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.

Paraoxon and Pyridostigmine Interfere with Neural Stem Cell Differentiation.

Acetylcholinesterase (AChE) inhibition has been described as the main mechanism of organophosphate (OP)-evoked toxicity. OPs represent a human health threat, because chronic exposure to low doses can damage the developing brain, and acute exposure can produce long-lasting damage to adult brains, despite post-exposure medical countermeasures. Although the main mechanism of OP toxicity is AChE inhibition, several lines of evidence suggest that OPs also act by other mechanisms. We hypothesized that rat neural progenitor cells extracted on embryonic day 14.5 would be affected by constant inhibition of AChE from chronic exposure to OP or pyridostigmine (a reversible AChE blocker) during differentiation. In this work, the OP paraoxon decreased cell viability in concentrations >50 µM, as measured with the MTT assay; however, this effect was not dose-dependent. Reduced viability could not be attributed to blockade of AChE activity, since treatment with 200 µM pyridostigmine did not affect cell viability, even after 6 days. Although changes in protein expression patterns were noted in both treatments, the distribution of differentiated phenotypes, such as the percentages of neurons and glial cells, was not altered, as determined by flow cytometry. Since paraoxon and pyridostigmine each decreased neurite outgrowth (but did not prevent differentiation), we infer that developmental patterns may have been affected.

Anti-hyperalgesic and anti-inflammatory effects of 4R-tobacco cembranoid in a mouse model of inflammatory pain.

4R is a tobacco cembranoid that binds to and modulates cholinergic receptors and exhibits neuroprotective and anti-inflammatory activity. Given the established function of the cholinergic system in pain and inflammation, we propose that 4R is also analgesic. Here, we tested the hypothesis that systemic 4R treatment decreases pain-related behaviors and peripheral inflammation via modulation of the alpha 7 nicotinic acetylcholine receptors (α7 nAChRs) in a mouse model of inflammatory pain. We elicited inflammation by injecting Complete Freund's Adjuvant (CFA) into the hind paw of male and female mice. We then assessed inflammation-induced hypersensitivity to cold, heat, and tactile stimulation using the Acetone, Hargreaves, and von Frey tests, respectively, before and at different time points (2.5 h - 8d) after a single systemic 4R (or vehicle) administration. We evaluated the contribution of α7 nAChRs 4R-mediated analgesia by pre-treating mice with a selective antagonist of α7 nAChRs followed by 4R (or vehicle) administration prior to behavioral tests. We assessed CFA-induced paw edema and inflammation by measuring paw thickness and quantifying immune cell infiltration in the injected hind paw using hematoxylin and eosin staining. Lastly, we performed immunohistochemical and flow cytometric analyses of paw skin in α7 nAChR-cre::Ai9 mice to measure the expression of α7 nAChRs on immune subsets. Our experiments show that systemic administration of 4R decreases inflammation-induced peripheral hypersensitivity in male and female mice and inflammation-induced paw edema in male but not female mice. Notably, 4R-mediated analgesia and anti-inflammatory effects lasted up to 8d after a single systemic administration on day 1. Pretreatment with an α7 nAChR-selective antagonist prevented 4R-mediated analgesia and anti-inflammatory effects, demonstrating that 4R effects are via modulation of α7 nAChRs. We further show that a subset of immune cells in the hind paw expresses α7 nAChRs. However, the number of α7 nAChR-expressing immune cells is unaltered by CFA or 4R treatment, suggesting that 4R effects are independent of α7 nAChR-expressing immune cells. Together, our findings identify a novel function of the 4R tobacco cembranoid as an analgesic agent in both male and female mice that reduces peripheral inflammation in a sex-dependent manner, further supporting the pharmacological targeting of the cholinergic system for pain treatment.

γ-Aminobutyric acid type A receptor inhibition triggers a nicotinic neuroprotective mechanism.

Nicotinic acetylcholine receptor (nAChR)-mediated neuroprotection has been implicated in the treatment of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases and hypoxic ischemic events as well as other diseases hallmarked by excitotoxic and apoptotic neuronal death. Several modalities of nicotinic neuroprotection have been reported. However, although this process generally involves α4ß2 and α7 subtypes, the underlying mechanisms are largely unknown. Interestingly, both activation and inhibition of α7 nAChRs have been reported to be neuroprotective. We have shown that inhibition of α7 nAChRs protects the function of acute hippocampal slices against excitotoxicity in an α4ß2-dependent manner. Neuroprotection was assessed as the prevention of the N-methyl-D-aspartate-dependent loss of the area of population spikes (PSs) in the CA1 area of acute hippocampal slices. Our results support a model in which α7 AChRs control the release of γ-aminobutyric acid (GABA). Blocking either α7 or GABA(A) receptors reduces the inhibitory tone on cholinergic terminals, thereby promoting α4ß2 activation, which in turn mediates neuroprotection. These results shed light on how α7 nAChR inhibition can be neuroprotective through a mechanism mediated by activation of α4ß2 nAChRs.

Memantine has a nicotinic neuroprotective pathway in acute hippocampal slices after an NMDA insult.

Memantine is a non-competitive antagonist with a moderate affinity to the N-methyl-d-Aspartate (NMDA) receptor. The present study assessed memantine's neuroprotective activity using electrophysiology of ex-vivo hippocampal slices. Interestingly, a nicotinic component was necessary for memantine's neuroprotection (NP). Memantine demonstrated a bell-shaped dose-response curve of NP against NMDA. Memantine was neuroprotective at concentrations below 3 µM, but the NP declined at higher concentrations (>3 µM) when memantine inhibits the NMDA receptor. Additional evidence that memantine NP is mediated by an alternate mechanism independent of the inhibition of the NMDA receptor is supported by its ability to protect neurons when applied before or after the NMDA insult and in the presence of D(-)-2-Amino-5-phosphonopentanoic acid (APV), the standard NMDA receptor inhibitor. We found several similarities between the memantine NP mechanism and the neuroprotective nicotinic drug, the 4R cembranoid. Memantine's NP requires the release of acetylcholine, the activation of α4ß2, and is independent of MEK/MAPK signaling. Both 4R and memantine require the activation of PI3K/AKT for NP against NMDA-mediated excitotoxicity, although at different concentrations. In conclusion, our studies show memantine is neuroprotective through a nicotinic pathway, similar to the nicotinic drug 4R. This information leads to a better understanding of memantine's mechanisms of action and explains its dose-dependent effectiveness in Alzheimer's and other neurological disorders.

4R-Cembranoid Improves Outcomes after 6-Hydroxydopamine Challenge in Both In vitro and In vivo Models of Parkinson's Disease.

(1S, 2E, 4R, 6R,-7E, 11E)-2, 7, 11-cembratriene-4, 6-diol (4R) is one of the cembranoids found in tobacco leaves. Previous studies have found that 4R protected acute rat hippocampal slices against neurotoxicity induced by N-methyl-D-aspartate (NMDA) and against the toxic organophosphorus compounds paraoxon and diisopropylfluorophosphate (DFP). Furthermore, in vivo, 4R reduced the infarct size in a rodent ischemic stroke model and neurodegeneration caused by DFP. The present study expanded our previous study by focusing on the effect of 4R in Parkinson's disease (PD) and elucidating its underlying mechanisms using 6-hydroxydopamine (6-OHDA)-induced injury models. We found that 4R exhibited significant neuroprotective activity in the rat unilateral 6-OHDA-induced PD model in vivo. The therapeutic effect was evident both at morphological and behavioral levels. 4R (6 and 12 mg/kg) treatments significantly improved outcomes of 6-OHDA-induced PD in vivo as indicated by reducing forelimb asymmetry scores and corner test scores 4 weeks after injection of 6-OHDA (p < 0.05). The therapeutic effect of 4R was also reflected by decreased depletion of tyrosine hydroxylase (TH) in the striatum and substantia nigra (SN) on the side injected with 6-OHDA. TH expression was 70.3 and 62.8% of the contralateral side in striatum and SN, respectively, after 6 mg/kg 4R treatment; furthermore, it was 80.1 and 79.3% after treatment with 12 mg/kg of 4R. In the control group, it was 51.9 and 23.6% of the contralateral striatum and SN (p < 0.05). Moreover, 4R also protected differentiated neuro-2a cells from 6-OHDA-induced cytotoxicity in vitro. The activation of p-AKT and HAX-1, and inhibition of caspase-3 and endothelial inflammation, were involved in 4R-mediated protection against 6-OHDA-induced injury. In conclusion, the present study indicates that 4R shows a therapeutic effect in the rat 6-OHDA-induced PD model in vivo and in 6-OHDA-challenged neuro-2a cells in vitro.

Pharmacokinetics and Metabolism of 4R-Cembranoid.

4R-cembranoid (4R) is a natural cyclic diterpenoid found in tobacco leaves that displays neuroprotective activity. 4R protects against NMDA, paraoxon (POX), and diisopropylfluorophosphate (DFP) damage in rat hippocampal slices and against DFP in rats in vivo. The purpose of this study was to examine the metabolism and pharmacokinetics of 4R as part of its preclinical development as a neuroprotective drug. 10 µM 4R was found to be very stable in plasma for up to 1 hr incubation. 4R metabolism in human microsomes was faster than in the rat. Ten metabolites of 4R were detected in the microsomal samples; 6 dihydroxy and 4 monohydroxy forms of 4R. Male rats received a single dose of 4R at 6 mg/kg i.v., i.m., or s.c. The i.v. group had the highest plasma concentration of 1017 ng/mL. The t1/2 was 36 min and reached the brain within 10 min. The brain peak concentration was 6516 ng/g. The peak plasma concentration in the i.m. group was 163 ng/mL compared to 138 ng/mL in the s.c. group. The t1/2 of 4R after i.m. and s.c. administration was approximately 1.5 hr. The brain peak concentration was 329 ng/g in the i.m. group and 323 ng/g for the s.c. group. The brain to plasma ratio in the i.v. group was 6.4, reached 10 min after dose, whereas in the i.m. and s.c. groups was 2.49 and 2.48, respectively, at 90 min after dose. Our data show that 4R crosses the BBB and concentrates in the brain where it exerts its neuroprotective effect.

Succinyl derivatives of N-tris (hydroxymethyl) methyl-2-aminoethane sulphonic acid: their effects on the frog neuromuscular junction.

Succinic anhydride (SA) dissolved in Ringer solution buffered with N-tris (hydroxymethyl) methyl-2-aminoethane sulphonic acid (SA-TES solution) potentiates the depolarizing action of acetylcholine (ACh, 10-40 microM) on frog muscle and the tension induced by bath application of this agonist. Applied from one side of a double-barrelled micropipette, SA-TES increases the amplitude of iontophoretically elicited ACh potentials. The potentiation of the effects of ACh by SA-TES does not involve changes in either the activity of the ACh esterase or the input resistance of the muscle membrane. For depolarizations of frog sartorius muscle, dose-response relationships obtained for ACh concentrations from 0.5 to 20 microM indicate that SA-TES increases the apparent affinity of ACh by a factor of 3. SA-TES exerts an "accelerating' effect on the responses elicited by bath-applied ACh; i.e., it increases the rate of depolarization when ACh is added to the bath and the rate of repolarization upon washing out. These effects are particularly marked in preparations treated with neostigmine (3 microM). SA-TES does not potentiate the depolarizing action of agonists which do not contain an ester group. Moreover, the time course of the responses elicited by these compounds is not influenced by SA-TES. SA-TES fails to influence significantly the effects of the neurally released transmitter. Only a 10% increase in the average amplitude of the endplate potentials was observed. SA hydrolyzes in about 30 min at room temperature; however the SA-TES solution retains its activity for several weeks. Succinate is inactive, and so is SA in Ringer buffered with phosphate. The SA-TES solution contains seven succinyl-TES derivatives, which were separated by ion-exchange chromatography and paper chromatography. At concentrations between 1 to 150 microM, these succinyl-TES derivatives affected the ACh-induced contraction of frog rectus abdominus muscle. The most abundant derivative potentiated the action of high doses of ACh, but was inhibitory at lower ones. The other derivatives were mostly inhibitory. These results are discussed in terms of two hypotheses. One postulates the presence of a diffusion barrier formed by groups that bind ACh and are saturated by SA-TES. The other assumes that SA-TES acts directly on the ACh receptor exerting its potentiating effect through a cooperative mechanism.

Spermine is neuroprotective against anoxia and N-methyl-D-aspartate in hippocampal slices.

Polyamines were implicated as either neurotoxic or neuroprotective in several models of stroke. Spermine augments the excitotoxicity mediated by the N-methyl-D-aspartate (NMDA) receptor because this receptor is activated at micromolar spermine concentrations. However, at higher concentrations, spermine could be neuroprotective because it blocks the NMDA receptor and voltage-activated Ca(2+) channels. In this work, acute hippocampal slices were exposed to 1 mM spermine and either 10 min of anoxia or 0.5 mM NMDA. The percent recovery of population spikes was the measure of neuroprotection. One millimolar spermine was robustly neuroprotective; however, 0.1 mM spermine and 1 mM putrescine were not. The neuroprotective concentration of spermine was higher than the physiological concentration of free spermine. However, during an excitotoxic episode, extracellular Ca(2+) is decreased, enabling the inhibitory activity of lower spermine concentration. In addition, several noxious stimuli trigger the release of intracellular spermine and could raise local levels of spermine. Therefore, it is possible that spermine has a neuroprotective role in vivo.

Spermine does not compete with omega-conotoxin GVIA in the striatum radiatum of the hippocampal slice.

The effect of spermine (Spm) and of omega-conotoxin GVIA (CTX) on the population excitatory postsynaptic potentials (pEPSP) in stratum radiatum of the CA1 area were compared. CTX decreased irreversibly the initial slope of pEPSP by 57%. Spm produced a maximum inhibition of 85% with an apparent dissociation constant of 0.85 mM and a maximum Hill coefficient larger than 3. The effect of Spm was mostly reversible. Preincubation with Spm did not protect the slice from the irreversible effect of CTX suggesting that they interact with different sites. Since CTX and Spm inhibited pEPSPs with very different affinities and reversibilities a kinetic model was developed to compare their effects. This model relates the inhibitors' binding to presynaptic voltage-activated Ca2+ channels (VACC) with inhibition of pEPSP. The model suggest that: all CTX and Spm effects can be explained by inhibition of VACC. Spm and CTX do not compete for the same site. CTX inhibits 20% (N-type) and Spm 40% of channels (probably the Q-type). More than three Spm molecules bind per one channel molecule, while one CTX is sufficient to inhibit channel function. The model also illustrates that the inhibitor concentration-pEPSP inhibition curves display a Hill coefficient similar to that for inhibitor binding.

Spermine increases paired-pulse facilitation in area CA1 of hippocampus in a calcium-dependent manner.

The effect of spermine on neurotransmission was studied in area CA1 of the hippocampal slice preparation. Paired-pulse stimulation (20 ms interpulse interval) was delivered to stratum radiatum; the evoked field potential responses were recorded simultaneously from stratum radiatum and from stratum pyramidale. At mM and sub-mM concentrations, spermine decreased the slope of pEPSP in stratum radiatum and the area of the conditioning population spike in stratum pyramidale. Short-latency paired-pulse inhibition of the population spike was converted to facilitation by spermine. These effects of spermine resembled those observed at low calcium concentration. In addition, dose-response and input-output curves determined at various Ca2+ concentrations demonstrated that the depressant effects of spermine were larger at low Ca2+ levels. The results support the notion that spermine competitively blocks presynaptic voltage-sensitive Ca2+ channels, thus causing a decreased release of neurotransmitter. Since spermine is present in brain, it is likely that it is a natural modulator of Ca2+ channels.

Spermine depresses NMDA, K/AMPA and GABAA-mediated synaptic transmission in the rat hippocampal slice preparation.

The effects of spermine, an endogenous polyamine, were examined in area CA1 of the rat hippocampal slice preparation. Spermine, at low millimolar concentrations, rapidly and potently depressed NMDA and K/AMPA-mediated population EPSPs, and GABA-mediated monosynaptic population IPSPs. These effects contrast with its well-known potentiation of NMDA currents at lower concentrations. Our results raise the possibility that the large intracellular stores of spermine that are released after various neural insults could act as an endogenous neuroprotective mechanism by limiting excessive calcium entry.

alpha-Difluoromethylornithine decreases inhibitory transmission in hippocampal slices independently of its inhibitory effect on ornithine decarboxylase.

We tested the effect of DL-alpha-(difluoromethyl)ornithine (DFMO), a specific inhibitor of ornithine decarboxylase (ODC), on recordings in area CA1 of rat hippocampal slices. In the concentration range in which it is used as an ODC inhibitor, DFMO increased neuronal excitability and blocked paired-pulse inhibition. The effect of DFMO was reversed by perfusing the slice with normal bathing solution. These effects were not attenuated by the simultaneous addition of putrescine; thus the activity of DFMO was not related to a decrease in putrescine caused by the inhibition of ODC. Mediation by the N-methyl-D-aspartate (NMDA) receptor was ruled out because DL-2-amino-5-phosphonovalerate (APV), an NMDA antagonist, did not block the effect of DFMO. Intracellular and extracellular recordings of pharmacologically isolated IPSPs supported the notion that DFMO depressed GABAergic transmission. DFMO has frequently been used as a tool to study the role of the ODC-polyamine system in neural preparations. This report suggests that the results from such studies must be interpreted with caution. In addition, our findings raise questions about the proposed use of DFMO as a neuroprotective agent against excitotoxicity.

Role of polyamines in experience-dependent brain plasticity.

Enriched experience increases brain growth, neuronal differentiation and learning abilities. Polyamines are modulators of growth and differentiation. We studied the effect of difluoromethylornithine (DFMO, an inhibitor of putrescine synthesis) on brain growth of rats exposed either to a complex or an impoverished environment. In both environmental conditions, DFMO decreased cortical putrescine by 50% and increased spermine by 13%; spermidine remained constant. Cortical RNA was not affected significantly by DFMO but DNA was decreased exclusively in rats exposed to the impoverished environment. Environmental complexity increased cortical weight, RNA and spermidine content. These differences were larger in DFMO-injected rats than in saline controls. Since stimulants such as amphetamines also enhance the environmental effects it was conceivable that DFMO might act as a stimulant. We have measured the effect of DFMO on rats' exploratory activity and found it decreased by the drug. Therefore the enhancing effect of DFMO cannot be explained by its behavioral activity. We propose that DFMO enhances the experience-dependent brain plasticity by facilitating differentiation of neurons.

Experience affects cortical but not subcortical polyamines.

The effect of brief periods of experience in an enriched environment (7 hours per day for 3 days), and of inhibition of polyamine synthesis was studied in four brain regions: occipital cortex, remaining cortex, subcortex and cerebellum plus medulla. Polyamine synthesis was inhibited by alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase. DFMO caused a 30-50% decrease in putrescine content in all brain areas, irrespective of the environmental treatment. Spermidine was decreased by the inhibitor in subcortex and in cerebellum plus medulla, while spermine was increased in remaining cortex. The regional differences in inhibitor effect suggest that the regulation of polyamine metabolism varies among the four brain areas. Experience increased the weight and spermidine content of remaining cortex and decreased putrescine content of occipital cortex. Noncortical areas were not affected. The effects of experience on polyamine levels were somewhat increased by DFMO. Therefore, experience did not have a generalized effect on polyamine levels; rather, each polyamine responded in a specific manner. In addition, polyamine levels were affected only in those brain areas which are known from previous studies to respond to environmental stimulation with weight increase. These facts suggest that polyamines might have a role in the regulation of experience-induced plasticity.

Putrescine decreases exploration of a black and white maze.

The effect of putrescine and cyclohexylamine on rat cortical polyamine concentration and on behavior in a black and white maze was studied. The levels of polyamines in brain cortex were determined 15 min, 2, 4, and 6 hours after injection of putrescine (200 or 400 mg/kg) or cyclohexylamine (380 mg/kg). Putrescine concentration increased 6-fold 15 min after injection of putrescine followed by a decline during the next 6 hours. Cyclohexylamine increased putrescine concentration doubling it 4 hours after injection. Spermidine and spermine concentrations did not change after either putrescine or cyclohexylamine injection. Behavior was studied in the Greek cross maze which provides the choice to enter either white or black compartments. Putrescine 200 mg/kg decreased entries into white but not black compartments, while putrescine 400 mg/kg decreased entries into both. The effect of cyclohexylamine was similar to putrescine 400 mg/kg. The behavioral effect of each treatment was independent of the time between injection and testing for up to 6 hours, while the levels of putrescine changed during the same period. Therefore, behavior was not directly related to total cortical putrescine.

alpha-Difluoromethylornithine does not antagonize the behavioral effects of putrescine.

alpha-Difluoromethylornithine (DFMO), a specific inhibitor of putrescine synthesis, is widely used in studies of polyamine function as well as clinically. We studied the effect of DFMO on the tendency to explore in the Greek cross maze that provides the rat with the choice to enter either white or black compartments. After a single injection of 400 mg/kg DFMO, the entries into white compartments were significantly decreased. A similar decrease had been observed previously with 200 mg/kg putrescine. Simultaneous administration of DFMO (400 mg/kg) and putrescine (200 mg/kg) resulted in decreased entries into both white and black compartments. When 400 mg/kg DFMO plus 400 mg/kg putrescine were injected, the entries into both compartments were further decreased and the time spent in white compartments was also decreased. This pattern mimicked that found with anxiogenic drugs. Injection of DFMO (400 mg/kg) produced no change in either putrescine, spermidine, or spermine concentration measured in brain cortex. Putrescine (200 mg/kg) plus DFMO produced the same transient increase in cortical putrescine as putrescine alone. We conclude that DFMO is mildly anxiogenic and that this activity is independent of its inhibition of putrescine synthesis.

Interaction with objects by group-living Saimiri sciureus.

We have studied the spontaneous interaction with small, inedible objects by group-living Saimiri. Twenty-one animals living in a large outdoor enclosure were presented with a set of 10 novel objects of diverse materials, colors, sizes, and shapes. We have used a sampling technique to record the number of monkeys interacting with each object; qualitative observations complemented these measurements. Next day the same objects were presented again and the observations continued. This experiment was repeated with some variations three times over a period of six months. The highest score for novel objects was obtained immediately upon objects presentation; afterwards the score sharply decreased for that day. On second presentation of the same objects the scores were more evenly distributed during the day, the cumulative total resembling that of day one. Objects' characteristics influenced the amount of interaction with them. The important factors seemed to be the material the objects were made of and their complexity. The objects that were contacted most frequently elicited intense investigative behavior and social play. These results differ from some previous works in which Saimiri were observed to interact only rarely with novel objects.

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.