Protection by pyridostigmine bromide of marmoset hemi-diaphragm acetylcholinesterase activity after soman exposure.

Pyridostigmine bromide (PB) was approved by the U.S. Food and Drug Administration (FDA) in 2003 as a pretreatment in humans against the lethal effects of the irreversible nerve agent soman (GD). Organophosphate (OP) chemical warfare agents such as GD exert their toxic effects by inhibiting acetylcholinesterase (AChE) from terminating the action of acetylcholine at postsynaptic sites in cholinergic nerve terminals (including crucial peripheral muscle such as diaphragm). As part of the post-marketing approval of PB, the FDA required (under 21CFR314, the "two animal rule") the study of a non-human primate model (the common marmoset Callithrix jacchus jacchus) to demonstrate increased survival against lethal GD poisoning, and protection of physiological hemi-diaphragm function after PB pretreatment and subsequent GD exposure. Marmosets (male and female) were placed in the following experimental groups: (i) control (saline pretreatment only), (ii) low dose PB (12.5 microg/kg), or (iii) high dose (39.5 microg/kg) PB. Thirty minutes after the PB dose, animals were challenged with either saline (control) or soman (GD, 45 microg/kg), followed 1 min later by atropine (2mg/kg) and 2-PAM (25mg/kg). After a further 16 min, animals were euthanized and the complete diaphragm removed; the right hemi-diaphragm was frozen immediately at -80 degrees C, and the left hemi-diaphragm was placed in a tissue bath for 4h (to allow for decarbamylation to occur), then frozen. AChE activities were determined using the automated WRAIR cholinesterase assay. Blood samples were collected for AChE activities prior to PB, before GD challenge, and after sacrifice. RBC-AChE was inhibited by approximately 18% and 50% at the low and high doses of PB, respectively, compared to control (baseline) activity. In the absence of PB pretreatment, the inhibition of RBC-AChE by GD was 98%. The recovery of hemi-diaphragm AChE activity after the 4h wash period (decarbamylation) was approximately 8% and 17%, at the low and high PB doses, respectively, compared with the baseline (control) AChE activity prior to PB pretreatment or soman exposure. The results suggest that PB pretreatment protects a critical fraction of AChE activity in the marmoset diaphragm, which is sufficient to allow the animal to breathe despite exposure to a dose of soman that is lethal in unprotected animals.

[+]-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.

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.

Celastrus paniculatus seed water soluble extracts protect against glutamate toxicity in neuronal cultures from rat forebrain.

Aqueous extracts of Celastrus paniculatus (CP) seed have been reported to improve learning and memory in rats. In addition, these extracts were shown to have antioxidant properties, augmented endogenous antioxidant enzymes, and decreased lipid peroxidation in rat brain. However, water soluble extracts of CP seed (CP-WSE) have not been evaluated for their neuroprotective effects. In the study reported here, we used enriched forebrain primary neuronal cell (FBNC) cultures to study the neuroprotective effects of three CP-WSE extracts (a room temperature, WF; a hot water, HF; and an acid, AF) on glutamate-induced toxicity. FBNC were pre-treated with the CP-WSE and then with glutamate to evaluate the protection afforded against excitatory amino acid-induced toxicity. The criteria for neuroprotection were based on the effects of CP-WSE on a mitochondrial function test following glutamate-induced neurotoxicity. Pre-treatment of neuronal cells with CP-WSE significantly attenuated glutamate-induced neuronal death. To understand the molecular mechanism of action of CP-WSE, we conducted electrophysiological studies using patch-clamp techniques on N-methyl-D-aspartate (NMDA)-activated whole-cell currents in FBNC. WSE significantly and reversibly inhibited whole-cell currents activated by NMDA. The results suggest that CP-WSE protected neuronal cells against glutamate-induced toxicity by modulating glutamate receptor function.

Celastrus paniculatus seed water soluble extracts protect cultured rat forebrain neuronal cells from hydrogen peroxide-induced oxidative injury.

The effects of aqueous extracts of Celastrus paniculatus (CP) seeds were shown to have antioxidant properties in rats. In the study reported here, we have investigated the free radical scavenging capacity of three aqueous extracts (WSEs) obtained from CP seeds: a room temperature extract (WF); a hot water extract (HF); an acid extract (AF). All the WSEs exhibited a dose-dependent free radical scavenging capacity for 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH) and also for superoxide-generated assays (in vitro assays). In addition, we used enriched forebrain primary neuronal cell (FBNC) cultures to evaluate the neuroprotective effects of the three CP-WSE extracts on H(2)O(2)-induced toxicity. FBNC were pre-treated with the CP-WSE and then with H(2)O(2) to evaluate the protection afforded against H(2)O(2)-induced toxicity. The criteria for neuroprotection by the WSEs were based on a mitochondrial function test following the H(2)O(2)-induced neurotoxicity. All the WSEs significantly attenuated H(2)O(2)-induced neuronal death, and AF was the most effective in protecting the neuronal cells against oxidative injury caused by H(2)O(2). In 10 day FBNC, cellular superoxide dismutase activity was not affected by the WSEs or H(2)O(2), but catalase activity was decreased and levels of malondialdehyde were increased by H(2)O(2) treatment. When the neuronal cells were treated with WSEs prior to H(2)O(2) exposure, catalase activity was increased and levels of malondialdehyde were decreased significantly. The data presented here suggest that CP seed WSEs protected neuronal cells in part by their free radical scavenging properties, by reducing lipid peroxidation, and also by their ability to induce the antioxidant enzyme catalase. Our results indicate that WSEs might exert neuroprotective effects against increased oxidative stress resulting from free radical damage that is associated with a number of neurodegenerative diseases.