TNFα G308A genotype, resilience to sleep deprivation, and the effect of caffeine on psychomotor vigilance performance in a randomized, double-blind, placebo-controlled, crossover study.

The TNFα G308A gene polymorphism has been reported to influence performance impairment during total sleep deprivation (TSD). We investigated this effect in a randomized, double-blind, crossover laboratory study of repeated exposure to 48 h TSD with caffeine administration at different doses. In a retrospective analysis, we replicated the finding that the A allele of TNFα G308A, found in 4 of 12 study participants, confers resilience to performance impairment during TSD. There was no evidence of an interaction of TNFα genotype with the beneficial effect of caffeine (200 or 300 mg) on performance during TSD, suggesting distinct underlying mechanisms.

Limited Efficacy of Caffeine and Recovery Costs During and Following 5 Days of Chronic Sleep Restriction.

Objectives: To investigate the effects of caffeine on psychomotor vigilance and sleepiness during sleep restriction and following subsequent recovery sleep. Methods: Participants were N = 48 healthy good sleepers. All participants underwent five nights of sleep satiation (time-in-bed [TIB]: 10 hours), followed by five nights of sleep restriction (TIB: 5 hours), and three nights of recovery sleep (TIB: 8 hours) in a sleep laboratory. Caffeine (200 mg) or placebo was administered in the form of chewing gum at 08:00 am and 12:00 pm each day during the sleep restriction phase. Participants completed hourly 10-minute psychomotor vigilance tests and a modified Maintenance of Wakefulness Test approximately every 4 hours during the sleep restriction and recovery phases. Results: Caffeine maintained objective alertness compared to placebo across the first 3 days of sleep restriction, but this effect was no longer evident by the fourth day. A similar pattern of results was found for Maintenance of Wakefulness Test sleep latencies, such that those in the caffeine group (compared to placebo) did not show maintenance of wakefulness relative to baseline after the second night of restriction. Compared to placebo, participants in the caffeine condition displayed slower return to baseline in alertness and wakefulness across the recovery sleep period. Finally, the caffeine group showed greater N3 sleep duration during recovery. Conclusions: Caffeine appears to have limited efficacy for maintaining alertness and wakefulness across 5 days of sleep restriction. Perhaps more importantly, there may be recovery costs associated with caffeine use following conditions of prolonged sleep loss.

Pro-2-PAM therapy for central and peripheral cholinesterases.

Novel therapeutics to overcome the toxic effects of organophosphorus (OP) chemical agents are needed due to the documented use of OPs in warfare (e.g. 1980-1988 Iran/Iraq war) and terrorism (e.g. 1995 Tokyo subway attacks). Standard OP exposure therapy in the United States consists of atropine sulfate (to block muscarinic receptors), the acetylcholinesterase (AChE) reactivator (oxime) pralidoxime chloride (2-PAM), and a benzodiazepine anticonvulsant to ameliorate seizures. A major disadvantage is that quaternary nitrogen charged oximes, including 2-PAM, do not cross the blood brain barrier (BBB) to treat brain AChE. Therefore, we have synthesized and evaluated pro-2-PAM (a lipid permeable 2-PAM derivative) that can enter the brain and reactivate CNS AChE, preventing seizures in guinea pigs after exposure to OPs. The protective effects of the pro-2-PAM after OP exposure were shown using (a) surgically implanted radiotelemetry probes for electroencephalogram (EEG), (b) neurohistopathology of brain, (c) cholinesterase activities in the PNS and CNS, and (d) survivability. The PNS oxime 2-PAM was ineffective at reducing seizures/status epilepticus (SE) in diisopropylfluorophosphate (DFP)-exposed animals. In contrast, pro-2-PAM significantly suppressed and then eliminated seizure activity. In OP-exposed guinea pigs, there was a significant reduction in neurological damage with pro-2-PAM but not 2-PAM. Distinct regional areas of the brains showed significantly higher AChE activity 1.5h after OP exposure in pro-2-PAM treated animals compared to the 2-PAM treated ones. However, blood and diaphragm showed similar AChE activities in animals treated with either oxime, as both 2-PAM and pro-2-PAM are PNS active oximes. In conclusion, pro-2-PAM can cross the BBB, is rapidly metabolized inside the brain to 2-PAM, and protects against OP-induced SE through restoration of brain AChE activity. Pro-2-PAM represents the first non-invasive means of administering a CNS therapeutic for the deleterious effects of OP poisoning by reactivating CNS AChE.

[+]-Huperzine A treatment protects against N-methyl-D-aspartate-induced seizure/status epilepticus in rats.

The toxicity of organophosphorous (OP) nerve agents is attributed to their irreversible inhibition of acetylcholinesterase (AChE), which leads to excessive accumulation of acetylcholine (ACh) and is followed by the release of excitatory amino acids (EAA). EAAs sustain seizure activity and induce neuropathology due to over-stimulation of N-methyl-d-aspartate (NMDA) receptors. Huperzine A (Hup A), a blood-brain barrier permeable selective reversible inhibitor of AChE, has been shown to reduce EAA-induced cell death by interfering with glutamate receptor-gated ion channels in primary neuronal cultures. Although [-]-Hup A, the natural isomer, inhibits AChE approximately 38-fold more potently than [+]-Hup A, both [-]- and [+]-Hup A block the NMDA channel similarly. Here, we evaluated the protective efficacy of [+]-Hup A for NMDA-induced seizure in a rat model. Rats implanted with radiotelemetry probes to record electroencephalography (EEG), electrocardiography (ECG), body temperature, and physical activity were administered various doses of [+]-Hup A (intramuscularly) and treated with 20 microg/kg NMDA (intracerebroventricular) 20-30 min later. For post-exposure, rats were treated with [+]-Hup A (3 mg/kg, intramuscularly) 1 min after NMDA (20 microg/kg). Our data showed that pre- and post-exposure, [+]-Hup A (3 mg/kg) protects animals against NMDA-induced seizures. Also, NMDA-administered animals showed increased survival following [+]-Hup A treatment. [+]-Hup A has no visible effect on EEG, heart-rate, body temperature, or physical activity, indicating a reduced risk of side effects, toxicity, or associated pathology. Our results suggest that [+]-Hup A protects against seizure and status epilepticus (SE) by blocking NMDA-induced excitotoxicity in vivo. We propose that [+]-Hup A, or a unique combination of [+]- and [-]-Hup A, may prove to be effective for pre- and post-exposure treatment of lethal doses of OP-induced neurotoxicity.

A rapid cation-exchange HPLC method for detection and quantification of pyridinium oximes in plasma and tissue.

A rapid and sensitive assay for pyridinium oximes in plasma and tissue was developed. The method was suitable for the analysis of mono- and di-pyridinium oximes and utilizes ultrafiltration followed by cation-exchange high-performance liquid chromatography with UV detection. The assay was originally developed for the measurement of the oxime MMB-4 in plasma for which the lower limit of detection was 0.0005 pg and the limit of quantitation was 0.001 to 2.5 microg. The assay required as little as 50 microL of whole blood or 30 pL of tissue homogenate, and it was used for a pharmacokinetic study from a single intramuscular injection of MMB-4 (dichloride or dimethylsulfonate salt) in the guinea pig. Both salts were found to have similar pharmacokinetic properties in the plasma with a T1/2 of about 34 to 42 min and the area-under-the-curve values increased dose dependently. MMB-4 tissue concentrations were much lower than the plasma. The tissue levels peaked at 5-20 min depending on the tissue. A rank of concentration was diaphragm > heart > thigh muscle.

Development of a rat pilocarpine model of seizure/status epilepticus that mimics chemical warfare nerve agent exposure.

We developed a rat pilocarpine seizure/status epilepticus (SE) model, which closely resembles 1.6-2.0 x LD50 soman exposure, to analyse the molecular mechanism of neuronal damage and to screen effective neuroprotectants against cholinergic agonist and chemical warfare nerve agent (CWNA) exposure. Rats implanted with radiotelemetry probes capable of recording electroencephalogram (EEG), electrocardiogram (ECG), temperature, and physical activity were treated with lithium chloride (5 mEq/kg, im), followed 24 h later by (ip) doses of pilocarpine hydrochloride. Based on radiotelemetry analysis, a dose of 240 mg/kg (ip) pilocarpine generated seizure/SE analogous to 1.6-2.0 x LD50 of soman. The model was refined by reducing the peripheral convulsions without affecting the central nervous system (CNS) by administering methylscopolamine bromide (1 mg/kg, ip), an anti-cholinergic that does not cross the blood-brain barrier. However, when methylscopolamine bromide was administered, a higher dose of pilocarpine (320 mg/kg, ip) was required to generate the equivalent seizure/SE. Histopathology data indicated that pilocarpine induces significant damage to the hippocampal region of the brain, with similar neuropathology to that of 1.6-2.0 x LD50 soman exposure. There was a reduction in body temperature after the administration of pilocarpine, as observed in organophosphate (OP) nerve agents exposure. The heart-rate of pilocarpine-treated animals increased compared to the normal range. The pilocarpine seizure/SE model was also reproducible in the absence of lithium chloride. These results support that pilocarpine seizure/SE model is useful in studying the molecular mechanisms of neuropathology and screening neuroprotectants following cholinergic agonist and CWNA exposure.