Multiple vaccine and pyridostigmine interactions: effects on EEG and sleep in the common marmoset.

Following active service during the 1990/1991 Gulf conflict, a number of UK and US veterans presented with a diverse range of symptoms, collectively known as Gulf Veterans' Illnesses (GVI). The administration of vaccines and/or the pretreatment against possible nerve agent poisoning, pyridostigmine bromide (PB), given to Armed Forces personnel during the Gulf conflict has been implicated as a possible factor in the aetiology of these illnesses. The possibility that long-term health effects may result from the administration of these vaccines (anthrax, pertussis, plague, yellow fever, polio, typhoid, tetanus, hepatitis B, meningococcal meningitis and cholera) and/or PB, have been investigated using a non-human primate model, the common marmoset. This paper reports the results from two aspects of the study, brain electrical activity (EEG, collected during performance of a touchscreen mediated discrimination task) and sleep. There were no marked long-term changes in EEG or sleep patterns that could be attributed to vaccines and/or PB administration. The changes that were detected were predominantly time related and independent of treatment. Where statistical differences were detected between treatments, the magnitudes of the difference were relatively minor and therefore not regarded as having long term biological significance.

Multiple vaccine and pyridostigmine interactions: effects on cognition, muscle function and health outcomes in marmosets.

Following active service during the 1990/1991 Gulf Conflict, a number of UK and US veterans presented with a diverse range of symptoms, collectively known as Gulf Veterans Illnesses (GVI). The administration of vaccines and/or the pretreatment against possible nerve agent poisoning, pyridostigmine bromide (PB), given to armed forces personnel during the Gulf Conflict has been implicated as a possible factor in the aetiology of these illnesses. The possibility that long-term health effects may result from the administration of these vaccines (anthrax, pertussis, plague, yellow fever, polio, typhoid, tetanus, hepatitis B, meningococcal meningitis and cholera) and/or PB, have been investigated using a non-human primate model, the common marmoset. This paper reports the results from three aspects of the study, cognitive behaviour (performance of a touchscreen mediated discrimination task), muscle function (performance of a simple strength test) and general health. There were no marked long-term changes in cognition, muscle function or health that could be attributed to vaccines and/or PB administration. Statistical differences related to treatments were only observed in two aspects of cognition and one of clinical chemistry. These changes were transient in nature and their magnitude were minor and, in consequence, was not regarded as having long-term biological significance.

A long-term study of the effects of diazinon on sleep, the electrocorticogram and cognitive behaviour in common marmosets.

The long-term sequelae of exposure to low doses of organophosphate compounds are ill defined, with effects variously reported on a range of indices of central nervous system functions such as sleep, cognitive performance and electroencephalogram (EEG). These indices were examined in common marmosets exposed to a range of doses of the organophosphorous sheep dip, diazinon. Cognitive performance was assessed by means of elements from the Cambridge Neuropsychological Test Automated Battery (CANTAB), and radiotelemetry techniques were employed to monitor the electrocorticogram and sleep patterns. Data were collected for 12 months following intramuscular administration of a single dose of diazinon (10, 90 or 130 mg.kg (-1)) or vehicle. Although high levels of erythrocyte acetylcholinesterase (AChE) inhibition (up to 82%) and short-term changes in sleep patterns were seen, there was no evidence of biologically significant long-term changes in any measures. The effects of multiple exposures, impurities or mixtures of OP compounds remain to be investigated.

A novel method for activity monitoring in small non-human primates.

Patterns of spontaneous activity are valuable reflections of well-being in animals and humans and, because of this, investigations have frequently incorporated some form of activity monitoring into their studies. It is widely believed that activity monitoring, alongside assessments of general behaviour, should be included in initial CNS safety pharmacology screening. As the number of marmoset studies having actimetry as their focus, or as an adjunct, is increasing, we wished to evaluate an alternative approach to those commonly used. The method is based on miniaturized accelerometer technologies, currently used for human activity monitoring.Actiwatch-Minis were used to monitor the activity of two groups of differently housed marmosets for 14 consecutive days. Group A consisted of four mixed-sex pairs of animals and group B comprised eight group-housed males. Activity profiles were generated for weekday and weekend periods. The devices captured quantifiable data which showed differences in total activity between the two differently housed groups and revealed intragroup variations in the temporal spread of activity between weekdays and weekends. The Actiwatch-Mini has been shown to generate retrospective, data-logged activity counts recorded from multiple animals in a single arena by means of non-invasive monitoring.

A simple method for assessing muscle function in common marmosets.

A novel method of assessing muscle function in the common marmoset was developed as part of a multidisciplinary long-term study. The method involved home cage presentation of a weight-pulling task. Over a 4-5 month period, 38 of 42 animals were successfully trained to displace weights of up to 920 g (mean 612+/-20 g). Performance, following initial training, was stable and independent of gender or body weight.

Assessment of a combination of physostigmine and scopolamine as pretreatment against the behavioural effects of organophosphates in the common marmoset (Callithrix jacchus).

RATIONALE: There is a requirement to ensure that UK armed forces are provided with the best possible medical countermeasures to prevent or mitigate the effects of exposure to nerve agents. When pretreatments are under consideration, it is of particular importance to ensure that they do not in themselves give rise to adverse effects and do not exacerbate the effects of agent exposure. OBJECTIVES: The present study was designed to address these considerations for a combination of physostigmine and scopolamine as a potential pretreatment regimen. METHODS: Common marmosets were trained to perform a two-choice discrimination serial reversal task, and baseline data were collected. Subjects received a dose of either soman or sarin after 2 weeks of pretreatment with either saline or physostigmine and scopolamine via miniosmotic pump. RESULTS: No effects of physostigmine and scopolamine were seen on task accuracy or response rates. Neither accuracy of reversal performance nor number of responses made were significantly changed by administration of either soman or sarin subsequent to pretreatment with physostigmine/scopolamine. In the groups pretreated with saline, performance of the behavioural task, in terms of responses made, was virtually abolished on the day the OP was administered, but a significant increase in accuracy of performance was seen over the 2- to 14-day period following administration. CONCLUSIONS: A combination of physostigmine and scopolamine, which is known to protect against nerve-agent lethality, offers protection against the effects of soman and sarin on behavioural performance, as measured by a discrimination reversal task. The improved performance observed following nerve agent requires further investigation.

Investigation of the sleep electrocorticogram of the common marmoset (Callithrix jacchus) using radiotelemetry.

OBJECTIVE: To evaluate the use of a totally implantable radiotelemetry system for recording the sleep electrocorticogram (ECoG) of a small new world primate, the common marmoset (Callithrix jacchus) without restraint during data collection. METHODS: Under anaesthesia a telemetry transmitter, which allowed the recording of a single ECoG channel, was implanted intraperitoneally. This system allowed ECoG data to be recorded overnight from animals living in pairs within their habitual laboratory environment over a period of 12 months. Data were subsequently scored using modified Rechtschaffen and Kales criteria (A manual of standardized terminology, techniques and scoring system for sleep stages of human subjects. Los Angeles, UCLA Brain Information Service/Brain Research Institute, 1968) into stages of waking, light sleep, deep sleep and probable rapid eye movement sleep (pREM). Concurrent video recording was used to assist in the categorising of pREM. RESULTS: Results showed that, as in man, the marmoset exhibits sleep cycles with stages alternating between non-REM (deep sleep and light sleep) and pREM sleep throughout the night. In common with other non-human primates the duration of each of the sleep stages was relatively short and punctuated with frequent waking. CONCLUSIONS: These data suggest that sleep in marmosets housed under laboratory conditions (a) can be recorded without restraint and (b) has potential to be used as a model for human sleep.

The effects of acutely administered low dose sarin on cognitive behaviour and the electroencephalogram in the common marmoset.

Previous studies have suggested that administration of a clinically sign-free dose of sarin to non-human primates gives rise to subtle changes in brain electrical activity as measured by electroencephalography (EEG) several months following exposure. The functional significances of such changes are unclear. The present study monitored EEG by using implantable radiotelemetry, and also assessed the performance of complex behavioural tasks, in non-human primates for up to 15 months following exposure to a low dose of sarin. Baselines of EEG and behaviour were shown to be stable over several months in control animals. The doses of sarin administered caused erythrocyte cholinesterase inhibitions of 36.4% to 67.1%. Overall, no significant changes in EEG patterns were observed although there were increases in beta 2 amplitude which approached significance (p=0.07). No deleterious effects on performance were seen on the touchscreen mediated discrimination tasks presented from the Cambridge Neuropsychological Test Automated Battery (CANTAB). This study illustrates the validity of the approach employed and makes an important contribution to the investigation of the long-term effects of organophosphorous compounds.

Home cage presentation of complex discrimination tasks to marmosets and rhesus monkeys.

The study reported here demonstrates the feasibility of presenting cognitive tests from the Cambridge Neuropsychological Test Automated Battery (CANTAB) to either marmosets or rhesus monkeys in their home cages. This location of testing offers opportunities for the measurement of additional indices, for example spontaneous behaviour (Prowse et al. 1995) and electrophysiology (Pearce et al. 1998) as well as facilitating repeated test presentation. Results from 12 marmosets and 4 rhesus monkeys which have completed several sequences of an eight-stage discrimination task involving simple discriminations, compound discriminations and reversals are reported. The paradigm developed has application in long-term studies. Tests from CANTAB have been used extensively in normal humans (Robbins et al. 1994) as well as a range of patient groups (Owen et al. 1992, Elliott et al. 1995) and to assess drug effects (Coull et al. 1996). Additionally some of these tests have been presented to marmosets (Roberts et al. 1988) to examine neuropsychological functioning. This comparative approach facilitates meaningful cross species comparison, particularly in the study of the effects of pharmacological intervention.

Concurrent monitoring of EEG and performance in the common marmoset: a methodological approach.

A model has been developed in a nonhuman primate, the common marmoset (Callithrix jacchus), which should enable the study of long term effects of compounds with potentially psychoactive properties. The technique facilitates concurrent monitoring of both behavioral and electrophysiological parameters while animals remain in their home cages. Subjects were trained to perform tests from a neuropsychological test battery (The Cambridge Neuropsychological Test Automated Battery, CANTAB) in which they learned to discriminate between pairs of stimuli presented on a touch sensitive computer screen. Single channel cortical electroencephalography (EEG) by radiotelemetry was simultaneously recorded while behavioral testing took place.

Protection from high pressure induced hyperexcitability by the AMPA/kainate receptor antagonists GYKI 52466 and LY 293558.

The neurophysiological effects of 2 novel AMPA/kainate receptor antagonists, GYKI 52466 and LY 293558, on the high pressure neurological syndrome have been investigated in the rat and baboon (GYKI 52466) and rat (LY 293558). Rats were exposed to increasing ambient pressures of helium and oxygen at 3 ATA/min, on one occasion each. GYKI 52466 at 20 mumol/kg i.v. immediately before, followed by 70 mumol/kg/hr i.v. during compression delayed tremor by 85% and myoclonus by 30%, compared with control vehicle, and no side effects were observed. Seizure activity was not affected by any of the doses used. LY 293558 at 36 mumol/kg i.p. delayed tremor and myoclonus (44% and 12%), LY 293558 72 mumol/kg additionally delayed seizure activity (21%). Side effects, principally tranquilization at the higher dose, were also noted. Six baboons were exposed to a maximum pressure of 91 ATA at 0.3 ATA/min, in the same environment, on two occasions. One exposure was treated with an i.v. infusion of GYKI 52466 15.2 mumol/kg/hr, the other with the same volume of control vehicle. Limb and face tremor and myoclonus were delayed and the severity of signs reduced. No seizures were observed in the drug treated group before 91 ATA. EEG changes associated with exposure to pressure were not affected. It is concluded that antagonism at the AMPA/kainate receptor by GYKI 52466 and LY 293558 beneficially alters HPNS signs but in a manner which is dependent on both the drug and species being studied.

Lack of effect of lamotrigine against HPNS in rodent and primate models.

The neurophysiological effects of the novel anticonvulsant lamotrigine on the high pressure neurological syndrome, HPNS, were investigated in the rat and nonhuman primate Papio anubis. Rats were exposed to pressure at a rate of 3 ATA per min in a helium/oxygen environment. They were pretreated with either lamotrigine isethionate 15, 30, or 60 mg/kg IP or control vehicle. After 15 and 30 mg/kg there were no changes in onset pressures for any of the grades of tremor or myoclonus. After 60 mg/kg, tremor was much slower, at 7-9 Hz, than the 15-20 Hz seen in controls. Four baboons were exposed to pressure at 0.33 ATA per min in the same environment and treated with lamotrigine isethionate at 7.5 mg/kg/h i.v. Each animal underwent a control and a drug-treated exposure. No changes in the onset or severity of HPNS behavioural signs were observed. However, an increase in alpha wave amplitude of the EEG was almost prevented. In both species sustained myoclonic jerking occurred at pressures similar to those at which seizure activity was observed in control exposures. It is concluded that although lamotrigine is protective in several models of neuronal excitation, it is ineffective in protecting against behavioural signs associated with high atmospheric pressure.

The orally active NMDA receptor antagonist CGP 39551 ameliorates the high pressure neurological syndrome in Papio anubis.

The neurophysiological effects of a novel, orally active, competitive N-methyl-D-aspartate (NMDA) receptor antagonist (DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid ethyl ester), CGP 39551, on the high pressure neurological syndrome (HPNS) were investigated in the non-human primate Papio anubis. Six animals were exposed to maximum pressures of 81 ATA in a helium and oxygen environment, on two occasions. One exposure was pretreated orally with CGP 39551 100 mg/kg 24 h before compression, the other pretreated with an equivalent volume of vehicle, in this case water. CGP 39551 significantly ameliorated the signs of HPNS, compared with controls, at pressures above 31 ATA and prevented the severe signs from occurring at the higher pressures. Onset pressures of the mild signs at low pressures were, however, unaffected. Among EEG changes, the pressure induced reduction in delta wave amplitude was prevented by CGP 39551, but the increase in the amplitude of the 7-9 Hz band was not. It is concluded that CGP 39551 may play an important role in the prophylactic treatment of HPNS.

Experimental pathology of intravenous polyurethane cannulae containing disinfectant.

Cannula tubing (1.6 mm external, 1 mm internal diameter) manufactured from medical grade polyurethane containing 2%, 2,4,4'-tri-chloro-2'-hydroxydiphenylether ('Irgasan', Ciba-Geigy) was found to have no effect other than that seen with control ('Irgasan'-free) tubing in the following test systems: (i) haemolysis, (ii) endothelial cell cultures, (iii) paravertebral muscle of rabbits, (iv) jugular vein of rabbits, (v) cannulation of baboons and (vi) clotting times of human platelet-rich plasma. However, the results from (iv) showed a significant amount of damage from both inpregnated and control cannulae and (v) showed that all detectable 'Irgasan' had been eluted from the portions of tubing retained within the animal before the end of the experiment, more rapidly than predicted from in-vitro studies. The rate of elution of 'Irgasan' in vivo needs to be further investigated, and consideration should be given to developing a plastic-disinfectant combination with a slower rate of loss of disinfectant.

Interactions of the beta carboline abecarnil with the high pressure neurological syndrome in a primate model.

The neurophysiological interactions between the high pressure neurological syndrome (HPNS) and a new beta carboline, abecarnil, were studied in the non-human primate Papio anubis. Abecarnil is a partial agonist at the benzodiazepine site on the GABA/benzodiazepine receptor. Six animals were exposed on two occasions to pressures of 91 ATA in an environment of helium and oxygen. One exposure was pretreated with a total dose of abecarnil 1.0 mg/kg, the other with an equivalent volume of vehicle. Treatment with abecarnil prevented the severe signs of HPNS occurring between 51 and 91 ATA. Onset pressures of the various signs were unaffected. Some signs, e.g. myoclonus, became more frequent when abecarnil was used. A residual protective effect of abecarnil was present 4 weeks after the dose was given, active at pressures less than 71 ATA. Changes with pressure in the EEG were recorded primarily from the frontal cortex, but were also present in the parietal and occipital areas of the left cortex. Amplitude and frequency spectra were calculated and changes with pressure in the four conventional wavebands, plus two others, analysed. The most striking change was the prevention by abecarnil of the pressure-induced 100% increase in alpha wave amplitude in the frontal region. It is concluded that modulation of GABA transmission is important in controlling the expression of HPNS.

The effects of the competitive NMDA receptor antagonist CPP on the high pressure neurological syndrome in a primate model.

Neurophysiological interactions between the competitive N-methyl-D-aspartate (NMDA) preferring receptor antagonist, CPP (3-((+-)-2-carboxypiperazine-4-yl)-propyl-1-phosphonate) and the high pressure neurological syndrome (HPNS) have been investigated in the non-human primate Papio anubis. Eight animals were exposed on two occasions to environmental pressures of 81 atmospheres absolute (ATA) in a hyperbaric chamber, using helium and oxygen. One exposure followed pretreatment with CPP (either 5 or 10 mg/kg i.v. plus 5 mg/kg/hr infusion), the other a saline control. Pretreatment with CPP delayed moderate signs of face tremor and myoclonus and abolished severe signs of whole body tremor and seizure activity. By 81 ATA, scores representing severity of HPNS were significantly reduced by CPP to a mean score, reflecting a level of just mild to moderate limb tremoring (P less than 0.001). Changes in the EEG were observed in channels associated with the frontal, parietal and occipital regions of the left cortex. Amplitude and frequency spectra were calculated and changes with pressure in the 4 conventional wavebands were analysed. The most striking change was the complete prevention by CPP of the 100% increase in the amplitude of alpha waves at 81 ATA in the frontal region (P less than 0.001). It is concluded that NMDA transmission has a major role in the expression of HPNS.

A chamber system for maintaining a hyperbaric environment for long-term animal studies.

An experimental hyperbaric chamber system is described whereby animals, including nonhuman primates, can be cared for under altered environmental conditions for periods in excess of 1 wk. The chamber itself is capable of a working pressure of 200 atm abs, used with various mixtures of gases which can be varied independently. The novel approach of vertical mounting enables cages to be lowered into position, and food and water can be supplied from above while excreta can be removed from below, irrespective of the internal pressure. The chamber has an integrated life support system such that temperature, both of the chamber and of the mass of gas inside, humidity, oxygen, carbon dioxide, and noise levels can be accurately and finely controlled, all within a pathogen-free environment.

The effects of MK801 on the high pressure neurological syndrome in the baboon (Papio anubis).

The in vivo neurophysiological interactions of the non-competitive NMDA receptor antagonist MK801 with the High Pressure Neurological Syndrome have been investigated in the primate Papio anubis. A hyperbaric chamber was used to achieve environmental pressures of 61 ATA (atmospheres absolute) over a period of 5 hr. Eight animals underwent 2 compressions each, one following pretreatment with 0.03 mg/kg (i.v.) MK801, the other a control. Half of the animals received MK801 on their first exposure. Mild signs of the high pressure neurological syndrome, e.g. paw and limb tremor were first observed between 10 and 20 ATA and more severe signs, e.g. whole body tremor, myoclonus and vomiting, appeared after 50 ATA. The onset pressures for the various signs were increased by 10-17 ATA when the animals received MK801 (P = 0.06) and the severity of the signs, over the whole range of pressures at which they appeared, was significantly reduced (P less than 0.001). Additional experiments showed that MK801 afforded considerable protection, at pressures up to 81 ATA, but doses larger than those used for the main experiment produced signs of tranquilisation and sedation. Changes in the EEG were observed in channels associated with the frontal, parietal and occipital regions. Amplitude and frequency spectra were calculated and trends with pressure in the 4 conventional wavebands were analysed. The most striking change was a decrease in amplitude of delta waves (P less than 0.001), which was ameliorated by MK801 (P less than 0.001).

Physiological effects of etorphine, acepromazine and xylazine in the scimitar horned oryx (Oryx dammah).

Scimitar horned oryx (Oryx dammah), kept under confined and unconfined conditions were immobilised with etorphine in combination with acepromazine or xylazine or both, and with xylazine alone. Both groups of animals were successfully sedated with etorphine and xylzine, with or without acepromazine, although hypothermia and mild hypoxaemia and a fall in packed cell volume were frequently noted. Xylazine alone produced a dose dependent degree of sedation in semitame subadult animals kept in confinement, but only slight depression in their wild, unconfined counterparts. If xylazine was not included in the immobilising mixture induction was traumatic and full sedation not achieved. Heart rates and arterial pressures (systemic and pulmonary) were also monitored but no remarkable changes were noted. The only abnormalities in blood biochemistry were raised aspartate transminase and creatine kinase. Ruminal regurgitation could be a major problem if endotracheal intubation was not achieved early in the procedure.

Physiological effects of etorphine, acepromazine and xylazine on the black fallow deer (Dama dama).

Confined and unconfined fallow deer (Dama dama) of the black variety kept at Whipsnade Park were sedated with etorphine in combination with acepromazine, or xylazine or both, with or without atropine. Induction, sedation and recovery times were recorded. During the period under sedation, systemic and pulmonary arterial pressures, electrocardiographs, body temperature, arterial blood gas pressures and pH, packed cell volume and plasma electrolytes and enzymes were monitored. In both groups, and with all drug combinations, heart rates (except where atropine was used and respiratory rates were depressed, pulmonary arterial pressures were elevated, and blood oxygen tension and pH lowered compared with interspecies norms. In confined animals, systemic arterial pressures were depressed. Unconfined animals and animals in which atropine was used showed higher and more varied systemic arterial pressures and more varied heart rates. Cardiac arrests occurred in eight animals, five during induction and three during sedation, one of which was revived with oxygen. Severe arrhythmia occurred in an animal in which atrioventricular block was confirmed and reversed by oxygen treatment.