U. Schaeppi

Pyridoxine neuropathy: Correlation of functional tests and neuropathology in beagle dogs treated with large doses of vitamin B6

Neurologic examination, electrophysiologic testing and microscopic post-mortem examination was used to study the neuropathy induced in the beagle dog by administration of excessive amounts of vitamin B6.Two female dogs received repeated daily oral doses of 3 g. The treatment was ceased when the dogs developed severe general morbidity, including uncoordinated gait and abnormal neurologic symptoms. The symptoms were most severe during and early after cessation of treatment, and in general they regressed during the subsequent 3 months of treatment-free observation. Sensory central and peripheral maximum nerve conduction velocity started to decrease after a considerable delay; the decrease progressed until late after termination of treatment and failed to fully regress. Morphologic lesions were confined to large, first order sensory neurons.The neurologic examination thus revealed the early changes, while electrodiagnostics and microscopic neuropathology were indicators of more advanced stages of toxic neuropathy and disclosed selective lesions in individuals whose gait appeared to be unremarkable.

Validation of a photobeam system for assessment of motor activity in rats

To evaluate the validity and sensitivity of test procedures for routine assessment of chemically induced changes in motor activity in rats, studies were carried out with a commercially available photocell cage system. Testing of single, untreated rats showed that motor activity decreased within 10 min to a steady level. Overall activity was the same when testing was repeated at weekly intervals for 6 weeks. In groups of each 10 rats tested immediately after i.p. injection, chlorpromazine HCl (6 or 2 mg/kg) and physostigmine salicylate (0.5 mg/kg) decreased activity. d-Amphetamine sulfate (1.0 mg/kg) caused an increase of virtually all parameters, whereas scopolamine HCl (1.0 mg/kg) and physostigmine salicylate (0.02 mg/kg) led to an increase of particular aspects of motor activity only. Data generated with a mechanical calibrator varied 1-5% between the cages tested. It is concluded that the above procedures of recording of selected parameters of motor activity in groups of 10 rats for periods of up to 20 min would comply with the guidelines recommended by EPA TOSCA.

Validation of a radial maze test for assessing learning and memory in rats

Choice behavior of rats in a radially symmetrical 6-arm maze, without food reward, was validated for assessing changes in learning and memory following treatment with 4 psychoactive agents. The test is designed for future use in routine toxicity studies with laboratory rodents. Each radial main arm of the maze leads to a T-shaped choice-point with a blind alley on the left and a long angled alley on the right. Order of choice of the radial main arms served to score within-session working memory, by evaluating relative recency of arm reentries. The choice between blind alley and long-angled alley at the T-intersections provided a measure of between-session reference memory. Maze behavior as an indicator for impairment in learning and memory was validated by testing rats treated with d-amphetamine, chlorpromazine, scopolamine and physostigmine. Based on the above evaluations, working memory was found to be severely impaired by 0.3 and 1.0 mg/kg scopolamine, and reference memory to be improved by 0.02 mg/kg physostigmine and 1.0 mg/kg amphetamine. Locomotor activity, in terms of the total number of arm choices per test session, was altered by all substances as expected from previous reports in the literature. The test appears to be a valid and sensitive method for assessing learning and memory in the rat without the use of food reward, and thus well suited for implementation in routine toxicity studies with rodents.

Differential vulnerability of 3 rapidly conducting somato-sensory pathways in the dog with vitamin B6 neuropathy

In anesthetized dogs with chronically implanted cortical electrodes somatic sensory-evoked potentials (SEPs) were produced by electrical stimulation at neural, muscular or cutaneous sites of the contralateral hind leg. Stimulation of the tibial nerve at the calcaneus or of the short flexor muscles of the hind paw caused SEPs having characteristics following activation of rapidly conducting afferents from muscle spindles. Stimulation of the glabrous skin of the central pad resulted in SEPs arriving after a more protracted latency evidently related to activation of afferents from Merkel cells, Krause and Pacinian corpuscles known to be located at these sites. Stimulation of the hairy skin from the dorsal surface of the hindpaw produced a further type of SEP presumably resulting from activation of afferents from receptors of tylotrich hair follicles.Vitamin B6-induced neuropathy involves the selective degeneration of the largest neurons in the spinal ganglia and of associated long peripheral and central neurites performing rapid impulse transmission. In the course of vitamin B6 neuropathy the relatively slow impulse transmission following stimulation of the central pad was more severely impaired than the faster one after activation of afferents from muscle spindles or receptors from hair follicles. This allows us to conclude that in the dog afferents from the glabrous skin of the central pad conduct centrally via the dorsal columns, susceptible to vitamin B6 intoxication, while muscle and hair receptor afferents ascend in the dorsal spinocerebellar and spinocervical tract, respectively, which are vitamin B6 resistant.

Procedures for routine clinical electroretinography (ERG) in dogs.

Following pupillary dilatation and immobilization of the dog with a cataleptic drug (I-Polamivet, Hoechst) the electroretinogram (ERG) was performed with the technique of Ganzfeld stimulation. The head of the dog was kept within a sphere of 60 cm diameter, the white inner surface of which could be indirectly illuminated with a stroboscope producing light flashes of 10 μsec duration. The ERG was recorded oscillographically by means of modified contact lenses. Dogs were tested for rod and cone function with luminance curves including white, blue and red stimuli and with trains of repetitive photic stimuli. Tests were performed under dark and light adaptation. The ERG of the dark-adapted dog, an indicator for the electrical activity of the rod system, was similar with that of man with respect to configuration and other characteristics. The electrical activity of the cone system was different from that of man by having a 10-fold lower sensitivity and a reduced capability for discrimination of red versus blue or white stimuli.

Electroretinography in rats

Basic mechanisms of the rat ERG were reinvestigated with contemporary methods of recording and photic stimulation via an optic fiber system connected with a contact lens. Flash stimulation and background illuminance were performed with photometrically defined light stimuli. ERGs recorded in darkness, from dark adapted rats, were similar with those observed in earlier work in which light flashes of much longer duration had been used. Flash stimulation carried out under stepwise increased background illuminance gave new information on the characteristics of oscillatory potentials of the ERG. In general the present observations agreed with the notion that the rods of the rat retina are as sensitive as in man, whereas the cones are functionally less efficient with respect to light sensitivity and temporal resolution. Differences in function of the cones from rat to man have to be kept in mind when using the rat ERG as a model for risk assessment in safety studies.

What can specific behavioural testing procedures contribute to the assessment of neurotoxicity in laboratory animals

Subchronic testing of laboratory animals in accordance with present regulatory guidelines involves maximum exposure with the chemical under investigation and serves for the evaluation of systemic toxicity as well as of lesions in organs and organ systems, including neurotoxicity. The primary assessment of neurotoxicity is essentially based on the overall observation of animal behaviour in the course of the customary toxicity studies and on the subsequent neuropathological evaluation with contemporary techniques. Under this maximum exposure the absence of symptoms and signs of neural abnormalities indicates that the material testd would be devoid of neurotoxicity.Any overt or suspicious symptoms for neurotoxicity appearing in the course of subchronic testing may be further characterized with additional functional tests such as neurological examination, electrodiagnostics and possibly with specific behavioural tests. The subsequent neuropathological investigation would have to be expanded to include a detailed evaluation of all neural structures possibly related with the above functional derangements.In case of relevant neurotoxicity subsequent specific behavioural tests might include the evaluation of complex neural functions such as integrated psycho-neuro-motor activity and memory. These behavioural tests might help to explain neurotoxicity and to assess behaviour at low levels of exposure. The implementation of such specific behavioural testing procedures beyond the scope of routine toxicity studies would require a group of investigators capable to earry out appropriate tests. The introduction of such testing will therefore have to be evaluated competitively regarding costs versus the advantages offered by the refinement of the other testing procedures for neurotoxicity including the neuropathological techniques. The performance of behavioural tests without preceding or concomitant toxicological evaluation is not considered to be a feasible approach.

Sensory and motor maximum nerve conduction velocity in the peripheral and central nervous system of the beagle dog

Sensory maximum nerve conduction velocity (MNCV) and motor MNCV were monitored in altogether 14 beagle dogs anaesthetized with pentobarbital. Sensory MNCV was determined by averaging cortically evoked potentials from somatosensory area I (SS I) in response to repeated electrical stimulation of digital paw pads, tibial nerve at calcaneus or sciatic nerve at trochanter.Sensory MNCV determined from paw to tibial nerve at calcaneous was 53 m/sec, from tibial nerve at calcaneus to sciatic nerve at trochanter 64 m/sec and from sciatic nerve at trochanter to cortex SS I 53 m/sec. Motor peripheral MNCV determined in the customary way from sciatic nerve at trochanter to tibial nerve at calcaneus was 68 m/sec and distal latency 3.6 msec. Motor central MNCV from motor cortex to the sciatic nerve at the trochanter in 5 unanaesthetized dogs was 57 m/sec.These testing procedures serve for quantitative assessment of possible impairment of impulse transmission in the central and peripheral sensory and motor pathway of beagle dogs used in routine toxicity studies.

Rod and cone components in the compound ERG of the beagle dog

In the dog ERG flash-induced activation of the rod receptors can be selectively investigated due to relatively poor photic excitability of the cone component.The ERG was produced in immobilized dogs using the method of Ganzfeld stimulation with white light flashes of 10 μsec duration. Relatively dim flashes ranging to about 100-fold above ERG threshold lead in the dark-adapted dog to selective activation of the rod receptors. This scotopic rod ERG consists of ERG positivity including ab wave with superimposed oscillations followed by a protracted potential, presumably thec wave.Light flashes exceeding 100-fold threshold luminance cause simultaneous activation of rod and cone receptors. ERG characteristics include appearance of an early negativity, thea wave, followed byb andc waves of reduced amplitudes.In the presence of a bright background luminance of 33 ftL bright light flashes cause an ERG resulting from selective cone activation. This photopic cone ERG consists of a smalla wave followed by a smallb wave with oscillatory potentials and by marked subsequent late negativity. With increase in background luminance the late negativity increases in amplitude and occurs progressively earlier.The characteristics of the dog ERG allow selective assessment of functional changes in rod and cone receptors.

Single fiber electromyography. A method for the evaluation of motor axonopathy during toxicity studies in dogs

In man, single fiber electromyography is used as a very sensitive indicator for the assessment of functional changes in motor nerves. The purpose of the present study was to adapt the above testing procedure to allow repeated investigations of dogs used in subchronic toxicity tests.Experiments were performed on anesthetized purebred beagle dogs. Action potentials from single muscle fibers in response to electrical stimulation of motor nerves were recorded with Medelec microelectrodes, amplified with a Medelec system and monitored on a Tektronix oscilloscope. Repeated clectrical stimulation with pulses of 0.03 msec and 1 p.p.s. produced characteristic action potentials of single muscle fibers consisting of a positive, followed by a negative, deflection having a duration of from 500 to 800 μsec altogether. Successive potentials occurred with a variable latency (the jitter) ranging from±5 to 15 μsec.Quantitative evaluation of the jitter will allow the clinical evaluation of motor axonopathies in dogs.