Neuroprotection Against Diisopropylfluorophosphate in Acute Hippocampal Slices.

Diisopropylfluorophosphate (DFP) is an irreversible inhibitor of acetylcholine esterase and a surrogate of the organophosphorus (OP) nerve agent sarin. The neurotoxicity of DFP was assessed as a reduction of population spike (PS) area elicited by synaptic stimulation in acute hippocampal slices. Two classical antidotes, atropine, and pralidoxime, and two novel antidotes, 4R-cembranotriene-diol (4R) and a caspase nine inhibitor, were tested. Atropine, pralidoxime, and 4R significantly protected when applied 30 min after DFP. The caspase inhibitor was neuroprotective when applied 5-10 min before or after DFP, suggesting that early synaptic apoptosis is responsible for the loss of PSs. It is likely that apoptosis starts at the synapses and, if antidotes are not applied, descends to the cell bodies, causing death. The acute slice is a reliable tool for mechanistic studies, and the assessment of neurotoxicity and neuroprotection with PS areas is, in general, pharmacologically congruent with in vivo results and predicts the effect of drugs in vivo. 4R was first found to be neuroprotective in slices and later we demonstrated that 4R is neuroprotective in vivo. The mechanism of neurotoxicity of OPs is not well understood, and there is a need for novel antidotes that could be discovered using acute slices.

4R-cembranoid protects against diisopropylfluorophosphate-mediated neurodegeneration.

Many organophosphorous esters synthesized for applications in industry, agriculture, or warfare irreversibly inhibit acetylcholinesterase, and acute poisoning with these compounds causes life-threatening cholinergic overstimulation. Following classical emergency treatment with atropine, an oxime, and a benzodiazepine, surviving victims often suffer brain neurodegeneration. Currently, there is no pharmacological treatment to prevent this brain injury. Here we show that a cyclic diterpenoid, (1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol (4R) ameliorates the damage caused by diisopropylfluorophosphate (DFP) in the hippocampal area CA1. DFP has been frequently used as a surrogate for the warfare nerve agent sarin. In rats, DFP is lethal at the dose used to cause brain damage. Therefore, to observe brain damage in survivors, the death rate was reduced by pre-administration of the peripherally acting antidotes pyridostigmine and methyl atropine or its analog ipratropium. Pyridostigmine bromide, methyl atropine nitrate, and ipratropium bromide were dissolved in saline and injected intramuscularly at 0.1mg/kg, 20mg/kg, and 23mg/kg, respectively. DFP (9mg/kg) dissolved in cold water was injected intraperitoneally. 4R (6mg/kg) dissolved in DMSO was injected subcutaneously, either 1h before or 5 or 24h after DFP. Neurodegeneration was assessed with Fluoro-Jade B and amino cupric silver staining; neuroinflammation was measured by the expression of nestin, a marker of activated astrocytes. Forty-eight hours after DFP administration, 4R decreased the number of dead neurons by half when injected before or after DFP. 4R also significantly decreased the number of activated astrocytes. These data suggest that 4R is a promising new drug that could change the therapeutic paradigm for acute poisoning with organophosphorous compounds by the implementation of a second-stage intervention after the classical countermeasure treatment.

Protective activity of (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol analogues against diisopropylfluorophosphate neurotoxicity: preliminary structure-activity relationship and pharmacophore modeling.

Diisopropylfluorophosphate (DFP) is an organophosphorous insecticide used as a surrogate for the more toxic chemical warfare nerve agent sarin. DFP produces neurotoxicity in vivo and irreversibly decreases the area of population spikes recorded from the CA1 region of acute hippocampal slices. (1S,2E,4R,6R,7E,11E)-2,7,11-Cembratriene-4,6-diol (1) is a neuroprotective natural cembranoid that reverses DFP-induced damage both in vivo and in the hippocampal slice. Cembranoid 1 acts by noncompetitive inhibition of the α7 nicotinic acetylcholine receptor. This study aims at establishing a preliminary structure-activity relationship to define the neuroprotective cembranoid pharmacophores using the hippocampal slice assay and pharmacophore modeling. Fourteen natural, semisynthetic, or biocatalytic cembranoid analogues 2-15 related to 1 were tested for their capacity to protect the population spikes from DFP-induced damage and intrinsic toxicity. Twelve cembranoids caused significant reversal of DFP toxicity; only 3 active analogues displayed minor intrinsic toxicity at 10 µM. The C-4 epimer of 1 (2) and the 4-O-methyl ether analogue of 1 (3), were totally devoid of neuroprotective activity. The results suggested a model for cembranoid binding where the hydrophobic ring surface binds to a hydrophobic (Hbic) patch on the receptor molecule and an electronegative atom (oxygen or sulfur) in proper spatial relationship to the ring surface interacts with an electropositive group in the receptor binding site. A pharmacophore model consisting of 1 hydrogen bond acceptor (HBA), 2 Hbic, and 10 exclusion spheres was established using HipHop-REFINE and supported the above mentioned pharmacophoric hypothesis.

Kinin-B2 receptor mediated neuroprotection after NMDA excitotoxicity is reversed in the presence of kinin-B1 receptor agonists.

BACKGROUND: Kinins, with bradykinin and des-Arg(9)-bradykinin being the most important ones, are pro-inflammatory peptides released after tissue injury including stroke. Although the actions of bradykinin are in general well characterized; it remains controversial whether the effects of bradykinin are beneficial or not. Kinin-B2 receptor activation participates in various physiological processes including hypotension, neurotransmission and neuronal differentiation. The bradykinin metabolite des-Arg(9)-bradykinin as well as Lys-des-Arg(9)-bradykinin activates the kinin-B1 receptor known to be expressed under inflammatory conditions. We have investigated the effects of kinin-B1 and B2 receptor activation on N-methyl-D-aspartate (NMDA)-induced excitotoxicity measured as decreased capacity to produce synaptically evoked population spikes in the CA1 area of rat hippocampal slices. PRINCIPAL FINDINGS: Bradykinin at 10 nM and 1 µM concentrations triggered a neuroprotective cascade via kinin-B2 receptor activation which conferred protection against NMDA-induced excitotoxicity. Recovery of population spikes induced by 10 nM bradykinin was completely abolished when the peptide was co-applied with the selective kinin-B2 receptor antagonist HOE-140. Kinin-B2 receptor activation promoted survival of hippocampal neurons via phosphatidylinositol 3-kinase, while MEK/MAPK signaling was not involved in protection against NMDA-evoked excitotoxic effects. However, 100 nM Lys-des-Arg(9)-bradykinin, a potent kinin-B1 receptor agonist, reversed bradykinin-induced population spike recovery. The inhibition of population spikes recovery was reversed by PD98059, showing that MEK/MAPK was involved in the induction of apoptosis mediated by the B1 receptor. CONCLUSIONS: Bradykinin exerted protection against NMDA-induced excitotoxicity which is reversed in the presence of a kinin-B1 receptor agonist. As bradykinin is converted to the kinin-B1 receptor metabolite des-Arg(9)-bradykinin by carboxypeptidases, present in different areas including in brain, our results provide a mechanism for the neuroprotective effect in vitro despite of the deleterious effect observed in vivo.

A cembranoid protects acute hippocampal slices against paraoxon neurotoxicity.

Many neurotoxic organophosphates (OPs) inhibit acetylcholinesterase (AChE) and as a result can cause a life threatening cholinergic crisis. Current medical countermeasures, which typically include atropine and oximes target the cholinergic crisis and are effective in decreasing mortality but do not sufficiently protect against delayed neurological deficits. There is, therefore, a need to develop neuroprotective drugs to prevent long-term neurological deficits. We used acute hippocampal slices to test the hypothesis that 4R,6R-cembratrienediol (4R) protects against functional damage caused by the OP paraoxon (POX). To assess hippocampal function, we measured synaptically evoked population spikes (PSs). Application of 4R reversed POX inhibition of PSs and the EC(50) of this effect was 0.8 µM. Atropine alone did not protect against POX neurotoxicity, but it did enhance protection by 4R. Pralidoxime partially regenerated AChE activity and protected against POX inhibition of PSs. 4R did not regenerate AChE suggesting that under our experimental conditions, the deleterious effect of POX on hippocampal function is not directly related to AChE inhibition. In conclusion, 4R is a promising neuroprotective compound against OP neurotoxins.