Otto L. Wolthuis

Behavioral performance, brain histology, and EEG sequela after immediate combined atropine/diazepam treatment of soman-intoxicated rats

It is known that rats poisoned with near-lethal doses of pinacolyl methylphosphonofluoridate (soman) develop brain lesions, particularly when convulsions are induced. When rats were intoxicated with a LD50 of soman and treated immediately thereafter with a combination of low doses of atropine and diazepam (LOW AS/DZ treatment), large decrements in performance of an earlier acquired shuttle-box task were found 6 days after intoxication. In contrast, no such decrements were found in soman-intoxicated animals treated similarly with a combination of high doses of these drugs (HIGH AS/DZ treatment). Surprisingly, surviving LOW AS/DZ animals acquired the same task again at a speed that was almost as fast as before intoxication. Similarly treated animals were examined light-microscopically 24 h after intoxication; in LOW-AS/DZ-treated animals, neuropathology was only observed in animals that had exhibited convulsions, whereas in HIGH AS/DZ animals neither convulsions nor brain damage were observed. Power spectra, obtained from electroencephalograms (EEGs) 6 days after intoxication, revealed significant differences between both treatment groups, particularly in the delta-, theta-, and beta-frequencies. After the HIGH AS/DZ treatment, a significant increase in delta activity was found compared to control values, suggestive of neuropathology. It is concluded that, in contrast with the LOW AS/DZ combination, HIGH AS/DZ prevents active avoidance deficits, convulsions, and light-microscopically detectable neuropathology after soman intoxication. However, the results of EEG measurements suggest that some aberrations may still remain even after the HIGH AS/DZ treatment.

The efficacy of some bis-pyridinium oximes as antidotes to soman in isolated muscles of several species including man

Previous results had shown that bis-pyridinium oximes, particularly HI-6 are quite effective therapeutically in soman-poisoned rats and mice in vivo and in the rat diaphragm preparation in vitro. The aim of the present study was to investigate the efficacy of bis-pyridinium oximes on soman-inhibited neuromuscular transmission in muscle preparations from several species including man. The muscles tested were preparations of rat diaphragm and intercostal muscle, guinea-pig diaphragm, dog external intercostal muscle and human external interscotal muscle. These muscles were stimulated indirectly with field stimulation. With a few exceptions the preparations were exposed to soman for 2.5 or 15 min. In some cases different exposure times were employed or the organophosphate sarin was administered instead of its analogue soman. After the degree of inhibition of neuromuscular transmission had been established, oximes were added to the bath fluid. After washout 15 min later, recovery of neuromuscular transmission was tested. Subsequently, a second dose of soman was administered to investigate whether the recovery observed had been caused by cholinesterase reactivation. The results of these experiments indicate that the oximes tested, mostly HI-6, were quite effective as soman antidotes in muscle preparations of rats, guinea-pigs and dogs. In the human preparation while these oximes were quite effective after sarin intoxication they were essentially without effect against soman.

Therapeutic efficacy of HI-6 in soman-poisoned marmoset monkeys.

The therapeutic efficacy of the oxime HI-6 against intoxication with the irreversible cholinesterase (ChE) inhibitor soman was tested in marmoset monkeys. Five out of six marmosets, intoxicated with 5 x LD50 soman and treated immediately with diazepam (0.2 mg.kg-1 iv) and 15 sec later with atropine (0.5 mg.kg-1 im) and HI-6 (50 mg.kg-1 im), survived for more than 24 hr. One of these animals died after 4 days. In the HI-6-treated marmosets blood ChE activity was inhibited at a rate slower than that in three animals treated similarly but with saline instead of HI-6. The latter marmosets died within 8 min after soman. HI-6 achieved its plasma peak 5 min after injection and was eliminated with a t1/2 of about 40 min. In a second experiment similarly treated marmosets were euthanized at 5 min (three saline-treated animals) or at 10 min (three HI-6-treated animals) after the soman intoxication to enable the determination of acetylcholinesterase (AChE) activities in diaphragm and brain tissue. In addition, in these animals blood AChE and butyrylcholine esterase (BuChE) activities were determined. Low AChE activities were encountered in diaphragms and brains. These levels were not significantly different between saline- and HI-6-treated marmosets. In vitro treatment with HI-6 at 40 min after soman still led to an increase of the AChE activity, which was significant in diaphragm, suggesting that postmortem AChE inhibition had occurred. The ratio of AChE to BuChE in blood was significantly enhanced in HI-6-treated animals, indicating that HI-6 preferentially reactivated AChE. It is concluded that (i) HI-6 is an effective treatment against soman poisoning in marmosets and (ii) AChE reactivation or protection by HI-6 contributed to the survival of the animals.

Evidence for an intramuscular depot of the cholinesterase inhibitor soman in the rat.

Isolated rat diaphragms were treated with the oxime HI-6, 25 min after the start of soman exposure for 5, 15, 20 or 25 min. Recovery of neuromuscular transmission (NMT) was smaller and the subsequent spontaneous failure of NMT greater when soman exposure was longer. Diaphragms taken from anaesthetized atropinized soman-poisoned rats treated with HI-6 again showed spontaneous failure of NMT when tested repeatedly in vitro. In vivo pretreatment with a soman simulator prevented reinhibition in vitro. The results are indicative of a simulator-sensitive soman depot in muscles.

The effect of ACTH-analogues on motor behavior and visual evoked responses in rats

Averaged visual evoked responses (VER) in cortical area 17 were recorded one hour after the administration of 7-l-phe ACTH4-10 or 7-d-phe ACTH4-10 to artificially ventilated rats, paralysed with gallamine. In addition, the effects of these peptides on spontaneous motor behavior were analysed. The results show that the latencies of all VER components remain unchanged and the amplitudes of the primary VER were unaffected. Measured at a wide variety of light intensities, however, the amplitudes of the VER afterdischarge were significantly and very similarly diminished by both peptides, the effect of 7-l-phe ACTH4-10 being somewhat stronger than that of 7-d-phe ACTH4-10. These results support the notion, advanced by others, that these peptides have an effect on a CNS vigilance regulating system, yet do not explain the reported opposite effects on active avoidance behavior of the two related peptides. The effects appear specific since spontaneous motor behavior, as index of changes in generalised arousal, is unaffected by these two peptides.

Automatically determined effects of lithium, scopolamine and methamphetamine on motor activity of rats

A device is described in which spontaneous motor activity of animals can be measured quantitatively in a capacitance field. By using multilevel detection in a homogeneous field the motions of the animals can be categorised according to their amplitudes and irrespective of the position of the animal in the cage. The effects of lithium ions, methamphetamine and scopolamine were assessed and compared with data in the literature.

Side effects of physostigmine as a pretreatment in guinea pigs

To prevent incapacitation following nerve agent intoxications, it is proposed to replace pyridostigmine by the centrally active carbamate physostigmine (PHY). Behavioral and neurophysiological effects of PHY were determined and whether these effects would be counteracted by scopolamine. In addition, we compared them with the effects of another reversible cholinesterase (ChE) inhibitor ethyl-p-nitrophenylphosphoramidate (PNF) At similar levels of blood AChE inhibition, PHY caused a larger shuttlebox performance decrement than PNF, which was antagonized by scopolamine (0.1 mg/kg). SCO enhanced the PHY-induced increase of the auditory startle response, whereas PNF, with or without scopolamine, had no effect. In the EEG, PHY led to a power increase at the theta 2-alpha 1 band, also found after PNF, and at the theta 1 band. SCO antagonized all EEG effects, but not the effects of PHY on visual evoked responses, in contrast to those of PNF. Based on the different effects of both inhibitors, it is suggested that at relevant doses several PHY-induced phenomena occur that are unrelated to AChE inhibition.

On the development of behavioral tolerance to organophosphates. I: Behavioral and biochemical aspects.

The development of tolerance to organophosphates (OPs) was investigated by SC injections of saline and sublethal doses of DFP or soman three times per week or every other day for at least 4 weeks. Shuttlebox performance was tested 1 hr and 24 hr after the injections. Notwithstanding a progressive inhibition of AChE to very low values in various organs, shuttlebox performance was virtually normal 24 hr after the OP injections. However, whereas the performance decrements measured 1 h after the injection of DFP practically disappeared in the course of weeks, the decrements 1 hr after soman remained approximately the same. These differences between the effects of DFP and soman cannot be explained: 1) by differences in inhibition or de novo synthesis of AChE in various regions of the CNS, the striated muscle or blood, 2) by differences in the reductions of the muscarinic receptors in various regions of the CNS, 3) by differences in the number of nicotinic receptors in the diaphragm muscle, or 4) by differences in phosphorylphosphatase (DFP-ase or somanase) activity in blood plasma or liver.

Automated TV-based system for open field studies: Effects of methamphetamine

A method is described whereby open field behaviour of rats can be automatically registered using a TV camera, a video converter, an X-Y recorder and a papertape puncher. Use is made of the scanning properties of the TV camera to obtain the X and Y coordinates of the rats position and to print this position on an X-Y recorder to obtain the running pattern. In addition, the X and Y coordinates at 1 sec intervals are punched on papertape. With computer processing of the tape, one can obtain--for any given period--the distance run, a frequency distribution of speeds, the number of entries into an inner field, the time spent in an inner field as well as the number of changes in corner positions. As an example the effects of 1 and 2 mg/kg methamphetamine are shown. This drug enhances all parameters measured in a dose-dependent fashion except the changes in corner positions which were not altered significantly.

The isolated blood-perfused pig ear: an inexpensive and animal-saving model for skin penetration studies☆

To overcome most of the disadvantages of current models to investigate percutaneous penetration of drugs or toxic substances, a model is proposed here based on the isolated pig ear, which is obtained at the slaughterhouse, and perfused with oxygenated blood from the same pig. To determine the viability of the preparations, we measured glucose consumption and lactate production as metabolic parameters, Na+ and K+ ions, as well as lactate dehydrogenase activity in blood as markers for cell damage, whereas vasomotor reactivity was assessed by administering noradrenaline and isoxsuprine. After 60 min of equilibration, only insignificant changes in these parameters were observed during the subsequent 3-hr test period (longer periods were not tested). A slight weight increase was noted during the total period 4 hr, presumably due to slight edema formation. On the basis of several types of measurements, such as in vivo blood flow and ear temperature and in vitro glucose metabolism, standard procedures were developed. It is concluded that this technique offers an easy to handle, cost-efficient, and animal-saving model for skin penetration studies that lacks most of the disadvantages of existing models.