Inbal Egoz

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.

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.

Efficacy Assessment of Various Anticholinergic Agents Against Topical Sarin-Induced Miosis and Visual Impairment in Rats

Eye exposure to the organophosphorus (OP) irreversible acetylcholinesterase inhibitor sarin results in long-term miosis and reduction in visual function. Anticholinergic drugs, such as atropine or homatropine, which are used topically in order to counter these effects may produce mydriasis and partial cycloplegia, which may worsen visual performance. This study was aimed to test the efficacy of short-acting anticholinergic drugs against sarin-induced miosis and visual impairment, which will minimally insult vision. Long-Evans rats, exposed topically to various sarin doses from 0 to 10 μg, showed a dose-dependent miosis, which returned to pre-exposure levels within 24-48 h. Tropicamide treatment rapidly widened the miotic effect to a different extent depending on time following treatment and dosage given. Cyclopentolate, however, showed a delayed response that finally widened the pupils in a dose-dependent manner. Atropine treatment showed a rapid widening of the pinpoint pupils exceeding baseline level finally causing mydriasis. Light reflex test showed that the contraction ability of the iris following atropine treatment was impaired, as opposed to the use of tropicamide which facilitated the iris contraction, similar to control. Finally, tropicamide and atropine treatments ameliorated the visual impairment, as opposed to cyclopentolate, which worsened visual performance. Considering that tropicamide treatment against sarin exposure did not cause mydriasis nor did it impair the iris contraction flexibility as a response to light, the use of this drug should be taken into consideration as a first-choice topical treatment against OP intoxication.

Efficacy assessment of a combined anticholinergic and oxime treatment against topical sarin‐induced miosis and visual impairment in rats

Eye exposure to the organophosphorus (OP) irreversible cholinesterase inhibitor sarin results in long‐term miosis and impaired visual function. We have previously shown that tropicamide is better at ameliorating this insult than topical atropine or cyclopentolate. However, to minimize side effects associated with repeated tropicamide applications and high treatment doses, we evaluated the effects of oximes (ChE re‐activators) alone and combined with tropicamide at ameliorating OP‐induced ocular impairments.

Retinal damage following exposure to pulsed Nd:YAG laser radiation in rabbits and its relation to physical parameters

The aim of the present study was to characterize permissible exposure limits (MPE) for safety analysis, with an emphasis on the immediate retinal damage, following Nd:YAG Q-switched laser radiation, and to test its correlation to physical parameters. Pigmented rabbits were exposed to Nd:YAG laser radiation (532nm, pulse duration: 20ns) in various energies. Exposures were conducted in retina tissue, very close to the optic nerve, with a total of 20 exposures per retina. Retinas were viewed during the first 10 min following exposure, using an on-line digital video camera. Thereafter, animals were sacrificed for histological evaluation. A part of the retinas were evaluated 24 hours post exposure. A quantitative analysis of the clinical findings, based on a severity score scale and a morphometric analysis of the extent of the lesions, was used to test the statistical relationship with the laser energy and number of pulses. In addition, hemorrhage threshold values were computed using Probit Analysis. Retinal damage, at various levels of severity, was observed immediately after exposure to energies above 10μJ, characterized by edema and subretinal hemorrhages. The appearance and severity of the lesions varied among animals, between fellow eyes and even within the same retina. The relationship between severity and extent of lesions, and energy levels and number of pulses was evaluated. The ED50 for various, immediate types of hemorrhage was determined, and correlated to physical parameters. Histological observations strengthened the clinical findings. The results were discussed in accordance with photomechanical and thermal theories of laser-tissue interactions.

Retinal damage following exposure to single pulses of Nd:YAG laser radiation in rabbits and its relation to energy levels

Purpose: The aim of the present study was to characterize permissible exposure limits (MPE) for safety analysis, with an emphasis on the immediate retinal damage following SHG of Nd:YAG Q-Switched laser radiation and to test its correlation to physical parameters. Methods: Pigmented rabbits (n=14) were exposed to single pulses of Nd:YAG laser radiation (532nm, pulse duration:8-12ns) in various energies ranging from 10 to 150 μJ. Exposures were conducted in retina tissue, very close to the optic nerve, with a total of 20 exposures per retina. Retinas were viewed during the first 15 min following exposure, using an on-line digital video camera. Thereafter, animals were sacrificed for histological evaluation. A quantitative analysis of the clinical findings, based on a severity score scale and a morphometric analysis of the extent of the lesions, was used to test the relationship with the laser energy. In addition, hemorrhage thresholds were computed using Probit Analysis. Results: Retinal damage, at various levels of severity, was observed immediately after exposure to energies above 26 μJ, characterized by edema and sub-retinal hemorrhages. The appearance and severity of the lesions varied among animals, between fellow eyes and even within the same retina. The ED50 for immediate pre-retinal hemorrhage was determined as 83μJ and the lesions’ diameter ranged from 141-640μ. A significant correlation (R=0.80, P<0.0001) was found between the extent of the lesions and energy levels. The diameter of the lesions showed a linear (P<0.008) increase with the laser energy. The histological observations indicated elevation of retinal layers and extensive damage in the outer segment of the photoreceptors and in the pigmented epithelial cells layer. Conclusions: A linear, laser-retinal tissue interaction was found immediately following exposure to single pulses of Nd:YAG laser radiation. It is suggested that unlike argon laser, which produces a thermal burn to the eye, Nd:YAG laser damage is a result of a combination of photo-mechanical and thermal mechanism.

Optimization of the Ocular Treatment Following Organophosphate Nerve Agent Insult

Eye exposure to organophosphate (OP) irreversible acetylcholinesterase inhibitors, results in long-term miosis and impaired visual function. The aim of this study was to find an anticholinergic antidote, which would counteract miosis and visual impairment induced by the nerve agents sarin and VX with minimal untoward side-effects. Rat pupil width and light reflex were evaluated from 15 min up to 2 weeks following topical OP exposure with or without topical ocular treatment of atropine or homatropine or with a combined intramuscular treatment of trimedoxime (TMB-4) and atropine (TA). Visual function following insult and treatment was assessed using a cued Morris water maze task. Topical VX exposure showed a dose-dependent miosis with a significant reduction in visual function similar to the effect seen following sarin exposure. Homatropine (2%; w/v) and atropine (0.1%; w/v) treatment ameliorated both sarin and VX-induced miosis and the resulting visual impairment. TA treatment was sufficient in ameliorating the sarin-induced ocular impairment while an additional ocular treatment with either 0.1% atropine or 2% homatropine was necessary following VX exposure. To conclude the use of 0.1% atropine or 2% homatropine was beneficial in ameliorating the ocular insult following VX or sarin ocular exposure and thus should be considered as universal treatments against this intoxication. The findings also emphasize the necessity of additional ocular treatment to the systemic treatment in visually impaired casualties following VX exposure.

Synergism Between Anticholinergic and Oxime Treatments Against Sarin-Induced Ocular Insult in Rats

Eye exposure to the extremely toxic organophosphorus sarin results in long-term miosis and visual impairment. As current treatment using atropine or homatropine eye drops may lead to considerable visual side effects, alternative combined treatments of intramuscular (im) oximes (16.8 µmol/kg, im) with atropine (0.5 mg/kg, im) or with the short acting antimuscarinic tropicamide (0.5%; w/v) eye drops were thus evaluated. The combined treatments efficacy following topical exposure to sarin (1 µg) was assessed by measuring pupil width and light reflex using an infra-red based digital photographic system. Results showed that the combined treatment of various oximes with atropine or with topical tropicamide eye drops rapidly reversed the sarin-induced miosis and presented a long-term improvement of 67-98% (oxime+tropicamide) or 84-109% (oxime+atropine) in pupil widening as early as 10-min following treatment. This recovery was shown to persist for at least 8-h following exposure. All combined treatments facilitated the ability of the iris to contract following sarin insult as tested by a light reflex response.Our findings emphasize the high efficacy of im oxime treatment combined with either atropine im or tropicamide eye drops in counteracting sarin-induced ocular insult. Therefore, in a mass casualty scenario the systemic combined treatment may be sufficient to ameliorate sarin-induced ocular insult with no need for additional, topical anticholinergic treatment at least in the initial stage of intoxication. For very mild casualties, who are unlikely to receive im treatment, the combined oxime (im) with topical tropicamide treatment may be sufficient in ameliorating the ocular insult.

Optical system for exposure of rabbit eyes to laser light and in situ assessment of retinal damage

An optical system designed for exposure of rabbit eyes to laser radiation and in-situ retinal damage assessment is presented. The laser radiation is of 2nd harmonic Q-switched Nd:YAG laser at 532 nm. The system is designed for multiple exposures at a regular grid array within a pre-determined region of the retina. Damage assessment is done in real time parallel to the exposure process. We present experimental results that demonstrate the versatility of the system for the determination of the threshold for laser-induced retinal damage in rabbit eye.

Histological aspects of retinal damage following exposure to pulsed Nd:YAG laser radiation in rabbits: indication for mechanism

The severity and characteristics of retinal injury following laser radiation derived from laser and tissue related factors. We have previously shown that retinal damage following Nd:YAG Q-switched laser radiation in rabbits was related to physical parameters, i.e. energy levels and number of pulses. Yet, an extremely large variability in the severity of the damage was found under similar exposure paradigms, even within the same retina. This emphasizes the role of the biological variables in the pathological mechanism of laser-induced retinal damage. The aim of the present study was to further study histological parameters of the injury in relation to retinal site and to elucidate their role in the initiation and characteristics of the damage, following various energy levels (10-50 &mgr;J) and number of pulses (1-4). Pigmented rabbits were exposed to Nd:YAG laser radiation (532nm, pulse duration: 20ns). Exposures were conducted in retina tissue, adjacent to the optic nerve, with a total of 20 exposures per retina. Animals were sacrificed 15 min or 24 hours post exposure, eyes enucleated and processed for paraffin embedding. 4&mgr;m thick serial sections, stained with hematoxylin and eosin, were examined under light microscopy. Two major types of retinal damage were observed: focal edema confined to the pigmented epithelium and the photoreceptor cells, and hemorrhages, associated with destruction of retinal tissue. While focal edema associated with slight elevation of the photoreceptor layer seems to depend on the pigmented epithelium, hemorrhages were related also to the choroid vasculature at the site of radiation. It is suggested that a thermo-mechanical mechanism is involved in laser induced retinal hemorrhages at energies above 10-30&mgr;J (2-1 pulses, respectively).