Eran Gilat

BENEFICIAL EFFECTS OF TOPICAL ANTI-INFLAMMATORY DRUGS AGAINST SULFUR MUSTARD-INDUCED OCULAR LESIONS IN RABBITS

Ocular injuries following sulfur mustard (HD) exposure are characterized by an inflammatory response, observed as eyelid swelling, conjunctivitis, corneal oedema and cellular infiltration starting 1–4 h after exposure, depending on dose. These effects heal partially during the first 1–2 weeks after exposure, with the later appearance of neovascularization, recurrent erosions and recurrent oedema of the cornea (delayed response). We have shown previously that topically applied steroid treatment, administered after HD exposure, attenuated the extent of neovascularization, one of the characteristics of delayed ocular pathology in rabbits.

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.

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

Abstract: 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; ο3 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.

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. Copyright

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.

Triggered activity induced by K(+)-free, Na(+)-deficient solution in guinea pig ventricular muscle: the effects of ouabain, lidocaine, and Ca2+ channel blockers.

Triggered activity induced by delayed afterdepolarizations has been suggested as a cellular mechanism for some arrhythmias. The development of triggered activity should be favored by conditions that increase myoplasmic Ca2+ and increase membrane resistance. A solution which could create these conditions was devised. After perfusion with normal Tyrodes solution, 24 trabeculae were exposed to the experimental solution. All preparations developed triggered activity after exposure to the experimental solution, 83% developed delayed afterdepolarizations before the onset of triggered activity, and triggered activity stopped in all trabeculae after reperfusion with normal Tyrodes solution. The interaction of ouabain, lidocaine, and three Ca2+ channel blockers with triggered activity induced by the experimental solution is described. Verapamil inhibited triggered activity but not underlying voltage oscillations, whereas nisoldipine and Mn2+ inhibited both triggered activity and voltage oscillations. Lidocaine did not inhibit afterdepolarizations or triggered activity. Exposure to ouabain for 10 min caused delayed afterdepolarizations but not triggered activity. Our results show that the experimental solution induced triggered activity, which was highly reliable and readily reversible. The high reproducibility of this activity enables the study of interactions of pharmacological agents with this triggered activity. This may contribute to the further understanding of the mechanism underlying some arrhythmias.

Protection of Guinea Pigs against Soman Inhalation by Pretreatment Alone with Human Butyrylcholinesterase

Multi-drug therapy (i. e. pyridostigmine, atropine, oximes, diazepam) is generally considered as effective against organophosphorus (OP) toxicity. However, this treatment raises several problems: 1. It is limited in its protection range. 2. It does not eliminate several post exposure symptoms, and 3. For fast acting OPs self injection of atropine and oximes may not be feasible. Prophylactic antidotes that will scavenge the OP in the blood may afford reasonable solutions for these limitations. Human butyrylcholinesterase (HuBChE) was previously evaluated by us both in vitro and in vivo as a single prophylactic antidote against the lethal effects of nerve agents. Remarkable protection has been demonstrated in Hu-BChE-treated mice (Raveh et al., 1993), rats (Raveh et al., 1993; Brandeis et al., 1993) and monkeys (Ashani et al., 1993) following an iv exposure to lethal doses of sarin, soman, VX and tabun. Since inhalation challenge is the most realistic simulation of exposure to nerve agents, the protection afforded by HuBChE was tested against inhaled soman. Awake animals caged in a whole body plethysmograph designed and built in our laboratory, were exposed for 45 to 75 sec to 417–450 μg/L soman. Five out of 8 animals with 28 to 45 nmol circulating HuBChE/animal were completely protected against 1.3–2.4×LD50 doses of inhaled soman. Two animals displayed slight tremors and ataxia while one guinea pig exposed to 2.6×LD50 died. A linear correlation (r =.946) was established between nmols of inhaled soman and the reduction in the levels of circulating HuBChE with a slope of 0.13. Since the soman to HuBChE ratio required to inhibit in vivo the exogenous enzyme is approximately 1.2, it is suggested that only 16% of inhaled soman reached the circulation.