Sarin-induced brain damage in rats is attenuated by delayed administration of midazolam.

Sarin poisoned rats display a hyper-cholinergic activity including hypersalivation, tremors, seizures and death. Here we studied the time and dose effects of midazolam treatment following nerve agent exposure. Rats were exposed to sarin (1.2 LD50, 108 µg/kg, im), and treated 1 min later with TMB4 and atropine (TA 7.5 and 5 mg/kg, im, respectively). Midazolam was injected either at 1 min (1 mg/kg, im), or 1 h later (1 or 5 mg/kg i.m.). Cortical seizures were monitored by electrocorticogram (ECoG). At 5 weeks, rats were assessed in a water maze task, and then their brains were extracted for biochemical analysis and histological evaluation. Results revealed a time and dose dependent effects of midazolam treatment. Rats treated with TA only displayed acute signs of sarin intoxication, 29% died within 24h and the ECoG showed seizures for several hours. Animals that received midazolam within 1 min survived with only minor clinical signs but with no biochemical, behavioral, or histological sequel. Animals that lived to receive midazolam at 1h (87%) survived and the effects of the delayed administration were dose dependent. Midazolam 5 mg/kg significantly counteracted the acute signs of intoxication and the impaired behavioral performance, attenuated some of the inflammatory response with no effect on morphological damage. Midazolam 1mg/kg showed only a slight tendency to modulate the cognitive function. In addition, the delayed administration of both midazolam doses significantly attenuated ECoG compared to TA treatment only. These results suggest that following prolonged seizure, high dose midazolam is beneficial in counteracting adverse effects of sarin poisoning.

Using MRI for the assessment of paraoxon-induced brain damage and efficacy of antidotal treatment.

Organophosphate intoxication induces neural toxicity as demonstrated in histological analysis of poisoned animals. Diffusion-weighted magnetic resonance imaging (DWMRI) enables early noninvasive characterization of biological tissues based on their water diffusion characteristics. Our objectives were to study the application of MRI for assessment of paraoxon-induced brain damage and the efficacy of antidotal treatments. Seventy-six rats were poisoned with paraoxon followed by treatment with atropine and obidoxime. The rats were then divided into five treatment groups consisting of midazolam after 1 or 30 min, scopolamine after 1 or 30 min and a no anticonvulsant treatment group. Five untreated rats served as controls. Animals underwent MRI on days 1, 8, 15, 29 and 50 post poisoning. Histological evaluation was performed on representative rat brains. Acute DWMRI effects, such as enhancement of temporal brain regions, and chronic effects such as ventricular enlargement and brain atrophy, depicted on T2-weighted MRI, were significantly more prominent in late anticonvulsant treatment groups. There was no significant difference between the neuroprotective effects of midazolam and scopolamine as shown by DWMRI. Early MRI abnormalities were found to correlate significantly with histological analysis of samples obtained 15 days post treatment. In conclusion, our results demonstrate the feasibility of using DWMRI for depiction of early cytotoxic response to paraoxon and T2-weighted MRI for later changes, thus enabling assessment of early/late brain damage as well as treatment efficacy in rats. The ability to depict these changes early and noninvasively may be applied clinically in the acute phase of organophosphate poisoning.

Subchronic exposure to low-doses of the nerve agent VX: physiological, behavioral, histopathological and neurochemical studies.

The highly toxic organophosphorous compound VX [O-ethyl-S-(isoporopylaminoethyl) methyl phosphonothiolate] undergoes an incomplete decontamination by conventional chemicals and thus evaporates from urban surfaces, e.g., pavement, long after the initial insult. As a consequence to these characteristics of VX, even the expected low levels should be examined for their potential to induce functional impairments including those associated with neuronal changes. In the present study, we developed an animal model for subchronic, low-dose VX exposure and evaluated its effects in rats. Animals were exposed to VX (2.25 microg/kg/day, 0.05 LD(50)) for three months via implanted mini osmotic pumps. The rapidly attained continuous and marked whole-blood cholinesterase inhibition (approximately 60%), fully recovered 96 h post pump removal. Under these conditions, body weight, blood count and chemistry, water maze acquisition task, sensitivity to the muscarinic agonist oxotremorine, peripheral benzodiazepine receptors density and brain morphology as demonstrated by routine histopathology, remained unchanged. However, animals treated with VX showed abnormal initial response in an Open Field test and a reduction (approximately 30%) in the expression of the exocytotic synaptobrevin/vesicle associate membrane protein (VAMP) in hippocampal neurons. These changes could not be detected one month following termination of exposure. Our findings indicate that following a subchronic, low-level exposure to the chemical warfare agent VX some important processes might be considerably impaired. Further research should be addressed towards better understanding of its potential health ramifications and in search of optimal countermeasures.

Efficacy of antidotal treatment against sarin poisoning: the superiority of benactyzine and caramiphen.

Sarin, a potent cholinesterase inhibitor, induces an array of toxic effects including convulsions and behavioral impairments. We report here on the protection provided by post-exposure antidotal treatments against a lethal dose of sarin (1.2xLD50) by scopolamine, benactyzine, trihexyphenidyl or caramiphen, administered 5, 10 or 20 min after the initiation of convulsions. A mixture of the oxime TMB4 and atropine (TA) was injected 1 min following poisoning a paradigm that may represent a scenario reminiscent of a terror incident. Surviving TA-treated rats exhibited marked tonic-clonic convulsions, weight loss, poor clinical status and abnormal cognitive performance as assessed by the Morris water maze. Additionally, a dramatic increase in the density of peripheral benzodiazepine receptors (PBRs), a faithful marker for neuronal damage, was noted. Animals treated 5 min after the development of toxic signs with benactyzine, trihexyphenidyl or caramiphen demonstrated control levels of PBR values, whereas scopolamine produced binding densities significantly above basal levels. Examined at the 10-min time point, scopolamine and trihexyphenidyl afforded no protection against brain damage and did not differ from TA-injected rats. All four drugs failed to significantly prevent the alterations when applied 20 min after onset of convulsions. Assessment of learning processes yielded similar results, where caramiphen exibited some protection at the 20-min time point. Our results show that caramiphen and benactyzine, agents with combined anticholinergic and antiglutamatergic pharmacological profiles, offer considerable shielding against sarin, even when their administration is delayed.

Efficacy of the bone injection gun in the treatment of organophosphate poisoning.

Immediate administration of antidotal treatment is crucial in severe organophosphate (OP) poisoning and the use of an open intravenous (i.v.) line might also be required. The state of casualties might prevent getting access to their veins. The bone injection gun (BIG) was established as a simple method for introducing an intraosseous (i.o.) line and could be applied while wearing a protective suit. The present study followed the pharmacokinetics of the anticonvulsive drug midazolam after i.o. administration in pigs compared with i.v. and the common intramuscular (i.m.) administration. A new method for monitoring midazolam concentrations in plasma was developed. Plasma concentrations following both i.v. and i.o. administrations peaked at 2 min post injection and only at 10 min following the i.m. route. In an antidotal treatment study against paraoxone poisoning, the anticonvulsive effect of midazolam appeared immediately following i.o. administration, while it took 5-10 min to exhibit a similar effect following i.m. administration. This study indicates that the use of i.o. administration after OP poisoning might provide the necessary fast response for rapid termination of convulsions. The BIG might offer a convenient method for treating casualties in the chemical arena by teams wearing full protective gear.

Extrapolating from animal studies to the efficacy in humans of a pretreatment combination against organophosphate poisoning.

The extrapolation from animal data to therapeutic effects in humans, a basic pharmacological issue, is especially critical in studies aimed to estimate the protective efficacy of drugs against nerve agent poisoning. Such efficacy can only be predicted by extrapolation of data from animal studies to humans. In pretreatment therapy against nerve agents, careful dose determination is even more crucial than in antidotal therapy, since excessive doses may lead to adverse effects or performance decrements. The common method of comparing dose per body weight, still used in some studies, may lead to erroneous extrapolation. A different approach is based on the comparison of plasma concentrations at steady state required to obtain a given pharmacodynamic endpoint. In the present study, this approach was applied to predict the prophylactic efficacy of the anticholinergic drug caramiphen in combination with pyridostigmine in man based on animal data. In two species of large animals, dogs and monkeys, similar plasma concentrations of caramiphen (in the range of 60-100 ng/ml) conferred adequate protection against exposure to a lethal-dose of sarin (1.6-1.8 LD(50)). Pharmacokinetic studies at steady state were required to achieve the correlation between caramiphen plasma concentrations and therapeutic effects. Evaluation of total plasma clearance values was instrumental in establishing desirable plasma concentrations and minimizing the number of animals used in the study. Previous data in the literature for plasma levels of caramiphen that do not lead to overt side effects in humans (70-100 ng/ml) enabled extrapolation to expected human protection. The method can be applied to other drugs and other clinical situations, in which human studies are impossible due to ethical considerations. When similar dose response curves are obtained in at least two animal models, the extrapolation to expected therapeutic effects in humans might be considered more reliable.

Subcellular alterations of protein kinase C isozymes in the rat brain after organophosphate poisoning.

The protein kinase C (PKC) signaling pathway has been associated with modulation of N-metyl-D-aspartate receptor activity, motor behavior, learning, and memory, all of which are severely impaired in organophosphate (OP) intoxication. Nevertheless, the role of PKC in OP intoxication is largely unknown. The present study attempted to characterize alterations in the immunoreactivity levels of PKC isozymes expressed in different brain areas in the rat following exposure to the nerve agent sarin (1x LD(50)). Furthermore, possible neuroprotective effect of selective PKC regulating peptide after such insult was evaluated. The results indicated that a significant reduction in the immunoreactivity level of the conventional betaII-PKC and the atypical zeta-PKC was observed in frontal cortex up to 24 h postsarin and in the striatum up to 5 days postsarin exposure. This reduction was in contrast to the increase in the immuno-reactivity level of both isozymes seen in the hippocampus or thalamus. Treatment with the anticonvulsant midazolam (0.5 mg/kg) 10 min postsarin exposure markedly reduced zeta-PKC immunoreactivity level and betaII-PKC in the membrane fractions in the hippocampus. betaII-PKC peptide (380 ng/kg), known to inhibit PKC translocation and activation, attenuated sarin-induced neuropathology. These observations suggest a role for both conventional and atypical PKC isozymes in OP-induced neuropathy in the rat and further support their involvement in cell death.

Monitoring drug-induced neurodegeneration by imaging of peripheral benzodiazepine receptors.

Several drugs of abuse are known to produce an array of deleterious effects, including alterations in neuronal circuitry and, ultimately, neuronal degeneration. For instance, methamphetamine was shown to induce substantial nigrostriatal dopaminergic terminal damage, including an increase in glial fibrillary acidic protein, a marker for astrocyte proliferation. Nevertheless, there was almost no attempt to define neurodegeneration by measuring the abundance of reactive microglia. In fact, some investigators fail to differentiate between astrocytes and microglia and claim glial fibrillary acidic protein to be a marker for gliosis. To date, there are numerous methods designed to assess brain neuropathologies resulting from a wide arsenal of insults. Regardless of the cause of neuronal damage, reactive glial cells always appear at and around the site of degeneration. These cells are distinguished by the exceptional abundance of peripheral benzodiazepine receptors (PBRs; omicron3 sites), particularly as compared to surrounding neurons. Measuring the binding of specific ligands to these PBRs (for example, [3H]PK 11195) offers a unique indirect marker for reliable impairment estimation in the central nervous system. Moreover, the availability of agents such as [11C]PK 11195 paved the road to in vivo animal and human brain positron emission tomography scanning, demonstrating inflammation-like processes in several diseases. Additionally, the measurement of increased binding of PBR ligands provides a faithful indicator for the behavioral and cognitive deficits accompanying neuronal injury.

Characterization of early plasma concentrations of midazolam in pigs after administration by an autoinjector.

The treatment of organophosphate-induced poisoning is based mainly on atropine and an oxime. Prompt anticonvulsive intervention is usually also required to terminate the ensuing seizure activity and to prevent delayed permanent brain damage. Midazolam, a water-soluble benzodiazepine agonist, has the advantage of rapid absorption following intramuscular administration. In mass casualty situations, the availability of an autoinjector, filled with midazolam, might be a further advantage. In the present study, the plasma pharmacokinetics of midazolam after administration by an autoinjector was compared with conventional intramuscular (i.m.) administration in two groups of four pigs each. During the first 15 min after injection, significantly higher plasma concentrations of midazolam were detected following autoinjector administration, compared with the i.m. injection. The physiological reflection of the accelerated midazolam absorption was a marked reduction in the time interval required for muscle relaxation, induced by midazolam. It is concluded that a midazolam autoinjector might be helpful in the mass casualty scenario following organophosphate poisoning.

Pharmacological modulation of cardiac gap junctions to enhance cardiac conduction: evidence supporting a novel target for antiarrhythmic therapy.

BACKGROUND: Disease-induced alterations of cardiac gap junctions lead to intercellular uncoupling, which is an important mechanism of arrhythmogenesis. Therefore, drugs that selectively open gap junctions potentially offer a novel strategy for antiarrhythmic therapy. Because the peptide ZP123 was found to increase conductance between paired myocytes, we hypothesized that ZP123 would suppress acidosis-induced gap junction closure in the intact heart. METHODS AND RESULTS: High-resolution optical mapping was used to measure conduction velocity (CV) and action potential duration from ventricular epicardium of Langendorff-perfused guinea pig hearts at baseline (pH 7.4) and during 45 minutes of perfusion with acidotic (pH 6.0) Tyrode's solution with (n=8) and without (control, n=7) ZP123 (80 nmol/L). Acidosis produced conduction slowing transverse (29.1+/-0.1 to 16.8+/-0.2 cm/s, P<0.0001) and longitudinal (47.2+/-2.4 to 33.2+/-4.8 cm/s, P<0.0001) to cardiac fibers. Importantly, ZP123 inhibited conduction slowing during acidosis by approximately 60%. The peak effect of ZP123 was achieved after 16 minutes of acidosis, consistent with inhibition of uncoupling. ZP123 did not affect Na+ current in isolated myocytes, additionally affirming that preservation of CV was attributable to the compound's action on gap junctions. ZP123 had no effect on CV in the absence of acidosis, suggesting that drug activity targets gap junctions under metabolic stress. Action potential duration heterogeneity was significantly reduced by ZP123 (6.7+/-0.8 ms) compared with controls (9.7+/-3.1 ms, P<0.05), presumably by enhancing cell-to-cell coupling. CONCLUSIONS: These data suggest that ZP123 significantly attenuates gap junction closure during acidosis. Preservation of intercellular coupling diminished CV slowing and heterogeneous repolarization, eliminating arrhythmogenic substrates.

Anticholinergic and antiglutamatergic agents protect against soman-induced brain damage and cognitive dysfunction.

Soman, a powerful inhibitor of acetylcholinesterase, causes an array of toxic effects in the central nervous system including convulsions, learning and memory impairments, and, ultimately, death. We report on the protection afforded by postexposure antidotal treatments, combined with pyridostigmine (0.1 mg/kg) pretreatment, against these consequences associated with soman poisoning. Scopolamine (0.1 mg/kg) or caramiphen (10 mg/kg) were administered 5 min after soman (1.2 LD50), whereas TAB (i.e., TMB4, atropine, and benactyzine, 7.5, 3, and 1 mg/kg, respectively) was injected in rats concomitant with the development of toxic signs. Atropine (4 mg/kg) was given to the two former groups at the onset of toxic symptoms. Caramiphen and TAB completely abolished electrographic seizure activity while scopolamine treatment exhibited only partial protection. Additionally, no significant alteration in the density of peripheral benzodiazepine receptors was noted following caramiphen or TAB administration, while scopolamine application resulted in a complex outcome: a portion of the animals demonstrated no change in the number of these sites whereas the others exhibited markedly higher densities. Cognitive functions (i.e., learning and memory processes) evaluated using the Morris water maze improved considerably by the three treatments when compared to soman-injected animals; the following rank order was observed: caramiphen > TAB > scopolamine. Additionally, statistically significant correlations (r = 0.72, r = 0.73) were demonstrated between two learning parameters and [3H]Ro5-4864 binding to brain membrane. These results show that drugs with a pharmacological profile consisting of anticholinergic and antiglutamatergic properties such as caramiphen and TAB, have a substantial potential as postexposure therapies against intoxication by organophosphates.