 3 weeks after the treatment the hens were submitted to fixation by whole body perfusion and their nervous system processed either to paraffin sections stained with Bodian's silver stain and luxol counterstain, or to semi-thin plastic sections stained with toluidine blue. The examined areas were the cerebellum, the spinal cord at upper cervical, thoracic and lumbar level, the sciatic nerve, and the posterior tibial nerve. The extent of nerve fiber degeneration was assessed independently by two pathologists using a semiquantitative scoring system. The most susceptible areas were the cerebellum and the tibial nerve, followed by the upper cervical spinal cord. Within the cerebellum the nerve fibers in the rostral lobules, especially IV and Va, were affected. Whereas the resolution of plastic section was superior to that of paraffin sections in the cerebellum (mid-longitudinal level) and the spinal cord (transverse level), in the peripheral nerves the lesions were best recognized in the longitudinal, paraffin sections. There was very good agreement between both pathologists with respect to detection and grading of lesions in the most susceptible areas, but poor agreement in the areas of low susceptibility, indicating the danger of false results when lesions are not very distinct. In the susceptible areas the lesions induced with 500 mg/kg were sufficiently prominent, indicating that this dose-level is acceptable as positive control. In the hen nervous system, examination of the most susceptible areas, especially the rostral cerebellar lobules, appears to be suitable for detection of any kind of organophosphorus induced, delayed neuropathy.

Detecting necrotic neurons with fluoro-jade stain.

Fluoro-jade, a novel stain for detection of neuropathic lesions by fluorescence microscopy, was validated on the models of toxic neuropathy induced with 3-acetylpyridine (3-AP) or with acrylamide (ACR). Groups of male and female albino rats of Wistar strain were either exposed to a single administration of 80 mg/kg i.p. 3-AP followed 5 hours later by 300 mg/kg of nicotinamide i.p. and examined at days 3 and 15, or to 15 daily doses of 30 mg/kg p.o. ACR and examined at day 15. Following in-life behavioral observations and measurements, the rats were fixed by perfusion with formalin. Additional animals treated with same dose of 3-AP and nicotinamide were submitted to purposeful autolysis for 4 or 16 hours before immersion fixation with formalin on test day 3. In-life observations showed in 3-AP-treated animals signs of severe general toxicity, sensorimotor dysfunction and decreased motor activity starting shortly after the treatment and persisting throughout the observation period. ACR-treated rats started to develop abnormal gait on test day 8 and by day 15 developed reduced grip strength, increased landing footsplay and decreased motor activity. Fluoro-jade, applied to paraffin sections of the nervous system, detected selectively and sensitively the necrotic neurons in the brain, especially those in the inferior olivary nucleus of animals treated with 3-AP, at test day 3, as well as the necrotic Purkinje cells in the cerebellum of ACR-treated animals at test day 15. Chromatolytic neurons in the dorsal root ganglia of ACR-treated animals did not stain positively, indicating that this kind of reversible neuronal remodeling is not detectable using fluoro-jade. Necrotic neurons were still stained by fluoro-jade after 4 hour autolysis, but following 16 hour autolysis the results became false negative. There was no false positive fluorescence in fresh or autolytic tissues, except that emitted by red blood cells in unperfused specimens. The study confirmed the validity of fluoro-jade as a stain suitable for detecting necrotic neurons in toxicological safety studies.

Differential effects of orally versus parenterally administered qinghaosu derivative artemether in dogs.

Artemether (AM) is an antimalarial drug derived from artemisinin (Qinghaosu), an extract of the herb Artemisia annua L., sweet wormwood. Its antiparasitic effect is that of a schizontocide and is explained by rapid uptake by parasitized erythrocytes and interaction with a component of hemoglobin degradation resulting in formation of free radicals. It has been shown to exhibit a high clinical cure rate. Previous animal safety studies with Qinghaosu derivatives revealed dose-dependent neurotoxicity with movement disturbances and neuropathic changes in the hindbrain of intramuscularly treated dogs, rats and monkeys. Such effects have not been seen in man. The objective of our present studies was to compare the effects of high levels of AM administered to dogs p.o. versus i.m. In a pilot study 20 mg/kg/day of AM was given i.m. to groups of 3 male Beagle dogs for 5 and 30 days, respectively. Clinical signs of neurotoxicity were noted in some individual dogs from test day 23 on. One dog had to be sacrificed pre-term. Hematologic findings indicated a hypochromic, microcytic anemia. Microscopic examination demonstrated neuropathic changes only at 30 days, but not at 5 days. The animals had neuronal and secondary axonal damage, most prominent in the cerebellar roof, pontine and vestibular nuclei, and in the raphe/paralemniscal region. The affected neurons showed loss of Nissl substance, cytoplasmic eosinophilia, shrinkage of the nucleus and in advanced stages scavenging by microglia. In a subsequent experiment, AM was administered to groups of 4 male and 4 female dogs, respectively, at 8 daily doses of 0, 20, 40 and 80 mg/kg i.m., or 0, 50, 150 and 600 mg/kg p.o. Neurologic signs were seen at high i.m. doses only. In most animals they were inconspicuous and consisted of reduced activity with convulsions seen in single dogs shortly before death. Neuronal damage occurred in all animals at 40 and 80 mg/kg following i.m. treatment. At 20 mg/kg minimal effects occurred in 5/8 dogs only, indicating that this level was close to tolerated exposure. No comparable lesions were observed after oral administration. Both i.m. and p.o. exposure at high dose levels was associated with a prolongation of mean QT interval of ECG, suggesting slowing of repolarization of the myocardium. Individual data indicated that in 1 of 4 females at 80 mg/kg i.m. this prolongation was above the 25% level considered as threshold for concern. After intramuscular administration pharmacokinetics indicated peak plasma levels of AM at 2 to 4 hours post-dose, slow elimination and a tendency to accumulate after repeated administration. Only low levels of the major metabolite, dihydroartemisinin (DHA), were found. AM levels in the cerebrospinal fluid (CSF) were < 10% of plasma levels. After oral administration AM concentrations were considerably lower than after i.m. administration. The concentration of DHA was high on day 1 but almost nil on day 7 indicating its fast inactivation in dogs. Two hours after the 8th oral administration neither AM nor DHA was detected in CSF which may explain the absence of neurotoxicity in dogs after oral administration of AM.

Functional and morphological characterization of neuropathy induced with 5-lipoxygenase inhibitor CGS 21595.

Peripheral toxic neuropathy induced in rats with a 5-lipoxygenase inhibitor CGS 21,595 was characterized using special functional tests and pathological procedures. Functional tests included measurement of grip strength, landing foot splay, assessment of sensorimotor and autonomic functions and monitoring of motor activity. Pathological procedures consisted of perfusion fixation, embedding in plastic, teasing of isolated nerve fibers, and light and electron microscopy. Male and female albino rats received the test article orally by gavage on 5 days per week. To characterize the development of the lesion animals treated with 1000 mg/kg were examined and sacrificed at 2-week intervals until termination at 10 weeks. In a separate study, the dose-effect relationship was examined in groups of animals treated with 50,200 or 1000 mg/kg for 10 weeks. Neurotoxicity occurred only in animals treated with 1000 mg/kg and was first detected following 4 weeks of treatment. Although there were no overt clinical signs of neurotoxicity, functional examination detected a reduction of grip strength, increased landing foot splay and reduced motor activity. Neuropathological examination revealed peripheral segmental demyelination affecting predominantly the Schwann cells in the ventral spinal nerve roots. Owing to its unusual localization in the nervous system and to subtlety of functional signs, peripheral segmental demyelination represents a special diagnostic challenge in toxicological safety studies.

[The effect of nicotine on body weight and feeding behavior of rats]. / Nikotinwirkungen auf Körpergewicht und Fressverhalten der Ratte

Daily nicotine injections for 6 weeks produced a transient anorexia with a concomitant reduction of the body weight. Upon nicotine withdrawal, the rats developed a compensatory hyperphagia and gained nearly control weight.

[Effect of nicotine and amphetamine on body-weight regulation]. / Wirkung von Nikotin und Amphetamine auf die Regulation des Körpergewichts.

Twice daily c.c. injections of nicotine or amphetamine, for 6 weeks, produced a stabilization of the body weight at a level below that of the controls. This is attained by a transient hypophagia in normal animals and by a brief hyperphagia in animals emaciated prior to the treatment period. These effects confirm the hypothesis of Powley and Keesey (2) that the set-point for body weight is regulated, and not food intake.

Sex differences in perceiving analgesic drug effects as measured by subjective pain ratings: a concealed signal detection theory analysis.

In a study using metamizole and placebo as equivalents of a strong and a weak stimulus in a signal detection device female headaches patients show better ability to discriminate the two drug stimuli than males, whereas there is no significant effect with respect to response bias.