Citrullinated human and murine MOG35-55 display distinct biophysical and biochemical behavior.

The peptide spanning residues 35 to 55 of the protein myelin oligodendrocyte glycoprotein (MOG) has been studied extensively in its role as a key autoantigen in the neuroinflammatory autoimmune disease multiple sclerosis. Rodents and nonhuman primate species immunized with this peptide develop a neuroinflammatory condition called experimental autoimmune encephalomyelitis, often used as a model for multiple sclerosis. Over the last decade, the role of citrullination of this antigen in the disease onset and progression has come under increased scrutiny. We recently reported on the ability of these citrullinated MOG35-55 peptides to aggregate in an amyloid-like fashion, suggesting a new potential pathogenic mechanism underlying this disease. The immunodominant region of MOG is highly conserved between species, with the only difference between the murine and human protein, a polymorphism on position 42, which is serine in mice and proline for humans. Here, we show that the biophysical and biochemical behavior we previously observed for citrullinated murine MOG35-55 is fundamentally different for human and mouse MOG35-55. The citrullinated human peptides do not show amyloid-like behavior under the conditions where the murine peptides do. Moreover, we tested the ability of these peptides to stimulate lymphocytes derived from MOG immunized marmoset monkeys. While the citrullinated murine peptides did not produce a proliferative response, one of the citrullinated human peptides did. We postulate that this unexpected difference is caused by disparate antigen processing. Taken together, our results suggest that further study on the role of citrullination in MOG-induced experimental autoimmune encephalomyelitis is necessary.

Progression and recovery of Parkinsonism in a chronic progressive MPTP-induction model in the marmoset without persistent molecular and cellular damage.

Chronic exposure to low-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in marmoset monkeys was used to model the prodromal stage of Parkinson's disease (PD), and to investigate mechanisms underlying disease progression and recovery. Marmosets were subcutaneously injected with MPTP for a period of 12weeks, 0.5mg/kg once per week, and clinical signs of Parkinsonism, motor- and non-motor behaviors were recorded before, during and after exposure. In addition, postmortem immunohistochemistry and proteomics analysis were performed. MPTP-induced parkinsonian clinical symptoms increased in severity during exposure, and recovered after MPTP administration was ended. Postmortem analyses, after the recovery period, revealed no alteration of the number and sizes of tyrosine hydroxylase (TH)-positive dopamine (DA) neurons in the substantia nigra. Also levels of TH in putamen and caudate nucleus were unaltered, no differences were observed in DA, serotonin or nor-adrenalin levels in the caudate nucleus, and proteomics analysis revealed no global changes in protein expression in these brain areas between treatment groups. Our findings indicate that parkinsonian symptoms can occur without detectable damage at the cellular or molecular level. Moreover, we show that parkinsonian symptoms may be reversible when diagnosed and treated early.

Exploring the neuroprotective effects of modafinil in a marmoset Parkinson model with immunohistochemistry, magnetic resonance imaging and spectroscopy.

Neuroprotective therapeutics stop or slow down the degeneration process in animal models of Parkinson's disease (PD). Neuronal survival in PD animal models is often measured by immunohistochemistry. However, dynamic changes in the pathology of the brain cannot be explored with this technique. Application of proton magnetic resonance (MR) imaging (MRI) and spectroscopy (MRS) can cover this lacuna as these techniques are non-invasive and can be repeated over time in the same animal. Therefore, the sensitivity of both techniques to measure changes in PD-pathology was explored in an experiment studying the neuroprotective effects of the vigilance enhancer modafinil in a marmoset PD model. Eleven marmoset monkeys were treated with the neurotoxin 1-methyl-1,2,3,6-tetrahydropyridine (MPTP). Six of these 11 animals, simultaneously, received a daily oral dose of modafinil (100 mg/kg) and five received vehicle for 27 days. MR experiments were performed at baseline and 1 and 3.5 weeks after the MPTP intoxication period after which brains were analyzed with immunohistochemistry. Tyrosine hydroxylase immunoreactive (TH-IR) staining of dopamine neurons of the substantia nigra pars compacta confirmed that modafinil was able to partially prevent the MPTP-induced neuronal damage. In MRS, N-acetylaspartate (NAA)/phosphocreatine (tCR) ratios confirmed the protective effect indicating that this is a sensitive measure to detect neuroprotection in the MPTP marmoset model. Furthermore, the number of TH-IR positive neurons and the NAA/tCR ratio were significantly correlated to behavioral observations indicating that the changes measured in the brain are also reflected in the behavior and vice versa.

Beneficial effects of TCP on soman intoxication in guinea pigs: seizures, brain damage and learning behaviour.

Poisoning with the potent nerve agent soman produces a cascade of central nervous system (CNS) effects characterized by severe convulsions and eventually death. In animals that survive a soman intoxication, lesions in the amygdala, piriform cortex, hippocampus and thalamus can be observed. In order to examine the mechanisms involved in the effects of soman and to evaluate possible curative interventions, a series of behavioural, electrophysiological and neuropathological experiments were carried out in the guinea pig using the NMDA antagonist N-[1-(2-thienyl)cyclohexyl] piperidine (TCP) in conjunction with atropine and pyridostigmine. The NMDA antagonist TCP appeared to be very effective in the treatment of casualties who suffered from soman-induced seizures for 30 min: (i)Seizures were arrested within minutes after the TCP injection, confirmed by quantitative electroencephalogram (EEG), after fast Fourier analysis. Three hours after TCP the quantitative EEGs were completely normal in all frequency bands and remained normal during the entire 3-week intoxication period. The power shift to the lower (delta) frequency bands, indicative for neuropathology and found in control animals intoxicated only by soman, was not observed in the soman-TCP group. (ii)The gross neuropathology found in soman control animals within 48 h after soman was prevented in soman-TCP animals and was still absent in 3-week survivors. Instead, ultrastructural changes were observed, indicative of defense mechanisms of the cell against toxic circumstances. (iii)Twenty-four hours after soman, soman-TCP animals were able to perform in the shuttle box and Morris water maze. The beneficial effects of TCP on the performance in these tests during the 3-week intoxication period were very impressive, notwithstanding (minor) deficits in memory and learning. (iv)The increase in excitability after TCP was confirmed by an increase in the acoustic startle response. Taken together, these results confirmed the involvement of NMDA receptors in the maintenance of soman-induced seizures and the development of brain damage. They underline the current hypothesis that cholinergic mechanisms are responsible for eliciting seizure activity after soman and that, most likely, the subsequent recruitment of other excitatory neurotransmitters and loss of inhibitory control are responsible for the maintenance of seizures and the development of subsequent brain damage.

Behavioral test systems in marmoset monkeys.

A number of neurobehavioral methods have been developed to test behavior in marmoset monkeys. These test systems are (1) the bungalow test, which quantifies spontaneous explorative behavior, (2) the hand-eye coordination test, which tests a learned task of coordinated motor behavior, and (3) the fear-potentiated startle response, which tests and quantifies pathological anxiety manifested by a response of fright. The test systems are extensively discussed, and the value of these test systems is exemplified by applying them to neurological disorders to register disease activity and drug efficacy.

Subchronic physostigmine pretreatment in marmosets: absence of side effects and effectiveness against soman poisoning with negligible postintoxication incapacitation.

Subchronic pretreatment with physostigmine (PHY) (0.0125 mg/kg/h) leading to a blood acetylcholinesterase inhibition of about 30% caused no side effects when applied to marmoset monkeys. This was evident on behavioral parameters and on EEG and cortical visual evoked response. Furthermore, this treatment regime, followed by atropine as postintoxication therapy, protected the marmosets against lethality after a 2 x LD50 dose of soman with negligible postintoxication incapacitation. These findings suggest that a symptom-free pretreatment with subchronic PHY could protect man sufficiently against severe soman intoxication.

Scopolamine augments the efficacy of physostigmine against soman poisoning in guinea pigs.

The efficacy of the subchronically administered cholinesterase-inhibitor physostigmine (PHY) (0.025 mg/kg/h) either with or without the muscarinergic receptor antagonist scopolamine (SCO) (0.018 mg/kg/h) in counteracting soman-induced lethality and incapacitation were determined in guinea pigs. This was tested in animals that either received atropine sulphate (AS, 17.4 mg/kg i.m.) or no postintoxication therapy. Behavioral and neurophysiological readout systems were used to measure postintoxication incapacitation. Only the pretreatment with PHY alone did not offer any protection against 2x LD50 soman intoxication. Animals that received the complete treatment (PHY + SCO + AS) did not show any abberations in the performance of learned behavior. The use of AS after soman intoxication resulted in an increase of the startle response, whereas the addition of SCO to the pretreatment led to a more persistent duration of the effect in time. In case one has to rely completely on the pretreatment, the addition of SCO to PHY is life-saving. However, some postintoxication incapacitation is still present. Therefore, the pretreatment regime may perhaps further be improved by the addition of a nicotinic antagonist.

Subchronic physostigmine pretreatment in guinea pigs: effective against soman and without side effects.

The behavioral and neurophysiological effects of the subchronically administered cholinesterase-inhibitor physostigmine (PHY) (0.025 mg/kg/h) either with or without the muscarinergic antagonist scopolamine (SCO) (0.018 mg/kg/h) were determined in guinea pigs. In contrast to a single injection of PHY, subchronic application by osmotic minipumps of PHY, even without SCO, caused no behavioral or neurophysiological side effects. Also, the efficacy of such a pretreatment in counteracting soman-induced lethality and apparent symptoms of intoxication were determined. After subchronically administered PHY or PHY + SCO, the treated animals were protected against a 3 x LD50 dose of soman.

Effects of physostigmine on the startle in guinea pigs: two mechanisms involved.

The effects of the acetylcholinesterase inhibitor physostigmine (PHY) on the auditory startle reflex in guinea pigs were studied. The dose-response curve of PHY appeared bell shaped, with a maximum effect dose of 0.3 mg/kg. In addition, PHY altered the shape of the startle response. The muscarinic antagonist scopolamine (SCO) increased the startle at PHY doses above 0.3 mg/kg without affecting the PHY-induced shape of the response. The decreasing part of the startle due to PHY could be mimicked by the cholinesterase inhibitor soman in combination with 0.3 mg/kg PHY. It appeared that the decreasing part of the dose-response curve at higher dose levels is caused by the cholinesterase inhibitory action of PHY and, in view of the SCO effect, is mediated by muscarinergic receptors. The increasing part of the curve is probably caused by an agonistic action of PHY on neuronal nicotinergic receptors, because the antagonist mecamylamine (20 mg/kg) antagonized the effects of 0.3 mg/kg PHY both on the deflection and shape of the startle.

Comparison of the efficacy of single or repeated HI-6 treatment following soman poisoning in guinea pigs and marmoset monkeys.

The therapeutic efficacy of single or repeated doses of HI-6, together with atropine, against soman poisoning were compared both in guinea pigs and in marmoset monkeys. In addition, the pharmacokinetics of HI-6 were determined after single or repeated injections. Both single and repeated HI-6 injections protected guinea pigs effectively against 2 x LD50 soman. The plasma levels of HI-6 after single HI-6 injection fitted a one-compartment elimination model. The plasma levels of HI-6 following repeated injections were in accordance with those predicted using the data obtained after single HI-6 injection. No evidence was found for any disturbance of the HI-6 elimination in guinea pigs following soman intoxication. Marmosets were intoxicated with 2 x LD50 soman (s.c.), followed after 1 min by i.m. injections of atropine and HI-6. One and 2 h later, four animals received additional HI-6 injections. The pharmacokinetics of HI-6 in plasma, after single and repeated HI-6 injections were similar to those found in the guinea pig. Furthermore, repeated HI-6 injections protected effectively against soman: four out of four animals survived, in fair condition. In contrast, only one out of four animals receiving single HI-6-treatment fully recovered within a few days. Two animals died, the fourth animal survived, but had to be euthanized 3 weeks after intoxication.

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.

Efficacy of HI-6 and HLö-7 in preventing incapacitation following nerve agent poisoning.

The therapeutic efficacy of the oximes HI-6 and HLö-7 (132.5 mumol/kg), in combination with atropine, in soman- or tabun-intoxicated guinea pigs was compared, particularly with respect to recovery of shuttlebox performance and electroencephalograms (EEGs). After 1.5 x LD50 soman SC, therapy with HI-6 or HLö-7 resulted in survival of 87.5% of the animals in each group. In both groups postintoxication performance decrements and EEG abnormalities lasted approximately 2 weeks after intoxication. After 3 x LD50 soman all HLö-7-treated animals died within 5 h; 70% of the HI-6-treated animals were still alive after 8 h; however, only 10% survived more than 24 h. After 2 x LD50 tabun 36% of the HI-6-treated animals died; HLö-7 prevented lethality and led to faster recovery of performance and EEG than after HI-6. Even after 7.5 x LD50 tabun, followed by HLö-7, full recovery was reached within 1 week in the surviving animals (82%). In soman-intoxicated guinea pigs HI-6 is therapeutically slightly more effective than HLö-7. HLö-7 is far more effective, under similar conditions, against tabun intoxication than HI-6.

A simple automated test to measure exploratory and motor activity of marmosets.

An automated device is described to test the exploratory and motor activity of common marmosets (Callithrix jacchus). The device consists of four boxes interconnected by PVC tubes. The presence of an animal in a box is detected by a photocell. Calibration takes place with an electric model train. Movements of the animal from one box to another are detected by disappearance from one and appearance in another box. The apparatus is linked to a PC. The effects of two doses of methamphetamine and of pentobarbital are shown.

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.

Active avoidance behavior in guinea pigs: effects of physostigmine and scopolamine.

Behavioral training of guinea pigs by conventional methods, such as used for rats and mice, appears difficult. Hence, only a few behavioral experiments with guinea pigs have been described in the literature. An active avoidance technique in an automated two-way shuttlebox is described using sound as a conditioned (CS) and a tactile stimulus (a stream of air ruffling their fur) as an unconditioned (UCS) stimulus. Acquisition is fairly rapid and reproducible. Doses of physostigmine that caused moderate blood acetylcholinesterase inhibition induced dose-dependent performance decrements. These decrements were counteracted by a sign-free dose of scopolamine.

On the development of behavioral tolerance to organophosphates. IV: EEGand visual evoked responses.

Several earlier studies showed that, in contrast with DFP, repeated injections with soman did not lead to behavioral tolerance in rats. The reason for the difference between the effects of these two organophosphate cholinesterase inhibitors was not clear and a neurophysiological approach was undertaken. Four experiments (A, B, C and D) were carried out, each consisting of three groups of rats, SC injected with saline, DFP (600 micrograms/kg) or soman (60 micrograms/kg) respectively. In Experiment B and D the rats were trained to criterion in a two-way shuttlebox. Thereafter, the animals of Experiment B were fitted with suitable electrodes and two days later their EEGs and visual evoked responses (VERs) were recorded, 1 and 24 h after a single dose of the above-mentioned compounds. In Experiment D the trained animals were subsequently injected 3 times per week for 4 weeks with the same doses and their performance was tested 5 days per week, 1 and 24 h after injection. After those 4 weeks, when the DFP-treated animals had developed behavioral tolerance, electrodes were fitted and EEGs and VERs were recorded after two days, again 1 and 24 h after injection, as in Experiment B. The difference with Experiments A and C was that these animals were not trained. Otherwise, treatment schedules and recording procedures of Experiment A were identical to those of Experiments B and of Experiment C to those of Experiment D. In all cases the EEGs and VERs were recorded from animals slowly walking in a rotating hollow transparent wheel. The results show a similar pattern in all four experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

On the development of behavioral tolerance to organophosphates. III: Behavioral aspects.

As part of a study on the mechanisms underlying behavioral tolerance to cholinesterase-inhibiting organophosphates (OP's) the present investigation was focussed on behavioral procedures affecting the development of tolerance. The effects of chronic administration of the OP's DFP (600 micrograms/kg SC) and soman (60 micrograms/kg SC) were compared in rats. These doses do not cause detectable effects upon close observation of the animals. As was found before, behavioral tolerance developed following DFP, but not following soman. Repeated behavioral testing affected the development of tolerance. Cross-tolerance between these two inhibitors was not found. Surprisingly, when DFP was administered 48 hr after soman, all animals were observationally normal, and when soman was given 48 hr after DFP the majority of the animals died. This indicates that the sequence in which these inhibitors were administered was of major importance. It is concluded that practice-related and/or state-dependent factors are important for the development of behavioral tolerance and that one should be careful in making generalizing statements about tolerance to cholinesterase-inhibiting OP's.

Side effects of therapeutic drugs against organophosphate poisoning.

The possible side effects of therapeutic drugs against organophosphate poisoning were investigated. First, dose-effect curves were obtained with atropine sulphate (AS), P2S, obidoxime, aprophen, N-methylatropine nitrate and HI-6. The first three drugs are currently used in the therapy of organophosphate poisoning, the others are potentially useful candidates. Automated tests measuring open field behavior, motor coordination and shuttlebox performance, as well as neurophysiological techniques such as the quantified EEG (qEEG) and visual evoked responses were used. The sign-free doses of these compounds were determined; it appeared that open field behavior and the qEEG were the most sensitive methods for these drugs. Subsequently, these two methods were used to investigate the effects of the combinations of AS and P2S, AS and obidoxime or AS and HI-6, each compound given in a sign-free dose. Synergistic or additive effects were found with the combination of AS and P2S, which were smaller with the combination of AS and obidoxime and absent with the combination of AS and HI-6. These results indicate that the untimely use (false alarm, panic) of the current drug combinations may cause undesirable side effects.